US009056899B2

(12) United States Patent (10) Patent No.: US 9,056,899 B2 Collins et al. (45) Date of Patent: Jun. 16, 2015

(54) ENGINEERED BACTERIOPHAGES AS FOREIGN PATENT DOCUMENTS ADUVANTS FOR ANTIMICROBAL AGENTS AND COMPOSITIONS AND METHODS OF EP O112803 4f1984 USE THEREOF OTHER PUBLICATIONS (75) Inventors: James J Collins, Newton Center, MA Alekshun et al. Molecular mechanisms of antibacterial multidrug (US); Timothy Kuan-Ta Lu, Boston, resistance. Cell 128, 1037-1050 (2007). MA (US) Avery, S.V. Microbial cell individuality and the underlying sources of heterogeneity. Nat Rev Microbiol 4, 577-587 (2006). (73) Assignees: Trustees of Boston University, Boston, Balaban et al. Bacterial persistence as a phenotypic Switch. Science MA (US); Massachusetts Institute of 305, 1622-1625 (2004). Technology, Cambridge, MA (US) Beaber et al. SOS response promotes horizontal dissemination of resistance genes. Nature 427, 72-74 (2004). Bergstrom et al. Ecological theory Suggests that (*) Notice: Subject to any disclaimer, the term of this cycling will not reduce antimicrobial resistance in hospitals. Proc patent is extended or adjusted under 35 Natl AcadSci U S A 101, 13285-13290 (2004). U.S.C. 154(b) by 744 days. Bonhoeffer et al. Evaluating treatment protocols to prevent antibiotic resistance. Proc Natl AcadSci U S A94, 12106-12111 (1997). (21) Appl. No.: 12/812,212 Brown et al., Antibiotic cycling or rotation: a systematic review of the evidence of efficacy. Journal of Antimicrobial , 55, 6-9 (22) PCT Filed: Jan. 12, 2009 (2005). Brissow, H. Phage therapy: the Escherichia coli experience. (86). PCT No.: PCT/US2009/030755 Microbiology 151, 2133-2140 (2005). S371 (c)(1), Chait et al. Antibiotic interactions that select against resistance. Nature 446, 668-671 (2007). (2), (4) Date: Aug. 31, 2010 Chang et al. with Vancomycin-resistant Staphylococcus aureus containing the VanA resistance gene, NE Journal of Medicine (87) PCT Pub. No.: WO2009/108406 348, 1342-1347 (2003). PCT Pub. Date: Sep. 3, 2009 Curtin et al. Using Bacteriophages to reduce formation of catheter associated biofilms by Staphylococcus epidermis, (2006) (65) Prior Publication Data Antimicrob. Agents Chemother. 50; 1268-1275. Dwyer, D.J., Kohanski, M.A., Hayete, B. & Collins, J.J. Gyrase US 2010/0322903A1 Dec. 23, 2010 inhibitors induce an oxidative damage cellular death pathway in Escherichia coli. Mol Syst Biol 3, 91 (2007). From the Centers for Disease Control and Prevention. Four pediatric Related U.S. Application Data deaths from community-acquired methicillin-resistant Staphylococ cus aureus—Minnesota and North Dakota, 1997-1999. JAMA. (60) Provisional application No. 61/020,197, filed on Jan. Hagens et al. Genetically modified filamentous phage as bactericidal 10, 2008. agents: a pilot study. Lett. Appl. Microbiol. 37, 318-323 (2003). (51) Int. Cl. Hagens et al. Augmentation of the antimicrobial efficacy of antibiot AOIN 63/00 (2006.01) ics by filamentous phage. Microb Drug Resist 12, 164-168 (2006). CI2N 7/01 (2006.01) (Continued) C07K I4/005 (2006.01) A6 IK35/76 (2015.01) Primary Examiner — Michael Burkhart CI2N IS/II3 (2010.01) (74) Attorney, Agent, or Firm — Nixon Peabody, LLP (52) U.S. Cl. CPC ...... C07K 14/005 (2013.01); A61 K35/76 (57) ABSTRACT (2013.01); C12N 15/I 13 (2013.01); C12N The present invention relates to the treatment and prevention 2320/32 (2013.01); C12N 2330/30 (2013.01); of bacteria and bacterial . In particular, the present CI2N2795/14 122 (2013.01); C12N invention relates to engineered bacteriophages used in com 2795/14132 (2013.01) bination with antimicrobial agents to potentiate the antimi (58) Field of Classification Search crobial effect and bacterial killing by the antimicrobial agent. None The present invention generally relates to methods and com See application file for complete search history. positions comprising engineered bacteriophages and antimi crobial agents for the treatment of bacteria, and more particu (56) References Cited larly to bacteriophages comprising agents that inhibit U.S. PATENT DOCUMENTS antibiotic resistance genes and/or cell Survival genes, and/or bacteriophages comprising repressors of SOS response genes 4,559,078 A 12/1985 Maier or inhibitors of antimicrobial defense genes and/or express 4,677,217 A 6, 1987 Maier ing an agent which increases the sensitivity of bacteria to an 4,678,750 A 7/1987 Vandenbergh et al. 6,335,012 B1 1/2002 Fischetti et al. antimicrobial agent in combination with at least one antimi 6,699,701 B1 3, 2004 Sulakvelidze et al. crobial agent, and their use thereof. 2005, 000403.0 A1 1/2005 Fischetti et al. 2012/03.01433 A1* 11/2012 Lu et al...... 424.93.2 20 Claims, 28 Drawing Sheets US 9,056,899 B2 Page 2

(56) References Cited Ubeda et al. Antibiotic-induced SOS response promotes horizontal dissemination of pathogenicity island-encoded virulence factors in OTHER PUBLICATIONS staphylococci. Mol. Microbiol. 56, 836-844 (2005). Hagens et al. Therapy of experimental pseudomonas infections with Vandenesch et al. Community-acquired methicillin-resistant a nonreplicating genetically modified phage. Antimicrob. Agents Staphylococcus aureus carrying Panton-Valentine leukocidin genes: Chemother. 48, 3817-3822 (2004). worldwide emergence. Emerg. Infect. Dis. 9, 978-984 (2003). Hall, B.G. Predicting the evolution of antibiotic resistance genes. Nat Vázquez-Laslop et al. Increased persistence in Escherichia coli Rev Microbiol 2, 430-435 (2004). caused by controlled expression of toxins or other unrelated proteins. Hall-Stoodley et al. Bacterial biofilms: from the natural environment J. Bacteriol. 188, 3494-3497 (2006). to infectious diseases. Nat Rev Microbiol 2,95-108 (2004). Walsh, C. Where will new come from? Nat Rev Microbiol Heitman et al. Phage Trojan horses: a conditional expression system 1, 65-70 (2003). for lethal genes. Gene 85, 193-197 (1989). Huff et al...Therapeutic Efficacy of Bacteriophage and Baytril Wang et al. Platensinycin is a selective FabF inhibitor with potent () Individually and in Combination to Treat Colibacil antibiotic properties. Nature 441, 358-361 (2006). losis in Broilers, Poultry Science, 83, 1994-1947 (2004). Wise, R. The relentless rise of resistance? J. Antimicrob. Chemother. Klevens et al. Invasive methicillin-resistant Staphylococcus aureus 54, 306–310 (2004). infections in the United States. JAMA 298, 1763-1771 (2007). Wiuff et al. Phenotypic tolerance: antibiotic enrichment of Kohanski et al. A common mechanism of cellular death induced by noninherited resistance in bacterial populations. Antimicrob. Agents bactericidal antibiotics. Cell 130, 797-810 (2007). Chemother. 49, 1483-1494 (2005). Korch et al. Ectopic overexpression of wild-type and mutant hipA Yacoby et al...Targeting antibacterial agents by using drug-carrying genes in Escherichia coli: effects on macromolecular synthesis and filamentous bacteriophages, Antimicrobial Agents and Chemo persister formation. J. Bacteriol. 188, 3826-3836 (2006). therapy, 50, 2087-2097 (2006). Levin et al. Cycling antibiotics may not be good for your health. Proc Yacoby et al., Targeted drug-carrying bacteriophages as antibacterial Natl AcadSci USA 101, 13101-13102 (2004). nanomedicines, Antimicrobial Agents and Chemotherapy, 51,2156 Levy et al. Antibacterial resistance worldwide: causes, challenges 2163 (2007). and responses. Nat. Med. 10, S122-S129 (2004). Hummel, A et al., "Characterisation and transfer of antibiotic resis Lewis, K. Persister cells and the riddle of biofilm Survival. Biochem tance genes from enterococci isolated from food.” Systematic and istry (Mosc). 70,267-274 (2005). Applied Microbiology 30:1-7, 2006. Lewis, K. Persister cells, dormancy and infectious disease. Nat Rev Kwon, NH et al., “Staphylococcal cassette chromosome mec Microbiol (2006). (SCCmec) characterization and molecular analysis for methicillin Loose et al. A linguistic model for the rational design of antimicrobial resistant Staphylococcus aureus and novel SCCmec Subtype IVg peptides. Nature 443, 867-869 (2006). isolated from bovine milk in Korea.” Journal of Antimicrobial Che Lorch, A. “Bacteriophages: An alternative to antibiotics?” motherapy 56:624-632, 2005. Biotechnology and Development Monitor, No. 39, pp. 14-17 (1999). Westwater, Cet al., “Use of Genetically Engineered Phage to Deliver Martinez et al. Mutation frequencies and antibiotic resistance. Antimicrobial Agents to Bacteria: an Alternative Therapy for Treat Antimicrob. Agents Chemother. 44, 1771-1777 (2000). ment of Bacterial Infections.” Antimicrobial Agents and Chemo Morens et al. The challenge of emerging and re-emerging infectious therapy 47(4): 1301-1307, 2003. diseases. Nature 430, 242-249 (2004). Lu, T Combating Biofilms and Antibiotic Resistance Using Synthetic Projan, S. Phage-inspired antibiotics? Nat. Biotechnol. 22, 167-168 Biology. DSPACE(aMIT, Dec. 11, 2008. (2004). Lu, TK "Curriculum Vitae.” Internet Article, pp. 1-8. Salyers et al. Human intestinal bacteria as reservoirs for antibiotic Yanisch-Perron C et al., “Improved M-13 Phage Cloning Vectors and resistance genes. Trends Microbiol. 12, 412-416 (2004). Host Strains Nucleotide Sequence of the M-13MP-18 and PUC-19 Schoolnik et al. Phage offer a real alternative. Nat. Biotechnol. 22. Vectors.” Database Biosis, Biosciences Information Service, Phila 505-507 (2004). delphia, PA. Database Accession No. PREV 198580021779, Gene Shah et al. Persisters: a distinct physiological state of E. coli. BMC 33(1): 103-119, 1985 (Abstract). Microbiol. 6, 53 (2006). Lu, TK and JJ Collins, “Dispersing biofilms with engineered enzy Soulsby, E.J. Resistance to in humans and animals. matic bacteriophage.” PNAS 104 (27): 11 197-11202, 2007. BMJ 331, 1219-1220 (2005). Lu, TK and JJ Collins, “Engineered bacteriophage targeting gene Soulsby, L. Antimicrobials and animal health: a fascinating nexus. J. networks as adjuvants for antibiotic therapy.” PNAS 106(12):4629 Antimicrob. Chemother. 60 Suppl 1, i77-i78 (2007). 4634, 2009. Summers, W.C. Bacteriophage therapy. Annu. Rev. Microbiol. 55. 437-451 (2001). * cited by examiner U.S. Patent Jun. 16, 2015 Sheet 1 of 28 US 9,056,899 B2

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US 9,056,899 B2 1. 2 ENGINEERED BACTERIOPHAGES AS developed resistance to Vancomycin, the only uniformly ADUVANTS FOR ANTMICROBAL AGENTS effective antibiotic against Staphylococci, by receiving van AND COMPOSITIONS AND METHODS OF comycin-resistance genes via conjugation from co-infecting USE THEREOF Enterococcus faecalis, which itself became completely resis tant to Vancomycin in nosocomial settings by 1988''''. CROSS REFERENCE TO RELATED Drugs such as that induce the SOS response can APPLICATIONS even promote the horizontal dissemination of antibiotic resis tance genes by mobilizing genetic elements''. For This application is a National Phase Entry Application example, Streptococcus pneumoniae and Neisseria gonor under 35 U.S.C. S371 of co-pending International Applica 10 rhoeae have also obtained resistance to antibiotics (Morens, tion PCT/US2009/030755, filed 12 Jan. 2009, which claims et al., (2004) Nature 430: 242-249). Sub-inhibitory concen benefit under 35 U.S.C. 119(e) of U.S. Provisional Patent trations or incomplete treatment courses can present evolu Application Ser. No. 61/020,197 filed 10 Jan. 2008, the con tionary pressures for the development of antibiotic resis tents of which are incorporated herein by reference in their tance'7. Use of antibiotics outside of clinical settings, for entirety. 15 example in livestockfor the agricultural industry, has contrib uted to the emergence of resistant organisms such as methi GOVERNMENT SUPPORT cillin-resistant staphylococci and is unlikely to abate due to This invention was made with the Government support economic reasons and modern farming practices''. Resis under Contract No. EF-0425719 awarded by the National tance genes that develop in non-clinical settings may be Sub Science Foundation (NSF) and Contract No. OD003644 sequently transmitted to bacterial populations which infect awarded by the National Institutes of Health (NIH). The humans, worsening the antibiotic resistance problem'. Government has certain rights in the invention. In addition to acquiring antibiotic-resistance genes, a small Subpopulation of cells known as persisters can Survive anti FIELD OF THE INVENTION 25 biotic treatment by entering a metabolically-dormant state’ 19.20. Persister cells do not typically carry genetic mutations The present invention relates to the field of treatment and but rather exhibit phenotypic resistance to antibiotics'. In prevention of bacteria and bacterial infections. In particular, Escherichia coli, the fraction of a population which repre the present invention relates to engineered bacteriophages sents persister cells increases dramatically in late-exponential used in combination with antimicrobial agents to potentiate 30 and stationary phases. Chromosomally-encoded toxins may the antimicrobial effect and bacterial killing of the antimicro be important contributors to the persister phenotype but the bial agent. underlying mechanisms that control the stochastic persis tence phenomena are not well understood’’. Persisters BACKGROUND constitute a reservoir of latent cells that can begin to regrow 35 once antibiotic treatment ceases and may be responsible for Bacteria rapidly develop resistance to antibiotic drugs the increased antibiotic tolerance observed in bacterial bio within years of first clinical use". Antibiotic resistance can be films”. By surviving treatment, persisters may play an acquired by horizontal gene transfer or result from persis important role in the development of mutations or acquisition tence, in which a small fraction of cells in a population exhib of genes that confer antibiotic resistance. its a non-inherited tolerance to antimicrobials. Since antimi 40 Several strategies have been proposed for controlling anti crobial drug discovery is increasingly lagging behind the biotic resistant infections. New classes of antibiotics would evolution of antibiotic resistance, there is a pressing need for improve the arsenal of drugs available to fight antibiotic new antibacterial therapies. resistant bacteria but few are in pharmaceutical pipelines. Bacterial infections are responsible for significant morbid Surveillance and containment measures have been instituted ity and mortality in clinical settings. Though the advent of 45 in government and hospitals so that problematic infections antibiotics has reduced the impact of bacterial diseases on are rapidly detected and isolated but do not address the fun human health, the constant evolution of antibiotic resistance damental evolution of resistance'. Cycling antibiotics is one poses a serious challenge to the usefulness of today's antibi method of controlling resistant organisms but is costly and otic drugs'. Infections that would have been easily cured by may not be efficacious’. Reducing the overprescribing of antibiotics in the past are now able to Survive to a greater 50 antibiotics has only moderately reduced antibiotic resis extent, resulting in sicker patients and longer hospitaliza tance’. Efforts have been also made to lessen the use of tions. The economic impact of antibiotic-resistant infec antibiotics in farming but some use is inevitable'. Using tions is estimated to be between US S5 billion and US S24 bacteriophage to kill bacteria has been in practice since the billion per year in the United States alone'. Resistance to early 20" century, particularly in Eastern Europe'''. Bac antibiotic drugs develops and spreads rapidly, often within a 55 teriophage can be chosen to lyse and kill bacteria or can be few years of first clinical use". However, the drug pipelines of modified to express lethal genes to cause cell death'. pharmaceutical companies have not kept pace with the evo However, bacteriophage which are directly lethal to their lution of antibiotic resistance'. bacterial hosts can also produce phage-resistant bacteria in Acquired antibiotic resistance results from mutations in short amounts of time'''''''. In addition to the aforemen antibacterial targets or from genes encoding conjugative pro 60 tioned approaches, novel methods for designing antimicro teins that pump antibiotics out of cells or inactivate antibiot bial drugs are becoming more important to extending the ics'. Horizontal gene transfer, which can occur via transfor lifespan of the antibiotic era. Combination therapy with mation, conjugative plasmids, or conjugative transposons, is different antibiotics or antibiotics with phage may enhance a major mechanism for the spread of antibiotic resistance bacterial cell killing and thus reduce the incidence of antibi genes''. For example, Staphylococcus aureus became 65 otic resistance, and reduce persisters'. Unmodified fila quickly resistant to Sulpha drugs in the 1940s, penicillin in the mentous bacteriophage have been shown to augment antibi 1950s, and methicillin in the 1980s'. In 2002, staphylococci otic efficacy'. Systems biology analysis can be employed to US 9,056,899 B2 3 4 identify pathways to target and followed by synthetic biology using natural bacteriophages that encode biofilm destroying to devise methods to attack those pathways. enzymes in general have been described. Art also provides a Bacterial biofilms are sources of contamination that are number of examples of lytic enzymes encoded by bacterioph difficult to eliminate in a variety of industrial, environmental ages that have been used as enzyme dispersion to destroy and clinical settings. Biofilms are polymer structures secreted 5 bacteria (U.S. Pat. No. 6,335,012 and U.S. Patent Application by bacteria to protect bacteria from various environmental Publication No. 2005/0004030). The Eastern European attacks, and thus result also in protection of the bacteria from research and clinical trials, particularly in treating human disinfectants and antibiotics. Biofilms can be found on any diseases, such as intestinal infections, has apparently concen environmental Surface where sufficient moisture and nutri trated on use of naturally occurring phages and their com ents are present. Bacterial biofilms are associated with many 10 bined uses (Lorch, A. (1999), “Bacteriophages: An alterna human and animal health and environmental problems. For tive to antibiotics? Biotechnology and Development instance, bacteria form biofilms on implanted medical Monitor, No. 39, p. 14-17). devices, e.g., catheters, heart Valves, replacements, and For example, non-engineered bacteriophages have been damaged tissue. Such as the lungs of cystic fibrosis patients. used as carriers to deliver antibiotics (such as chloroampheni Bacteria in biofilms are highly resistant to antibiotics and host 15 col) (Yacoby et al., Antimicrobial agents and chemotherapy, defenses and consequently are persistent sources of infection. 2006:50: 2087-2097). Non-engineered bacteriophages have Biofilms also contaminate Surfaces such as water pipes and also had aminoglycosides antibiotics, such as chloroam the like, and render also other industrial surfaces hard to phenicol, attached to the outside of filamentous non-engi disinfect. For example, catheters, in particular central venous neered bacteriophage (Yacoby et al., Antimicrobial agents catheters (CVCs), are one of the most frequently used tools 20 and chemotherapy, 2007: 51; 2156-2163). M13 non-lytic for the treatment of patients with chronic or critical illnesses bacteriophages have also been engineered to carry lethal cell and are inserted in more than 20 million hospital patients in death genes Gefand ChpBK. However, these phages have not the USA each year. Their use is often severely compromised been used, or Suggested to be useful in combination with as a result of bacterial biofilm infection which is associated antimicrobial or antibiotic agents (Westwater et al., 2003, with significant mortality and increased costs. Catheters are 25 Antimicrobial agents and chemotherapy, 47: 1301-1307). associated with infection by many biofilm forming organisms Non-engineered filamentous Pf3 bacteriophages have also Such as Staphylococcus epidermidis, Staphylococcus aureus, been administered with low concentration of gentamicin, Pseudomonas aeruginosa, Enterococcus faecalis and Can where neither the filamentous Pf3 or the gentamicin could dida albicans which frequently result in generalized eliminate the bacterial infection alone (Hagens et al. Microb. stream infection. Approximately 250,000 cases of CVC-as- 30 Drug resistance, 2006: 12; 164-8). The non-engineered bac sociated bloodstream infections occur in the US each year teriophage and the antibiotic enrofloxacin have been admin with an associated mortality of 12%-25% and an estimated istered simultaneously, although the use of the antibiotic was cost of treatment per episode of approximately $25,000. more effective than the combination of the antibiotic and Treatment of CVC-associated infections with conventional bacteriophage (see Table 1 in Huffet al., 2004: Poltry Sci, 83: antimicrobial agents alone is frequently unsuccessful due to 35 1994-1947). the extremely high tolerance of biofilms to these agents. Once Constant evolutionary pressure will ensure that antibiotic CVCs become infected the most effective treatment still resistance bacteria will continue to grow in number. The involves removal of the catheter, where possible, and the dearth of new antibacterial agents being developed in the last treatment of any Surrounding tissue or systemic infection 25-30 years certainly bodes poorly for the future of the anti using antimicrobial agents. This is a costly and risky proce- 40 biotic era (Wise, R (2004) J Antimicrob Chemother 54:306 dure and re-infection can quickly occur upon replacement of 310). Thus, new methods for combating bacterial infections the catheter. are needed in order to prolong the antibiotic age. For example, Bacteriophages (often known simply as “phages') are bacteriophage therapy or synthetic antibacterial peptides viruses that grow within bacteria. The name translates as have been proposed as potential solutions (Loose et al., “eaters of bacteria' and reflects the fact that as they grow, the 45 (2006) Nature 443: 867-869; Curtin, et al., (2006) Antimicrob majority of bacteriophages kill the bacterial host in order to Agents Chemother 50: 1268-1275). release the next generation of bacteriophages. Naturally Because antibiotic resistance in treating bacterial infec occurring bacteriophages are incapable of infecting anything tions and biofilms poses a significant hurdle to eliminating or other than specific strains of the target bacteria, undermining controlling or inhibiting bacteria and biofilms with conven their potential for use as control agents. 50 tional antimicrobial drugs, new anti-biofilm strategies. Such Bacteriophages and their therapeutic uses have been the as phage therapy, should be explored. Novel synthetic biol Subject of much interest since they were first recognized early ogy technologies are needed to enable the engineering of in the 20th century. Lytic bacteriophages are viruses that natural phage with biofilm-degrading enzymes to produce infect bacteria exclusively, replicate, disrupt bacterial libraries of enzymatically-active phage, which can comple and destroy the cell upon release of phage prog- 55 ment efforts to screen for new biofilm-degrading bacterioph eny in a process known as lysis. These bacteriophages have ages in the environment. very effective antibacterial activity and in theory have several advantages over antibiotics. Most notably they replicate at the SUMMARY site of infection and are therefore available in abundance where they are most required; no serious or irreversible side 60 The inventors have discovered a two pronged strategy to effects of phage therapy have yet been described and selecting significantly reduce or eliminate a bacterial infection. In par alternative phages against resistant bacteria is a relatively ticular, the inventors have engineered bacteriophages to be rapid process that can be carried out in days or weeks. used in combination with an antimicrobial agent, such that the Bacteriophage have been used in the past for treatment of engineered bacteriophage functions as an adjuvant to the plant diseases, such as fireblight as described in U.S. Pat. No. 65 antimicrobial agent. In particular, the inventors have engi 4,678,750. Also, Bacteriophages have been used to destroy neered bacteriophages to specifically disable (or deactivate) biofilms (e.g., U.S. Pat. No. 6,699,701). In addition, systems the bacteria's natural resistance mechanisms to the antimi US 9,056,899 B2 5 6 crobial agents and/or phage infection. Accordingly, one RecB. RecC. Spot or RelA. In another embodiment, an aspect of the present invention generally relates to engineered inhibitor-engineered bacteriophages can comprise at least 2, bacteriophages which have been modified or engineered to (i) 3, 4, 5 or more, for example 8 different nucleic acids encoding inhibit at least one bacterial resistance gene, or (ii) to inhibit inhibitors to antibiotic resistance genes or cell Survival repair at least one SOS response gene or bacterial defense gene in 5 genes, such as at least 2, 3, 4, 5 or more selected from the bacteria, or (iii) to express a protein which increases the group, but not limited to, cat, VanA, mecD, RecA, RecB. Susceptibility of a bacterial cell to an antimicrobial agent. Any RecC, Spot or RelA and other antibiotic resistance genes or one of these engineered bacteriophages, used alone, or in any cell Survival repair genes. In some embodiments of this aspect combination can be used with an antimicrobial agent. and all aspects described herein, an agent encoded by the Accordingly, the inventors have discovered a method to pre 10 nucleic acid of an inhibitor-engineered bacteriophage is a vent the development of bacterial resistance to antimicrobial protein which inhibits an antibiotic resistance gene and/or agents and the generation of persistent bacteria by inhibiting cell survival gene or encodes an RNA-inhibitor (RNAi) agent the local bacterial synthetic machinery which normally cir which inhibits the translation and expression of an antibiotic cumvents the antimicrobial effect, by engineering bacte resistance gene and/or cell Survival gene. riophages to be used in conjunction (or in combination with) 15 Another aspect of the present invention relates to an engi an antimicrobial agent, where an engineered bacteriophage neered bacteriophage which comprises a nucleic acid encod can inhibit an antimicrobial resistance gene, or inhibit a SOS ing a repressor protein, or fragment thereof of a bacterial SOS response gene or a non-SOS bacterial defense gene, or response gene, or an agent (such as a protein) which inhibits express a protein to increase the Susceptibility of a bacterial a non-SOS pathway bacterial defense gene and are referred to cell to an antimicrobial agent. herein as “repressor-engineered bacteriophages. In some Accordingly, one aspect of the present invention relates to embodiments, the repressor of an SOS response gene is, for the engineered bacteriophages as discussed herein for use in example but not limited to, lex A, or modified version thereof. conjunction with (i.e. in combination with) at least one anti In some embodiments, the SOS response gene is, for example microbial agent, and that the engineered bacteriophages serve but is not limited to marFRAB, arcAB and lexO. In some as adjuvants to Such antimicrobial agents. Another aspect of 25 embodiments of this aspect and all other aspects described the present invention relates to a method for inhibiting bac herein, an inhibitor of a non-SOS pathway bacterial defense teria and/or removing bacterial biofilms in environmental, gene is soxR, or modified version thereof. In some embodi industrial, and clinical settings by administering a composi ments of this aspect and all other aspects described herein, an tion comprising at least one engineered bacteriophages as inhibitor of a non-SOS pathway bacterial defense gene is discussed herein with at least one antimicrobial agent. 30 selected from the group of marr, arc, SOXR, fur, crp, iccdA or One aspect of the present invention relates to methods of cra A or omp A or modified version thereof. In other embodi using engineered bacteriophages in combination with antimi ments of this aspect of the invention, an agent encoded by the crobial agents to potentiate the antimicrobial effect of bacte nucleic acid of a repressor engineered bacteriophage which rial killing (i.e. eliminating or inhibiting the growth or con inhibits a non-SOS defense gene can inhibit any gene listed in trolling the bacteria) by the antimicrobial agent. Accordingly, 35 Table 2. In some embodiments, a repressor-engineered bac the present invention relates to the discovery of an engineered teriophage which inhibits a non-SOS defense gene can be bacteriophage as an antibiotic adjuvant. In some embodi used in combination with selected antimicrobial agents, for ments, an engineeredbacteriophage as discussed herein func example, where the repressor-engineered bacteriophage tions as an antibiotic adjuvant for an aminglycoside antimi encodes an agent which inhibits a gene listed in Table 2A, crobial agent, Such as but not limited to, gentamicin, as an 40 Such a repressor-engineered bacteriophage can be used in antibiotic adjuvant for B-lactam antibiotics. Such as but not combination with a ciprofloxacin antimicrobial agent or a limited to, amplicillin, and as antibiotic adjuvants for quino variant or analogue thereof. Similarly, in other embodiments lones antimicrobial agents, such as but not limited to, ofloxa a repressor-engineered bacteriophage which inhibits a non cin. SOS defense gene can encode an agent which inhibits a gene Another aspect of the present invention relates to an engi 45 listed in Table 4B can be used in combination with a Vanco neered bacteriophage which comprises a nucleic acid encod mycin antimicrobial agent or a variant or analogue thereof. ing an agent which inhibits at least one gene involved in Similarly, in other embodiments a repressor-engineered bac antibiotic resistance. In Such and embodiment of this aspect teriophage which inhibits a non-SOS defense gene can of the invention, an engineered bacteriophage can comprise at encode an agent which inhibits a gene listed in Table 2C, 2D, least 2, 3, 4.5 or even more, for example 10 different nucleic 50 2E, 2F and 2G can be used in combination with a acids which inhibit at least one gene involved in antibiotic antimicrobial agent, or a amplicillin antimicrobial agent or a resistance. In an alternative embodiment, an engineered bac SulfmethaxaZone antimicrobial agent or a gentamicin antimi teriophage can comprise a nucleic acid encoding an agent crobial agent or a antimicrobial agent, respec which inhibits at least one gene involved in cell survival tively, or a variant or analogue thereof. repair. In another embodiment, an engineered bacteriophage 55 Another aspect of the present invention relates to an engi can comprise at least 2, 3, 4, 5 or even more, for example 10 neered bacteriophage which comprises a nucleic acid encod different nucleic acids which inhibit at least one gene ing an agent, such as but not limited to a protein, which involved in cell survival repair. Such engineered bacterioph increases the Susceptibility of a bacteria to an antimicrobial ages as disclosed herein which comprise a nucleic acid agent. Such herein engineered bacteriophage which com encoding an agent which inhibits at least one gene involved in 60 prises a nucleic acid encoding an agent which increases the bacterial antibiotic resistance and/or cell Survival gene are Susceptibility of a bacteria to an antimicrobial agent can be referred to herein as “inhibitor-engineered bacteriophages”. referred to herein as an “susceptibility agent-engineered bac In some embodiments, the agent inhibits the gene expression teriophage' but are also encompassed under the definition of and/or protein function of antibiotic resistance genes such as, a “repressor-engineered bacteriophage'. In some embodi but not limited to cat, VanA or mecD. In some embodiments, 65 ments of this aspect, and all other aspects described herein, the agent inhibits the gene expression and/or protein function Such an agent which increases the Susceptibility of a bacteria of a cell Survival repair gene Such as, but not limited to RecA to an antimicrobial agent is referred to as a “susceptibility US 9,056,899 B2 7 8 agent” and refers to any agent which increases the bacteria's cat, VanA, mecD, RecA, RecB. RecC, Spot or RelA. In susceptibility to the antimicrobial agent by at least about 10% another aspect, an engineered bacteriophage can express an or at least about 15%, or at least about 20% or at least about repressor, or fragment thereof, of at least one, or at least two 30% or at least about 50% or more than 50%, or any integer or at least three SOS response genes, such as, but not limited between 10% and 50% or more, as compared to the use of the to leXA, marr, arc, SOXR, fur, crp, iccdA, craA or omp A. antimicrobial agent alone. In one embodiment, a Susceptibil The inventors also demonstrated that a repressor-engi ity agent is an agent which specifically targets a bacteria cell. neered bacteriophage and/or an inhibitor-engineered bacte In another embodiment, a Susceptibility agent modifies (i.e. riophage and/or a susceptibility agent-engineered bacte inhibits or activates) a pathway which is specifically riophage can reduce the number of antibiotic-resistant expressed in bacterial cells. In one embodiment, a suscepti 10 bility agent is an agent which has an additive effect of the bacteria in a population and act as a strong adjuvant for a efficacy of the antimicrobial agent (i.e. the agent has an addi variety of other bactericidal antibiotics, such as for example, tive effect of the killing efficacy or inhibition of growth by the but not limited to gentamicin and amplicillin. antimicrobial agent). In a preferred embodiment, a suscepti In some embodiments of all aspects of the invention, any bility agent is an agent which has a synergistic effect on the 15 engineered bacteriophage disclosed herein, such as repres efficacy of the antimicrobial agent (i.e. the agent has a syn sor-engineered bacteriophage and/or an inhibitor-engineered ergistic effect of the killing efficacy or inhibition of growth by bacteriophage and/or a Susceptibility agent-engineered bac the antimicrobial agent). teriophage as discussed herein can additionally comprise a In one embodiment, a Susceptibility agent increases the least one of the degrading enzymes effective at degrading entry of an antimicrobial agent into a bacterial cell, for bacteria biofilms, such as effective EPS-degrading enzymes example, a Susceptibility agent is a porin orporin-like protein, specific to the target biofilm, particularly, for example, dis such as but is not limited to, protein OmpF, and Beta barrel persin B (DspB) which is discussed in PCT application PCT/ porins, or other members of the outer membrane porin US2005/032365 and U.S. application Ser. No. 12/337,677, (OMP)) functional superfamily which include, but are not which are incorporated herein by reference. limited to those disclosed in world wide web site: “//biocy 25 Also discussed herein is the generation of a diverse library c.org/ECOLI/NEW-IMAGE?object=BC-4.1.B”, or a OMP of engineered bacteriophages described herein, Such as a family member listed in Table 3 as disclosed herein, or a library of repressor-engineered bacteriophage and/or an variant or fragment thereof. In another embodiment, a sus inhibitor-engineered bacteriophage and/or a Susceptibility ceptibility agent is an agent, such as but not limited to a agent-engineered bacteriophages which are capable of acting protein, which increases iron-sulfur clusters in the bacteria 30 as adjuvants or to enhance antimicrobial agents, which is cell and/or increases oxidative stress or hydroxyl radicals in advantageous than trying to isolate such bacteriophages that the bacteria. Examples of a susceptibility agent which function as adjuvants from the environment. By multiplying increases the iron-sulfur clusters include agents which modu within the bacterial colony or biofilm and hijacking the bac late (i.e. increase or decrease) the Fenton reaction to form terial machinery, inhibitor engineered bacteriophages hydroxyl radicals, as disclosed in Kahanski et al., Cell, 2007, 35 130; 797-810, which is incorporated herein by reference in its achieves high local concentrations of both enzyme and lytic entirety. Examples of a Susceptibility agent to be expressed by phage to target multiple biofilm components, even with Small a Susceptibility-engineered bacteriophage include, for initial phage inoculations. example, those listed in Table 4, or a fragment or variant Rapid bacteriophage (also referred to as “phage’ herein) thereof or described in world-wide-web site “biocyc.org/ 40 replication with Subsequent bacterial lysis and expression of ECOLI/NEW-IMAGE?type=COMPOUND&object=CPD inhibitors of SOS genes renders this a two-pronged attack 7: strategy for use in combination with antimicrobial agents for In some embodiments, a Susceptibility agent is not a che an efficient, autocatalytic method for inhibiting bacteria and/ motherapeutic agent. In another embodiment, a Susceptibility or removing bacterial biofilms in environmental, industrial, agent is not a toxin protein, and in another embodiment, a 45 and clinical settings. Susceptibility agent is not a bacterial toxin protein or mol Also disclosed herein is a method for the combined use of ecule. an inhibitor-engineered bacteriophage and/or a repressor-en Accordingly, the inventors have developed a modular gineered bacteriophage and/or Susceptibility agent-engi design strategy in which bacteriophages are engineered to neered bacteriophage with at least one antimicrobial agent. have enhanced capacity to kill bacteria to disable or deacti 50 The inventors have demonstrated that the combined use of an vate the bacteria's natural resistance genes to antimicrobial inhibitor-engineered bacteriophage and/or a repressor-engi agents or phage infection. In some embodiments, the bacte neered bacteriophage and/or Susceptibility agent-engineered riophages can be engineered or modified to express (i) at least bacteriophage is at least 4.5 orders of magnitude more effi one inhibitor to at least one bacterial resistance gene and/or cient than use of the antimicrobial agent alone, and at least cell Survival gene, or (ii) at least one inhibitor (Such as, but not 55 two orders of magnitude more efficient at killing or eliminat limited to a repressor) at least one SOS response gene or ing the bacteria as compared to use of an antimicrobial agent bacterial defense gene in bacteria, or (iii) a Susceptibility with a non-engineered bacteriophage alone (i.e. an antimicro agent which increases the susceptibility of a bacterial cell to bial agent in the presence of a bacteriophage which is not an an antimicrobial agent. inhibitor-engineeredbacteriophage or a repressor-engineered In some embodiments, any one of these engineered bacte 60 bacteriophage or Susceptibility agent-engineered bacterioph riophages, used alone, or in any combination can be used with age). Thus, the inventors have demonstrated a significant and at least one antimicrobial agent. For example, one aspect Surprising improvement over the combined use of non-engi discussed herein relates to an engineered bacteriophage neered bacteriophages and antimicrobial agents as therapies which expresses a nucleic acid inhibitor, such as an antisense described in prior art. The inventors have also demonstrated nucleic acid inhibitor or antisense RNA (asRNA) which 65 that use of Such engineered bacteriophages as disclosed inhibits at least one, or at least two or at least three antibiotic herein, Such as the inhibitor-engineered bacteriophages or genes and/or a cell Survival gene. Such as, but not limited to repressor-engineered bacteriophages are very effective at US 9,056,899 B2 10 reducing the number of antibiotic resistant bacterial cells encodes at least one agent that increases the Susceptibility of which can develop in the presence of sub-inhibitory antimi a bacterial cell to an antimicrobial gene. crobial drug concentrations. In some embodiments, an antibiotic resistance gene is Also, one significant advantage of the present invention as selected from the group comprising cat, VanA or mec) or compared to methods using non-engineered bacteriophages variants thereof. In some embodiments, a cell Survival gene is in combination with antimicrobial agents is that the use of the selected from the group comprising RecA, RecB. RecC., spot, engineered bacteriophages as disclosed herein with antimi RelA or variants thereof. crobial agents allows one to significantly reduce or eliminate In some embodiments of all aspects described herein, a a population of persister cells. For example, the administra bacteriophage can comprise an agent which is selected from tion or application of an engineered bacteriophage as dis 10 a group comprising, siRNA, antisense nucleic acid, asRNA, closed herein after initial treatment with an antimicrobial RNAi, miRNA and variants thereof. In some embodiments, agent can reduce or eliminate a population of persister cells. the bacteriophage comprises an as RNA agent. Furthermore, the inventors have discovered that an engi In some embodiments, the bacteriophage comprises a neered bacteriophage as disclosed herein, such as an inhibi nucleic acid encoding at least two agents that inhibit at least tor-engineered bacteriophage or a repressor-engineered bac 15 two different cell survival repair genes, for example but not teriophage or Susceptibility agent-engineered bacteriophage limited to, at least two agents that inhibit at least two of RecA can reduce the number of antibiotic resistant mutant bacteria RecB or RecC. that Survive in a bacterial population exposed to one or more In some embodiments, the repressor of a SOS response antimicrobial agents, and therefore the engineered bacte gene is selected from the group comprisingleXA, marr, arck, riophages described herein are effective at reducing the num SOXR, fur, crp, iccdA, craA, ompl or variants or fragments ber of antibiotic resistant cells which develop in the presence thereof. In some embodiments, the repressor is Lex A and in of Sub-inhibitory antimicrobial agent drug concentrations. Some embodiments, the repressor is cSrA or omF, and in some Another advantage of the present invention is that it allows embodiments the bacteriophage can comprise the nucleic one to reduce or eliminate multiple applications of the com acid encoding a mixture of Lex A, cSrA or omF in any com position during the treatment of a Surface having a bacterial 25 bination. For example, in some embodiments, the bacterioph biofilm. age can comprise the nucleic acid encoding at least two dif One aspect of the present invention relates to engineering ferent repressors of at least one SOS response gene, such as, or modification of any bacteriophage strain or species to but not limited to the bacteriophage can comprise the repres generate the engineered bacteriophages disclosed herein. For sors cSrA and ompl or variants or homologues thereof. example, an inhibitor-engineered bacteriophage or a repres 30 Another aspect of the present invention relates to a method sor-engineered bacteriophage or Susceptibility agent-engi to inhibit or eliminate a bacterial infection comprising admin neered bacteriophage can be any bacteriophage known by a istering to a surface infected with bacteria: (i) a bacteriophage skilled artisan. For example, in one embodiment, the bacte comprising a nucleic acid operatively linked to a bacterioph riophage is a lysogenic bacteriophage, for example but not age promoter, wherein the nucleic acid encodes at least one limited to a M13 bacteriophage. In another embodiment, the 35 agent that inhibits an antibiotic resistance gene and/or a cell bacteriophage is a lytic bacteriophage such as, but not limited Survival repair gene, and (ii) at least one antimicrobial agent. to T7 bacteriophage. In another embodiment, the bacterioph Another aspect of the present invention relates to a method age is a phage Kora Staphylococcus phage K for use against to inhibit or eliminate a bacterial infection comprising admin bacterial infections of methicillin-resistant S. aureus. istering to a surface infected with bacteria: (i) a bacteriophage One aspect of the present invention relates to an engineered 40 comprising a nucleic acid operatively linked to a bacterioph lysogenic M13 bacteriophage comprising a nucleic acid age promoter, wherein the nucleic acid encodes at least one operatively linked to a M13 promoter, wherein the nucleic repressor of a SOS response gene, and (ii) at least one anti acid encodes at least one agent that inhibits an antibiotic microbial agent. resistance gene and/or a cell Survival repair gene. Another aspect of the present invention relates to a method Another aspect of the present invention relates to an engi 45 to inhibit or eliminate a bacterial infection comprising admin neered lysogenic M13 bacteriophage comprising a nucleic istering to a surface infected with bacteria: (i) a bacteriophage acid operatively linked to a M13 promoter, wherein the comprising a nucleic acid operatively linked to a bacterioph nucleic acid encodes at least one repressor of a SOS response age promoter, wherein the nucleic acid encodes at least one gene and/or an inhibitor to a non-SOS bacterial defense gene. agent which increases the susceptibility of a bacterial cell to Another aspect of the present invention relates to an engi 50 aantimicrobial agent, and (ii) at least one antimicrobial agent. neered lysogenic M13 bacteriophage comprising a nucleic In some embodiments of all aspects described herein, a acid operatively linked to a M13 promoter, wherein the bacteriophage useful in the methods disclosed herein and nucleic acid encodes at least one agent that increases the used to generate an engineered bacteriophage, such as a Susceptibility of a bacterial cell to an antimicrobial gene. inhibitor-engineeredbacteriophage or a repressor-engineered Another aspect of the present invention relates to an engi 55 bacteriophage or a susceptibility-engineeredbacteriophage is neered lytic T7 bacteriophage comprising a nucleic acid any bacteriophage know by a skilled artisan. A non-limiting operatively linked to a T7 promoter, wherein the nucleic acid list of examples of bacteriophages which can be used are encodes at least one agent that inhibits at least one antibiotic disclosed in Table 5 herein. In one embodiment, the bacte resistance gene and/or at least one cell Survival repair gene. riophage is a lysogenic bacteriophage such as, for example a Another aspect of the present invention relates to an engi 60 M13 lysogenic bacteriophage. In alternative embodiments, a neered lytic T7 bacteriophage comprising a nucleic acid bacteriophage useful in all aspects disclosed herein is a lytic operatively linked to a T7 promoter, wherein the nucleic acid bacteriophage, for example but not limited to a T7 lytic bac encodes at least one repressor of a SOS response gene and/or teriophage. In one embodiment, a bacteriophage useful in all an inhibitor to a non-SOS bacterial defense gene. aspects disclosed herein is a SP6 bacteriophage or a phage K. Another aspect of the present invention relates to an engi 65 or a staphylococcus phage K bacteriophage. neered lytic T7 bacteriophage comprising a nucleic acid In some embodiments, administration of any engineered operatively linked to a T7 promoter, wherein the nucleic acid bacteriophage as disclosed herein and the antimicrobial agent US 9,056,899 B2 11 12 occurs simultaneously, and in alternative embodiments, the lin, amplicillin, penicillin derivatives, cephalosporins, mono administration of a engineered-bacteriophage occurs prior to bactams, carbapenems, B-lactamase inhibitors or variants or the administration of the antimicrobial agent. In other analogues thereof. embodiments, the administration of an antimicrobial agent In some embodiments, the composition comprises at least occurs prior to the administration of a engineered-bacterioph- 5 one inhibitor-engineered bacteriophage and/or at least one age. repressor-engineered bacteriophage as disclosed herein. In some embodiments, antimicrobial agents useful in the Another aspect of the present invention relates to a kit methods as disclosed herein are quinolone antimicrobial comprising a lysogenic M13 bacteriophage comprising the agents, for example but not limited to, antimicrobial agents nucleic acid operatively linked to a M13 promoter, wherein selected from a group comprising ciprofloxacin, levofloxa 10 the nucleic acid encodes at least one agent that inhibits an antibiotic resistance gene and/or a cell Survival repair gene. cin, and , , , , Another aspect of the present invention relates a kit compris , , , , ing a lysogenic M13 bacteriophage comprising the nucleic or variants or analogues thereof. In some acid operatively linked to a M13 promoter, wherein the embodiments, an antimicrobial agents useful in the methods 15 nucleic acid encodes at least one repressor of a SOS response. as disclosed herein is ofloxacin or variants or analogues Another aspect of the present invention relates a kit com thereof. prising a lytic T7 bacteriophage comprising the nucleic acid In some embodiments, antimicrobial agents useful in the operatively linked to a T7 promoter, wherein the nucleic acid methods as disclosed herein are aminoglycoside antimicro encodes at least one agent that inhibits an antibiotic resistance bial agents, for example but not limited to, antimicrobial 20 gene and/or a cell Survival repair gene. Another aspect of the agents selected from a group consisting of amikacin, genta present invention relates a kit comprising a lytic T7 bacte mycin, tobramycin, metromycin, Streptomycin, kanamycin, riophage comprising the nucleic acid operatively linked to a paromomycin, neomycin or variants or analogues thereof. In T7 promoter, wherein the nucleic acid encodes at least one Some embodiments, an antimicrobial agent useful in the repressor of a SOS response. methods as disclosed herein is gentamicin or variants orana- 25 In some embodiments, the methods and compositions as logues thereof. disclosed herein are administered to a Subject. In some In some embodiments, antimicrobial agents useful in the embodiments, the methods to inhibit or eliminate a bacterial methods as disclosed herein are B-lactam antibiotic antimi infection comprising administering the compositions as dis crobial agents, such as for example but not limited to, anti closed herein to a subject, wherein the bacteria are present in microbial agents selected from a group consisting of penicil 30 the Subject. In some embodiments, the Subject is a mammal, lin, amplicillin, penicillin derivatives, cephalosporins, for example but not limited to a human. In some embodiments, any of the bacteriophages as dis monobactams, carbapenems, B-lactamase inhibitors or vari closed herein are useful in combination with at least one ants or analogues thereof. In some embodiments, an antimi antimicrobial agent to reduce the number of bacteria as com crobial agent useful in the methods as disclosed herein is 35 pared to use of the antimicrobial agent alone. In some ampicillin or variants or analogues thereof. embodiments, any of the bacteriophages as disclosed herein Another aspect of the present invention relates to a com are useful in combination with at least one antimicrobial position comprising a lysogenic M13 bacteriophage compris agent to inhibit or eliminate a bacterial infection, such as for ing a nucleic acid operatively linked to a M13 promoter, example inhibit or eliminate a bacteria present a biofilm. wherein the nucleic acid encodes at least one agent that inhib- 40 In some embodiments, the present invention relates to its an antibiotic resistance gene and/or a cell Survival repair methods to inhibit or eliminate a bacterial infection compris gene and at least one antimicrobial agent. Another aspect of ing administering to a surface infected with bacteria; (i) a the present invention relates to a composition comprising a bacteriophage comprising a nucleic acid operatively linked to lysogenic M13 bacteriophage comprising a nucleic acid a bacteriophage promoter, wherein the nucleic acid encodes operatively linked to a M13 promoter, wherein the nucleic 45 at least one repressor of a SOS response gene, and (ii) at least acid encodes at least one repressor of a SOS response gene one antimicrobial agent. In some embodiments, the bacteria and at least one antimicrobial agent. is in a biofilm. Another aspect of the present invention relates to a com position comprising a lytic T7 bacteriophage comprising a BRIEF DESCRIPTION OF FIGURES nucleic acid operatively linked to a T7 promoter, wherein the 50 nucleic acid encodes at least one agent that inhibits an anti FIGS. 1A-1E show engineered (p bacteriophage biotic resistance gene and/or a cell Survival repair gene and at enhances killing of wild-type E. coli EMG2 bacteria by bac least one antimicrobial agent. Another aspect of the present tericidal antibiotics. FIG. 1A shows a schematic of combina invention relates to a composition a lytic T7 bacteriophage tion therapy with engineered phage and antibiotics. Bacteri comprising a nucleic acid operatively linked to a T7 promoter, 55 cidal antibiotics induce DNA damage via hydroxyl radicals, wherein the nucleic acid encodes at least one repressor of a leading to induction of the SOS response. SOS induction SOS response gene and at least one antimicrobial agent. results in DNA repair and can lead to survival (Kohanski et In some embodiments, the composition comprises antimi al., 2007, Cell 130,797-8108). Engineered phage carrying the crobials agents such as, for example but not limited to, qui leXA3 gene (cps) under the control of the synthetic pro nolone antimicrobial agents and/or aminoglycoside antimi- 60 moter PLtetO and a ribosome-binding sequence (Lutz et al., crobial agents and/or f3-lactam antimicrobial agent, for 1997, Nucleic Acids Res 25, 1203-121027) acts as an antibi example, but not limited to, antimicrobial agents selected otic adjuvant by Suppressing the SOS response and increasing from a group comprising ciprofloxacin, , and cell death. FIG. 1B shows a killing curves for no phage ofloxacin, gatifloxacin, norfloxacin, lomefloxacin, trova (diamonds), unmodified phage (p., (squares), and engi floxacin, moxifloxacin, sparfloxacin, gemifloxacin, paZu- 65 neered phage (ps (circles) with 60 ng/mL ofloxacinoflox floxacin, amikacin, gentamycin, tobramycin, netromycin, (solid lines, closed symbols). 10 PFU/mL phage was used. A streptomycin, kanamycin, paromomycin, neomycin, penicil growth curve for E. coli EMG2 with no treatment is shown for US 9,056,899 B2 13 14 comparison (dotted line, open Symbols). (ps greatly cin-based killing by disrupting regulation of the oxidative enhanced killing by ofloxacin by 4 hours of treatment. FIG. stress response. FIG. 5B show killing curves for no phage 1C is a ofloxacin dose response showing that (ps (circles (diamonds), unmodified phage (p., (squares), and engi with solid line) increases killing even at low levels of drug neered phage (p (downwards-facing triangles) with 60 compared with no phage (diamonds with dash-dotted line) ng/mL ofloxacin (solid lines, closed symbols). 10s PFU/mL and (punmod (squares with dashed line). 10 PFU/mL phage phage was used. The killing curve for funmod and a growth was used. FIG. 1D shows killing curves for no phage (dia curve for E. coli EMG2 with no treatment (dotted line, open monds), (p,f(squares), and (pets (circles) with 5 Lig/mL symbols) are reproduced from FIG. 1B for comparison and gentamicingent). 10 PFU/mL phage was used. (pus phage show that p, enhances killing by ofloxacin. FIG. 5CCsrA greatly increases killing by gentamicin. FIG.1E shows killing 10 suppresses the biofilm state in which bacterial cells tend to be curves for no phage (diamonds), p. (squares), and (Pets more resistant to antibiotics (Jackson et al., 2002, J. Bacteriol. (circles) with 5ug/mL ampicillin amp. 10 PFU/mL phage 184,290-30135). Ompl is a porin used by quinolones to enter was used. (p. phage greatly increases killing by amplicillin. bacterial cells (Hirai K, et al., 1986, Antimicrob. Agents FIG.2 shows that engineered (p bacteriophage enhances Chemother. 29, 535-53837). Engineered phage producing killing of quinolone-resistant E. coli RFS289 bacteria by 15 both CsrA and OmpF simultaneously (cps) enhances ofloxacin. Killing curves for no phage (diamonds), unmodi antibiotic penetration via OmpF and represses biofilm forma fied phage funmod (squares), and engineered phage (ps tion and antibiotic tolerance via CSrA to produce an improved (circles) with 1 ug/mL ofloxacinoflox (Solid lines, closed dual targeting adjuvant for ofloxacin. FIG. 5D shows killing symbols). 10 PFU/mL phage was used. (ps greatly curves for cps (diamonds), per (squares), and ps-, enhanced killing by ofloxacin by 1 hour of treatment. (upwards-facing triangles) with 60 ng/mL ofloxacin. 10 FIGS. 3A-3B show that engineered (p bacteriophage PFU/mL phage was used. Phage expressing both csrA and increases survival of mice infected with bacteria. FIG. 3A ompF (cp.se) is a better adjuvant for ofloxacin than shows a schematic of a female Charles River CD-1 mice phage expressing cSrA (cps) or ompF alone (cp). inoculated with intraperitoneal injections of 8.8x10 CFU/ FIGS. 6A-6D show engineered bacteriophage targeting mouse E. coli EMG2bacteria. After 1 hour, the mice received 25 non-SOS systems in E. coli as adjuvants for ofloxacin treat either no treatment or intravenous treatment with no phage, ment oflox. FIG. 6A shows a killing curves for no phage unmodified phage (p, or engineered phage (p, with 0.2 (black diamonds), 10 PFU/mL unmodified M13mp 18 (i.e. mg/kg ofloxacin. 10 PFU/mouse phage was used. The mice (p) (squares), and 10 PFU/mL M13mp18-soxR (i.e. were observed for 5 days and deaths were recorded at the end (psi) (downwards-facing triangles) without ofloxacin (dot of each day to generate survival curves. FIG. 3B shows sur 30 ted lines, open symbols) or with 60 ng/mL ofloxacin (solid vival curves for infected mice treated with phage and/or lines, closed symbols). Killing curves for no phage and ofloxacin demonstrate that engineered phage (p, plus unmodified m13mp18 phage (p) are reproduced from ofloxacin (closed circles with solid line) significantly FIG. 1B for comparison and demonstrate that M13mp 18 increases Survival of mice compared with unmodified phage soxR (i.e. (p) enhances killing by ofloxacin. 10 PFU/mL funmod plus ofloxacin (closed squares with Solid line), no 35 represents an MOI of approximately 1:10. FIG. 6B shows a phage plus ofloxacin (closed diamonds with Solid line), and killing curves for 10 PFU/mL M13 mp 18-csrA (cps) (black no treatment (open diamonds with dashed line). diamonds), 10 PFU/mL M13mp 18-ompF (per) (squares). FIGS. 4A-4B show box-and-whisker plot of the total num and 10 PFU/mL M13mp18-csrA-ompF (cp.4-ompF) (up ber of E. coli EMG2 cells in 60 observations that were resis wards-facing triangles) without ofloxacin (dotted lines, open tant to 100 ng/mL ofloxacin after growth under various con 40 symbols) or with 60 ng/mL ofloxacin (solid lines, closed ditions (bars indicate medians, diamonds represent outliers). symbols). Phage expressing both csrA and ompl (M13mp18 FIG. 4A shows cells grown with no phage and no ofloxacin cSrA-ompF or (p) is a better adjuvant for ofloxacin for 24 hours had very low numbers of antibiotic-resistant than phage expressing cSrA alone (M13mp 18-cSrA. (p.) or cells. Cells grown with no phage and 30 ng/mL ofloxacin for ompf alone (M13mp 18-ompF; (P). 10 PFU/mL repre 24 hours had high numbers of resistant cells due to growth in 45 sents an MOI of approximately 1:10. FIG. 6C shows a phage subinhibitory drug concentrations (Martinez et al., 2000, dose response which demonstrates that both M13mp18-soxR Antimicrob. Agents Chemother. 44, 1771-177730). Cells (downwards-facing triangles with solid line) and M13mp18 grown with no phage and 30 ng/mL ofloxacin for 12 hours cSrA-ompF (upwards-facing triangles with Solid line) are followed by 10 PFU/mL unmodified phage funmod and 30 effective as adjuvants for ofloxacin (60 ng/mL) over a wide ng/mL ofloxacin for 12 hours exhibited a modest level of 50 range of initial inoculations. Phage dose response curves for antibiotic-resistant bacteria. Cells grown with no phage and no phage (dash-dotted line) and unmodified M13mp 18 phage 30 ng/mL ofloxacin for 12 hours followed by 10 PFU/mL (squares with dashed line) are reproduced from FIG. 1c for (p and 30 ng/mL ofloxacin for 12 hours exhibited a low comparison. FIG. 6D shows a Ofloxacin dose response with level of antibiotic-resistant bacteria, close to the numbers 10 PFU/mL that shows that both M13mp 18-soxR (down seen with no ofloxacin and no phage. FIG. 4B shows a 55 wards-facing triangles with solid line) and M13mp 18-csrA Zoomed-in version of box-and-whisker plot in (a) for ompl (upwards-facing triangles with solid line) improve kill increased resolution around low total resistant cell counts ing throughout a range of drug concentrations. Ofloxacin confirms that p with 30 ng/mL ofloxacin treatment dose response curves for no phage (diamonds with dash reduced the number of resistant cells to levels similar to that dotted line) and unmodified M13mp 18 phage (squares with of no ofloxacin with no phage. 60 dashed line) are reproduced from FIG. 1D for comparison. FIGS. 5A-5D show engineered bacteriophage targeting FIGS. 7A-7D show histograms of the total number of E. single and multiple gene networks (other than the SOS net coli cells in 60 observations that were resistant to 100 ng/mL work) as adjuvants for ofloxacin treatment oflox. FIG. 5A ofloxacin after growth under various conditions. FIG. 7A show Ofloxacin stimulates Superoxide generation, which is shows cells grown with no phage and no ofloxacin for 24 normally countered by the oxidative stress response, coordi 65 hours had very low numbers of antibiotic-resistant cells. Inset nated by SoxR (Kohanski et al., 2007, Cell 130, 797-8108). of FIG. 8A shows the distribution of observations with total Engineered phage producing SoxR (cp) enhances ofloxa resistant cells between 0 and 50 for increased resolution and US 9,056,899 B2 15 16 demonstrates that many observations were devoid of antibi FIG. 11 shows persister killing assay demonstrates that otic-resistant bacteria. FIG. 7B shows cells grown with no engineered bacteriophage can be applied to a previously phage and 30 ng/mL ofloxacin for 24 hours had high numbers drug-treated population to increase killing of Surviving per of resistant cells, demonstrating a large increase in antibiotic sister cells. After 3 hours of 200 ng/mL ofloxacin treatment, resistance due to growth in Subinhibitory drug concentra no phage, 10 PFU/mL control M13mp 18 phage, or 10 PFU/ tions'. No inset is shown because no observations had less mL engineered M13mp 18-lex A3 phage were added to the than 50 resistant cells. FIG. 7C shows cells grown with no previously drug-treated cultures. Three additional hours later, phage and 30 ng/mL ofloxacin for 12 hours followed by 10 viable cell counts were obtained and demonstrated that PFU/mL unmodified M13mp 18 phage and 30 ng/mL ofloxa M13mp18-lex A3 was able to reduce persister cell levels bet cin for 12 hours exhibited a modest level of antibiotic-resis 10 ter than no phage or control M13mp8 phage. tant bacteria. Inset of FIG.7C shows the distribution of obser FIG. 12 shows paired-termini design from Nakashima, etal vations with total resistant cells between 0 and 50 for (2006) Nucleic Acids Res 34: e138, in which the antisense increased resolution and demonstrates that no observations RNA is cloned between the flanking restriction sites at the top were devoid of antibiotic-resistant bacteria. FIG. 7D shows of the stem. Reprinted from Nakashima, etal (2006) Nucleic 15 Acids Res 34, e138. cells grown with no phage and 30 ng/mL ofloxacin for 12 FIG. 13 shows autoregulated negative-feedback module hours followed by 10 PFU/mL M13mp 18-lex A3 and 30 with lexA repressing PlexO from Morens, et al., (2004) ng/mL ofloxacin for 12 hours exhibited a low level of antibi Nature 430: 242-249, can increase the level of lexA expres otic-resistant bacteria compared to no phage and 30 ng/mL sion when lex A is cleaved by recA in response to DNA ofloxacin in FIG. 7D, and unmodified M13mp 18 and 30 damage by agents such as ofloxacin. ng/mL ofloxacin in FIG. 8C. Inset of FIG. 7D shows the FIG. 14 shows persistence assay for various constructs in distribution of observations with total resistant cells between wild-type E. coli EMG2 cells after 8 hours of growth in the 0 and 50 for increased resolution and demonstrates that presence of 1 mM IPTG followed by 8 hours of treatment M13mp 18-lex A3 treatment reduced the number of resistant with 5 lug/mL ofloxacin. Greatly improved cell killing was cells under 30 ng/mL ofloxacinto levels similar to that of 0 25 generated by the double knockouts, especially PtetO-recB ng/mL ofloxacin in FIG. 8A. asRNA/PlacO-recA-asRNA and PtetO-recC-asRNA/ FIGS. 8A-8B shows engineered M13mp 18-lexA3 bacte PlacO-recB-askNA. pZE1L-leXA also reduced the number riophage enhances killing by other bactericidal drugs. FIG. of surviving cells compared with wild-type E. coli EMG2. 8A shows killing curves for no phage (diamonds), 10 PFU/ FIG. 15 shows engineered (p bacteriophage enhances mL unmodified M13mp18 (squares), and 10 PFU/mL 30 killing of wild-type E. coli EMG2 bacteria by bactericidal M13mp 18-lex A3 (circles) with 5 lug/mL gentamicin gent. antibiotics. Phage dose response shows that p (blue Engineered M13mp 18-lex A3 phage greatly improved killing circles with solid line) is a strong adjuvant for ofloxacin (60 by gentamicin. 10 PFU/mL represents an MOI of approxi ng/mL) over a wide range of initial inoculations compared mately 1:1. FIG. 8B shows a killing curves for no phage with no phage (black dash-dotted line) and (p (red (diamonds), 10 PFU/mL unmodified M13mp 18 (squares), 35 squares with dashed line). The starting concentration of bac and 10 PFU/mL M13mp 18-lex A3 (circles) with 5 g/mL teria was about 10 CFU/mL (data not shown). ampicillin amp. Engineered M13mp 18-lex A3 phage FIG. 16 shows persister killing assay demonstrates that greatly improved killing by amplicillin 10 PFU/mL repre engineered bacteriophage can be applied to a previously sents an MOI of approximately 1:1. drug-treated population to increase killing of Surviving per FIGS.9A-9F show genomes of unmodified M13mp 18 bac 40 sister cells. After 3 hours of 200 ng/mL ofloxacin treatment, teriophage and engineered bacteriophage. Engineered bacte no phage (black bar), 10 PFU/mL unmodified phage (p, riophage were constructed by inserting genetic modules (red bar), or 10 PFU/mL engineered phage (p3 (blue bar) under the control of a synthetic promoter (PtetO) and ribo were added to the previously drug-treated cultures. Three some-binding sequence (RBS) in between Sad and Pvul additional hours later, viable cell counts were obtained and restriction sites. A terminator (Term) ends transcription of 45 demonstrated that (pl. was able to reduce persister cell the respective gene(s). FIG.9A shows unmodified M13mp 18 levels better than no phage or (p, (p) contains lacz to allow blue-white screening of engi FIG. 17 shows mean killing with 60 ng/mL ofloxacin after neered bacteriophage. FIG.9B shows engineered M13mp 18 12 hours of treatment of E. coli EMG2 biofilms pregrown for bacteriophage expressing lexA3 (cp). FIG. 9C shows 24 hours. Where indicated, 10 PFU/mL of (r) lex A3 bacte engineered M13mp 18 bacteriophage expressing SOXR 50 riophage was used. (cp). FIG. 9D shows engineered M13mp 18 bacteriophage FIG. 18 shows the mean killing with 60 ng/mL ofloxacin expressing cSrA (cp). FIG. 9E shows engineered after 12 hours of treatment of E. coli EMG2 biofilms pre M13mp 18 bacteriophage expressing ompf (p). FIG. 9F grown for 24 hours. Where indicated, 10 PFU/mL of ps, shows engineered M13mp 18 bacteriophage expressing cSrA (P. Or (pset bacteriophage was used. and omple (Posra-ompF). 55 FIG. 19 shows an example of a promoter which can be used FIGS. 10A-10E show flow cytometry of cells with an SOS to express the nucleic acid in the engineered bacteriophage. responsive GFP plasmid exposed to no phage (black lines), FIG. 19 shows a P- (SEQID NO:32), P. (SEQID unmodified phage (p, (red lines), or engineered phage NO: 33), P - (SEQ ID NO. 34) and P (SEQ ID (ps (blue lines) for 6 hours with varying doses of ofloxacin. NO: 35) promoters which can be used. 10 plaque forming units per mL (PFU/mL) of phage were 60 applied. Cells exposed to no phage or punmod showed similar DETAILED DESCRIPTION SOS induction profiles, whereas cells with p exhibited significantly suppressed SOS responses. FIG. 10A shows 0 As disclosed herein, the inventors have discovered a two ng/mL ofloxacin treatment. FIG. 10B shows 20 ng/mL pronged strategy to significantly reduce or eliminate a bacte ofloxacin treatment. FIG. 10C show 60 ng/mL ofloxacin 65 rial infection. In particular, the inventors have engineered treatment. FIG. 10D show 100 ng/mL ofloxacin treatment. bacteriophages to be used in combination with an antimicro FIG. 10E shows 200 ng/mL ofloxacin treatment. bial agent. Such that the engineered bacteriophage functions US 9,056,899 B2 17 18 as an adjuvant to the antimicrobial agent. Thus, the inventors bacterial gene involved in antibiotic resistance and/or at least have engineered bacteriophages to be used in combination one bacterial gene involved in cell survival are referred to with an antimicrobial agent, such that the engineered bacte herein as “inhibitor-engineered bacteriophages'. In some riophage functions as an adjuvant to at least one antimicrobial embodiments of this aspect and all aspects discussed herein, agent. In particular, the inventors have engineered bacte an agent which inhibits an antibiotic resistance bacterial gene riophages to specifically disable (or deactivate) the bacteria's can inhibit the gene expression and/or protein function of natural resistance mechanisms to the antimicrobial agents antibiotic resistance genes such as, but not limited to cat, and/orphage infection. Accordingly, one aspect of the present VanA or mecD. In some embodiments of this aspect and all invention generally relates to engineered bacteriophages aspects discussed herein, an agent which inhibits a bacterial which have been modified or engineered to (i) inhibit at least 10 cell Survival gene can inhibit the gene expression and/or one bacterial resistance gene, or (ii) to inhibit at least one SOS protein function of a cell Survival repair gene Such as, but not response gene or bacterial defense gene in bacteria, or (iii) to limited to RecA, RecB. RecC, Spot or RelA. express a protein which increases the Susceptibility of a bac In some embodiments of this aspect and all aspects terial cell to an antimicrobial agent. Any one of these engi described herein, an inhibitor-engineered bacteriophage can neered bacteriophages, used alone, or in any combination can 15 comprise a nucleic acid encoding an agent which inhibits at be used with an antimicrobial agent. Accordingly, the inven least one gene involved in antibiotic resistance and/or cell tors have discovered a method to prevent the development of Survival repair. In one embodiment of this aspect and all bacterial resistance to antimicrobial agents and the generation aspect described herein, an inhibitor-engineered bacterioph of persistent bacteria by inhibiting the local bacterial syn age can comprise at least 2, 3, 4, 5 or even more, for example thetic machinery which normally circumvents the antimicro 10 different nucleic acids which inhibit at least one gene, for bial effect, by engineering bacteriophages to be used in con example, 2, 3, 4, 5 or up to 10 genes involved in antibiotic junction (or in combination with) an antimicrobial agent, resistance and/or cell Survival repair. In some embodiment of where an engineered bacteriophage can inhibit an antimicro this aspect, an inhibitor-engineered bacteriophage can com bial resistance gene, or inhibit a SOS response gene or a prise at least 2,3,4, 5 or more, for example 8 different nucleic non-SOS bacterial defense gene, or express a protein to 25 acids encoding inhibitors to at least one antibiotic resistance increase the Susceptibility of a bacterial cell to an antimicro gene or to at least one cell Survival repair gene. Such as at least bial agent. 2, 3, 4, 5 or more selected from the group, but not limited to, Accordingly, one aspect of the present invention relates to cat, VanA, mec), RecA, RecB. RecC. Spot or RelA and other the engineered bacteriophages as discussed herein for use in antibiotic resistance genes or cell Survival repair genes. In conjunction with (i.e. in combination with) at least one anti 30 some embodiments, any or all different combinations of microbial agent, and that the engineered bacteriophages serve inhibitors of antibiotic resistance genes and/or cell survival as adjuvants to such antimicrobial agents. repair genes can be present in an inhibitor-engineered bacte One aspect of the present invention relates to a method to riophage. potentiate the bacterial killing effect of an antimicrobial In another aspect of the present invention, an engineered agent. In particular, one aspect of the present invention relates 35 bacteriophage can comprise at least one nucleic acid encod to methods and compositions comprising engineered bacte ing a repressor protein, or fragment thereof of a bacterial SOS riophages for use in combination with an antimicrobial agent response gene, or an agent (such as a protein) which inhibits to potentiate the antimicrobial effect and bacterial killing of a non-SOS pathway bacterial defense gene and are referred to the antimicrobial agent. Another aspects relates to the use of herein as “repressor-engineered bacteriophages. In some an engineered bacteriophage as an antibiotic adjuvant. In 40 embodiments, the repressor of an SOS response gene is, for Some embodiments of this and all aspects described herein, an example but not limited to, lex A, or modified version thereof. engineered bacteriophage can be used as an antibiotic adju In some embodiments, the SOS response gene is, for example vant for anaminglycoside antimicrobial agent, such as but not but is not limited to marFRAB, arcAB and lexO. In some limited to, gentamicin, as antibiotic adjuvants for a B-lactam embodiments of this aspect and all other aspects described antibiotic, such as but not limited to, amplicillin, and as an 45 herein, an inhibitor of a non-SOS pathway bacterial defense antibiotic adjuvant for a quinolone antimicrobial agent. Such gene can be any agent, such as but not limited to a protein or as but not limited to, ofloxacin. In one embodiment of this an RNAi agent, Such as antisense to a non-SOS gene Such as, aspect and all aspects described herein, an engineered bacte for example but not limited to soxR, or modified version riophage can function as an antimicrobial adjuvant or antibi thereof. In some embodiments of this aspect and all other otic adjuvant for at least 2, at least 3, at least 4, at least 5, least 50 aspects described herein, an repressor, Such as an agent which 6, at least 7, at least 8, at least 9 or at least 10 or more different inhibits a non-SOS pathway bacterial defense gene inhibits, antimicrobial agents at any one time. In some embodiments, for example genes selected from the group of marr, arc, any of the engineered bacteriophages as disclosed herein can SOXR, fur, crp, iccdA or craA or ompA or modified version used in combination with at least one or more antimicrobial thereof. In other embodiments of this aspect of the invention, agent, for example an engineered bacteriophage as disclosed 55 a nucleic acid of a repressor engineered bacteriophage is an herein can used in combination with at least 2, 3, 4, 5, 6, 7, 8, agent which inhibits a non-SOS defense gene, for example 9 or 10 or more different antimicrobial agents. the repressor agent can inhibit any gene, or any combination In one aspect of the present invention, an engineered bac of genes listed in Table 2. In some embodiments, a repressor teriophage as disclosed herein can comprise a nucleic acid engineered bacteriophage which inhibits a non-SOS defense encoding an agent which inhibits at least one bacterial gene 60 gene can be used in combination with selected antimicrobial involved in the development of antibiotic resistance. In agents, for example, where the repressor-engineered bacte another embodiment of this aspect and all aspects described riophage encodes an agent which inhibits a gene listed in herein, an engineered bacteriophage can comprise a nucleic Table 2A, such a repressor-engineered bacteriophage can be acid encoding an agent which inhibits at least one gene used in combination with a ciprofloxacin antimicrobial agent involved in bacterial cell survival repair. As discussed previ 65 or a variant or analogue thereof. Similarly, in other embodi ously, such engineered bacteriophages which comprise a ments a repressor-engineered bacteriophage which inhibits a nucleic acid encoding an agent which inhibits at least one non-SOS defense gene can encode an agent which inhibits a US 9,056,899 B2 19 20 gene listed in Table 4B can be used in combination with a efficacy of antimicrobial agents. An inhibitor-engineered Vancomycin antimicrobial agent or a variant or analogue bacteriophages and/or a repressor engineered bacteriophage thereof. Similarly, in other embodiments a repressor-engi and/or a susceptibility-engineered bacteriophage is consid neered bacteriophage which inhibits a non-SOS defense gene ered to potentiate the effectiveness of an antimicrobial agent can encode an agent which inhibits a gene listed in Table 2C. if the amount of antimicrobial agent used in combination with 2D, 2E, 2F and 2G can be used in combination with a rifampi an engineered bacteriophage as disclosed herein is reduced cin antimicrobial agent, or a amplicillin antimicrobial agent or by at least about 10% without adversely affecting the result, a sulfmethaxaZone antimicrobial agent or a gentamicin anti for example, without adversely effecting the level of antimi microbial agent or a metronidazole antimicrobial agent, crobial activity. In another embodiment, the criteria used to respectively, or a variant or analogue thereof. 10 select an inhibitor-engineered bacteriophage and/or a repres In some embodiments of this aspect an all other aspects Sor engineered bacteriophage and/or a susceptibility-engi discussed herein, a repressor is, for example but not limited neered bacteriophage that potentiates the activity of an anti to, leXA, marr, arc, SOXR, fur, crp, iccdA, craA or ompA or a microbial agent is a reduction of at least about 10%, ... or at modified version thereof. In some embodiments, the SOS least about 15%, ... or at least about 20%, ... or at least about response gene is, for example but is not limited to marr AB, 15 25%, ... or at least about 35%, ... or at least about 50%, ... arc AB and lexO. or at least about 60%, ... or at least about 90% and all integers In some embodiments of this aspect and all other aspects in between 10-90% of the amount of the antimicrobial agent described herein, a repressor-engineered bacteriophage can without adversely effecting the antimicrobial effect when comprise at least 2, 3, 4, 5 or more, for example 8 different compared to the similar amount without the addition of an nucleic acids encoding different repressors of SOS response inhibitor-engineered bacteriophage and/or repressor engi genes, such as at least 2, 3, 4, 5 or more selected from the neered bacteriophage and/or a susceptibility-engineered bac group, but not limited to, lex A, marr AB, arc AB andlexO and teriophage. Stated another way, an inhibitor-engineered bac other repressors of SOS response genes, or least 2, 3, 4, 5 or teriophage and/or repressor engineered bacteriophage and/or more, for example 8 different nucleic acids encoding different a Susceptibility-engineered bacteriophage is effective as an repressors (i.e. inhibitors) of non-SOS defense genes. In some 25 adjuvant to an antimicrobial agent when the combination of embodiments, a repressor engineered bacteriophage can the antimicrobial agent and the engineered bacteriophage comprise any or all different combinations of repressors of results in about the same level (i.e. within about 10%) of SOS genes described herein and/or any and all different com antimicrobial effect at reducing the bacterial infection or binations of inhibitors non-SOS defense genes listed in killing the bacteria with the reduction in the dose (i.e. the Tables 2 and 2A-2G can be present in a repressor-engineered 30 amount) of the antimicrobial agent. Such a reduction in anti bacteriophage. microbial dose can be, for example by about 10%, or about In another aspect of the present invention, an engineered 15%, . . . or about 20%, . . . or about 25%, . . . or about bacteriophage can comprise at least one nucleic acid encod 35%, ... or about 50%, ... or about 60%, ... or more than 60% ing an agent, such as but not limited to a protein, which with the same level of antimicrobial efficacy. increases the Susceptibility of a bacteria to an antimicrobial 35 The inventors herein have demonstrated that the engi agent. Such herein engineered bacteriophage which com neered bacteriophage can target gene networks that are not prises a nucleic acid encoding an agent which increases the directly attacked by antibiotics and by doing so, greatly Susceptibility of a bacteria to an antimicrobial agent can be enhanced the efficacy of antibiotic treatment in bacteria, such referred to herein as an “susceptibility agent-engineered bac as Escherichia coli. The inventors demonstrated that Sup teriophage' but are also encompassed under the definition of 40 pressing or inhibiting the bacterial SOS response network a “repressor-engineered bacteriophage'. In some embodi with a repressor-engineered bacteriophage can enhance kill ments of this aspect, and all other aspects described herein, ing by an antimicrobial agent such as an antibiotic, for Such an agent which increases the Susceptibility of a bacteria example but not limited to, ofloxacin, a quinolone drug, by to an antimicrobial agent is referred to as a “susceptibility over 2.7 orders of magnitude as compared with a control agent” and refers to any agent which increases the bacteria's 45 bacteriophage (i.e. non-engineered bacteriophages) plus susceptibility to the antimicrobial agent by at least about 10% ofloxacin, and over 4.5 orders of magnitude compared with or at least about 15%, or at least about 20% or at least about ofloxacin alone. 30% or at least about 50% or more than 50%, or any integer The inventors have also demonstrated herein in Examples between 10% and 50% or more, as compared to the use of the 6-8 that a repressor-engineered bacteriophage, which com antimicrobial agent alone. In one embodiment, a Susceptibil 50 prises at least one inhibitor to one or more non-SOS genetic ity agent is an agent which specifically targets a bacteria cell. networks are also effective antibiotic adjuvants. The inven In another embodiment, a Susceptibility agent modifies (i.e. tors also demonstrated that repressor-engineered bacterioph inhibits or activates) a pathway which is specifically age and/or inhibitor-engineered bacteriophage can reduce the expressed in bacterial cells. In one embodiment, a suscepti number of antibiotic-resistant bacteria in a population and act bility agent is an agent which has an additive effect of the 55 as a strong adjuvant for a variety of other bactericidal antibi efficacy of the antimicrobial agent (i.e. the agent has an addi otics, such as for example, but not limited to gentamicin and tive effect of the killing efficacy or inhibition of growth by the ampicillin Thus, the inventors have demonstrated that by antimicrobial agent). In a preferred embodiment, a suscepti selectively targeting gene networks with bacteriophage, one bility agent is an agent which has a synergistic effect on the can enhance killing by antibiotics, thus discovering a highly efficacy of the antimicrobial agent (i.e. the agent has a syn 60 effective new antimicrobial strategy. ergistic effect of the killing efficacy or inhibition of growth by Definitions the antimicrobial agent). For convenience, certain terms employed in the entire Accordingly, another aspect of the invention relates to the application (including the specification, examples, and use of an inhibitor-engineered bacteriophage and/or a repres appended claims) are collected here. Unless defined other sor-engineered bacteriophage and/or a susceptibility-engi 65 wise, all technical and scientific terms used herein have the neered bacteriophage to potentiate the killing effect of anti same meaning as commonly understood by one of ordinary microbial agents or stated another way, to enhance the skill in the art to which this invention belongs. US 9,056,899 B2 21 22 As used herein, the term “adjuvant” as used herein refers to includes any agent (Such as a protein or RNAi agent) which an agent which enhances the pharmaceutical effect of another increases the bacteria's susceptibility to the antimicrobial agent. As used herein, the bacteriophages as disclosed herein agent by at least about 10% or at least about 15%, or at least function as adjuvants to antimicrobial agents, such as, but not about 20% or at least about 30% or at least about 50% or more limited to antibiotic agents, by enhancing the effect of the than 50%, or any integer between 10% and 50% or more, as antimicrobial agents by at least. .. 5%, ... at least 10%. . . . compared to the use of the antimicrobial agent alone. In one at least 15%, ... at least 20%, ... at least 25%, ... at least embodiment, a susceptibility agent is an agent which specifi 35%, ... at least 50%, ... at least 60%, ... at least 90% and cally targets a bacteria cell. In another embodiment, a Suscep all amounts in-between as compared to use of the antimicro tibility agent modifies (i.e. inhibits or activates) a pathway bial agent alone. Accordingly, the engineered bacteriophages 10 which is specifically expressed in bacterial cells. In one as disclosed herein, such as the inhibitor-engineered bacte embodiment, a Susceptibility agent is an agent which has an riophage and/or repressor engineered bacteriophage function additive effect of the efficacy of the antimicrobial agent (i.e. as antimicrobial agent adjuvants. the agent has an additive effect of the killing efficacy or As used herein, the term “inhibitor-engineered bacterioph inhibition of growth by the antimicrobial agent). In a pre age” refers to a bacteriophage that have been genetically 15 ferred embodiment, a susceptibility agent is an agent which engineered to comprise a nucleic acid which encodes an agent has a synergistic effect on the efficacy of the antimicrobial which inhibits at least one gene involved in antibiotic resis agent (i.e. the agent has a synergistic effect of the killing tance and/or cell Survival. Such engineered bacteriophages as efficacy or inhibition of growth by the antimicrobial agent). disclosed herein are termed “inhibitor-engineered bacte The term “engineered bacteriophage' as used herein refer riophages' as they comprise a nucleic acid which encodes at to any one, or a combination of an inhibitor-engineered bac least one inhibitor genes, such as but not limited to antibiotic teriophage or a repressor-engineered bacteriophage or a Sus resistance genes such as, but not limited to cat, VanA or mecD. ceptibility-engineered bacteriophage as these phrases are or cell Survival repair gene Such as, but not limited to RecA defined herein. RecB. RecC, Spot or RelA. Naturally, one can engineer a The term “additive' when used in reference to a suscepti bacteriophage to comprise at least one nucleic acid which 25 bility agent, or an engineered bacteriophage Such as an Sus encodes more than one inhibitor, for example, two or more ceptibility-bacteriophage having an additive effect of the effi inhibitors to the same gene or to at least two different genes cacy of the antimicrobial agent refers to refers to a total which can be used in the methods and compositions as dis increase in antimicrobial efficacy (i e killing, or reducing the closed herein. viability of a bacterial population or inhibiting growth of a As used herein, the term “repressor-engineered bacte 30 bacterial population) with the combination of the antimicro riophage” refers to bacteriophages that have been genetically bial agent and the Susceptibility-engineered bacteriophage engineered to comprise at least one nucleic acid which components of the invention, over their single efficacy of each encodes a repressor protein, or fragment thereof, where the component alone. An additive effect to increase total antimi repressor protein function to prevent activation of a gene crobial effectiveness can be a result of an increase in antimi involved in a SOS response. Alternatively, the term repressor 35 crobial effect of both components (i.e. the antimicrobial agent engineeredbacteriophage refers to a bacteriophage which has and the Susceptibility-engineered bacteriophage) or alterna been genetically engineered to comprise at least one nucleic tively, it can be the result of the increase inactivity of only one acid which encodes a repressor protein, such as an inhibitors of the components (i.e. the antimicrobial agent or the Suscep (including but not limited to RNAi agents) which inhibits a tibility-engineered bacteriophage). For clarification by way non-SOS bacterial defense. Such engineered bacteriophages 40 of a non-limiting illustrative example of a additive effect, if an as disclosed herein are referred to herein as “repressor-engi antimicrobial agent is effective at reducing a bacterial popu neered bacteriophages' as they comprise a nucleic acid lation by 30%, and a susceptibility-engineered bacteriophage encoding a repressor protein, for example, but not limited to, was effective at reducing a bacterial population by 20%, an leXA, or soxR, or modified version thereof. In some embodi additive effect of a combination of the antimicrobial agent ments, a SOS response gene is, for example but is not limited 45 and the Susceptibility-engineered bacteriophage could be, for to marr AB, arc AB and lexO. One can engineer a repressor example 35%. Stated another way, in this example, any total engineered bacteriophage to comprise at least one nucleic effect greater than 30% (i.e. greater than the highest antimi acid which encodes more than one repressor, for example at crobial efficacy (i.e. 30% which, in this example is displayed least 2, 3, 4 or more repressors to the same or different SOS by the antimicrobial agent) would be indicative of an additive response gene, in any combination, can be used in the meth 50 effect. In some embodiments of the present invention, the ods and compositions as disclosed herein. Similarly, one can antimicrobial agent and Susceptibility-engineered bacte also engineer a repressor-engineered bacteriophage to com riophage component show at least some additive anti-patho prise at least one nucleic acid which encodes more than one genic activity. An additive effect of the combination of an repressor, for example at least 2, 3, 4 or more repressors. Such antimicrobial agent with an engineered bacteriophage can be as inhibitors which inhibits any number and any combination 55 an increase in at least about 10% or at least about 20% or at of non-SOS bacterial defense genes listed in Table 2, and can least about 30% or at least about 40% or at least about 50% or be used in any combination, can be used in the methods and more anti-pathogenic (or antimicrobial) efficacy as compared compositions as disclosed herein. The term “repressor-engi to the highest antimicrobial effect achieved with either the neered bacteriophage' also encompasses Susceptibility-engi antimicrobial agent alone or the engineered bacteriophage neered bacteriophages as that term is defined herein. 60 alone. As used herein, the term “susceptibility-engineered bacte The term “synergy' or “synergistically are used inter riophage” refers to a bacteriophage that has been genetically changeably herein, and when used in reference to a suscepti engineered to comprise at least one nucleic acid which bility agent, or an engineered bacteriophage Such as an Sus encodes at least one agent which increases the Susceptibility ceptibility-bacteriophage having a synergistic effect of the of a bacterial cell to an antimicrobial agent. An agent which 65 efficacy of the antimicrobial agent refers to a total increase in increases the Susceptibility of a bacteria to an antimicrobial antimicrobial efficacy (i.e. killing, or reducing the viability of agent is referred to herein as a “susceptibility agent” and a bacterial population or inhibiting growth of a bacterial US 9,056,899 B2 23 24 population) with the combination of the antimicrobial agent functional SOS response (i.e. in the presence of repressors as and the Susceptibility-engineered bacteriophage components disclosed herein), cells are sensitive to DNA damaging of the invention, over their single and/or additive efficacy of agents. McKenzie et al., PNAS, 2000; 6646-6651; Michel, each component alone. A synergistic effect to increase total PLoS Biology, 2005; 3: e255, and which are incorporated in antimicrobial effectiveness can be a result of an increase in 5 their entirety herein by reference. A “non-SOS gene' also antimicrobial effect of both components (i.e. the antimicro includes a “bacterial defense gene' and refers to genes bial agent and the Susceptibility-engineered bacteriophage) expressed by a bacteria or a microorganism which serve pro or alternatively, it can be the result of the increase in activity tect the bacteria or microorganism from cell death, for of only one of the components (i.e. the antimicrobial agent or example from being killed or growth Suppressed by an anti the Susceptibility-engineered bacteriophage). For clarifica 10 microbial agent. Typically, inhibition or knocking out Such tion by way of a non-limiting illustrative example of a syner non-SOS defense genes increases the susceptibility of a gistic effect, if an antimicrobial agent is effective at reducing microorganism Such as bacteria to an antimicrobial agent. A (i.e. killing) a bacterial population by 15%, and a Susceptibil non-SOS gene' or “bacterial defense gene' is not part of the ity-engineered bacteriophage was effective at reducing a bac SOS-response network, but still serve as protective functions terial population by 10%, a synergistic effect of a combina 15 to prevent microorganism cell death. In certain conditions, tion of the antimicrobial agent and the Susceptibility Some non-SOS genes and/or bacterial defense genes can be engineered bacteriophage could be 50%. Stated another way, expressed (i.e. upregulated) on DNA damage or in stressful in this example, any total effect greater than 25% (i.e. greater conditions. Examples of a non-SOS gene is soxS, which is than the sum of the antibacterial agent alone (i.e. 15%) and the repressed by SOXR, and examples of defense genes are any susceptibility agent alone (i.e. 10%) would be indicative of a gene listed in Table 2. synergistic effect. In some embodiments of the present inven The term “repressor as used herein, refers to a protein that tion, the antimicrobial agent and Susceptibility-engineered binds to an operator of a gene preventing the transcription of bacteriophage component show at least some synergistic anti the gene. Accordingly, a repressor can effectively "suppress' pathogenic activity. A synergistic effect of the combination of or inhibit the transcription of a gene. The binding affinity of an antimicrobial agent with an engineered bacteriophage can 25 repressors for the operator can be affected by other mol be an increase in at least about 10% or at least about 20% or ecules, such as inducers, which bind to repressors and at least about 30% or at least about 40% or at least about 50% decrease their binding to the operator, while co-repressors or more anti-pathogenic (or antimicrobial) efficacy as com increase the binding. The paradigm of repressor proteins is pared to the sum of the antimicrobial effect achieved with use the lactose repressorprotein that acts on the lac operon and for of the antimicrobial agent alone or the engineered bacterioph 30 which the inducers are B-galactosides such as lactose, it is a age alone. polypeptide of 360 amino acids that is active as a tetramer. The term “bidirectional synergy” refers to the increase in Other examples are the lambda repressor protein of lambda activity of each component (i.e. the antimicrobial agent and bacteriophage that prevents the transcription of the genes the engineered bacteriophage) when used in combination required for the lytic cycle leading to lysogeny and the cro with each other, and not merely an increase in activity of one 35 protein, also of lambda, which represses the transcription of of the antimicrobial components. In some embodiments, an the lambda repressor protein establishing the lytic cycle. Both antimicrobial agent and engineered bacteriophage show at of these are active as dimers and have a common structural least synergistic antimicrobial activity. In some embodi feature the helix turn helix motif that is thought to bind to ments, an antimicrobial agent and engineered bacteriophage DNA with the helices fitting into adjacent major grooves. show bidirectional synergistic antimicrobial activity. Stated 40 Useful repressors according to the present invention include, in other words, for example, bidirectional Synergy means an but are not limited to leXA, marr, arc, SOXR, fur, crp, iccdA, or engineered bacteriophage enhances the activity of an antimi craA or modified version thereof. crobial agent and Vice versa, an antimicrobial agent can be The term “antimicrobial agent” as used herein refers to any used to enhance the activity of the engineered bacteriophage. entity with antimicrobial activity, i.e. the ability to inhibit the The term “SOS used in the context of "SOS response” or 45 growth and/or kill bacterium, for example gram positive- and “SOS response genes' as used herein refers to an inducible gram negative bacteria. An antimicrobial agent is any agent DNA repair system that allows bacteria to survive sudden which results in inhibition of growth or reduction of viability increases in DNA damage. SOS response genes are repressed of a bacteria by at least about 30% or at least about 40%, or at to differ rent degrees under normal growth conditions. With least about 50% or at least about 60% or at least about 70% or out being bound by theory, the SOS response is a postrepli 50 more than 70%, or any integer between 30% and 70% or cation DNA repair system that allows DNA replication to more, as compared to in the absence of the antimicrobial bypass lesions or errors in the DNA. One example is the SOS agent. Stated another way, an antimicrobial agent is any agent repressor RecA protein. The RecA protein, stimulated by which reduces a population of antimicrobial cells, such as single-stranded DNA, is involved in the inactivation of the bacteria by at least about 30% or at least about 40%, or at least Lex A repressor thereby inducing the response. The bacterial 55 about 50% or at least about 60% or at least about 70% or more SOS response, studied extensively in Escherichia coli, is a than 70%, or any integer between 30% and 70% as compared global response to DNA damage in which the cell cycle is to in the absence of the antimicrobial agent. In one embodi arrested and DNA repair and mutagenesis are induced. SOS is ment, an antimicrobial agent is an agent which specifically the prototypic cell cycle check-point control and DNA repair targets a bacteria cell. In another embodiment, an antimicro system. A central part of the SOS response is the de-repres 60 bial agent modifies (i.e. inhibits or activates or increases) a sion of more than 20 genes under the direct and indirect pathway which is specifically expressed in bacterial cells. In transcriptional control of the Lex A repressor. The Lex A regu Some embodiments, an antimicrobial agent does not include lon includes recombination and repair genes recA, recN, and the following agents; chemotherapeutic agent, a toxin protein ruvAB, nucleotide excision repair genesuvraBanduvrD, the expressed by a bacteria or other microorganism (i.e. a bacte error-prone DNA polymerase (pol) genes dinB (encoding pol 65 rial toxin protein) and the like. An antimicrobial agent can IV) and umul C (encoding polV), and DNA polymerase II in include any chemical, peptide (i.e. an antimicrobial peptide), addition to many other genes functions. In the absence of a peptidomimetic, entity or moiety, or analogues of hybrids US 9,056,899 B2 25 26 thereof, including without limitation synthetic and naturally classes. This includes those microorganisms that have more occurring non-proteinaceous entities. In some embodiments, resistance than those that are resistant to three or more anti an antimicrobial agent is a small molecule having a chemical biotics in a single antibiotic class. This also includes micro moiety. For example, chemical moieties include unsubsti organisms that are resistant to a wider range of antibiotics, i.e. tuted or substituted alkyl, aromatic or heterocyclyl moieties microorganisms that are resistant to one or more classes of including macrollides, leptomycins and related natural prod antibiotics. ucts or analogues thereof. Antimicrobial agents can be any The term “persistent cell' or “persisters' are used inter entity known to have a desired activity and/or property, or can changeably herein and refer to a metabolically dormant Sub be selected from a library of diverse compounds. population of microorganisms, typically bacteria, which are The term 'agent’ as used herein and throughout the appli 10 not sensitive to antimicrobial agents such as antibiotics. Per cation is intended to refer to any means such as an organic or sisters typically are not responsive (i.e. are not killed by the inorganic molecule, including modified and unmodified antibiotics) as they have non-lethally downregulated the path nucleic acids Such as antisense nucleic acids, RNAi Such as ways on which the antimicrobial agents act i.e. the persister siRNA or shRNA, peptides, peptidomimetics, receptors, cells have down regulated the pathways which are normally ligands, and antibodies, aptamers, polypeptides, nucleic acid 15 inhibited or corrupted by the antimicrobial agents, such as the analogues or variants thereof. transcription, translation, DNA replication and cell wall bio The term “antimicrobial peptide' as used herein refers to synthesis pathways. Persisters can develop at non-lethal (or any peptides with antimicrobial activity, i.e. the ability to Sub-lethal) concentrations of the antimicrobial agent. inhibit the growth and/or kill bacterium, for example gram The term “analog as used herein refers to a composition positive- and gram negative bacteria. The term antimicrobial that retains the same structure or function (e.g., binding to a peptides encompasses all peptides that have antimicrobial receptor) as a polypeptide or nucleic acid herein. Examples of activity, and are typically, for example but not limited to, short analogs include peptidomimetics, peptide nucleic acids, proteins, generally between 12 and 50 amino acids long, Small and large organic or inorganic compounds, as well as however larger proteins with Such as, for example lysozymes derivatives and variants of a polypeptide or nucleic acid are also encompassed as antimicrobial peptides in the present 25 herein. The term “analog as used herein refers to a compo invention. Also included in the term antimicrobial peptide are sition that retains the same structure or function (e.g., binding antimicrobial peptidomimetics, and analogues or fragments to a receptor) as a polypeptide or nucleic acid herein. thereof. The term “antimicrobial peptide also includes all The term “infection' or “microbial infection' which are cyclic and non-cyclic antimicrobial peptides, orderivatives or used interchangeably herein refers to in its broadest , variants thereof, including tautomers, see Li et al. JACS, 30 any infection caused by a microorganism and includes bac 2006, 128: 5776-85 and world-wide-web at //aps.unmc.edu, terial infections, fungal infections, yeast infections and pro at/AP/main.php for examples, which are incorporated herein toZomal infections. in their entirety by reference. In some embodiments, the The term “treatment and/prophylaxis' refers generally to antimicrobial peptide is a lipopeptide, and in some embodi afflicting a subject, tissue or cell to obtain a desired pharma ments the lipopeptide is a cyclic lipopeptide. The lipopeptides 35 cologic arid/or physiologic effect, which in the case of the include, for example but not limited to, the polymyxin class of methods of this invention, include reduction or elimination of antimicrobial peptides. microbial infections or prevention of microbial infections. The term “microorganism’ includes any microscopic The methods as disclosed herein can be used prophylactically organism or taxonomically related macroscopic organism for example in instances where an individual is Susceptible within the categories algae, bacteria, fungi, yeast and proto 40 for infections or re-infection with a particular bacterial strain Zoa or the like. It includes Susceptible and resistant microor or a combination of Such strains. For example, microbial ganisms, as well as recombinant microorganisms. Examples infections such as bacterial infections such as biofilms can of infections produced by Such microorganisms are provided occur on any Surface where sufficient moisture and nutrients herein. In one aspect of the invention, the antimicrobial are present. One such surface is the Surface of implanted agents and enhancers thereof are used to target microorgan 45 medical devices, such as catheters, heart valves and joint isms in order to prevent and/or inhibit their growth, and/or for replacements. In particular, catheters are associated with their use in the treatment and/or prophylaxis of an infection infection by many biofilm forming organisms such as Staphy caused by the microorganism, for example multi-drug resis lococcus epidermidis, Staphylococcus aureus, Pseudomonas tant microorganisms and gram-negative microorganisms. In aeruginosa, Enterococcus faecalis and Candida albicans Some embodiments, gram-negative microorganisms are also 50 which frequently result in generalized blood stream infection. targeted. In a subject identified to have a catheter infected with bacte The anti-pathogenic aspects of the invention target the rial. Such as for example, a bacterial infected central venous broader class of “microorganism' as defined herein. How catheter (CVC), the subject can have the infected catheter ever, given that a multi-drug resistant microorganism is so removed and can be treated by the methods and compositions difficult to treat, the antimicrobial agent and inhibitor-engi 55 as disclosed herein comprising an engineered bacteriophage neered bacteriophage and/or repressor-engineered bacte and antimicrobial agent to eliminate the bacterial infection. riophage in the context of the anti-pathogenic aspect of the Furthermore, on removal of the infected catheter and its invention is Suited to treating all microorganisms, including replacement with a new catheter, the Subject can also be for example multi-drug resistant microorganisms, such as administered the compositions comprising engineered bacte bacterium and multi-drug resistant bacteria. 60 riophages and antimicrobial agents as disclosed herein on a Unless stated otherwise, in the context of this specification, prophylaxis basis to prevent re-infection or the re-occurrence the use of the term “microorganism” alone is not limited to of the bacterial infection. Alternatively, a subject can be “multi-drug resistant organism’, and encompasses both drug administered the compositions as disclosed herein compris Susceptible and drug-resistant microorganisms. The term ing engineered bacteriophages and antimicrobial agents on a “multi-drug resistant microorganism” refers to those organ 65 prophylaxis basis on initial placement of the catheter to pre isms that are, at the very least, resistant to more than two vent any antimicrobial infection such as a bacterial biofilm antimicrobial agents such as antibiotics in different antibiotic infection. The effect can be prophylactic in terms of com US 9,056,899 B2 27 28 pletely or partially preventing a disease or sign or symptom As used herein, “gene silencing or “gene silenced in thereof, and/or can be therapeutic in terms of a partial or reference to an activity of in RNAi molecule, for example a complete cure of a disease. siRNA or miRNA refers to a decrease in the mRNA level in a As used herein, the term “effective amount' is meant an cell for a target gene by at least about 5%, about 10%, about amount of antimicrobial agent and/or inhibitor-engineered 20%, about 30%, about 40%, about 50%, about 60%, about bacteriophages or repressor-engineered bacteriophages 70%, about 80%, about 90%, about 95%, about 99%, about effective to yield a desired decrease in bacteria or increase to 100% of the mRNA level found in the cell without the pres increase the efficacy of antimicrobial agent as compared to ence of the miRNA or RNA interference molecule. In one the activity of the antimicrobial agent alone (i.e. without the preferred embodiment, the mRNA levels are decreased by at engineered bacteriophages as disclosed herein). The term 10 least about 70%, about 80%, about 90%, about 95%, about “effective amount’ as used herein refers to that amount of 99%, about 100%. composition necessary to achieve the indicated effect, i.e. a As used herein, the term “RNAi refers to any type of reduction of the number of viable microorganisms, such as interfering RNA, including but not limited to, siRNAi, shR bacteria, by at reduction of least 5%, at least 10%, by at least NAi, endogenous microRNA and artificial microRNA. For 20%, by at least 30%... at least 35%, ... at least 50%, ... at 15 instance, it includes sequences previously identified as least 60%, ... at least 90% or any reduction of viable micro siRNA, regardless of the mechanism of down-stream pro organism in between. As used herein, the effective amount of cessing of the RNA (i.e. although siRNAs are believed to have the bacteriophage as disclosed herein is the amount Sufficient a specific method of in vivo processing resulting in the cleav to enhance the effect of the antimicrobial agents by at age of mRNA, such sequences can be incorporated into the least . . . 5%, at least 10%, . . . at least 15%, . . . at least vectors in the context of the flanking sequences described 20%, ... at least 25%, ... at least 35%, ... at least 50%, ... herein). at least 60%, ... at least 90% and all amounts in-between as As used herein an “siRNA refers to a nucleic acid that compared to use of the antimicrobial agent alone. Or alterna forms a double stranded RNA, which double stranded RNA tively result in the same efficacy of the antimicrobial effect has the ability to reduce or inhibit expression of a gene or with less (i.e. for example by about 10%, or about 15%, ... or 25 target gene when the siRNA is present or expressed in the about 20%, ... or about 25%, ... or about 35%, ... or about same cell as the target gene, for example Lp-PLA. The 50%, ... or about 60%, ... or more than 60% less) amount or double stranded RNA siRNA can be formed by the comple dose of the antimicrobial agents as compared to its use alone mentary strands. In one embodiment, a siRNA refers to a to achieve the same efficacy of antimicrobial effect. The nucleic acid that can form a double stranded siRNA. The “effective amount’ or “effective dose' will, obviously, vary 30 sequence of the siRNA can correspond to the full length target with Such factors, in particular, the Strain of bacteria being gene, or a Subsequence thereof. Typically, the siRNA is at treated, the strain of bacteriophage being used, the genetic least about 15-50 nucleotides in length (e.g., each comple modification of the bacteriophage being used, the antimicro mentary sequence of the double stranded siRNA is about bial agent, as well as the particular condition being treated, 15-50 nucleotides in length, and the double stranded siRNA is the physical condition of the Subject, the type of subject being 35 about 15-50 base pairs in length, preferably about 19-30 base treated, the duration of the treatment, the route of administra nucleotides, preferably about 20-25 nucleotides in length, tion, the type of antimicrobial agent and/or enhancer of anti e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in microbial agent, the nature of concurrent therapy (if any), and length). the specific formulations employed, the ratio of the antimi As used herein “shRNA” or “small hairpin RNA' (also crobial agent and/or enhancers antimicrobial agent compo 40 called stem loop) is a type of siRNA. In one embodiment, nents to each other, the structure of each of these components these shRNAs are composed of a short, e.g. about 19 to about or their derivatives. The term “effective amount' when used 25 nucleotide, antisense strand, followed by a nucleotide loop in reference to administration of the compositions comprising of about 5 to about 9 nucleotides, and the analogous sense an antimicrobial agent and a engineered bacteriophage as Strand. Alternatively, the sense strand can precede the nucle disclosed herein to a subject refers to the amount of the 45 otide loop structure and the antisense Strand can follow. compositions—to reduce or stop at least one symptom of the The terms “microRNA or “miRNA are used inter disease or disorder, for example a symptom or disorder of the changeably herein are endogenous RNAS, Some of which are microorganism infection, such as bacterial infection. For known to regulate the expression of protein-coding genes at example, an effective amount using the methods as disclosed the posttranscriptional level. Endogenous microRNA are herein would be considered as the amount sufficient to reduce 50 small RNAs naturally present in the genome which are a symptom of the disease or disorder of the bacterial infection capable of modulating the productive utilization of mRNA. by at least 10%. An effective amount as used herein would The term artificial microRNA includes any type of RNA also include an amount Sufficient to prevent or delay the sequence, other than endogenous microRNA, which is development of a symptom of the disease, alter the course of capable of modulating the productive utilization of mRNA. a symptom disease (for example but not limited to, slow the 55 MicroRNA sequences have been described in publications progression of a symptom of the disease), or reverse a symp such as Lim, et al., Genes & Development, 17, p. 991-1008 tom of the disease. (2003), Limetal Science 299, 1540 (2003), Lee and Ambros As used herein, a “pharmaceutical carrier is a pharmaceu Science, 294, 862 (2001), Lau et al., Science 294, 858-861 tically acceptable solvent, Suspending agent or vehicle for (2001), Lagos-Quintana et al. Current Biology, 12, 735-739 delivering the combination of antimicrobial agent and/or 60 (2002), Lagos Quintana et al. Science 294, 853-857 (2001), inhibitor-engineered bacteriophages or repressor-engineered and Lagos-Quintana etal, RNA, 9, 175-179 (2003), which are bacteriophages to the surface infected with bacteria or to a incorporated by reference. Multiple microRNAs can also be subject. The carrier can be liquid or solid and is selected with incorporated into a precursor molecule. Furthermore, the planned manner of administration in mind. Each carrier miRNA-like stem-loops can be expressed in cells as a vehicle must be pharmaceutically “acceptable' in the sense of being 65 to deliver artificial miRNAs and short interfering RNAs (siR compatible with other ingredients of the composition and non NAS) for the purpose of modulating the expression of endog injurious to the Subject. enous genes through the miRNA and or RNAi pathways. US 9,056,899 B2 29 30 As used herein, “double stranded RNA or “dsRNA refers tance gene protein in the presence of an inhibitor), or any to RNA molecules that are comprised of two strands. Double decrease in biological activity of the protein (i.e. of an anti stranded molecules include those comprised of a single RNA biotic resistance gene protein) between 10-100% as com molecule that doubles back on itself to form a two-stranded pared to a in the absence of an inhibitor. structure. For example, the stem loop structure of the progeni The terms 'activate” or “increased' or “increase' as used tor molecules from which the single-stranded miRNA is in the context of biological activity of a protein (i.e. activation derived, called the pre-miRNA (Bartel et al. 2004. Cell 116: of a SOS response gene) herein generally means an increase 281-297), comprises a dsRNA molecule. in the biological function of the protein (i.e. SOS response The terms “patient”, “subject' and “individual are used protein) by a statically significant amount relative to in a interchangeably herein, and refer to an animal, particularly a 10 control condition. For the avoidance of doubt, an “increase' human, to whom treatment including prophylaxis treatment of activity, or “activation of a protein means a statistically is provided. The term “subject' as used herein refers to human significant increase of at least about 10% as compared to the and non-human animals. The term “non-human animals' and absence of an agonist or activator agent, including an increase “non-human mammals' are used interchangeably herein of at least about 20%, at least about 30%, at least about 40%, includes all vertebrates, e.g., mammals, such as non-human 15 at least about 50%, at least about 60%, at least about 70%, at primates, (particularly higher primates), sheep, dog, rodent least about 80%, at least about 90%, at least about 100% or (e.g. mouse or rat), guinea pig, goat, pig, cat, rabbits, cows, more, including, for example at least 2-fold, at least 3-fold, at and non-mammals such as chickens, amphibians, reptiles etc. least 4-fold, at least 5-fold, at least 10-fold increase or greater In one embodiment, the Subject is human. In another embodi as compared to in a control condition. ment, the Subject is an experimental animal or animal Substi The term “nucleic acid' or "oligonucleotide' or “poly tute as a disease model. Suitable mammals also include mem nucleotide' used herein can mean at least two nucleotides bers of the orders Primates, Rodentla, Lagomorpha, Cetacea, covalently linked together. As will be appreciated by those in Homo sapiens, Carnivora, Perissodactyla and Artiodactyla. the art, the depiction of a single strand also defines the Members of the orders Perissodactyla and Artiodactyla are sequence of the complementary strand. Thus, a nucleic acid included in the invention because of their similar biology and 25 also encompasses the complementary Strand of a depicted economic importance, for example but not limited to many of single strand. As will also be appreciated by those in the art, the economically important and commercially importantani many variants of a nucleic acid can be used for the same mals such as goats, sheep, cattle and pigs have very similar purpose as a given nucleic acid. Thus, a nucleic acid also biology and share high degrees of genomic homology. encompasses Substantially identical nucleic acids and The term "gene' used herein can be a genomic gene com 30 complements thereof. As will also be appreciated by those in prising transcriptional and/or translational regulatory the art, a single strand provides a probe for a probe that can sequences and/or a coding region and/or non-translated hybridize to the target sequence understringent hybridization sequences (e.g., introns, 5'- and 3'-untranslated sequences conditions. Thus, a nucleic acid also encompasses a probe and regulatory sequences). The coding region of agene can be that hybridizes under stringent hybridization conditions. a nucleotide sequence coding for an amino acid sequence or 35 Nucleic acids can be single stranded or double stranded, or a functional RNA, such as tRNA, rRNA, catalytic RNA, can contain portions of both double stranded and single siRNA, miRNA and antisense RNA. A gene can also be an stranded sequence. The nucleic acid can be DNA, both mRNA or cDNA corresponding to the coding regions (e.g. genomic and cDNA, RNA, or a hybrid, where the nucleic acid exons and miRNA) optionally comprising 5'- or 3' untrans can contain combinations of deoxyribo- and ribo- nucle lated sequences linked thereto. A gene can also be an ampli 40 otides, and combinations of bases including uracil, adenine, fied nucleic acid molecule produced in vitro comprising all or thymine, cytosine, guanine, inosine, Xanthine hypoxanthine, a part of the coding region and/or 5'- or 3'-untranslated isocytosine and isoguanine. Nucleic acids can be obtained by sequences linked thereto. chemical synthesis methods or by recombinant methods. The term “gene product(s) as used herein refers to include A nucleic acid will generally contain phosphodiester RNA transcribed from a gene, or a polypeptide encoded by a 45 bonds, although nucleic acid analogs can be included that can gene or translated from RNA. have at least one different linkage, e.g., phosphoramidate, The term “inhibit or “reduced or “reduce’ or “decrease phosphorothioate, phosphorodithioate, or O-methylphospho as used herein generally means to inhibit or decrease the roamidite linkages and peptide nucleic acid backbones and expression of a gene or the biological function of the protein linkages. Other analog nucleic acids include those with posi (i.e. an antibiotic resistance protein) by a statistically signifi 50 tive backbones; non-ionic backbones, and non-ribose back cantamount relative to in the absence of an inhibitor. The term , including those described in U.S. Pat. Nos. 5.235,033 “inhibition” or “inhibit” or “reduce” when referring to the and 5,034,506, which are incorporated by reference. Nucleic activity of an antimicrobial agent or composition as disclosed acids containing one or more non-naturally occurring or herein refers to prevention of, or reduction in the rate of modified nucleotides are also included within one definition growth of the bacteria. Inhibition and/or inhibit when used in 55 of nucleic acids. The modified nucleotide analog can be the context to refer to an agent that inhibits an antibiotic located for example at the 5'-end and/or the 3'-end of the resistance gene and/or cell Survival refers to the prevention or nucleic acid molecule. Representative examples of nucle reduction of activity of a gene or gene product, that when otide analogs can be selected from Sugar- or backbone-modi inactivated potentiates the activity of an antimicrobial agent. fied ribonucleotides. It should be noted, however, that also However, for avoidance of doubt, “inhibit” means statisti 60 nucleobase-modified ribonucleotides, i.e. ribonucleotides, cally significant decrease inactivity of the biological function containing a non naturally occurring nucleobase instead of a of a protein by at least about 10% as compared to in the naturally occurring nucleobase such as uridines or cytidines absence of an inhibitor, for example a decrease by at least modified at the 5-position, e.g. 5-(2-amino)propyl uridine, about 20%, at least about 30%, at least about 40%, at least 5-bromo uridine; adenosines and guanosines modified at the about 50%, or least about 60%, or least about 70%, or least 65 8-position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7 about 80%, at least about 90% or more, up to and including a deaza-adenosine; O- and N-alkylated nucleotides, e.g. 100% inhibition (i.e. complete absence of an antibiotic resis N6-methyl adenosine are suitable. The 2'OH-group can be US 9,056,899 B2 31 32 replaced by a group selected from H. O.R. R. halo, SH, SR, or hydrophobic amino; acid with alanine), or alternatively, in NH, NHR, NR, or CN, wherein R is C-C6 alkyl, alkenyl or which a naturally-occurring amino acid is substituted with a non-conventional amino acid. In some embodiments amino alkynyl and halo is F, Cl, Br or I. Modifications of the ribose acid Substitutions are conservative. phosphate backbone can be done for a variety of reasons, e.g., The articles “a” and “an are used herein to refer to one or to increase the stability and half-life of such molecules in to more than one (i.e., at least one) of the grammatical object physiological environments or as probes on a biochip. Mix of the article. By way of example, “an element’ means one tures of naturally occurring nucleic acids and analogs can be element or more than one element. Thus, in this specification made; alternatively, mixtures of different nucleic acid ana and the appended claims, the singular forms “a” “an and logs, and mixtures of naturally occurring nucleic acids and “the include plural references unless the context clearly 10 dictates otherwise. Thus, for example, reference to a pharma analogs can be made. ceutical composition comprising “an agent' includes refer As used herein, the terms “administering, and “introduc ence to two or more agents. ing” are used interchangeably and refer to the placement of As used herein, the term "comprising means that other the bacteriophages and/or antimicrobial agents as disclosed elements can also be present in addition to the defined ele herein onto the surface colonized by bacteria or into a subject, ments presented. The use of "comprising indicates inclusion such as a subject with a bacterial infection or other microor 15 rather than limitation. The term “consisting of refers to com ganism infection, by any method or route which results in at positions, methods, and respective components thereof as least partial localization of the engineered-bacteriophages described herein, which are exclusive of any element not and/or antimicrobial agents at a desired site. The composi recited in that description of the embodiment. As used herein tions as disclosed herein can be administered by any appro the term “consisting essentially of refers to those elements priate route which results in the effective killing, elimination required for a given embodiment. The term permits the pres or control of the growth of the bacteria. ence of elements that do not materially affect the basic and The term “vectors' is used interchangeably with "plasmid novel or functional characteristic(s) of that embodiment of to refer to a nucleic acid molecule capable of transporting the invention. another nucleic acid to which it has been linked A vector can Other than in the operating examples, or where otherwise be a plasmid, bacteriophage, bacterial artificial chromosome 25 indicated, all numbers expressing quantities of ingredients or or yeast artificial chromosome. A vector can be a DNA or reaction conditions used herein should be understood as RNA vector. A vector can be either a self replicating extrach modified in all instances by the term “about.” The term romosomal vector or a vector which integrate into a host "about when used in connection with percentages can mean genome. Vectors capable of directing the expression of genes 1%. and/or nucleic acid sequence to which they are operatively 30 This invention is further illustrated by the following linked are referred to herein as “expression vectors'. In gen examples which should not be construed as limiting. The contents of all references cited throughout this application, as eral, expression vectors of utility in recombinant DNA tech well as the figures and tables are incorporated herein by niques are often in the form of "plasmids” which refer to reference. circular double stranded DNA loops which, in their vector It should be understood that this invention is not limited to form are not bound to the chromosome. Other expression 35 vectors can be used in different embodiments of the invention, the particular methodology, protocols, and reagents, etc., for example, but are not limited to, plasmids, episomes, bac described herein and as such can vary. The terminology used teriophages or viral vectors, and Such vectors can integrate herein is for the purpose of describing particular embodi into the host’s genome or replicate autonomously in the par ments only, and is not intended to limit the scope of the ticular cell. Otherforms of expression vectors known by those present invention, which is defined solely by the claims. skilled in the art which serve the equivalent functions can also 40 Other features and advantages of the invention will be appar be used. Expression vectors comprise expression vectors for ent from the following Detailed Description, the drawings, stable or transient expression encoding the DNA. and the claims. The term “analog as used herein refers to a composition Inhibitor-engineered Bacteriophages that retains the same structure or function (e.g., binding to a One aspect of the present invention relates to an engineered receptor) as a polypeptide or nucleic acid herein. Examples of 45 bacteriophage which comprise a nucleic acid which encodes analogs include peptidomimetics, peptide nucleic acids, an agent which inhibits at least one antibiotic resistance gene Small and large organic or inorganic compounds, as well as or at least one cell Survival gene, thereby gene silencing Such derivatives and variants of a polypeptide or nucleic acid genes and preventing the development of antibiotic resistance herein. The term “analog as used herein refers to a compo and/or increased cell viability of the bacteria in the presence sition that retains the same structure or function (e.g., binding 50 of the antimicrobial agent. As discussed herein, such engi to a receptor) as a polypeptide or nucleic acid herein. neered bacteriophages which comprise a nucleic acid encod The term "derivative' or “variant' as used herein refers to ing an agent which inhibits at least one gene involved in a peptide, chemical or nucleic acid that differs from the natu antibiotic resistance and/or at least one cell Survival gene as disclosed herein are referred to herein as “inhibitor-engi rally occurring polypeptide or nucleic acid by one or more neered bacteriophages'. amino acid or nucleic acid deletions, additions, Substitutions 55 or side-chain modifications. Amino acid Substitutions include In some embodiments, an inhibitor-engineered bacterioph alterations in which an amino acid is replaced with a different age can comprise a nucleic acid encoding any type of inhibi naturally-occurring or a non-conventional amino acid resi tor, such as a nucleic acid inhibitor. Nucleic acid inhibitors due. Such substitutions may be classified as “conservative'. include, for example but are not limited to antisense nucleic in which case an amino acid residue contained in a polypep acid inhibitors, oligonucleosides, RNA interference (RNAi) tide is replaced with another naturally occurring amino acid 60 and paired termini (PT) antisense and variants thereof. of similar character either in relation to polarity, side chain In some embodiments of this aspect of the invention, an functionality or size. inhibitor-engineered bacteriophage can encode an agent Substitutions encompassed by the present invention may which inhibits the gene expression and/or protein function of also be “non conservative', in which an amino acid residue any bacterial antibiotic resistance genes commonly known by which is present in a peptide is Substituted with an amino acid 65 persons of ordinary skill in the art, Such as, but not limited to having different properties, such as naturally-occurring cat (SEQID NO:1), van A (SEQID NO:2) or mecD (SEQID amino acid from a different group (e.g., Substituting a charged NO:3). In alternative embodiments, an agent can inhibit the US 9,056,899 B2 33 34 gene expression and/or protein function of any bacterial cell Table 1 provides the accession numbers and Gene ID num Survival repair gene commonly known by persons of ordinary bers for examples of antibiotic resistance genes and cell Sur skill in the art such as, but not limited to RecA, RecB, RecC. Spot or RelA. vival genes which can be inhibited in the methods of the For reference, RecA (recombinase A) can be identified by 5 present invention, as well examples of as repressors which Accession number: P03017 and Gene ID Seq ID GI: 132224. one can use in repressor-engineered bacteriophages. TABLE 1. Gene ID numbers and SEQ ID SEQ ID Gene NO: Other Aliases: Annotation Gene ID: Other Designations: ptsG 1 b1101, CR, NC OOO913.2 945651 fused glucose-specific PTS (cat) ECK1087, (1157092... 1158525) enzymes: IIB JW1087, car, cat, component IIC component glCA, tgl, umg, umgC VanA M97297 479085 Vancomycin-resistant protein mecA 3 X52593 46610 Penicillin binding protein II recA b2699, NC OOO913.2 947 170 ECK2694, (2820730... 2821791, JW2669, lexB, complement) rech, rnmB, Srf, if, umuB, umuR, Zab rec b2820, NC OOO913.2 947286 exonuclease V (RecBCD ECK2816, (295.0483 ... 2954.025, complex), beta subunit JW2788, ior, complement) ror A recC b2822, NC OOO913.2 947294 exonuclease V (RecBCD ECK2818, (2957082... 2960450, complex), gamma chain JW 2790 complement) spoT b3650, NC OOO913.2 94.8159 bifunctional (p)ppGpp ECK3640, (38.20423... 3822531) Synthetase IIguanosine JW3625 3',5'-bis pyrophosphate 3'- pyrophosphohydrolase relA b2784, NC OOO913.2 947244 (p)ppGpp synthetase IGTP ECK2778, (2909439... 2911673, pyrophosphokinase JW2755, RC complement) lexA b4043, NC OOO913.2 948.544 DNA-binding ECK4035, (4255138... 4255746) transcriptional repressor of JW4003, exra, SOS regulon recA, spr, ts, umuA b1530, NC OOO913.2 94582S DNA-binding ECK1523, (1617144. ... 1617578) transcriptional repressor of JW5248, cfxB, multiple antibiotic resistance inaR, SOXQ P22gp18 NC 002371.2 1262.795 Arc; transcriptional (14793 ... 15022) repressor SoxR b4063, NC OOO913.2 94.8566 DNA-binding ECK4055, (4275492 ... 4275956) transcriptional dual JW4024, marC regulator, Fe—S center for redox-sensing fur b0683, NC OOO913.2 945295 DNA-binding ECKO671, (709423 ... 709869, transcriptional dual JWO669 complement) regulator of siderophore biosynthesis and transport crp b3357, NC OOO913.2 94.7867 DNA-binding ECK3345, (3484142... 3484774) transcriptional dual JW5702, cap, regulator CS icod b1136, NC OOO913.2 945,702 e14 prophage; isocitrate ECK1122, (1194346 ... 1195596) dehydrogenase, specific for JW1122, iccdA, NADP iccE cSrA b2696, NC OOO913.2 947176 pleiotropic regulatory ECK2691, (2816983... 2817168, protein for carbon source JW2666, ZfA complement) metabolism omp A b0957, NC OOO913.2 945571 Outer membrane protein A ECKO948, (1018236... 1019276, (3a; II*; G: d) JWO940, con, complement) olG, tut US 9,056,899 B2 35 36 In some embodiments, one can use a modular design strat target and disable the bacteria's antimicrobial resistance egy in which bacteriophage kill bacteria in a species-specific mechanism by inhibiting the bacterial resistance genes or manner are engineered to express at least one inhibitor of at expressing a repressor to a SOS gene. least one antibiotic gene and/or a cell Survival gene, or An inhibitor to any antimicrobial resistance genes known express at least one repressor of a SOS response gene. For 5 to one or ordinary skill in the art is encompassed for use in the example, in Some embodiments, the bacteriophage can inhibitor-engineered bacteriophages disclosed herein. In express an nucleic acid inhibitor, Such as an antisense nucleic addition to the antibiotic resistance genes discussed herein, acid inhibitor or antisense RNA (asRNA) which inhibits at other such antibiotic resistance genes which can be used least one, or at least two or at least three antibiotic genes include, for example, are katG, rpoB, rpsL, ampC, beta-lac and/or a cell survival gene, such as, but not limited to cat (SEQ 10 tamases, aminoglycoside kinases, mex A. mexB, oprM. ID NO:1), van A (SEQ ID NO:2) mecD (SEQ ID NO:3), ermA, carA, Imra, ereA, VgbA, InVA, mph A. tetA, tetB, RecA (SEQID NO:4), RecB (SEQID NO:5), RecC (SEQID van H, van R, VanX, VanY. vanZ, folC, folE, folP, and folk NO:6), Spot (SEQ ID NO:7) or RelA (SEQID NO:8). which are disclosed in U.S. Pat. No. 7,125,622, which is Some aspects of the present invention are directed to use of incorporated herein in its entity by reference. a inhibitor-engineered bacteriophage as an adjuvants to an 15 Repressor-engineered Bacteriophages antimicrobial agent, where an inhibitor-engineered bacte In another aspect of the present invention, an engineered riophage encodes at least one inhibitor to an antimicrobial or bacteriophage can comprise a nucleic acid encoding a repres antibacterial resistance gene in the bacteria. Previous uses of Sor, or fragment thereof, of a SOS response gene or a non antibiotic resistance genes have been used to increase the SOS defense gene and as discussed previously, are referred to susceptibility of bacteria to antimicrobial agents. For herein as “repressor-engineered bacteriophages.” example, US patent application US2002/0076722 discusses a In some embodiments of this aspect and all aspects method of improving susceptibility of bacteria to antibacte described herein, a repressor-engineered bacteriophage can rial agents by identifying gene loci which decrease the bac comprises a nucleic acid encoding a repressor protein, or terium’s Susceptibility to antibacterial agents, and identify fragment thereof of a bacterial SOS response gene, or an OftX, WbbL, Slt, and Wza as such loci. However, in contrast 25 agent (such as a protein) which inhibits a non-SOS pathway to the present application, US2002/0076722 does not teach bacterial defense gene. method to inhibit the loci to increase the bacterial suscepti Without wishing to be limited to theory, the SOS response bility to antibacterial agents. Similarly, U.S. Pat. No. 7,125, in bacteria is an inducible DNA repair system which allows 622 discusses a method to identify bacterial antibiotic resis bacteria to survive sudden increases in DNA damage. For tance genes by analyzing pools of bacterial genomic 30 instance, whenbacteria are exposed to stress they produce can fragments and selecting those fragments which hybridize or defense proteins from genes which are normally in a have high homology (using computer assisted in silico meth repressed state and allow repair of damaged DNA and reac odologies) to numerous known bacterial resistance genes. tivation of DNA synthesis. The SOS response is based upon The U.S. Pat. No. 7,125,622 discloses a number of bacterial the paradigm that bacteria play an active role in the mutation resistance genes, including, katG, rpoB, rpsL, ampC, beta 35 of their own genomes by inducing the production of proteins lactamases, aminoglycoside kinases, meXA, mexB, oprM. during stressful conditions which facilitate mutations, includ ermA, carA, Imra, ereA, VgbA, InVA, mph A. tetA, tetB, ing Pol II (PolB), Pol IV (dinB) and PolV (umulDandumuC). pp-cat, VanA. VanH. vanR, vanX, vanY. Vanz, folC, folE, folP. Inhibition of these proteins, such as Pol II, Pol IV and Pol V and folk, which are encompassed as targets for the inhibitors or prevention of their derepression by inhibition of Lex A in an inhibitor-engineered bacteriophage as discussed herein. 40 cleavage is one strategy to prevent the development of anti However, in contrast to the present application, U.S. Pat. No. biotic-resistant bacteria. The SOS response is commonly trig 7,125,622 does not teach method to inhibit the bacterial resis gered by single-stranded DNA, which accumulates as a result tance genes using an inhibitor-engineered bacteriophage of of either DNA damage or problematic replication or on bac the present invention, or their inhibition by such an inhibitor teriophage infection. In some situations antibiotics trigger the engineered bacteriophage in combination with an antimicro 45 SOS response, as some antibiotics, such as fluoroquinolones bial agent. Similarly, International Application WO2008/ and B-lactams induce antibiotic-mediated DNA damage. The 110840 discusses the use of six different bacteriophages SOS response is discussed in Benedicte Michel, PLoS Biol (NCIMB numbers 41174-41179) to increase sensitivity of ogy, 2005; 3: 1174-1176; Janion et al., Acta Biochemica bacteria to antibiotics. However, WO2008/110840 but does Polonica, 2001; 48: 599-610 and Smith et al., 2007, 9; 549 not teach genetically modifying Such bacteriophages to 50 555, and Cirz et al., PLoS Biology, 2005; 6: 1024-1033, and inhibit bacterial resistance genes or repressing SOS genes. are incorporated herein in their entirety by reference. While there are some reports of modifying bacteriophages to In some embodiments, the repressor of an SOS response increase their effectiveness of killing bacteria, previous stud gene is, for example but not limited to, lex A (SEQID NO:9), ies have mainly focused on optimizing method to degrade or modified version thereof. In other embodiments of this bacteria biofilms, such as, for example introducing a lysase 55 aspect of the invention, a SOS response gene is, for example enzyme Such as alginate lyse (discussed in International but is not limited to marr AB (SEQID NO:18), arc AB (SEQ Application WOO4/062677); or modifying bacteriophages to ID NO:19) and lexO (SEQID NO:20). inhibit the cell which propagates the bacteriophage. Such In some embodiments of this aspect and all other aspects introducing a KIL gene Such as the Holin gene in the bacte described herein, an inhibitor of a non-SOS pathway bacterial riophage (discussed in International Application WO02/ 60 defense gene is soxR (SEQ ID NO: 12), or modified version 034892 and WO04/046319), or introducing bacterial toxin thereof. In some embodiments of this aspect and all other genes such as pGef or ChpBK and Toxin A (discussed in U.S. aspects described herein, an inhibitor of a non-SOS pathway Pat. No. 6,759.229 and Westwater et al., Antimicrobial agents bacterial defense gene is selected from the group of marr and Chemotherapy, 2003., 47: 1301-1307). However, unlike (SEQ ID NO:10), arc (SEQ ID NO:11), soxR (SEQ ID the present invention the modified bacteriophages discussed 65 NO:12), fur (SEQ ID NO:13), crp (SEQ ID NO:14), iccdA in WOO4/062677, WO02/034892, WOO4/046319, U.S. Pat. (SEQ ID NO:15), craA (SEQID NO:16) or ompA (SEQ ID No. 6,759,229 and Westwater et al., have not been modified to NO:17) or modified version thereof. In some embodiments, a US 9,056,899 B2 37 38 non-SOS repressor expressed by a repressor-engineered bac TABLE 2-continued teriophage is soxR (SEQ ID NO: 12) which represses soxS Examples of non-SOS defense genes which can be inhibited by a and protects against oxidative stress. repressor or an inhibitor expressed by a In other embodiments of this aspect of the invention, a repressor-engineered bacteriophage. repressor-engineered bacteriophage can express an repressor, Table 2: Examples of non-SOS defense genes which can or fragment thereof, of at least one, or at least two or at least be inhibited by an repressor or inhibitor expressed by a three or more SOS response genes, such as, but not limited to repressor-engineered bacteriophage leXA, marr, arc, SOXR, fur, crp, iccdA, craA or omp A. Other nudB repressors known by a skilled artisan are also encompassed oxyR 10 pal for use in repressor-engineered bacteriophages. In some pal embodiments, repressor-engineered bacteriophages are used pgmB in combination with antimicrobial agents which trigger the phoP plsX SOS response, or trigger DNA damage. Such as, for example ppiB fluoroquinolones, ciprofloxacin and B-lactams. prf In other embodiments of this aspect of the invention, an 15 proW agent encoded by the nucleic acid of a repressor engineered pstA pstS bacteriophage which inhibits a non-SOS defense gene can qmcA inhibit any gene listed in Table 2. recA rec TABLE 2 recC recC Examples of non-SOS defense genes which can be inhibited by a recs repressor or an inhibitor expressed by a recC) repressor-engineered bacteriophage. r Table 2: Examples of non-SOS defense genes which can be inhibited by an repressor or inhibitor expressed by a 25 repressor-engineered bacteriophage :s aG acrA acrB atp A bdm 30 BW251.13 l t ced A e cysB piA dacA bI dapF bmE dcd 35 mF didIB pm dedD boN degP deoT bsU dinB dkSA K dnaK 40 ruvA ela) ruvC emtA sapC envC secC envZ fab Smp A 45 suf SurA tat B tatC tolC toIR 50 tonB trxA tusC tus) typA ubiG 55 uvrA hirpA uwrC hscA uwrD hscE XapR ihfA XSeA JW 5115 XseB JW5360 ybcN JW5474 60 ybdN lon ybeD lpdA ybeY lipp ybgC lptB ybgF mircB ybhT msbB 65 ybjL nagA ycbR US 9,056,899 B2 39 40 TABLE 2-continued referred to herein as an “susceptibility agent-engineered bac teriophage' or “susceptibility-engineered bacteriophage' but Examples of non-SOS defense genes which can be inhibited by a are also encompassed under the definition of a “repressor repressor or an inhibitor expressed by a repressor-engineered bacteriophage. engineered bacteriophage'. In some embodiments of this Table 2: Examples of non-SOS defense genes which can aspect, and all other aspects described herein, Such an agent be inhibited by an repressor or inhibitor expressed by a which increases the Susceptibility of a bacteria to an antimi repressor-engineered bacteriophage crobial agent is referred to as a 'susceptibility agent' and yceD refers to any agent which increases the bacteria's Susceptibil ych ity to the antimicrobial agent by about at least 10% or about at yciM 10 least 15%, or about at least 20% or about at least 30% or about at least 50% or more than 50%, or any integer between 10% and 50% or more, as compared to the use of the antimicrobial agent alone. In one embodiment, a Susceptibility agent is an agent which specifically targets a bacteria cell. In another 15 embodiment, a susceptibility agent modifies (i.e. inhibits or activates) a pathway which is specifically expressed in bac terial cells. In one embodiment, a Susceptibility agent is an agent which has an additive effect of the efficacy of the antimicrobial agent (i.e. the agent has an additive effect of the killing efficacy or inhibition of growth by the antimicrobial agent). In a preferred embodiment, a susceptibility agent is an agent which has a synergistic effect on the efficacy of the antimicrobial agent (i.e. the agent has a synergistic effect of the killing efficacy or inhibition of growth by the antimicro bial agent). In some embodiments, a repressor-engineered bacterioph 25 In one embodiment, a susceptibility agent increases the age which inhibits a non-SOS defense gene can be used in entry of an antimicrobial agent into a bacterial cell, for combination with selected antimicrobial agents, for example, example, a susceptibility agent is a porin orporin-like protein, where the repressor-engineered bacteriophage encodes an such as but is not limited to, protein Omp, and Beta barrel agent which inhibits a gene listed in Table 2A, Such a repres porins, or other members of the outer membrane porin sor-engineered bacteriophage can be used in combination 30 (OMP)) functional superfamily which include, but are not with a ciprofloxacin antimicrobial agent or a variant or ana limited to those disclosed in world wide web site: “//biocy logue thereof. Similarly, in other embodiments a repressor c.org/ECOLI/NEW-IMAGE?object=BC-4.1.B”, or a OMP engineered bacteriophage which inhibits a non-SOS defense family member listed in Table 3 as disclosed herein, or a variant or fragment thereof. gene can encode an agent which inhibits a gene listed in Table 35 2B can be used in combination with a Vancomycin antimicro bial agent or a variant or analogue thereof. Similarly, in other TABLE 3 embodiments a repressor-engineered bacteriophage which Examples of members of the Outer Membrane Porin (OMP) Superfamily inhibits a non-SOS defense gene can encode an agent which which can be expressed as a susceptibility agent by a Susceptibility-agent inhibits a gene listed in Table 2C, 2D, 2E, 2F and 2G can be 40 engineered bacteriophage. Table 3: Members of The Outer Membrane Porin (OMP) used in combination with a rifampicin antimicrobial agent, or Functional Superfamily a amplicillin antimicrobial agent or a SulfmethaxaZone anti microbial agent or a gentamicin antimicrobial agent or a bglH (carbohydrate-specific Outer membrane porin, cryptic), btuB (outer membrane receptor for transport of B12, Ecolicins, metronidazole antimicrobial agent, respectively, or a variant and bacteriophage BF23), or analogue thereof. In some embodiments, other non-SOS 45 fadL (long-chain fatty acid outer membrane transporter; sensitivity response genes which can be inhibited or repressed in a to phage T2), repressor-engineered bacteriophage includes, for example, fecA (outer membrane receptor; citrate-dependent iron transport, Outer membrane receptor), but not limited to genes induced by DNA damage, such as fepA (FepA, outer membrane receptor for ferric enterobactin DinD, DinF, DinG, Dinl, DinP, OraA, PolB, RecA, RecN, (enterochelin) and colicins B and D), RuvA. RuvB, SbmC, Ssb, SulA, UmuC, UmuD, UvrA, 50 fhuA (FhuA outer membrane protein receptor for ferrichrome, colicin M, and phages T1, T5, and phi80), UvrB, and UvrD, as discussed in Dwyer et al., Mol Systems fhuE (outer membrane receptor for ferric iron uptake), Biology, 2007: 3: 1-15, which is incorporated herein in its fiu (putative Outer membrane receptor for iron transport), entirety by reference. In another embodiment, other non-SOS lamB, response genes which can be inhibited or repressed in a mdtQ (putative channel/filament protein), repressor-engineered bacteriophage includes, for example, 55 omp A (outer membrane protein 3a (II*; G; d)), ompC, but not limited to genes induced by oxidative damage, such as ompF, MarA, MarB, MarFR, SodA and SoxS, as discussed in Dwyer ompG (outer membrane porin OmpG), et al., Mol Systems Biology, 2007: 3: 1-15, which is incorpo ompIL (predicted outer membrane porin L.), ompN (Outer membrane pore protein N, non-specific), rated herein in its entirety by reference. ompW (OmpW, Outer membrane protein), Susceptibility Agent-engineered Bacteriophages 60 pgaA (partially N-deacetylated poly-2-1,6-N-acetyl-D-glucosamine Another aspect of the present invention relates to an engi Outer membrane porin), neered bacteriophage which comprises a nucleic acid encod phoE ing an agent, such as but not limited to a protein, which toIB tolC (TolCouter membrane channel), increases the Susceptibility of a bacteria to an antimicrobial tSX (nucleoside channel; receptor of phage T6 and colicin K), agent. Such herein engineered bacteriophage which com 65 yncD (probable TonB-dependent receptor prises a nucleic acid encoding an agent which increases the Susceptibility of a bacteria to an antimicrobial agent can be US 9,056,899 B2 41 42 In another embodiment, a susceptibility agent is an agent, modified to comprise nucleic acids which encode enzymes Such as but not limited to a protein, which increases iron which assist in breaking down or degrading the biofilm sulfur clusters in the bacteria cell and/or increases oxidative matrix, for example any phage resistant gene known as a stress or hydroxyl radicals in the bacteria. Examples of a biofilm degrading enzyme by persons of ordinary skill in the Susceptibility agent which increases the iron-sulfur clusters art, such as, but not limited to Dispersin D aminopeptidase, include agents which modultate (i.e. increase or decrease) the amylase, carbohydrase, carboxypeptidase, catalase, cellu Fenton reaction to form hydroxyl radicals, as disclosed in lase, chitinase, cutinase, cyclodextrin glycosyltransferase, Kahanski et al., Cell, 2007, 130; 797-810, which is incorpo deoxyribonuclease, esterase, alpha-galactosidase, beta-ga rated herein by reference in its entirety. Examples of a sus lactosidase, glucoamylase, alpha-glucosidase, beta-glucosi ceptibility agent to be expressed by a susceptibility-engi 10 dase, haloperoxidase, invertase, laccase, lipase, mannosi neered bacteriophage include, for example, those listed in dase, oxidase, pectinolytic enzyme, peptidoglutaminase, Table 4, or a fragment or variant thereof or described in peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, world-wide-web site “biocyc.org/ECOLI/NEW ribonuclease, transglutaminase, Xylanase or lyase. In other IMAGE?type=COMPOUND&object=CPD-7”. Examples embodiments, the enzyme is selected from the group consist of susceptibility agents which increases iron-sulfur clusters in 15 ing of cellulases, such as glycosyl hydroxylase family of the bacteria cell include, for example but not limited to IscA, cellulases, such as glycosylhydroxylase 5 family of enzymes IscR, IscS and IscU. Examples of susceptibility agents which also called cellulase A; polyglucosamine (PGA) depoly increase iron uptake and utilization and can be used as Sus merases; and colonic acid depolymerases, such as 1,4-L- ceptibility agents include, for example but not limited to fucodise hydrolase (see, e.g., Verhoef R. et al., Characteriza EntC, ExbB, ExbD, Fecl, Feck, FepB, FepC, Fes, FhuA, tion of a 1,4-beta-fucoside hydrolase degrading collanic acid, FhuB, FhuC, FhuF, NrdH, Nrdl, SodA and TonB, as dis Carbohydr Res. 2005 Aug. 15:340(11):1780-8), depolymer cussed in Dwyer et al., Mol Systems Biology, 2007: 3: 1-15, azing alginase, and DNase I, or combinations thereof, as which is incorporated herein in its entirety by reference. disclosed in the methods as disclosed in U.S. patent applica tion Ser. No. 1 1/662,551 and International Patent Application TABLE 4 25 Wo2006/137847 and provisional patent application 61/014, 518, which are specifically incorporated herein in their Examples of genes which can be expressed as a Susceptibility agent by a Susceptibility-engineered bacteriophage to increase entirety by reference. iron cluster formation in bacteria. In another embodiment, the inhibitor-engineered bacte Table 4: Example of susceptibility agents which increase iron clusters riophages and/or repressor-engineered bacteriophages and/or 30 a susceptibility-engineered bacteriophage can be further be Cofactor of: serine deaminase, L-serine deaminase, L-serine deaminase, pyruvate formate-lyase activating enzyme, 2,4-dienoyl-CoA reductase modified in a species-specific manner, for example, one can Prosthetic Group of biotin synthase, dihydroxy-acid dehydratase, modify or select the bacteriophage on the basis for its infec dihydroxy-acid dehydratase, lysine 2,3-aminomutase, NADH:ubiquinone tivity of specific bacteria. oxidoreductase, sulfite reductase-(NADPH), aconitase B, fumarase A, A bacteriophage to be engineered or developed into an aconitase, fumarase B, anaerobic coproporphyrinogen III oxidase, Succinate dehydrogenase, nitrate reductase, flavin reductase, 35 inhibitor-engineered bacteriophage or repressor-engineered aconitase B, fumarate reductase bacteriophage or a Susceptibility-engineered bacteriophage Cofactor or Prosthetic Group of: quinolinate synthase, ribonucleoside can be any bacteriophage as known by a person of ordinary triphosphate reductase activase, 23S ribosomal RNA 5-methyluridine skill in the art. In some embodiments, an inhibitor-engineered methyltransferase bacteriophage or a repressor-engineered bacteriophage or a 40 Susceptibility-engineered bacteriophage is derived from any In some embodiments, a Susceptibility agent is an agent or a combination of bacteriophages listed in Table 5. such as CsrA, which is described in world-wide web site: In some embodiments, a bacteriophage which is engi “biocyc.org/ECOLI/NEW neered to become an engineered bacteriophage as disclosed IMAGE?type=ENZYME&object=CPLX0-1041. herein is a lytic bacteriophage or lysogenic bacteriophage, or In some embodiments, a Susceptibility agent is not a che 45 any bacteriophage that infects E. coli, P. aeriginosa, S. motherapeutic agent. In another embodiment, a Susceptibility aureaus, E. facalis and the like. Such bacteriophages are well agent is not a toxin protein, and in another embodiment, a known to one skilled in the art and are listed in Table 5, and Susceptibility agent is not a bacterial toxin protein or mol include, but are not limited to, lambda phages, M13, T7, T3, ecule. and T-even and T-even like phages, such as T2, and T4, and Modification of Inhibitor-engineered Bacteriophages, 50 RB69; also phages such as Pfl. Pfa, Bacteroides fragilis phage Repressor-engineered Bacteriophages and Susceptibility B40-8 and coliphage MS-2 can be used. For example, lambda agent Engineered Bacteriophages phage attacks E. coli by attaching itself to the outside of the In another embodiment, an inhibitor-engineered bacte bacteria and injecting its DNA into the bacteria. Once injected riophage and/or a repressor-engineered bacteriophage and/or into its new host, a bacteriophage uses E. coli's genetic a Susceptibility-engineered bacteriophage can be further be 55 machinery to transcribe its genes. Any of the known phages modified to comprise nucleic acids which encode phage resis can be engineered to express an agent that inhibits an antibi tant genes, for example any phage resistant gene known by otic resistance gene or cell Survival gene, or alternatively persons of ordinary skill in the art, such as, but not limited to express a repressor agent oran inhibitor of a non-SOS defense AbiZ (as disclosed in U.S. Pat. No. 7,169,911 which is incor gene for a repressor-engineered bacteriophage, or express a porated herein by reference), sie2009, Sierraoo, Sier 72.4, orf2. 60 Susceptibility agent for a Susceptibility-engineered bacte orf258, orf2(M), olf D, orf304, orfB, orf142, orf203, orf3 p. riophage as described herein. orf2 gp34. gp33, gp32, gp25, glo, orfl. SieA, SieB, imm, In some embodiments, bacteriophages which have been sim, rexB (McGrath et al., Mol Microbiol, 2002, 43:509 engineered to be more efficient cloning vectors or naturally 520). lack a gene important in infecting all bacteria, Such as male In another embodiment, the inhibitor-engineered bacte 65 and female bacteria can be used to generate engineered bac riophages and/or repressor-engineeredbacteriophages and/or teriophages as disclosed herein. Typically, bacteriophages a Susceptibility-engineered bacteriophage can be further be have been engineered to lack genes for infecting all variants US 9,056,899 B2 43 44 and species of bacteria can have reduced capacity to replicate Nucleic Acid Inhibitors of Antibiotic Resistance Genes and/ in naturally occurring bacteria thus limiting the use of Such or Cell Survival Genes for Inhibitor-engineered Bacterioph phages in degradation of biofilm produced by the naturally ages or Nucleic Acid Inhibitors of Non-SOS Defense Genes occurring bacteria. in Repressor-engineered Bacteriophages. For example, the capsid protein of phage T7 gene 10, In some embodiments of aspects of the invention involving comes in two forms, the major product 10A (36 kDa) and the inhibitor-engineered bacteriophages, agents that inhibit an minor product 10B (41 kDa) (Condron, B. G., Atkins, J. F., antibiotic resistance gene and/or a cell Survival gene is a and Gesteland, R. F. 1991. Frameshifting in gene 10 of bac nucleic acid. In another embodiments, repressor-engineered teriophage T7. J. Bacteriol. 173:6998-7003). Capsid protein bacteriophages comprise nucleic acids which inhibit non 1OB is produced by frameshifting near the end of the coding 10 SOS defense genes, such as those listed in Table 2, and Tables 2A-2F. An antibiotic resistance gene and/or cell Survival gene region of 10A. NOVAGENR modified gene 10 in T7 to and/or non-SOS defense gene can be inhibited by inhibition remove the frameshifting site so that only 10B with the of the expression of such antibiotic resistance proteins and/or attached user-introduced peptide for Surface display is pro cell survival polypeptide or non-SOS defense gene or by duced (U.S. Pat. No. 5,766,905. 1998. Cytoplasmic bacte 15 'gene silencing methods commonly known by persons of riophage display system, which is incorporated in its entirety ordinary skill in the art. A nucleic acid inhibitor of an antibi herein by reference). The 10B-enzyme fusion product is too otic resistance gene and/or a cell Survival gene or non-SOS large to make up the entire phage capsid because the enzymes defense gene, includes for example, but is not limited to, RNA that are typically introduced into phages, such as T7, are large interference-inducing (RNAi) molecules, for example but are (greater thana few hundredamino acids). As a result, T7 select not limited to siRNA, dsRNA, stRNA, shRNA, miRNA and 10-3b must be grown in host bacterial strains that produce modified versions thereof, where the RNA interference mol wild-type 10A capsid protein, such as BLT5403 or BLT5615, ecule gene silences the expression of the antibiotic resistance so that enough 10A is available to be interspersed with the gene and/or cell Survival gene non SOS-defense gene. In 1OB-enzyme fusion product to allow replication of phage some embodiments, the nucleic acid inhibitor of an antibiotic (U.S. Pat. No. 5,766.905. 1998. Cytoplasmic bacteriophage 25 resistance gene and/or cell Survival gene and/or non-SOS display System, which is incorporated in its entirety herein by defense gene is an anti-sense oligonucleic acid, or a nucleic reference). However, because most biofilm-forming E. coli acid analogue, for example but are not limited to DNA, RNA, do not produce wild-type 10A capsid protein, this limits the peptide-nucleic acid (PNA), pseudo-complementary PNA ability of T7 select 10-3b displaying large enzymes on their (pc-PNA), or locked nucleic acid (LNA) and the like. In Surface to propagate within and lyse some important Strains of 30 alternative embodiments, the nucleic acid is DNA or RNA, E. coli. Accordingly, in some embodiments, the present and nucleic acid analogues, for example PNA, pcPNA and invention provides genetically engineered phages that in LNA. A nucleic acid can be single or double stranded, and can addition to comprising inhibitors to cell Survival genes or be selected from a group comprising nucleic acid encoding a antibiotic resistance genes, or nucleic acids encoding repres protein of interest, oligonucleotides, PNA, etc. Such nucleic Sor proteins, also express all the essential genes for virus 35 acid inhibitors include for example, but are not limited to, a replication in naturally occurring bacterial strains. In one nucleic acid sequence encoding a protein that is a transcrip embodiment, the invention provides an engineered T7 select tional repressor, or an antisense molecule, or a ribozyme, or a 10-3b phage that expresses both cellulase and 10A capsid Small inhibitory nucleic acid sequence Such as a RNAi, an protein. shRNAi, an siRNA, a micro RNAi (miRNA), an antisense It is known that wild-type T7 does not productively infect 40 oligonucleotide etc. male (F plasmid-containing) E. coli because of interactions In some embodiments, a nucleic acid inhibitor of an anti between the F plasmid protein PifA and T7 genes 1.2 or 10 biotic resistance gene and/or a cell Survival gene and/or non (Garcia, L. R., and Molineux, I.J. 1995. Incomplete entry of SOS defense gene can be for example, but not are limited to, bacteriophage T7 DNA into F plasmid-containing Escheri paired termini antisense, an example of which is disclosed in chia coli. J. Bacteriol. 177:4077-4083.). F plasmid-contain 45 FIG. 8 and disclosed in Nakashima, et al., (2006) Nucleic ing E. coli infected by T7 die but do not lyse or release large Acids Res 34: e138, which in incorporated herein in its numbers of T7 (Garcia, L. R., and Molineux, I. J. 1995. entirety by reference. Incomplete entry of bacteriophage T7 DNA into F plasmid In some embodiments of this aspect and all aspects containing Escherichia coli. J. Bacteriol. 177:4077-4083). described herein, a single-stranded RNA (ssRNA), a form of Wild-type T3 grows normally on male cells because of T3's 50 RNA endogenously found in eukaryotic cells can be used to gene 1.2 product (Garcia, L. R., and Molineux, I.J. 1995. Id.). forman RNAi molecule. Cellular ssRNA molecules include When T3 gene 1.2 is expressed in wild-type T7. T7 is able to messenger RNAS (and the progenitor pre-messenger RNAS), productively infect male cells (Garcia, L. R., and Molineux, I. small nuclear RNAs, Small nucleolar RNAs, transfer RNAs J. 1995. Id). and ribosomal RNAs. Double-stranded RNA (dsRNA) Because many biofilm-producing E. coli contain the F 55 induces a size-dependent immune response such that dsRNA plasmid (Ghigo, et al., 2001. Natural conjugative plasmids larger than 30 bp activates the interferon response, while induce bacterial biofilm development. Nature. 412:442-445), shorter dsRNAs feed into the cells endogenous RNA inter it is important, although not necessary, for an engineered ference machinery downstream of the Dicer enzyme. bacteriophage to be able to productively infect also male RNA interference (RNAi) provides a powerful approach cells. Therefore, in addition to engineering the phage to dis 60 for inhibiting the expression of selected target polypeptides. play a biofilm degrading enzyme on its Surface, one can also RNAi uses small interfering RNA (siRNA) duplexes that engineer it to express the gene necessary for infecting the target the messenger RNA encoding the target polypeptide male bacteria. For example, one can use the modification for selective degradation. SiRNA-dependent post-transcrip described by Garcia and Molineux (Garcia, L. R., and tional silencing of gene expression involves cutting the target Molineux, I.J. 1995. Incomplete entry of bacteriophage T7 65 messenger RNA molecule at a site guided by the siRNA. DNA into F plasmid-containing Escherichia coli. J. Bacte RNA interference (RNAi) is an evolutionally conserved riol. 177:4077-4083) to express T3 gene 1.2 in T7. process whereby the expression or introduction of RNA of a US 9,056,899 B2 45 46 sequence that is identical or highly similar to a target gene context, the term "homologous is defined as being Substan results in the sequence specific degradation or specific post tially identical, Sufficiently complementary, or similar to the transcriptional gene silencing (PTGS) of messenger RNA target mRNA, or a fragment thereof, to effect RNA interfer (mRNA) transcribed from that targeted gene (see Coburn, G. ence of the target. In addition to native RNA molecules, RNA and Cullen, B. (2002) J. of Virology 76(18): 9225), thereby suitable for inhibiting or interfering with the expression of a inhibiting expression of the target gene. In one embodiment, target sequence include RNA derivatives and analogs. Pref the RNA is double stranded RNA (dsRNA). This process has erably, the siRNA is identical to its target. been described in plants, invertebrates, and mammaliancells. The siRNA preferably targets only one sequence. Each of In nature, RNAi is initiated by the dsRNA-specific endonu the RNA interfering agents, such as siRNAs, can be screened clease Dicer, which promotes processive cleavage of long 10 for potential off-target effects by, for example, expression dsRNA into double-stranded fragments termed siRNAs. siR profiling. Such methods are known to one skilled in the art NAs are incorporated into a protein complex (termed “RNA and are described, for example, in Jackson et al. Nature Bio induced silencing complex,” or “RISC) that recognizes and technology 6:635-637, 2003. In addition to expression pro cleaves target mRNAs. RNAi can also be initiated by intro filing, one can also screen the potential target sequences for ducing nucleic acid molecules, e.g., synthetic siRNAS or 15 similar sequences in the sequence databases to identify poten RNA interfering agents, to inhibit or silence the expression of tial sequences which can have off-target effects. For example, a target genes, such an antibiotic resistance gene and/or cell according to Jackson et al. (Id.) 15, or perhaps as few as 11 Survival gene and/or non-SOS defense gene. As used herein, contiguous nucleotides of sequence identity are sufficient to "inhibition of target gene expression' includes any decrease direct silencing of non-targeted transcripts. Therefore, one in expression or protein activity or level of the target gene (i.e. can initially screen the proposed siRNAs to avoid potential antibiotic resistance gene) or protein encoded by the target off-target silencing using the sequence identity analysis by gene (i.e. antibiotic resistance protein) as compared to the any known sequence comparison methods. Such as BLAST level in the absence of an RNA interference (RNAi) molecule. (Basic Local Alignment Search Tool available from or at The decrease in expression or protein level as result of gene NIBI). silencing can be of at least 30%, 40%, 50%, 60%, 70%, 80%, 25 siRNA molecules need not be limited to those molecules 90%. 95% or 99% or more as compared to the expression of containing only RNA, but, for example, further encompasses a target gene or the activity or level of the protein (i.e. expres chemically modified nucleotides and non-nucleotides, and sion of the antibiotic resistance gene or antibiotic resistance also include molecules wherein a ribose Sugar molecule is protein) encoded by a target gene which has not been targeted substituted for another sugar molecule or a molecule which and gene silenced by an RNA interfering (RNAi) agent. 30 performs a similar function. Moreover, a non-natural linkage As used herein, the term "short interfering RNA (siRNA), between nucleotide residues can be used. Such as a phospho also referred to herein as “small interfering RNA is defined rothioate linkage. For example, siRNA containing D-arabino as an agent which functions to inhibit expression of a target furanosyl Structures in place of the naturally-occurring D-ri gene, e.g., by RNAi. An siRNA can be chemically synthe bonucleosides found in RNA can be used in RNAi molecules sized, can be produced by in vitro transcription, or can be 35 according to the present invention (U.S. Pat. No. 5,177,196, produced within a host cell. In one embodiment, siRNA is a which is incorporated herein by reference). Other examples double stranded RNA (dsRNA) molecule of about 15 to about include RNA molecules containing the o-linkage between the 40 nucleotides in length, preferably about 15 to about 28 Sugar and the heterocyclic base of the nucleoside, which nucleotides, more preferably about 19 to about 25 nucleotides confers nuclease resistance and tight complementary Strand in length, and more preferably about 19, 20, 21, 22, or 23 40 binding to the oligonucleotidesmolecules similar to the oli nucleotides in length, and can contain a 3' and/or 5' overhang gonucleotides containing 2'-O-methyl ribose, arabinose and on each strand having a length of about 0, 1, 2, 3, 4, or 5 particularly D-arabinose (U.S. Pat. No. 5,177,196, which is nucleotides. The length of the overhang is independent incorporated herein in its entirety by reference). between the two strands, i.e., the length of the overhang on The RNA strand can be derivatized with a reactive func one strand is not dependent on the length of the overhang on 45 tional group of a reporter group, such as a fluorophore. Par the second strand. In some embodiments, the siRNA is ticularly useful derivatives are modified at a terminus or ter capable of promoting RNA interference through degradation mini of an RNA strand, typically the 3' terminus of the sense or specific post-transcriptional gene silencing (PTGS) of the strand. For example, the 2'-hydroxyl at the 3' terminus can be target messenger RNA (mRNA). readily and selectively derivatized with a variety of groups. siRNAs also include small hairpin (also called stem loop) 50 Other useful RNA derivatives incorporate nucleotides hav RNAs (shRNAs). In one embodiment, these shRNAs are ing modified carbohydrate moieties, such as 2'O-alkylated composed of a short (e.g., about 19 to about 25 nucleotide) residues or 2'-O-methyl ribosyl derivatives and 2'-O-fluoro antisense strand, followed by a nucleotide loop of about 5 to ribosyl derivatives. The RNA bases can also be modified. Any about 9 nucleotides, and the analogous sense Strand. Alterna modified base useful for inhibiting or interfering with the tively, the sense Strand can precede the nucleotide loop struc 55 expression of a target sequence can be used. For example, ture and the antisense strand can follow. These shRNAs can halogenated bases, such as 5-bromouracil and 5-iodouracil be contained in plasmids, retroviruses, and lentiviruses and can be incorporated. The bases can also be alkylated, for expressed from, for example, the pol III U6 promoter, or example, 7-methylguanosine can be incorporated in place of another promoter (see, e.g., Stewart, et al. (2003) RNA Apr; a guanosine residue. Non-natural bases that yield Successful 9(4):493-501, incorporated by reference herein in its 60 inhibition can also be incorporated. entirety). The most preferred siRNA modifications include 2'-deoxy Typically a target gene or sequence targeted by gene silenc 2'-fluorouridine or locked nucleic acid (LNA) nucleotides ing by an RNA interfering (RNAi) agent can be a cellular and RNA duplexes containing either phosphodiester or vary gene or genomic sequence encoding an antibiotic resistant ing numbers of phosphorothioate linkages. Such modifica protein or a cell Survival protein. In some embodiments, an 65 tions are known to one skilled in the art and are described, for siRNA can be substantially homologous to the target gene or example, in Braasch et al., Biochemistry, 42: 7967-7975, genomic sequence, or a fragment thereof. As used in this 2003. Most of the useful modifications to the siRNA mol US 9,056,899 B2 47 48 ecules can be introduced using chemistries established for of ordinary skill in the art determining the viability of a antisense oligonucleotide technology. Preferably, the modi bacteria expressing Such a RNAi agent in the presence of an fications involve minimal 2'-O-methyl modification, prefer antimicrobial agent. In some embodiments, bacterial cell ably excluding such modification. Modifications also prefer viability can be determined by using commercially available ably exclude modifications of the free 5'-hydroxyl groups of 5 kits. Others can be readily prepared by those of skill in the art the siRNA. based on the known sequence of the target mRNA. To avoid siRNA and miRNA molecules having various “tails' doubt, the nucleic acid sequence which can be used to design covalently attached to either their 3'- or to their 5'-ends, or to nucleic acid inhibitors for inhibitor-engineered bacterioph both, are also known in the art and can be used to stabilize the ages as disclosed herein can be based on any antibiotic resis siRNA and miRNA molecules delivered using the methods of 10 tance gene or any SOS gene or any non-SOS defense gene the present invention. Generally speaking, intercalating listed in Tables 2 or 2A-2F as disclosed herein. groups, various kinds of reportergroups and lipophilic groups siRNA sequences are chosen to maximize the uptake of the attached to the 3' or 5' ends of the RNA molecules are well antisense (guide) strand of the siRNA into RISC and thereby known to one skilled in the art and are useful according to the maximize the ability of the inhibitor to target RISC to target methods of the present invention. Descriptions of syntheses 15 antibiotic resistance gene or cell survival gene mRNA for of 3'-cholesterol or 3'-acridine modified oligonucleotides degradation. This can be accomplished by Scanning for applicable to preparation of modified RNA molecules useful sequences that have the lowest free energy of binding at the according to the present invention can be found, for example, 5'-terminus of the antisense strand. The lower free energy in the articles: Gamper, H. B., Reed, M. W., Cox, T., Virosco, leads to an enhancement of the unwinding of the 5'-end of the J. S., Adams, A. D., Gall, A., Scholler, J. K., and Meyer, R. B. antisense strand of the siRNA duplex, thereby ensuring that (1993) Facile Preparation and Exonuclease Stability of the antisense strand will be taken up by RISC and direct the 3'-Modified Oligodeoxynucleotides. Nucleic Acids Res. 21 sequence-specific cleavage of the targeted mRNA. 145-150; and Reed, M. W., Adams, A. D., Nelson, J. S., and RNA interference molecules and nucleic acid inhibitors Meyer, R. B., Jr. (1991) Acridine and Cholesterol-Derivatized useful in the methods as disclosed herein can be produced Solid Supports for Improved Synthesis of 3'-Modified Oligo 25 using any known techniques such as direct chemical synthe nucleotides. Bioconjugate Chem. 2217-225 (1993). sis, through processing of longer double stranded RNAS by Other siRNAs useful for targeting Lp-PLA expression can exposure to recombinant Dicer protein or Drosophila embryo be readily designed and tested. Accordingly, siRNAs useful lysates, through an in vitro system derived from S2 cells, for the methods described herein include siRNA molecules of using phage RNA polymerase, RNA-dependant RNA poly about 15 to about 40 or about 15 to about 28 nucleotides in 30 merase, and DNA based vectors. Use of cell lysates or in vitro length. Preferably, the siRNA molecules have a length of processing can further involve the Subsequent isolation of the about 19 to about 25 nucleotides. More preferably, the siRNA short, for example, about 21-23 nucleotide, siRNAs from the molecules have a length of about 19, 20, 21, or 22 nucleotides. lysate, etc. Chemical synthesis usually proceeds by making The siRNA molecules can also comprise a 3' hydroxyl group. two single stranded RNA-oligomers followed by the anneal The siRNA molecules can be single-stranded or double 35 ing of the two single stranded oligomers into a double Stranded; Such molecules can be blunt ended or comprise stranded RNA. Other examples include methods disclosed in overhanging ends (e.g., 5' 3"). In specific embodiments, the WO 99/32619 and WO 01/68836, which are incorporated RNA molecule is double stranded and either blunt ended or herein by reference, teach chemical and enzymatic synthesis comprises overhanging ends. of siRNA. Moreover, numerous commercial services are In one embodiment, at least one strand of the RNA mol 40 available for designing and manufacturing specific siRNAS ecule has a 3' overhang from about 0 to about 6 nucleotides (see, e.g., QIAGEN Inc., Valencia, Calif. and AMBION Inc., (e.g., pyrimidine nucleotides, purine nucleotides) in length. Austin, Tex.) In other embodiments, the 3' overhang is from about 1 to In one embodiment, the nucleic acid inhibitors of antibiotic about 5 nucleotides, from about 1 to about 3 nucleotides and resistance genes and/or cell Survival genes can be obtained from about 2 to about 4 nucleotides in length. In one embodi 45 synthetically, for example, by chemically synthesizing a ment the RNA molecule is double stranded—one strand has a nucleic acid by any method of synthesis known to the skilled 3' overhang and the other strand can be blunt-ended or have an artisan. The synthesized nucleic acid inhibitors of antibiotic overhang. In the embodiment in which the RNA molecule is resistance genes and/or cell Survival genes can then be puri double stranded and both Strands comprise an overhang, the fied by any method known in the art. Methods for chemical length of the overhangs can be the same or different for each 50 synthesis of nucleic acids include, but are not limited to, in strand. In a particular embodiment, the RNA of the present vitro chemical synthesis using phosphotriester, phosphate or invention comprises about 19, 20, 21, or 22 nucleotides which phosphoramidite chemistry and Solid phase techniques, or via are paired and which have overhangs of from about 1 to about deoxynucleoside H-phosphonate intermediates (see U.S. Pat. 3, particularly about 2, nucleotides on both 3' ends of the No. 5,705,629 to Bhongle). RNA. In one embodiment, the 3' overhangs can be stabilized 55 In some circumstances, for example, where increased against degradation. In a preferred embodiment, the RNA is nuclease stability is desired, nucleic acids having nucleic acid stabilized by including purine nucleotides, such as adenosine analogs and/or modified internucleoside linkages can be pre or guanosine nucleotides. Alternatively, Substitution of pyri ferred. Nucleic acids containing modified internucleoside midine nucleotides by modified analogues, e.g., Substitution linkages can also be synthesized using reagents and methods of uridine 2 nucleotide 3' overhangs by 2'-deoxythymidine is 60 that are well known in the art. For example, methods of tolerated and does not affect the efficiency of RNAi. The synthesizing nucleic acids containing phosphonate phospho absence of a 2 hydroxyl significantly enhances the nuclease rothioate, phosphorodithioate, phosphoramidate methoxy resistance of the overhang in tissue culture medium. ethyl phosphoramidate, formacetal, thioformacetal, diisopro In some embodiments, assessment of the expression and/or pylsilyl, acetamidate, carbamate, dimethylene-Sulfide knock down of antibiotic resistance gene and/or cell Survival 65 (—CH2—S CH), diinethylene-sulfoxide ( CH, SO— gene protein and/or non-SOS defense genes using Such RNAi CH), dimethylene-sulfone (—CH2—SOCH). 2'-O-alkyl, agents such as antisense RNA can be determined by a person and 2'-deoxy-2'-fluoro' phosphorothioate internucleoside US 9,056,899 B2 49 50 linkages are well known in the art (see Uhlmann et al., 1990, sized as the complement to nucleotide positions 1 to 21 of the Chem. Rev. 90:543-584; Schneider et al., 1990, Tetrahedron 23 nucleotide sequence motif. The use of symmetric 3' TT Lett. 31:335 and references cited therein). U.S. Pat. Nos. overhangs can be advantageous to ensure that the Small inter 5,614,617 and 5,223,618 to Cook, et al., U.S. Pat. No. 5,714, fering ribonucleoprotein particles (siRNPs) are formed with 606 to Acevedo, etal, U.S. Pat. No. 5,378,825 to Cook, et al., approximately equal ratios of sense and antisense target U.S. Pat. Nos. 5,672,697 and 5,466,786 to Buhr, et al., U.S. RNA-cleaving siRNPs (Elbashir et al. (2001) supra and Pat. No. 5,777,092 to Cook, et al., U.S. Pat. No. 5,602,240 to Elbashir et al. 2001 supra). Analysis of sequence databases, De Mesmacker, et al., U.S. Pat. No. 5,610,289 to Cook, et al. including but are not limited to the NCBI, BLAST, Derwent and U.S. Pat. No. 5,858,988 to Wang, also describe nucleic and GenSeq as well as commercially available oligosynthesis acid analogs for enhanced nuclease stability and cellular 10 software such as OLIGOENGINE(R), can also be used to uptake. select siRNA sequences against EST libraries to ensure that Synthetic siRNA molecules, including shRNA molecules, only one gene is targeted. can be obtained using a number of techniques known to those Accordingly, the RNAi molecules functioning as nucleic of skill in the art. For example, the siRNA molecule can be acid inhibitors of antibiotic resistance genes and/or cell Sur chemically synthesized or recombinantly produced using 15 vival genes as disclosed herein are for example, but are not methods known in the art, such as using appropriately pro limited to, unmodified and modified double stranded (ds) tected ribonucleoside phosphoramidites and a conventional RNA molecules including short-temporal RNA (stRNA), DNA/RNA synthesizer (see, e.g., Elbashir, S.M. et al. (2001) small interfering RNA (siRNA), short-hairpin RNA Nature 411:494-498: Elbashir, S. M., W. Lendeckel and T. (shRNA), microRNA (miRNA), double-stranded RNA Tuschl (2001) Genes & Development 15:188-200; Harborth, (dsRNA), (see, e.g. Baulcombe, Science 297:2002-2003, J. et al. (2001).J. Cell Science 1 14:4557-4565; Masters, J. R. 2002). The dsRNA molecules, e.g. siRNA, also can contain 3' et al. (2001) Proc. Natl. Acad. Sci., USA 98:8012-8017; and overhangs, preferably 3'UU or 3TT overhangs. In one Tuschl, T. etal. (1999) Genes & Development 13:3191-3197). embodiment, the siRNA molecules of the present invention Alternatively, several commercial RNA synthesis suppliers do not include RNA molecules that comprise ssRNA greater are available including, but are not limited to, Proligo (Ham 25 than about 30-40 bases, about 40-50 bases, about 50 bases or burg, Germany), Dharmacon Research (Lafayette, Colo. more. In one embodiment, the siRNA molecules of the USA), Pierce Chemical (part of Perbio Science, Rockford, present invention are double stranded for more than about Ill., USA), Glen Research (Sterling, Va., USA), ChemGenes 25%, more than about 50%, more than about 60%, more than (Ashland, Mass., USA), and Cruachem (Glasgow, UK). As about 70%, more than about 80%, more than about 90% of such, siRNA molecules are not overly difficult to synthesize 30 their length. In some embodiments, a nucleic acid inhibitor of and are readily provided in a quality suitable for RNAi. In antibiotic resistance genes and/or cell Survival genes is any addition, dsRNAs can be expressed as stem loop structures agent which binds to and inhibits the expression of antibiotic encoded by plasmid vectors, retroviruses and lentiviruses resistance genes and/or cell Survival gene mRNA, where the (Paddison, P. J. et al. (2002) Genes Dev. 16:948-958; expression of the antibiotic resistance genes and/or cell Sur McManus, M.T. etal. (2002) RNA 8:842-850; Paul, C. P. etal. 35 vival mRNA or a product of transcription of nucleic acid (2002) Nat. Biotechnol. 20:505-508; Miyagishi, M. et al. encoded by antibiotic resistance genes and/or cell Survival (2002) Nat. Biotechnol. 20:497-500; Sui, G. et al. (2002) gene is inhibited. Proc. Natl. Acad. Sci., USA99:5515-5520; Brummelkamp, In another embodiment of the invention, agents inhibiting T. et al. (2002) Cancer Cell 2:243; Lee, N. S., et al. (2002) antibiotic resistance genes and/or cell Survival genes are cata Nat. Biotechnol. 20:500-505; Yu, J. Y., et al. (2002) Proc. 40 lytic nucleic acid constructs, such as, for example ribozymes, Natl. Acad. Sci., USA99:6047-6052; Zeng, Y., et al. (2002) which are capable of cleaving RNA transcripts and thereby Mol. Cell. 9:1327-1333; Rubinson, D. A., et al. (2003) Nat. preventing the production of wildtype protein. Ribozymes are Genet. 33:401–406; Stewart, S.A., et al. (2003) RNA 9:493 targeted to and anneal with a particular sequence by virtue of 501). These vectors generally have a polIII promoter two regions of sequence complementary to the target flanking upstream of the dsRNA and can express sense and antisense 45 the ribozyme catalytic site. After binding, the ribozyme RNA strands separately and/or as a hairpin structures. Within cleaves the target in a site specific manner. The design and cells, Dicer processes the short hairpin RNA (shRNA) into testing of ribozymes which specifically recognize and cleave effective siRNA. sequences of the gene products described herein, for example The targeted region of the siRNA molecule of the present for cleavage of antibiotic resistance genes and/or cell Survival invention can be selected from a given target gene sequence, 50 genes or homologues or variants thereof can be achieved by e.g., an antibiotic resistance genes and/or cell Survival genes techniques well known to those skilled in the art (for example coding sequence, beginning from about 25 to 50 nucleotides, Lleber and Strauss, (1995) Mol Cell Biol 15:540.551, the from about 50 to 75 nucleotides, or from about 75 to 100 disclosure of which is incorporated herein by reference). nucleotides downstream of the start codon. Nucleotide Promoters of the Engineered Bacteriophages sequences can contain 5" or 3' UTRs and regions nearby the 55 In some embodiments of all aspects described herein, an start codon. One method of designing a siRNA molecule of engineered bacteriophage comprises a nucleic acid which the present invention involves identifying the 23 nucleotide expresses an inhibitor to an antibiotic resistance gene (such as sequence motif AA(N19)TT (where N can be any nucle in inhibitor-engineered bacteriophages) or a repressor to a otide), and selecting hits with at least 25%, 30%, 35%, 40%, SOS gene or a repressor (or inhibitor) to a non-SOS defense 45%, 50%, 55%, 60%, 65%, 70% or 75% G/C content. The 60 gene (in the case of repressor-engineered bacteriophages) or “TT portion of the sequence is optional. Alternatively, if no a susceptibility agent (in a case of a Susceptibility-agent engi Such sequence is found, the search can be extended using the neered bacteriophage). In each instance, gene expression motif NA(N21), where N can be any nucleotide. In this situ from the nucleic acid is regulated by a promoter to which the ation, the 3' end of the sense siRNA can be converted to TT to nucleic acid is operatively linked to. In some embodiments, a allow for the generation of a symmetric duplex with respect to 65 promoter is a bacteriophage promoter. One can use any bac the sequence composition of the sense and antisense 3' over teriophage promoter known by one of ordinary skill in the art, hangs. The antisense siRNA molecule can then be synthe for example but not limited to, any promoter listed in Table 6 US 9,056,899 B2 51 52 or disclosed in world-wide web site “partsregistry.org/cgi/ GATAAATTATCTCTGGCGGTGTTGACATAAATACC partsdb/pgroup.cgi?pgroup other regulator&show=1. ACTGGCGGTtATAaTGAGCACATCAGCAGG//GTATG In some embodiments, an agent is protein or polypeptide or CAAAGGA (SEQ ID NOS: 39-40) and modified variants RNAi agent that inhibits expression of antibiotic resistance thereof. genes and/or cell Survival gene, or a non-SOS defense genes. Modification of Engineered Bacteriophages. In Such embodiments bacteriophage cells can be modified In some embodiments of all aspects described herein, an (e.g., by homologous recombination) to provide increased engineered bacteriophage can also be designed for example, expression of Such an agent, for example by replacing, in for optimal enzyme activity or to delay cell lysis or using whole or in part, the naturally occurring bacteriophage pro multiple phage promoters to allow for increased enzyme pro moter with all or part of a heterologous promoter so that the 10 duction, or targeting multiple biofilm EPS components with bacteriophage and/or the bacteriophage infected-host cell different proteins. In some embodiments, one can also target expresses a high level of the inhibitor agent of antibiotic multi-species biofilm with a cocktail of different species resistance genes and/or cell Survival gene or a repressor oran specific engineered enzymatically-active phage, and combi inhibitor to a non-SOS defense gene or a susceptibility agent. nation therapy with other agents other than antimicrobial In some embodiments, a heterologous promoter is inserted in 15 agent that are well known to one skilled in the art and phage such a manner that it is operatively linked to the desired to improve the efficacy of both types of treatment. nucleic acid encoding the agent. See, for example, PCT Inter In some embodiments of all aspects described herein, an national Publication No. WO 94/12650 by Transkaryotic engineered bacteriophage can also be used together with Therapies, Inc., PCT International Publication No. WO other antibacterial or bacteriofilm degrading agents or chemi 92/20808 by Cell Genesys, Inc., and PCT International Pub cals such as EGTA, a calcium-specific chelating agent, lication No. WO 91/09955 by Applied Research Systems, effected the immediate and substantial detachment of a P which are incorporated herein in their entirety by reference. aeruginosa biofilm without affecting microbial activity, In some embodiments, bacteriophages can be engineered NaCl, CaCl, or MgCl, surfactants and urea. as disclosed herein to express an endogenous gene, such as a Phage therapy or bacteriophage therapy has begun to be repressor protein, or a nucleic acid inhibitor of an antibiotic 25 accepted in industrial and biotechnological settings. For resistance gene or cell Survival gene comprising the agent example, the FDA has previously approved the use of phage under the control of inducible regulatory elements, in which targeted at Listeria monocytogenes as a food additive. Phage case the regulatory sequences of the endogenous gene can be therapy has been used Successfully for therapeutic purposes replaced by homologous recombination. Gene activation in Eastern Europe for over 60 years. The development and use techniques are described in U.S. Pat. No. 5.272,071 to Chap 30 of phage therapy in clinical settings in Western medicine, in pel; U.S. Pat. No. 5,578.461 to Sherwin et al.: PCT/US92/ particular for treating mammals such as humans has been 09627(WO93/09222) by Seldenet al.; and PCT/US90/06436 delayed due to the lack of properly designed clinical trials to (WO91/06667) by Skoultchietal, which are all incorporated date as well as concerns with (i) development of phage resis herein in their entirety by reference. tance, (ii) phage immunogenicity in the human body and Other exemplary examples of promoter which can be used 35 clearance by the reticuloendothelial system (RES), (iii) the include, for example but not limited, Anhydrotetracycline release of toxins upon bacterial lysis, and (iv) phage specific (aTc) promoter, PLtetO-1 (Pubmed Nucleotide# U66309), ity. Many of these concerns are currently being studied and Arabinose promoter (PBAD), IPTG inducible promoters addressed, such as the isolation and development of long PTAC (in vectors such as Pubmed Accession #EU546824), circulating phage that can avoid RES clearance for increased PTrc-2. Plac (in vectors such as Pubmed Accession 40 in vivo efficacy. Accordingly, in all aspects described herein, #EU546816), PLlacO-1, PAllacO-1, and Arabinose and the methods of the present invention are applicable to human IPTG promoters, such as Placfara-a. Examples of these pro treatment as the engineered bacteriophages can be designed moters are as follows: to prevent the development of phage resistance in bacteria. A Anhydrotetracycline (aTc) promoter, such as PLtetO-1 skilled artisan can also develop and carry out an appropriate (Pubmed Nucleotide# U66309): GCATGCTCCCTAT 45 clinical trial for use in clinical applications, such as therapeu CAGTGATAGAGATTGACATCCCTAT tic purposes as well as in human Subjects. In some instances, CAGTGATAGAGATACTGAGCAC ATCAGCAGGACG a skilled artisan could establish and set up a clinical trial to CACTGACCAGGA (SEQ ID NO:36); Arabinose promoter establish the specific tolerance of the engineered bacterioph (PBAD): or modified versions which can be found at world age in human Subjects. The inventors have already demon wide web site: partsregistry.org/wiki/index.php?title=Part: 50 strated herein that inhibitor-engineered bacteriophage and BBa I 13453." AAGAAACCAATTGTCCATATTGCATCA repressor-engineered bacteriophages and Susceptibility-engi GACATTGCCGTCACTGCGTCTTTTACTGGCTCTT neered bacteriophages are effective at increasing the efficacy CTCGCTAACCAAACCGGTAACCCCGCT of antimicrobial agents, and are effective in dispersing bio TATTAAAAGCATTCTGTAACAAAGCGGGACCAA films, including biofilms present in human organs, such as AGCCATGACAAAAACGCGTAACAAAAGT 55 colon or lungs and other organs in a Subject prone to bacterial GTCTATAATCACGGCAGAAAAGTCCACATTG infection such as bacterial biofilm infection. ATTATTTGCACGGCGTCACACTTTGC Another aspect relates to a pharmaceutical composition TATGCCATAGCATTTTTATCCATAAGATTAGCGGA comprising at least one engineered bacteriophage and at least TCCTACCTGACGCTTTTTATCG one antimicrobial agent. In some embodiments of this and all CAACTCTCTACTGTTTCTCCATA (SEQ ID NO: 37): 60 aspects described herein, the composition can be adminis IPTG promoters: (i) PTAC (in vectors such as Pubmed Acces tered as a co-formulation with one or more other non-antimi sion #EU546824, which is incorporated herein by reference), crobial or therapeutic agents. (ii) PTrc-2. CCATCGAATGGCTGAAATGAGCTGTTGA In a further embodiment, the invention provides methods CAATTAATCATCCGGCTCGTATAATGTGTGGA of administration of the compositions and/or pharmaceutical ATTGTGAGCGGATAACAATTTCACACAGGA (SEQ ID 65 formulations of the invention and include any means com NO: 38) and temperature sensitive promoters such as monly known by persons skilled in the art. In some embodi PLSlcon, GCATGCACAGATAACCATCTGCGGT ments, the Subject is any organism, including for example a US 9,056,899 B2 53 54 mammalian, avian or plant. In some embodiments, the mam and PCT/US90/06436 (WO91/06667) by Skoultchi et al., malian is a human, a domesticated animal and/or a commer which are all incorporated herein in their entirety by refer cial animal. CCC. While clearance issue is not significant in treatment of Furthermore, rational engineering methods with new syn chronic diseases, the problem of phage clearance is an impor 5 thesis technologies can be employed to broaden the engi tant one that needs to be solved as it can make phage therapy neered bacteriophage host range. For example, T7 can be more useful for treating transient infections rather than modified to express K1-5 endosialidase, allowing it to effec chronic ones. Non-lytic and non-replicative phage have been tively replicate in E. coli that produce the K1 polysaccharide engineered to kill bacteria while minimizing endotoxin capsule. In some embodiments, the gene 1.2 from phage T3 release. Accordingly, the present invention encompasses 10 can be used to extend the bacteriophages as disclosed herein modification of the inhibitor-engineered and/or repressor to be able to transfect a host range to include E. coli that engineered bacteriophage and/or Susceptibility engineered contain the Fplasmid, thus demonstrating that multiple modi bacteriophage with minimal endotoxin release or toxin-free fications of a phage genome can be done without significant bacteriophage preparation. 15 impairment of the phage's ability to replicate. Bordetella The specificity of phage for host bacteria is both an advan bacteriophage use a reverse-transcriptase-mediated mecha tage and a disadvantage for phage therapy. Specificity allows nism to produce diversity in host tropism which can also be human cells as well as innocuous bacteria to be spared, poten used according to the methods of the present invention to tially avoiding serious issues such as drug toxicity. Antibiotic create a phage that encodes an agent which inhibits antibiotic therapy is believed to alter the microbial flora in the colon due resistance genes and/or cell Survival genes, or alternatively to lack of target specificity, and in Some instances allowing encodes repressors of SOS response genes, and is lytic to the resistant C. difficile to proliferate and cause disease such as target bacterium or bacteria. The many biofilm-promoting diarrhea and colitis. The inhibitor-engineered bacteriophage factors required by E. coli K-12 to produce a mature biofilm and repressor-engineered bacteriophages and/or Susceptibil are likely to be shared among different biofilm-forming bac ity engineered bacteriophage as disclosed herein are capable 25 terial strains and are thus also targets for engineered enzy of inhibiting the local bacterial synthetic machinery which matic bacteriophage as disclosed herein. normally circumvent antimicrobial effect to result in persis Antimicrobial Agents tent bacteria. One aspect of the present invention relates to the killing or For host specificity (i.e. bacteria specific inhibitor or inhibiting the growth of bacteria using a combination of an repressor-engineered bacteriophages), a well-characterized 30 inhibitor-engineered bacteriophage and/or a repressor engi library of phage must be maintained so that an appropriate neered bacteriophage and/or a susceptibility engineered bac inhibitor-engineered bacteriophage or repressor-engineered teriophage with at least one antimicrobial agent. Accordingly, one aspect of the present invention relates to methods and bacteriophage and/or Susceptibility engineered bacterioph compositions comprising engineered bacteriophages for use age therapy can be designed for each individual bacterial 35 in combination with antimicrobial agents to potentiate the infection. The diversity of bacterial infections implies that it antimicrobial effect and bacterial killing function or inhibi may be difficult for any one particular engineered phage to be tion of growth function of the antimicrobial agent. an effective therapeutic solution for a wide range of biofilms. Accordingly in Some embodiments of this aspect of the Accordingly, in one embodiment, the invention provides use present invention relates to the use of a inhibitor-engineered of a variety of different engineered bacteriophages in combi 40 bacteriophage and/or a repressor engineered bacteriophage nation (i.e. a cocktail of engineered bacteriophages discussed and/or Susceptibility engineered bacteriophage to potentiate herein) to cover a range of target bacteria. the killing effect of antimicrobial agents. Stated another way, One skilled in the art can generate a collection or a library the inhibitor-engineered or repressor-engineered bacterioph of the inhibitor-engineered bacteriophage and/or repressor age or Susceptibility engineered bacteriophage can be used to engineered bacteriophage and/or Susceptibility engineered 45 enhance the efficacy of at least one antimicrobial agent. bacteriophage as disclosed herein by new cost-effective, An inhibitor-engineered bacteriophages and/or a repressor large-scale DNA sequencing and DNA synthesis technolo engineered bacteriophage and/or a Susceptibility engineered gies. Sequencing technologies allows the characterization of bacteriophage is considered to potentiate the effectiveness of collections of natural phage that have been used in phage the antimicrobial agent if the amount of antimicrobial agent typing and phage therapy for many years. Accordingly, a 50 used in combination with the engineered bacteriophages as skilled artisan can use synthesis technologies as described disclosed herein is reduced by at least 10% without adversely hereinto add different inhibitors to antibiotic resistance genes affecting the result, for example, without adversely effecting or cell survival genes, and/or different repressors to different the level of antimicrobial activity. In another embodiment, the SOS response genes or non-SOS defense genes or Suscepti criteria used to select inhibitor-engineered bacteriophages bility agents to produce a variety of new inhibitor-engineered 55 and/or a repressor engineered bacteriophage and/or a suscep bacteriophage and repressor-engineered bacteriophages and/ tibility engineered bacteriophage that can potentiate the or Susceptibility engineered bacteriophage respectively. activity of an antimicrobial agent is an engineeredbacterioph In particular embodiments, the engineered bacteriophages age which enables a reduction of at least about 10%, ... or at as described herein can be engineered to express an endog least about 15%, ... or at least about 20%, ... or at least about enous gene. Such as a repressor protein, or a nucleic acid 60 25%, ... or at least about 35%, ... or at least about 50%, ... inhibitor of an antibiotic resistance gene or cell Survival gene or at least about 60%, ... or at least about 90% and all integers comprising the agent under the control of inducible regula inbetween 10-90% of the amount (i.e. dose) of the antimicro tory elements, in which case the regulatory sequences of the bial agent without adversely effecting the antimicrobial effect endogenous gene can be replaced by homologous recombi when compared to the similar amount in the absence of an nation. Gene activation techniques are described in U.S. Pat. 65 inhibitor-engineered bacteriophage and/or a repressor engi No. 5.272,071 to Chappel; U.S. Pat. No. 5,578.461 to Sher neered bacteriophage and/or a susceptibility engineered bac win et al.; PCT/US92/09627 (WO93/09222) by Selden et al.: teriophage. US 9,056,899 B2 55 56 In some embodiments, any antimicrobial agent can be used kacin, gentamycin, tobramycin, metromycin, Streptomycin, which is know by persons of ordinary skill in the art can be kanamycin, paromomycin, and neomycin. used in combination with an inhibitor-engineered bacterioph Carbapenems are a class of broad spectrum antibiotics that age or a repressor-engineered bacteriophage and/or a suscep are used to fight gram-positive, gram-negative, and anaerobic tibility engineered bacteriophage. In some embodiments an microorganisms. Carbapenems are available for intravenous antimicrobial agent is an antibiotic. Thus, in some embodi administration, and as such are used for serious infections ments, the engineered bacteriophages as disclosed herein which oral drugs are unable to adequately address. For function as antibiotic adjuvants foraminglycoside antimicro example, carbapenems are often used to treat serious single or bial agents, such as but not limited to, gentamicin, amikacin, mixed bacterial infections, such as lower respiratory tract gentamycin, tobramycin, netromycin, streptomycin, kana 10 infections, urinary tract infections, intra-abdominal infec mycin, paromomycin, neomycin. In some embodiments, the tions, gynecological and postpartum infections, septicemia, engineered bacteriophages as disclosed herein function as and joint infections, skin and skin structure infections, antibiotic adjuvants for B-lactam antibiotics, such as but not and meningitis. Examples of carbapenems include imi limited to, amplicillin, penicillin, penicillin derivatives, penem/cilastatin Sodium, meropenem, ertapenem, and cephalosporins, monobactams, carbapenems and B-lacta 15 panipenem/betamipron. mase inhibitors. In some embodiments, the engineered bac Cephalosporins and cephems are broad spectrum antibiot teriophages as disclosed herein function as antibiotic adju ics used to treat gram-positive, gram-negative, and spirocha vants for quinolones antimicrobial agents, such as, but not etal infections. Cephems are considered the next generation limited to, ofloxacin, ciproflaxacin, levofloxacin, gatifloxa Cephalosporins with newer drugs being stronger against cin, norfloxacin, lomefloxacin, trovafloxacin, moxifloxacin, gram negative and older drugs better against gram-positive. sparfloxacin, gemifloxacin, and paZufloxacin. Cephalosporins and cephems are commonly Substituted for In alternative embodiments, an antimicrobial agent can be, penicillin allergies and can be used to treat common urinary for example, but not limited to, a Small molecule, a peptide, a tract infections and upper respiratory infections (e.g., phar peptidomimetic, a chemical, a compound and any entity that yugitis and tonsillitis). inhibits the growth and/or kills a microorganism. In some 25 Cephalosporins and cephems are also used to treat otitis embodiments, an antimicrobial agent can include, but is not media, Some skin infections, bronchitis, lower respiratory limited to; antibodies (polyclonal or monoclonal), neutraliz infections (pneumonia), and bone infection (certain; mem ing antibodies, antibody fragments, chimeric antibodies, bers), and are a preferred antibiotic for Surgical prophylaxis. humanized antibodies, recombinant antibodies, peptides, Examples of Cephalosporins include cefixime, cefpodoxime, proteins, peptide-mimetics, aptamers, oligonucleotides, hor 30 ceftibuten, cefdinir, cefaclor, cefprozil, loracarbef, mones, Small molecules, nucleic acids, nucleic acid ana cefadroxil, cephalexin, and cephradineze. Examples of ceph logues, carbohydrates or variants thereof that function to ems include cefepime, cefpirome, cefataxidime pentahy inactivate the nucleic acid and/or protein of the gene products drate, ceftazidime, ceftriaxone, ceftazidime, cefotaxime, identified herein, and those as yet unidentified. Nucleic acids cefteram, cefotiam, cefuroxime, cefamandole, cefuroxime include, for example but not limited to, DNA, RNA, oligo 35 axetil, cefotetan, cefazolin Sodium, cefazolin, cefalexin. nucleotides, peptide nucleic acid (PNA), pseudo-comple Fluoroquinolones/quinolones are antibiotics used to treat mentary-PNA (pcPNA), locked nucleic acid (LNA), RNAi, gram-negative infections, though some newer agents have microRNAi, siRNA, shRNA etc. The an antimicrobial agent activity against gram-positive bacteria and anaerobes. Fluo inhibitors can be selected from a group of a chemical, Small roquinolones/quinolones are often used to treat conditions molecule, chemical entity, nucleic acid sequences, nucleic 40 Such as urinary tract infections, sexually transmitted diseases acid analogues or protein or polypeptide or analogue or frag (e.g., gonorrhea, chlamydial urethritisficerviciitis, pelvic ment thereof. inflammatory disease), gram-negative gastrointestinal infec In Some embodiments, an antimicrobial agent is an antimi tions, Soft tissue infections, pphthalmic infections, dermato crobial peptide, for example but not limited to, mefloquine, logical infections, sinusitis, and respiratory tract infections Venturicidin A, antimycin, myxothiazol, Stigmatellin, diuron, 45 (e.g., bronchitis, pneumonia, and tuberculosis). Fluoroquino iodoacetamide, potassium tellurite hydrate, a L-Vinylgly lones/quinolones are used in combination with other antibi cine, N-ethylmaleimide, L-allyglycine, diarycuinoline, otics to treat conditions, such as multi-drug resistant tubercu betaine aldehyde chloride, acivcin, psicofuraine, buthionine losis, neutropenic cancer patients with fever, and potentially Sulfoximine, diaminopemelic acid, 4-phospho-D-erythron anthrax. Examples of fluoroquinolones/quinolones include hydroxamic acid, motexafin gadolinium and/or Xycitrin or 50 ciproflaxacin, levofloxacin, and ofloxacin, gatifloxacin, nor modified versions or analogues thereof. floxacin, lomefloxacin, trovafloxacin, moxifloxacin, spar In some embodiments, an antimicrobial agent useful in floxacin, gemifloxacin, and paZufloxacin. combination with an inhibitor-engineered or repressor-engi Glycopeptides and streptogramins represent antibiotics neered bacteriophage described herein includes, but are not that are used to treat bacteria that are resistant to other anti limited to aminoglycosides, carbapenemes, cephalosporins, 55 biotics, such as methicillin-resistant Staphylococcus aureus cephems, glycoproteins fluoroquinolones/quinolones, (MRSA). They are also be used for patients who are allergic oxazolidinones, penicillins, streptogramins, Sulfonamides to penicillin Examples of glycopeptides include Vancomycin, and/or tetracyclines. teicoplanin, and daptomycin. Aminoglycosides are a group of antibiotics found to be B-lactam antibiotics are a broad class of antibiotics which effective against gram-negative. Aminoglycosides are used to 60 include penicillin derivatives, cephalosporins, monobactams, treat complicated urinary tract infections, septicemia, perito carbapenems and B-lactamase inhibitors; basically, any anti nitis and other severe intra-abdominal infections, severe pel bioticor agent or antimicrobial agent which contains a 3-lac Vic inflammatory disease, endocarditis, mycobacterium tam nucleus in its molecular structure. Without being bound infections, neonatal sepsis, and various ocular infections. by theory, B-Lactam antibiotics are bactericidal, and act by They are also frequently used in combination with penicillins 65 inhibiting the synthesis of the peptidoglycan layer of bacterial and cephalosporins to treat both gram-positive and gram cell walls. The peptidoglycan layer is important for cell wall negative bacteria. Examples of aminoglycosides includeami structural integrity, especially in Gram-positive organisms. US 9,056,899 B2 57 58 The final transpeptidation step in the synthesis of the pepti icillin plus clavulonic acid; apalcillin, apramycin; astromicin; doglycan is facilitated by transpeptidases known as penicillin arbekacin; aspoxicillin; azidozillin; azlocillin; aztreonam, binding proteins (PBPs). B-lactam antibiotics are analogues bacitracin; benzathine penicillin; benzylpenicillin: clarithro of D-alanyl-D-alanine—the terminal amino acid residues on mycin, carbencillin, cefaclor, cefadroxil, cefalexin, cefaman the precursor NAM/NAG-peptide subunits of the nascent dole; cefaparin, cefatrizine, cefazolin, cefbuperaZone, cef peptidoglycan layer. The structural similarity between 3-lac capene; cefdinir, cefditoren, cefepime, cefetamet, cefixime; tamantibiotics and D-alanyl-D-alanine facilitates their bind cefinetazole; cefiminox, cefoperaZone, ceforanide, cefo ing to the active site of penicillin binding proteins (PBPs). taxime; cefotetan, cefotiam, cefoxitin; ce?pimizole; ce?pira The B-lactam nucleus of the molecule irreversibly binds to mide; cefpodoxime; cefprozil; cefradine, cefroxadine, cefsu (acylates) the Ser403 residue of the PBP active site. This 10 irreversible inhibition of the PBPs prevents the final lodin, ceftazidime; ceftriaxone, cefuroxime; cephalexin; crosslinking (transpeptidation) of the nascent peptidoglycan chloramphenicol; chlortetracycline; ciclacillin; ; layer, disrupting cell wall synthesis. Under normal circum clemizole penicillin; cleocin, cleocin-T, cloxacillin; corifam; stances peptidoglycan precursors signal a reorganization of daptomycin; daptomycin; demeclocycline; descuinolone; the bacterial cell wall and consequently trigger the activation 15 dibekacin; dicloxacillin; dirithromycin; doxycycline; enoxa of autolytic cell wall hydrolyses. Inhibition of cross-linkage cin; epicillin; ; gemifloxacin; fenampicin; by B-lactams causes a build-up of peptidoglycan precursors finamicina; ; flomoxef flucloxacillin; ; which triggers the digestion of existing peptidoglycan by flurithromycin; fosfomycin; fosmidomycin; fusidic acid; autolytic hydrolases without the production of new pepti gatifloxacin; gemifloxaxin, isepamicin; ; josamy doglycan. This as a result further enhances the bactericidal cin; kanamycin; kasugamycin; kitasamycin; kalrifam, lata action off-lactam antibiotics. moxef levofloxacin, levofloxacing lincomycin; lineZolid; Carbapenems are used to treat gram-positive, gram-nega lomefloxacin; loracarbaf, lymecycline; mecillinam; meth tive, and/or anaerobes. acycline; methicillin; metronidazole; mezlocillin; mideca Oxazolidinones are commonly administered to treatgram mycin; minocycline; miokamycin; moxifloxacin, nafcillin; positive infections. Oxazolidinones are commonly used as an 25 nafcillin, ; neomycin; netilmicin; norfloxacin; alternative to other antibiotic classes for bacteria that have , oflaxacin; oleandomycin; oxacillin, oxolinic developed resistance. Examples of oxazolidinones include acid; oxytetracycline; paromycin; paZufloxacin; ; linezolid. penicillin g; penicillin V; phenethicillin; phenoxymethyl Penicillins are broad spectrum used to treat gram-positive, penicillin; ; piperacillin and taZobactam com gram-negative, and spirochaetal infections. Conditions that 30 bination; ; procaine penicillin; propicillin; are often treated with penicillins include pneumococcal and pyrimethamine; rifadin: : rifamide; rifampin: rifap meningococcal meningitis, dermatological infections, ear entene: rifomycin; rimactane, rofact; rokitamycin; rollitetra infections, respiratory infections, urinary tract infections, cycline; roXithromycin; ; ; sparfloxa acute sinusitis, pneumonia, and Lyme disease. Examples of cin; spectinomycin; spiramycin; ; ; penicillins include penicillin, amoxicillin, amoxicillin-clavu 35 ; sisomicin; streptomycin; Sulfamethox lanate, amplicillin, ticarcillin, piperacillin-taZobactam, carbe azole; Sulfisoxazole; quinupristan-dalfopristan; teicoplanin; nicillin, piperacillin, meZocillin, benzathin penicillin G peni temocillin; gatifloxacin, tetracycline; ; tellithro cillin V potassium, methicillin, nafcillin, oxacillin, mycin; thiamphenicol; ticarcillin; tigecycline; tobramycin; cloxacillin, and dicloxacillin. to Sufloxacin; ; trimetrexate; trovafloxacin; Van Streptogramins are antibiotics developed in response to 40 comycin; Verdamicin; azithromycin; and lineZolid. bacterial resistance that diminished effectiveness of existing Uses of the Engineered Bacteriophages antibiotics. Streptogramins are avery small class of drugs and Accordingly, the inventors have demonstrated that an anti are currently very expensive. Examples of streptogramins microbial agent when used in combination with an inhibitor include quinupristin/dafopristin and pristinamycin. engineered bacteriophage (which expresses an inhibitor to an Sulphonamides are broad spectrum antibiotics that have 45 antibiotic resistance gene or a cell Survival gene) and/or in had reduced usage due to increase in bacterial resistance to combination with a repressor-engineered bacteriophage them. Sulphonamides are commonly used to treat recurrent (which expresses at least one repressor to a SOS response attacks of rheumatic fever, urinary tract infections, prevention gene, or at least one inhibitor or repressor to a non-SOS of infections of the throat and chest, traveler's diarrhea, defense gene) and/or in combination with a susceptibility whooping cough, meningococcal disease, sexually transmit 50 engineered bacteriophage is effective at killing bacteria, Such ted diseases, toxoplasmosis, and rhinitis. Examples of Sul as a bacterial infection or a bacteria biofilm than use of the fonamides include co-trimoxazole, Sulfamethoxazole trime antimicrobial alone or the use of the antimicrobial agent used thoprim, Sulfadiazine, Sulfadoxine, and trimethoprim. in combination with a non-engineered bacteriophage. The Tetracyclines are broad spectrum antibiotics that are often inventors have also discovered that engineered bacterioph used to treat gram-positive, gram-negative, and/or spirocha 55 ages can be adapted to work with a variety of different anti etal infections. Tetracyclines are often used to treat mixed microbial agents as well as be modified to express other infections, such as chronic bronchitis and peritonitis, urinary biofilm-degrading enzymes to target a wide range of bacteria tract infections, rickets, chlamydia, gonorrhea, Lyme disease, and bacteria biofilms. In some embodiments, an antimicro and periodontal disease. Tetracyclines are an alternative bial agent is used in combination with at least one engineered therapy to penicillin in syphilis treatment and are also used to 60 bacteriophage as disclosed herein, and optionally an addition treat acne and anthrax. Examples of tetracyclines include bacteriophage which is not an inhibitor-engineered or repres tetracycline, demeclocycline, minocycline, and doxycycline. sor-engineered bacteriophage or a susceptibility engineered Other antimicrobial agents and antibiotics contemplated bacteriophage, but a bacteriophage which is modified to herein useful in combination with the engineered bacterioph express atherapeutic gene oratoxin gene or a biofilm degrad ages as disclosed herein according to the present invention 65 ing gene. Such bacteriophages are well known in the art and (some of which can be redundant with the list above) include, are encompassed for use in the methods and compositions as but are not limited to; abrifam; acrofloxacin; aptecin, amoX disclosed herein. US 9,056,899 B2 59 60 Bacterial Infections lia recurrentis, Borrelia burgdorfii and Leptospira icterohe One aspect of the present invention relates to the use of the morrhagiae and other miscellaneous bacteria, including methods and compositions comprising an inhibitor-engi Actinomyces and Nocardia. neered and/or repressor-engineered bacteriophage and/or a In some embodiments, the microbial infection is caused by Susceptibility engineered bacteriophage in combination with gram-negative bacterium, for example, P. aeruginosa, A. an antimicrobial agent to inhibit the growth and/or kill (or bunannii, Salmonella spp., Klebsiella pneumonia, Shigeila reduce the cell viability) of a microorganism, such as a bac spp. and/or Stenotrophomonas maltophilia. Examples of teria. In some embodiments of this aspect and all aspects microbial infections include bacterial wound infections, described herein, a microorganism is a bacterium. In some mucosal infections, enteric infections, septic conditions, embodiments, the bacteria are gram positive and gram nega 10 pneumonia, trachoma, onithosis, trichomoniasis and salmo tive bacteria. In some embodiments, the bacteria are multi nellosis, especially in Veterinary practice. drug resistant bacterium. In further embodiments, the bacte ria are polymyxin-resistant bacterium. In some embodiments, Examples of infections caused by P. aeruginosa include: the bacterium is a persister bacteria. Examples of gram-nega A) Nosoconial infections, 1. Respiratory tract infections in tive bacteria are for example, but not limited to Paeruginosa, 15 cystic fibrosis patients and mechanically-ventilated patients; A. bumannii, Salmonella spp., Klebsiella pneumonia, Shigeila 2. Bacteraemia and sepsis; 3. Wound infections, particularly spp. and/or Stenotrophomonas maltophilia. In one embodi in burn wound patients; 4. Urinary tract infections: 5. Post ment, the bacteria to be targeted using the phage of the inven Surgery infections on invasive devises 5. Endocarditis by tion include E. coli, S. epidermidis, Yersina pestis and intravenous administration of contaminated drug solutions; 7. Pseudomonas fluorescens. Infections in patients with acquired immunodeficiency syn In some embodiments, the methods and compositions as drome, cancer chemotherapy, steroid therapy, hematological disclosed herein can be used to kill or reduce the viability of malignancies, organ transplantation, renal replacement a bacterium, for example a bacterium Such as, but not limited therapy, and other situations with severe neutropenia. B) to: Bacillus cereus, Bacillus anbhracis, Bacillus cereus, Community-acquired infections; 1. Community-acquired Bacillus anthracia, Clostridium botulinum, Clostridium dif 25 respiratory tract infections; 2. Meningitis; 3. Folliculitis and ficle, Clostridium tetani, Clostridium perfingens, Coryne infections of the ear canal caused by contaminated waters; 4. bacteria diptheriae, Enterococcus (Streptococcus D), Liet Malignant otitis externa in the elderly and diabetics; 5. Osteo eria monocytogenes, Pneumoccoccal infections myelitis of the caleaneus in children; Eye infections com (Streptococcus pneumoniae), Staphylococcal infections and monly associated with contaminated contact lens; 6. Skin Streptococcal infections; Gram-negative bacteria including 30 infections such as nail infections in people whose hands are Bacteroides, Bordetella pertussis, Brucella, Campylobacter frequently exposed to water; 7. infec infections, enterohaemorrhagic Escherichia coli (EHEC/E. tions; 8. Muscoskeletal system infections. coli O157:17), enteroinvasive Escherichia coli (EIEC), Examples of infections caused by A. baumannii include: A) enterotoxigenic Escherichia coli (ETEC), Haemophilus Nosoconial infections 1. Bacteraemia and sepsis, 2. respira influenzae, Helicobacter pylori, Klebsiella pneumoniae, 35 tory tract infections in mechanically ventilated patients; 3. Legionella spp., Moraxella catarrhalis, Neisseria gonnor Post-Surgery infections on invasive devices; 4. wound infec rhoeae, Neisseria meningitidis, Proteus spp., Pseudomonas tious, particularly in burn wound patients; 5. infection in aeruginosa, Salmonella spp., Shigella spp., Vibrio cholera patients with acquired immunodeficiency syndrome, cancer and Yersinia; acid fast bacteria including Mycobacterium chemotherapy, steroid therapy, hematological malignancies, tuberculosis, Mycobacterium avium-intracellulars, Myobac 40 organ transplantation, renal replacement therapy, and other terium johnei, Mycobacterium leprae, atypical bacteria, situations with severe neutropenia; 6. urinary tract infections; Chlamydia, Myoplasma, Rickettsia, Spirochetes, Treponema 7. Endocarditis by intravenous administration of contami pallidum, Borrelia recurrentis, Borrelia burgdorfii and Lep nated drug solutions; 8. Cellulitis. B) Community-acquired to spira icterohemorrhagiae, Actinomyces, Nocardia, P infections; a. community-acquired pulmonary infections; 2. aeruginosa, A. bumannii, Salmonella spp., Klebsiella pneu 45 Meningitis; Cheratitis associated with contaminated contact monia, Shigeila spp. and/or Stenotrophomonas maltophilia lens; 4. War-Zone community-acquired infections. C) Atypi and other miscellaneous bacteria. cal infections: 1. Chronic gastritis. Bacterial infections include, but are not limited to, infec Examples of infections caused by Stenotrophomonas mal tions caused by Bacillus cereus, Bacillus anbhracis, Bacillus tophilia include Bacteremia, pneumonia, meningitis, wound cereus, Bacillus anthracia, Clostridium botulinum, 50 infections and urinary tract infections. Some hospital breaks Clostridium difficle, Clostridium tetani, Clostridium perfrin are caused by contaminated disinfectant solutions, respira gens, Corynebacteria diptheriae, Enterococcus (Streptococ tory devices, monitoring instruments and ice machines. cus D), Lieteria monocytogenes, Pneumoccoccal infections Infections usually occur in debilitated patients with impaired (Streptococcus pneumoniae), Staphylococcal infections and host defense mechanisms. Streptococcal infections/Gram-negative bacteria including 55 Examples of infections caused by Klebsiella pneumoniae Bacteroides, Bordetella pertussis, Brucella, Campylobacter include community-acquired primary lobar pneumonia, par infections, enterohaemorrhagic Escherichia coli (EHEC/E. ticularly in people with compromised pulmonary function coli O157:17) enteroinvasive Escherichia coli (EIEC), entero and alcoholics. It also caused wound infections, soft tissue toxigenic Escherichia coli (ETEC), Haemophilus influenzae, infections and urinary tract infections. Helicobacter pylori, Klebsiella pneumoniae, Legionella spp., 60 Examples of infections caused by Salmonella app. are Moraxella catarrhalis, Neisseria gonnorrhoeae, Neisseria acquired by eating contaminated food products. Infections meningitidis, Proteus spp., Pseudomonas aeruginosa, Sal include enteric fever, enteritis and bacteremia. monella spp., Shigella spp., Vibrio cholera and Yersinia; acid Examples of infections caused by Shigella spp. include fast bacteria including Mycobacterium tuberculosis, Myco gastroenteritis (shigellosis). bacterium avium-intracellulars, Myobacterium johnei, 65 The methods and compositions as disclosed herein com Mycobacterium leprae, atypical bacteria, Chlamydia, Myo prising an inhibitor-engineered or repressor-engineered bac plasma, Rickettsia, Spirochetes, Treponema pallidum, Borre teriophage and at least one antimicrobial agent can also be US 9,056,899 B2 61 62 used in various fields as where antiseptic treatment or disin dia psittaci, Chlamydia trachonzatis, Bordetella pertcsis, fection of materials it required, for example, Surface disinfec Shigella spp., Campylobacter jejuni; Proteus spp., Citro tion. bacter spp.; Enterobacter spp., Pseudomonas aeruginosa, The methods and compositions as disclosed herein com Propionibacterium spp.; Bacillus anthracia; Pseudomonas prising an inhibitor-engineered or repressor-engineered bac syringae; Spirrilum minus; Neisseria meningitidis, Listeria teriophage and at least one antimicrobial agent can be used to monocytogenes, Neisseria gonorrheae, Treponema palli treat microorganisms infecting a cell, group of cells, or a dum, Francisella tularensis, Brucella spp.; Borrelia recur multi-cellular organism. rentis, Borrelia hennsii; Borrelia turicatue, Borrelia burg In one embodiment, an antimicrobial agent and an engi dorferi, Mycobacterium avium, Mycobacterium Smegmatis; neered bacteriophage as described herein can be used to 10 reduce the rate of proliferation and/or growth of microorgan Methicillin-resistant Staphyloccus aureus; Vanomycin-resis isms. In some embodiments, the microorganism are either or tant enterococcus; and multi-drug resistant bacteria (e.g., both gram-positive or gram-negative bacteria, whether Such bacteria that are resistant to more than 1, more than 2, more bacteria are cocci (spherical), rods, vibrio (comma shaped), than 3, or more than 4 different drugs). or spiral. 15 Of the cocci bacteria, micrococcus and Staphylococcus TABLE 7 species are commonly associated with the skin, and Strepto Examples of bacteria. coccus species are commonly associated with tooth enamel Table 7: Examples of Bacteria and contribute to tooth decay. Of the rods family, bacteria Staphyloccoctisatiretts Bacillus species produce endospores seen in various stages of Bacilius anthracis development in the photograph and B. cereus cause a rela Bacilius cereus tively mild food poisoning, especially due to reheated fried Bacilius subtilis Streptococci is phenonia food. Of the vibrio species, V. cholerae is the most common Streptococci is pyogenes bacteria and causes cholera, a severe diarrhea disease result Cliostridium tetani ing from a toxin produced by bacterial growth in the gut. Of 25 Listeria monocytogenes the spiral bacteria, rhodospirillum and Treponema pallidum Mycobacterium tuberculosis are the common species to cause infection (e.g., Treponema Staphyloccoctis epidermidis Nisseria menigintidis pallidum causes syphilis). Spiral bacteria typically grow in Nisseria gonerrhoeae shallow anaerobic conditions and can photosynthesize to Vibrio choierae obtain energy from Sunlight. 30 Escherichia coli K12 Bartoneia henseiae Moreover, the present invention relates to use of or meth Haemophilus influenzae ods comprising an antimicrobial agent and an engineered Salmonella typhi bacteriophage as disclosed herein can be used to reduce the Shigella dysentriae rate of growth and/or kill either gram positive, gram negative, Yeriniisa pestis or mixed flora bacteria or other microorganisms. In one 35 Pseudomona aeruginosa Helicbacter pylori embodiment, the composition consists essentially of at least Legionella pnemophia one antimicrobial agent and at least one engineered bacte Borrelia burgdorferi riophage, such as an inhibitor-engineered bacteriophage or Ehrlichia chafeensis repressor-engineered bacteriophage or a Susceptibility engi Treponema pallidiin Chlamydia trachomatis neered bacteriophage as disclosed herein for the use to reduce 40 the rate of growth and/or kill either gram positive, gram negative, or mixed flora bacteria or other microorganisms. In In some embodiments, antimicrobial agent and engineered another embodiment, the composition contains at least one bacteriophages described herein can be used to treat an antimicrobial agent and at least one engineered bacterioph already drug resistant bacterial strain such as Methicillin age. Such as an inhibitor-engineered bacteriophage or repres 45 resistant Staphylococcus aureus (MRSA) or Vancomycin sor-engineered bacteriophage or a Susceptibility engineered resistant enterococcus (VRE) of variant strains thereof. bacteriophage as disclosed herein for the use to reduce the In some embodiments, the present invention also contem rate of growth and/or kill either gram positive, gram negative, plates the use and methods of use of an antimicrobial agent or mixed flora bacteria or other microorganisms. and an engineered bacteriophage as described herein in all Such bacteria are for example, but are not limited to, listed 50 combinations with other antimicrobial agents and/or antibi in Table 7. Further examples of bacteria are, for example but otics to fight gram-positive bacteria that maintain resistance not limited to Baciccis Antracis; Enterococcus faecalis, to certain drugs. Corynebacterium, diphtheriae, Escherichia coli, Strepto In some embodiments, an antimicrobial agents and an coccus coelicolor, Streptococcus pyogenes, Streptobacillus engineered bacteriophage as disclosed herein can be used to moniliformis, Streptococcus agalactiae, Streptococcus 55 treat infections, for example bacterial infections and other pneurmoniae, Salmonella typhi; Salmonella paratyphi; Sal conditions such as urinary tract infections, ear infections, monella schottmulleri, Salmonella hirshieldii; Staphylococ sinus infections, bacterial infections of the skin, bacterial cus epidermidis, Staphylococcus aureus, Klebsiella pneumo infections of the lungs, sexually transmitted diseases, tuber niae, Legionella pneumophila, Helicobacter pylori; culosis, pneumonia, Lyme disease, and Legionnaire's dis Mycoplasma pneumonia, Mycobacterium tuberculosis, 60 ease. Thus any of the above conditions and other conditions Mycobacterium leprae, Yersinia enterocolitica, Yersinia pes resulting from a microorganism infection, for example a bac tis, Vibrio cholerae, Vibrio parahaemolyticus, Rickettsia pro terial infection or a biofilm can be prevented or treated by the wOzekii, Rickettsia rickettsii, Rickettsia akari. Clostridium compositions of the invention herein. difficile, Clostridium tetani; Clostridium perfingens, Biofilms Clostridianz novyi, Clostridianz Septicum, Clostridium 65 Another aspect of the present invention relates to the use of botulinum, Legionella pneumophila, Hemophilus influenzue; an inhibitor-engineered bacteriophage and/or a repressor-en Hemophilus parainfluenzue, Hemophilus aegyptus, Chlamy gineered bacteriophage and/or a Susceptibility engineered US 9,056,899 B2 63 64 bacteriophage in combination with any antimicrobial agent to or a repressor-engineered bacteriophage and/or Susceptibility eliminate or reduce a bacterial biofilm, for example a bacte engineered bacteriophage. In some embodiments, an antimi rial biofilm in a medical, or industrial, or biotechnological crobial agent can be formulated to a specific time-release for Setting. activity, Such as the antimicrobial agent is present in a time For instance, some bacteria, including P aeruginosa, release capsule. In Such embodiments, an antimicrobial agent actively form tightly arranged multi-cell structures in vivo that is formulated for time-release can be administered to a known as biofilm. The production of biofilm is important for Subject at the same time, concurrent with, or prior to, or after the persistence of infectious processes Such as seen in the administration of an inhibitor-engineered bacteriophage pseudomonal lung-infections in patients with cystic fibrosis and/or a repressor-engineered bacteriophage and/or Suscep and diffuse panbronchiolitis and many other diseases. A bio 10 tibility engineered bacteriophage. Methods of formulation of film is typically resistant to phagocytosis by host immune an antimicrobial agent for release in a time-dependent man cells and the effectiveness of antibiotics at killing bacteria in ner are disclosed herein as “sustained release pharmaceutical biofilm structures can be reduced by 10 to 1000 fold. Biofilm compositions' in the section entitled “pharmaceutical formu production and arrangement is governed by quorum sensing lations and compositions.” Accordingly, in Such embodi systems. The disruption of the quorum sensing system in 15 ments, a time-release antimicrobial agent can be adminis bacteria Such as Paeruginosa is an important anti-pathogenic tered to a subject at the same time (i.e. concurrent with), prior activity as it disrupts the biofilm formation and also inhibits to or after the administration of an engineered bacteriophage alginate production independent to the time to which the antimicrobial agent Selection of Subjects Administered a Composition Compris becomes active. In some embodiments, an antimicrobial ing an Engineered Bacteriophage agent can be administered prior to the administration of the In some embodiments, a subject amenable for the method engineered bacteriophage, and the time at which the antimi described herein or for the administration with a composition crobial agent is released from the time-release capsule coin comprising at least one antimicrobial agent and an inhibitor cides with the time of the administration of the engineered engineered bacteriophage and/or a repressor-engineered bac bacteriophage. teriophage and/or a susceptibility engineered bacteriophage 25 In some embodiments, an antimicrobial agent can be a is selected based on the desired treatment regime. For pro-drug, where it is activated by a second agent. Accord instance, a subject is selected for treatment if the Subject has ingly, in Such embodiments, an antimicrobial pro-drug agent a bacterial infection where the bacteria form a biofilm, or can be administered to a subject at the same time, concurrent where the Subject has been non-responsive to prior therapy or with, or prior to, or after the administration of an inhibitor administration with an antimicrobial agent. 30 engineeredbacteriophage and/or repressor-engineered bacte Accordingly, in some embodiments, a Subjects is adminis riophage and/or Susceptibility engineered bacteriophage, and tered a combination of at least one antimicrobial agent and at administration of an agent which activates the pro-drug into least one inhibitor-engineered bacteriophage and/or a repres its active form can be administered the same time, concurrent sor-engineered bacteriophage and/or a susceptibility engi with, or prior to, or after the administration of the inhibitor neered bacteriophage to potentiate the effect of the antimi 35 engineeredbacteriophage and/or repressor-engineered bacte crobial agent. riophage and/or Susceptibility engineered bacteriophage. In some embodiments, a Subject can be administered a In some embodiments, a Subject is selected for the admin composition comprising at least one antimicrobial agent, for istration with the compositions as disclosed herein by identi example at least 2, 3, or 4 or as many of 10 different antimi fying a subject that needs a specific treatment regimen of an crobial agents and at least one engineered bacteriophage as 40 antimicrobial agent, and is administered an antimicrobial disclosed herein, for example, for example at least 2, 3, or 4 or agent concurrently with, or prior to, or after administration as many of 10 different engineered bacteriophages as dis with an inhibitor-engineered bacteriophage and/or a repres closed herein. In some embodiments, the composition can sor-engineered bacteriophage and/or Susceptibility engi comprise an antimicrobial agent and at least one or a variety neered bacteriophage as disclosed herein. of different repressor-engineered bacteriophages with at least 45 Using a Subject with cystic fibrosis as an exemplary one or a variety of different inhibitor-engineered bacterioph example, a Subject could be administered an antimicrobial ages and/or with at least one or a variety of Susceptibility agent to avoid chronic endobronchial infections, such as engineered bacteriophages. In alternative embodiments, the those caused by pseudomonas aeruginosis or Steintrophomo composition can comprise at least two, or at least 3, 4, 5 or as nas maltophilia. One such antimicrobial agent which can be many of 10 different inhibitor-engineered bacteriophages, 50 used is colistin, however, administration of colistin at the wherein each of the inhibitor-engineered bacteriophages doses and the duration required to efficiently prevent Such comprise a nucleic acid which encodes at least one inhibitor endobronchial infections in Subjects is highly toxic and in to a different antibiotic resistance gene and/or cell survival Some instances fatal. Accordingly, in some embodiments, repair gene. In alternative embodiments, the composition can Such a subject selected for a treatment regimen would be comprise at least two, or at least 3, 4, 5 or as many of 10 55 administered compositions as disclosed herein comprising an different repressor-engineered bacteriophages, wherein each antimicrobial agent and an inhibitor-engineered bacterioph of the repressor-engineered bacteriophages comprise a age and/or a repressor-engineered bacteriophage and/or Sus nucleic acid which encodes at least one repressor to a differ ceptibility engineered bacteriophage. Thus in Such embodi ent SOS response gene and/or at least one repressor or inhibi ments, an antimicrobial agent can be used at a lower dose torto a non-SOS defense gene. Any combination and mixture 60 when used in combination with an inhibitor-engineered bac of antimicrobial agents and mixture of inhibitor-engineered teriophage and/or repressor-engineered bacteriophage and/or bacteriophages and/or repressor-engineered bacteriophages Susceptibility engineered bacteriophage as compared to the and/or Susceptibility engineered bacteriophages are useful in use of Such an antimicrobial agent alone. Thus one aspect of the compositions and methods of the present invention. the invention relates to methods to reduce or decrease the dose In some embodiments, an antimicrobial agent is adminis 65 of an antimicrobial agent while maintaining efficacy of Such tered to a subject at the same time, prior to, or after the an antimicrobial agent, and thus reduce toxic side affects administration of an inhibitor-engineered bacteriophage and/ associated with higher doses. US 9,056,899 B2 65 66 Pharmaceutical Formulations and Compositions one or more of the utilities disclosed herein. They can be The inhibitor-engineered bacteriophage and repressor-en administered in vitro to cells in culture, in vivo to cells in the gineered bacteriophages as disclosed herein can be formu body, or ex vivo to cells outside of a subject that can later be lated in combination with one or more pharmaceutically returned to the body of the same subject or another subject. acceptable anti-microbial agents. In some embodiments, Such cells can be disaggregated or provided as solid tissue in combinations of different antimicrobial agents can be tailored tissue transplantation procedures. to be combined with a specific inhibitor-engineered bacte Compositions comprising at least one antimicrobial agent riophage and a repressor-engineered bacteriophage and/or and at least one engineered bacteriophage (i.e. an inhibitor Susceptibility engineered bacteriophage, where the inhibitor engineered and/or repressor-engineered bacteriophage and/ engineeredbacteriophage and/or repressor-engineered bacte 10 or Susceptibility engineered bacteriophage) as disclosed riophages and/or Susceptibility engineered bacteriophage are herein can be used to produce a medicament or other phar designed to target different (or the same) microorganisms or maceutical compositions. Use of the compositions as dis bacteria, which contribute towards morbidity and mortality. A closed herein which comprise a combination of at least one pharmaceutically acceptable composition comprising an antimicrobial agents and an engineered bacteriophage can inhibitor-engineered bacteriophage and/or a repressor-engi 15 further comprise a pharmaceutically acceptable carrier. The neered bacteriophage and/or susceptibility engineered bacte composition can further comprise other components or riophage and an antimicrobial agent as disclosed herein, are agents useful for delivering the composition to a Subject are Suitable for internal administration to an animal, for example known in the art. Addition of Such carriers and other compo human. nents to the agents as disclosed herein is well within the level In some embodiments, an inhibitor-engineered bacterioph of skill in this art. age and/or a repressor-engineered bacteriophage and/or Sus In some embodiments, the composition is a composition ceptibility engineered bacteriophage as disclosed herein can for sterilization of a physical object, that is infected with be used for industrial sterilizing, sterilizing chemicals such as bacteria, Such as Sterilization of hospital equipment, indus detergents, disinfectants, and ammonium-based chemicals trial equipment, medical devices and food products. In (e.g. quaternary ammonium compounds such as QUATAL, 25 another embodiment, the compositions are a pharmaceutical which contains 10.5% N-alkyldimethyl-benzlammonium composition useful to treat a bacterial infection in a Subject, HCl and 5.5% gluteraldehyde as active ingredients, Eco for example a human or animal Subject. chimie Ltée, Quebec, Canada), and can be used in concur In some embodiments, a pharmaceutical composition as rently with, or prior to or after the treatment or administration disclosed herein can be administered as a formulation of an antimicrobial agent. Such sterilizing chemicals are typi 30 adapted for passage through the blood-brain barrier or direct cally used in the art for sterilizing industrial work surfaces contact with the endothelium. In some embodiments, the (e.g. in food processing, or hospital environments), and are pharmaceutical compositions can be administered as a for not suitable for administration to an animal. mulation adapted for systemic delivery. In some embodi In another aspect of the present invention relates to a phar ments, the compositions can be administered as a formulation maceutical composition comprising an inhibitor-engineered 35 adapted for delivery to specific organs, for example but not bacteriophage and/or repressor-engineered bacteriophage limited to the liver, bone marrow, or systemic delivery. and/or Susceptibility engineered bacteriophage and an anti Alternatively, pharmaceutical compositions can be added microbial agent and a pharmaceutically acceptable excipient. to the culture medium of cells ex vivo. In addition to the Suitable carriers for the engineered bacteriophages of the antimicrobial agent and engineered bacteriophages. Such invention, and their formulations, are described in Reming 40 compositions can contain pharmaceutically-acceptable carri ton's Pharmaceutical Sciences, 16" ed., 1980, Mack Publish ers and other ingredients or agents known to facilitate admin ing Co., edited by Oslo et al. Typically an appropriate amount istration and/or enhance uptake (e.g., Saline, dimethylsulfox of a pharmaceutically acceptable salt is used in the formula ide, lipid, polymer, affinity-based cell specific-targeting tion to render the formulation isotonic. Examples of the car systems). In some embodiments, a pharmaceutical composi rier include buffers such as Saline, Ringer's solution and 45 tion can be incorporated in a gel, Sponge, or other permeable dextrose solution. The pH of the solution is preferably from matrix (e.g., formed as pellets or a disk) and placed in proX about 5 to about 8, and more preferably from about 7.4 to imity to the endothelium for sustained, local release. The about 7.8. Further carriers include sustained release prepara composition can be administered in a single dose or in mul tions such as semipermeable matrices of Solid hydrophobic tiple doses which are administered at different times. polymers, which matrices are in the form of shaped articles, 50 Pharmaceutical compositions can be administered to a Sub e.g. liposomes, films or microparticles. It will be apparent to ject by any known route. By way of example, the composition those of skill in the art that certain carriers can be more can be administered by a mucosal, pulmonary, topical, or preferable depending upon for instance the route of adminis other localized or systemic route (e.g., enteral and tration and concentration of the an engineered bacteriophage parenteral). The phrases “parenteral administration' and being administered. 55 “administered parenterally as used herein means modes of Administration to human can be accomplished by means administration other than enteral and topical administration, determined by the underlying condition. For example, if the usually by injection, and includes, without limitation, intra engineered bacteriophage is to be delivered into lungs of an venous, intramuscular, intraarterial, intrathecal, intraven individual, inhalers can be used. If the composition is to be tricular, intracapsular, intraorbital, intracardiac, intradermal, delivered into any part of the gut or colon, coated tablets, 60 intraperitoneal, transtracheal, Subcutaneous, Subcuticular, Suppositories or orally administered liquids, tablets, caplets intraarticular, Sub capsular, Subarachnoid, intraspinal, intrac and so forth can be used. A skilled artisan will be able to erebro spinal, and intrasternal injection, infusion and other determine the appropriate way of administering the phages of injection or infusion techniques, without limitation. The the invention in view of the general knowledge and skill in the phrases “systemic administration.” “administered systemi art. 65 cally”, “peripheral administration' and “administered Compounds as disclosed herein, can be used as a medica peripherally as used herein mean the administration of the ment or used to formulate a pharmaceutical composition with agents as disclosed herein Such that it enters the animals US 9,056,899 B2 67 68 system and, thus, is subject to metabolism and other like chemical markers in a Subject, preventing one or more symp processes, for example, Subcutaneous administration. toms from worsening or progressing, promoting recovery or The phrase “pharmaceutically acceptable' is employed improving prognosis, and/or preventing disease in a subject herein to refer to those compounds, materials, compositions, who is free therefrom as well as slowing or reducing progres and/or dosage forms which are, within the scope of Sound sion of existing disease. medical judgment, Suitable for use in contact with the tissues In some embodiments, efficacy of treatment can be mea of human beings and animals without excessive toxicity, irri Sured as an improvement in morbidity or mortality (e.g., tation, allergic response, or other problem or complication, lengthening of Survival curve for a selected population). Pro commensurate with a reasonable benefit/risk ratio. phylactic methods (e.g., preventing or reducing the incidence The phrase “pharmaceutically acceptable carrier as used 10 of relapse) are also considered treatment. herein means a pharmaceutically acceptable material, com Dosages, formulations, dosage Volumes, regimens, and position or vehicle. Such as a liquid or Solid filler, diluent, methods for analyzing results aimed at reducing the number excipient, solvent or encapsulating material, involved in car of viable bacteria and/or activity can vary. Thus, minimum rying or transporting the Subject agents from one organ, or and maximum effective dosages vary depending on the portion of the body, to another organ, or portion of the body. 15 method of administration. Suppression of the clinical Each carrier must be “acceptable' in the sense of being com changes associated with bacterial infections or infection with patible with the other ingredients of the formulation, for a microorganism can occur within a specific dosage range, example the carrier does not decrease the impact of the agent which, however, varies depending on the organism receiving on the treatment. In other words, a carrier is pharmaceutically the dosage, the route of administration, whether the antimi inert. crobial agents are administered in conjunction with the engi Suitable choices in amounts and timing of doses, formula neered bacteriophages as disclosed herein, and in some tion, and routes of administration can be made with the goals embodiments with other co-stimulatory molecules, and the of achieving a favorable response in the subject with a bacte specific regimen administration. For example, in general, rial infection or infection with a microorganism, for example, nasal administration requires a Smaller dosage than oral, a favorable response is killing or elimination of the microor 25 enteral, rectal, or vaginal administration. ganism or bacteria, or control of, or inhibition of growth of the For oral or enteral formulations for use with the present bacterial infection in the subject or a subject at risk thereof invention, tablets can be formulated in accordance with con (i.e., efficacy), and avoiding undue toxicity or other harm ventional procedures employing solid carriers well-known in thereto (i.e., safety). Therefore, “effective' refers to such the art. Capsules employed for oral formulations to be used choices that involve routine manipulation of conditions to 30 with the methods of the present invention can be made from achieve a desired effect or favorable response. any pharmaceutically acceptable material. Such as gelatin or Abolus of the pharmaceutical composition can be admin cellulose derivatives. Sustained release oral delivery systems istered to a Subject over a short time, such as once a day is a and/or enteric coatings for orally administered dosage forms convenient dosing schedule. Alternatively, the effective daily are also contemplated, such as those described in U.S. Pat. dose can be divided into multiple doses for purposes of 35 No. 4,704,295, “Enteric Film-Coating Compositions issued administration, for example, two to twelve doses per day. Nov. 3, 1987; U.S. Pat. No. 4,556,552, “Enteric Film-Coating Dosage levels of active ingredients in a pharmaceutical com Compositions,” issued Dec. 3, 1985; U.S. Pat. No. 4,309.404, position can also be varied so as to achieve a transient or “Sustained Release Pharmaceutical Compositions, issued Sustained concentration of the composition in the Subject, Jan.5, 1982; and U.S. Pat. No. 4,309,406, “Sustained Release especially in and around the area of the bacterial infection or 40 Pharmaceutical Compositions, issued Jan. 5, 1982, which infection with a microorganism, and to result in the desired are incorporated herein in their entirety by reference. therapeutic response or protection. It is also within the skill of Examples of Solid carriers include starch, Sugar, bentonite, the art to start doses at levels lower than required to achieve silica, and other commonly used carriers. Further non-limit the desired therapeutic effect and to gradually increase the ing examples of carriers and diluents which can be used in the dosage until the desired effect is achieved. 45 formulations of the present invention include Saline, syrup, The amount of the pharmaceutical compositions to be dextrose, and water. administered to a Subject is dependent upon factors known to Practice of the present invention will employ, unless indi a persons of ordinary skill in the art such as bioactivity and cated otherwise, conventional techniques of cell biology, cell bioavailability of the antimicrobial agent (e.g., half-life in the culture, molecular biology, microbiology, recombinant DNA, body, stability, and metabolism of the engineered bacterioph 50 protein chemistry, and immunology, which are within the age); chemical properties of the antimicrobial agent (e.g., skill of the art. Such techniques are described in the literature. molecular weight, hydrophobicity, and solubility); route and See, for example, Molecular Cloning: A Laboratory Manual, scheduling of administration, and the like. It will also be 2nd edition. (Sambrook, Fritsch and Maniatis, eds.), Cold understood that the specific dose level of the composition Spring Harbor Laboratory Press, 1989; DNA Cloning, Vol comprising antimicrobial agents and engineered bacterioph 55 umes I and II (D. N. Glover, ed), 1985; Oligonucleotide ages as disclosed herein to be achieved for any particular Synthesis, (M. J. Gait, ed.), 1984; U.S. Pat. No. 4,683,195 Subject can depend on a variety of factors, including age, (Mullis et al.); Nucleic Acid Hybridization (B. D. Hames and gender, health, medical history, weight, combination with one S.J. Higgins, eds.), 1984; Transcription and Translation (B. or more other drugs, and severity of disease, and bacterial D. Hames and S. J. Higgins, eds.), 1984; Culture of Animal strain or microorganism the Subject is infected with, such as 60 Cells (R.I. Freshney, ed). Alan R. Liss, Inc., 1987: Immobi infection with multi-resistant bacterial strains. lized Cells and Enzymes, IRL Press, 1986: A Practical Guide The term “treatment', with respect to treatment of a bac to Molecular Cloning (B. Perbal), 1984; Methods in Enzy terial infection or bacterial colonization, interalia, preventing mology, Volumes 154 and 155 (Wu et al., eds), Academic the development of the disease, or altering the course of the Press, New York; Gene Transfer Vectors for Mammalian Cells disease (for example, but not limited to, slowing the progres 65 (J. H. Miller and M. P. Calos, eds.), 1987, Cold Spring Harbor sion of the disease), or reversing a symptom of the disease or Laboratory: Immunochemical Methods in Cell and Molecu reducing one or more symptoms and/or one or more bio lar Biology (Mayer and Walker, eds.), Academic Press, Lon US 9,056,899 B2 69 70 don, 1987; Handbook of Experiment Immunology, Volumes 19. The bacteriophage of any of paragraphs 14 to 15, wherein I-IV (D. M. Weir and C. C. Blackwell, eds.), 1986; Manipu the agent which increases the Susceptibility of a bacteria lating the Mouse Embryo, Cold Spring Harbor Laboratory cell to an antimicrobial agent is craA or variants or frag Press, 1986. ments thereof. In some embodiments of the present invention may be 5 20. The bacteriophage of any of paragraphs 14 to 15, wherein defined in any of the following numbered paragraphs: the agent which increases the Susceptibility of a bacteria 1. An engineered bacteriophage comprising a nucleic acid cell to an antimicrobial agent is craA or variants or frag operatively linked to a promoter, wherein the nucleic acid ments thereof. encodes at least one agent that inhibits an antibiotic resis 21. The bacteriophage of any of paragraphs 14 to 15, wherein tance gene and/or a cell Survival repair gene. 10 the agent which increases the Susceptibility of a bacteria 2. The bacteriophage of any of paragraph 1, wherein the cell to an antimicrobial agent modifies a pathway specifi antibiotic resistance gene is selected from the group com cally expressed in a bacterial cell. prising cat, VanA or mec) or variants thereof. 22. The bacteriophage of any of paragraphs 14 to 15 or 21, 3. The bacteriophage of any of paragraphs 1 or 2, wherein the 15 wherein modification is inhibition or activation of a path cell Survival gene is selected from the group comprising way specifically expressed in a bacterial cell. RecA, RecB. RecC, spot, RelA or variants thereof. 23. The bacteriophage of any of paragraphs 14 to 15, wherein 4. The bacteriophage of any of paragraphs 1 to 3, wherein the the agent which increases iron-Sulfur clusters in the bacte agent is selected from a group comprising, siRNA, anti rial cell. sense nucleic acid, asRNA, RNAi, miRNA and variants 24. The bacteriophage of any of paragraphs 14 to 15, wherein thereof. the agent which increases oxidative stress in a bacterial cell 5. The bacteriophage of any of paragraphs 1 to 4, wherein the or increases hydrozyl radicals in a bacterial cell. agent is an antisense RNA (asRNA). 25. The bacteriophage of any of paragraphs 14 to 24, wherein 6. The bacteriophage of any of paragraphs 1 to 5, wherein the the agent is not substantially toxic a bacterial cell in the bacteriophage comprises a nucleic acid encoding at least 25 absence of an antimicrobial agent. two agents that inhibit at least two different cell survival 26. The bacteriophage of any of paragraphs 14 to 25, wherein repair genes. the agent is not a chemotherapeutic agent or an protein 7. The bacteriophage of any of paragraphs 1 to 6, wherein the toxin. bacteriophage comprises a nucleic acid encoding at least 27. The bacteriophage of any of paragraphs 14 to 26, wherein two agents that inhibit at least two of RecA, RecB or RecC. 30 the bacteriophage comprises a nucleic acid encoding at 8. An engineered bacteriophage comprising a nucleic acid least two different proteins which increase the susceptibil ity of a bacteria cell to an antimicrobial agent. operatively linked to a promoter, wherein the nucleic acid 28. The bacteriophage of any of paragraphs 14 to 27, wherein encodes at least one repressor of a SOS response gene the proteins are csrA and omp or variants or fragments and/or bacterial defense gene. 35 thereof. 9. The bacteriophage of any of paragraphs 8, wherein the 29. The bacteriophage of any of paragraphs 1 to 28, wherein repressor of a SOS response gene is lexA. the bacteriophage is a lysogenic bacteriophage. 10. The bacteriophage of any of paragraphs 8 or 9, wherein 30. The bacteriophage of any of paragraphs 1 to 29, wherein the repressor of a defense gene is SoxR. the lysogenic bacteriophage is a M13 bacteriophage. 11. The bacteriophage of any of paragraphs 8 to 10, wherein 40 31. The bacteriophage of any of paragraphs 1 to 29, wherein the repressor is selected from the group consisting of the bacteriophage is a lytic bacteriophage. marr, arck, fur, crp, iccdA or variants or fragments thereof. 32. The bacteriophage of any of paragraphs 1 to 29, or 31 12. The bacteriophage any of paragraphs 8 to 11, wherein the wherein the lytic bacteriophage is a T7 bacteriophage. bacteriophage comprises a nucleic acid encoding at least 33. A method to inhibit or eliminate a bacterial infection two different repressors of at least one SOS response gene. 45 comprising administering to a Surface infected with bacte 13. The bacteriophage any of paragraphs 8 to 12, wherein the ria; (a) a bacteriophage comprising a nucleic acid opera bacteriophage comprises a nucleic acid encoding at least tively linked to a bacteriophage promoter, wherein the two different repressors of at least one bacterial defense nucleic acid encodes at least one agent that inhibits an gene. antibiotic resistance gene and/or a cell Survival repair gene, 14. An engineered bacteriophage comprising a nucleic acid 50 and (b) at least one antimicrobial agent. operatively linked to a promoter, wherein the nucleic acid 34. A method to inhibit or eliminate a bacterial infection encodes at least one agent which increases the Susceptibil comprising administering to a Surface infected with bacte ity of a bacteria cell to an antimicrobial agent. ria; (a) a bacteriophage comprising a nucleic acid opera 15. The bacteriophage of paragraph 14, wherein the agent tively linked to a bacteriophage promoter, wherein the 55 nucleic acid encodes at least one repressor of a SOS which increases the susceptibility of a bacteria cell to an response gene or a bacterial-defense gene, and (b) at least antimicrobial agent increases the efficacy of the antimicro one antimicrobial agent. bial effect of the antimicrobial agent by at least 10%. 35. A method to inhibit or eliminate a bacterial infection 16. The bacteriophage any of paragraphs 14 or 15, wherein comprising administering to a Surface infected with bacte the agent which increases the Susceptibility of a bacteria 60 ria; (a) a bacteriophage comprising nucleic acid opera cell to an antimicrobial agent increases the entry of an tively linked to a bacteriophage promoter, wherein the antimicrobial agent to a bacterial cell. nucleic acid a encodes at least one agent which increases 17. The bacteriophage of any of paragraphs 14 to 16, wherein the susceptibility of a bacteria cell to an antimicrobial the agent which increases the entry of an antimicrobial agent, and (b) at least one antimicrobial agent. agent to a bacterial cell is a porin. 65 36. The method of paragraph 33, wherein the bacteriophage is 18. The bacteriophage of any of paragraphs 14 to 17, wherein a bacteriophage according to any of paragraphs 1 to 7 or the porin is ompl or variants or fragments thereof. 29-32. US 9,056,899 B2 71 72 37. The method of paragraph34, wherein the bacteriophage is 57. A composition comprising a bacteriophage comprising a a bacteriophage according to any of paragraphs 8 to 13 or nucleic acid operatively linked to a promoter, wherein the 29-32. nucleic acid encodes at least one protein which increases 38. The method of paragraph 35, wherein the bacteriophage is the susceptibility of a bacteria cell to an antimicrobial a bacteriophage according to any of paragraphs 14 to 32. 5 agent and at least one antimicrobial agent. 39. The method of any of paragraphs 33 to 38, wherein the 58. The composition of any of paragraphs 55 to 57, wherein administration of the bacteriophage and the antimicrobial the antimicrobial agent is a quinolone antimicrobial agent, agent occurs simultaneously. oraminoglycoside antimicrobial agent or B-lactamantimi 40. The method of any of paragraphs 33 to 38, wherein the crobial agent. administration of the bacteriophage occurs prior to the 10 59. The composition of any of paragraphs 55 or 58, wherein administration of the antimicrobial agent. the bacteriophage is according to any paragraphs 1-7 or 41. The method of any of paragraphs 33 to 38, wherein the 29-32. administration of the antimicrobial agent occurs prior to 60. The composition of paragraphs 56 or 58, wherein the the administration of the bacteriophage. 15 bacteriophage is according to any paragraphs 8 to 13 or 42. The method of any of paragraphs of any of paragraphs 33 29-32. to 38, wherein the antimicrobial agent is a quinolone anti 61. The composition of paragraphs 57 or 58, wherein the microbial agent. bacteriophage is according to any paragraphs 14 to 32. 43. The method of paragraph 33 to 42, wherein the antimi 62. A kit comprising a bacteriophage comprising the nucleic crobial agent is selected from a group consisting of acid operatively linked to a promoter, wherein the nucleic ciproflaxacin, levofloxacin, and ofloxacin, gatifloxacin, acid encodes at least one agent that inhibits an antibiotic norfloxacin, lomefloxacin, trovafloxacin, moxifloxacin, resistance gene and/or a cell Survival repair gene. sparfloxacin, gemifloxacin, paZufloxacin or variants or 63. A kit comprising a bacteriophage comprising the nucleic analogues thereof. acid operatively linked to a promoter, wherein the nucleic 44. The method of any of paragraphs 33 to 38, wherein the 25 acid encodes at least one repressor of a SOS response oran antimicrobial agent is ofloxacin or variants or analogues antimicrobial defense gene. thereof. 64. A kit comprising a bacteriophage comprising the nucleic 45. The method of any of paragraphs 33 to 38, wherein the acid operatively linked to a promoter, wherein the nucleic antimicrobial agent is an aminoglycoside antimicrobial acid encodes at least one protein which increases the Sus agent. 30 ceptibility of a bacteria cell to an antimicrobial agent and at 46. The method of paragraph 45, wherein the antimicrobial least one antimicrobial agent. agent is selected from a group consisting of amikacin, 65. The use of a bacteriophage according to any of paragraphs gentamycin, tobramycin, netromycin, Streptomycin, kana 1 to 23 in combination with an antimicrobial agent to mycin, paromomycin, neomycin or variants or analogues reduce the number of bacteria as compared to use of the thereof. 35 antimicrobial agent alone. 47. The method of any of paragraphs 33 to 38, wherein the 66. The use of any of the paragraphs 62-65, wherein the antimicrobial agent is gentamicin or variants or analogues bacteria is in a biofilm. thereof. 67. A combination of at least two bacteriophages of any of 48. The method of any of paragraphs 33 to 38, wherein the paragraphs 1 to 23 with at least one antimicrobial agent. antimicrobial agent is an B-lactam antibiotic antimicrobial 40 68. The combination of paragraph 67, wherein the antimicro agent. bial agent is a quinolone antimicrobial agent. 49. The method of any of paragraphs 33 to 38, wherein the 69. The combination of paragraph 67, wherein the antimicro antimicrobial agent is selected from a group consisting of bial agent is selected from a group consisting of ciproflaxa penicillin, amplicillin, penicillin derivatives, cephalospor cin, levofloxacin, and ofloxacin, gatifloxacin, norfloxacin, ins, monobactams, carbapenems, B-lactamase inhibitors or 45 lomefloxacin, trovafloxacin, moxifloxacin, sparfloxacin, variants or analogues thereof. gemifloxacin, paZufloxacin or variants or analogues 50. The method of any of paragraphs 33 to 38, wherein the thereof. antimicrobial agent is amplicillin or variants or analogues 70. The combination of paragraph 67, wherein the antimicro thereof. bial agent is ofloxacin or variants or analogues thereof. 51. The method of any of paragraphs 33 to 38, wherein the 50 71. The combination of paragraph 67, wherein the antimicro bacteria is present in a Subject. bial agent is an aminoglycoside antimicrobial agent. 52. The method of any of paragraphs 33 to 51, wherein the 72. The combination of paragraph 67, wherein the antimicro Subject is a mammal. bial agent is selected from a group consisting of amikacin, 53. The method of any of paragraph 33 to 52, wherein the gentamycin, tobramycin, netromycin, streptomycin, kana mammal is a human. 55 mycin, paromomycin, neomycin or variants or analogues 54. The method of any of paragraphs 33 to 53, wherein the thereof. bacteria is in a biofilm. 73. The combination of paragraph 67, wherein the antimicro 55. A composition comprising a bacteriophage comprising a bial agent is gentamicin or variants or analogues thereof. nucleic acid operatively linked to a promoter, wherein the 74. The combination of paragraph 67, wherein the antimicro nucleic acid encodes at least one agent that inhibits an 60 bial agent is an B-lactam antibiotic antimicrobial agent. antibiotic resistance gene and/or a cell Survival repair gene 75. The combination of paragraph 67, wherein the antimicro and at least one antimicrobial agent. bial agent is selected from a group consisting of penicillin, 56. A composition comprising a bacteriophage comprising a ampicillin, penicillin derivatives, cephalosporins, mono nucleic acid operatively linked to a promoter, wherein the bactams, carbapenems, B-lactamase inhibitors or variants nucleic acid encodes at least one repressor of a SOS 65 or analogues thereof. response gene or a antimicrobial defense gene and at least 76. The combination of paragraph 67, wherein the antimicro one antimicrobial agent. bial agent is amplicillin or variants or analogues thereof. US 9,056,899 B2 73 74 77. The combination of paragraph 67, wherein the composi was isolated using the QIAPREP SPIN Miniprep kit tion comprises a combination of any of the antimicrobial (QIAGEN, Valencia, Calif.). All other chemicals and materi agents according to paragraphs 68-76. als were purchased from Fisher Scientific, Inc. (Hampton, 78. Use of a bacteriophage of any of claims 1 to 32 with at N.H.). least one antimicrobial agent. Engineering M13mp 18 bacteriophage to target genetic 79. Use of a combination of at least two of any the bacterioph networks. To construct engineered phage, leXA3, SOXR, cSrA, ages of claims 1 to 32 with at least one antimicrobial agent. and omple genes were first placed under the control of the 80. The use of a bacteriophage of claim 78 or 79 or any to PtetO promoter in the p7E11G vector''. Using PCR with claims 1 to 32 to inhibit or eliminate a bacterial infection. primers 5'ttatcaggtaccatgAAAGCGTTAACGGCC 3' (SEQ 81. The use of a bacteriophage of claim 78 or 79, wherein the 10 bacteria is present in a Subject. ID NO: 18) and 5'atacataagctt TTACAGCCA GTCGCCG 3' 82. The use of a bacteriophage of claim 81, wherein the (SEQ ID NO: 19), lexA3 was cloned between the KpnI and Subject is a mammal. HindIII sites of pZE11G to form pZE11-leXA3. Since soxR 83. The use of a bacteriophage of claim 82, wherein the has an internal KpnI site, the inventors built a synthetic RBS mammal is a human. 15 by sequential PCR using 5' agaggagaaa gg.tacc atg 84. The use of a bacteriophage of claim 78 or 79, wherein the GAAAAGAAATTACCCCG 3' (SEQ ID NO: 20) and 5' bacteria is in a biofilm. atacataagctt TTAGTTTTGTTCATCTTCCAG 3' (SEQ ID 85. Use of a composition of any of claims 55 to 57 to inhibit NO: 21) followed by 5' agtaga gaattic attaaagaggagaaaggtacc or eliminate a bacterial infection. atg3' (SEQID NO: 22) and 5' atacataagctt TTAGTTTTGT 86. The use of the composition of claim 85, wherein the TCATCTTCCAG3' (SEQID NO. 23). The resulting EcoRI bacteria is present in a Subject. RBS-soxR-HindIII DNA was ligated to an XhoI-P, tetO 87. The use of the composition of claim 86, wherein the EcoRI fragment excised from pZE11G and the entire DNA Subject is a mammal. fragment was ligated into pZE11G between XhoI and HindIII 88. The use of the composition of claim 87, wherein the to form p7E11-soxR''. Primers for csrA for cloning into mammal is a human. 25 pZE11G in between KpnI and HindIII to form pZE 11-csrA 89. The use of the composition of claim 85, wherein the were 5' agaggagaaaggtacc atgCTGATTC TGACTCGT 3' bacteria is in a biofilm. (SEQ ID NO: 24) and 5' atacat aagctt TTAGTA ACTG The following Examples are provided to illustrate the GACTG CTGG 3' (SEQ ID NO: 25); and for omple to form present invention, and should not be construed as limiting pZE11-ompF, 5' agaggagaaa gg.tacc atgATGAAG C thereof. 30 GCAATATTCT 3' (SEQ ID NO: 26) and 5' atacat aagctt TTAGAACTG GTAAACGATACC3' (SEQID NO: 27). To EXAMPLES express csrA and omple simultaneously under the control of PtetO, we PCR amplified RBS-ompF DNA from p7E11 The examples presented herein relate to the methods and ompF using 5'ccagtcaagctt attaaagaggagaaaggtacc 3' (SEQ compositions comprising inhibitor-engineered bacterioph 35 ID NO: 28) and 5' atacat GGATCC TTAGAACTG ages, repressor-engineered bacteriophages or Susceptibility GTAAACGATA CC 3' (SEQ ID NO: 29) and cloned the agent engineered bacteriophages and antimicrobial agents. product in between HindIII and BamHI in pZE1 1-csrA to Throughout this application, various publications are refer form p7E1 1-csrA-ompF. The resulting plasmids were trans enced. The disclosures of all of the publications and those formed into E. coli XL-10 cells. references cited within those publications in their entireties 40 All P, tetO-gene constructs followed by terminator T1 of are hereby incorporated by reference into this application in the rrnB operon and preceded by a stop codon were PCR order to more fully describe the state of the art to which this amplified from the respective pZE 11 plasmids with primers 5 invention pertains. The following examples are not intended aataca GAGCTC cTAA tecctatcagtgatagagattg 3' (SEQ ID to limit the scope of the claims to the invention, but are rather NO:30) and 5' taatct CGATCG totaggg.cgg.cggat 3' (SEQID intended to be exemplary of certain embodiments. Any varia 45 NO:31) and cloned into the Sad and Pvul sites of M13mp 18 tions in the exemplified methods which occur to the skilled (FIG. 5)'''. Resulting phage genomes were transformed artisan are intended to fall within the scope of the present into XL-10 cells, mixed with 200 uL overnight XL-10 cells in invention. 3 mL top agar, 1 mM IPTG, and 40 uL of 20 mg/mL X-gal, Methods and poured onto LBagar-chloramphenicol (30 ug/mL) plates Bacterial strains, bacteriophage, and chemicals. E. coli 50 for plaque formation and blue-white screening. After over K-12 EMG2 cells, which lack 0 antigens, were obtained from night incubation of plates at 37° C., white plaques were the Yale Coli Genetic Stock Center (CGSC #4401). E. coli scraped and placed into 1:10 dilutions of overnight XL-10 RFS289 cells, which contain a gyra 111 mutation rendering cells and grown for 5 hours. Replicative form (RF) M13mp 18 them resistant to quinolones, were obtained from the Yale DNA was collected by DNA minipreps of the bacterial cul Coli Genetic Stock Center (CGSC #5742). M13mp 18 bacte 55 tures. All insertions into M13mp 18 were verified by PCR and riophage was purchased from New England Biolabs, Inc. restriction digests of RF DNA. Infective bacteriophage solu (Ipswich, Mass.). E. coli XL-10 cells used for cloning, ampli tions were obtained by centrifuging infected cultures for 5 fying phage, and plating phage were obtained from Strat minutes at 16,100xg and collecting Supernatants followed by agene (La Jolla, Calif.). filtration through Nalgene #190-25200.2 um filters (Nalge T4DNA ligase and all restriction enzymes were purchased 60 Nunc International, Rochester, N.Y.). from New England Biolabs, Inc. (Ipswich, Mass.). PCR reac Determination of plaque forming units. To obtain plaque tions were carried out using PCR SUPERMIXHIGH FIDEL forming units, we added serial dilutions of bacteriophage ITY from INVITROGEN (Carlsbad, Calif.) or PHUSION performed in 1xRBS to 200 uL of overnight XL-10 cells in 3 HIGH FIDELITY from New England Biolabs, Inc. (Ipswich, mL top agar, 1 mMIPTG, and 40 u, of 20 mg/mL X-gal, and Mass.). Purification of PCR reactions and restriction digests 65 poured the mixture onto LB agar-chloramphenicol (30 was carried out with the QIAQUICKGEL Extraction or PCR ug/mL) plates. After overnight incubation at 37°C., plaques Purification kits (QIAGEN, Valencia, Calif.). Plasmid DNA were counted. US 9,056,899 B2 75 76 Determination of colony forming units. To obtain CFU and Collins, 2007). Briefly, lids containing plastic pegs counts, 150 uL of relevant cultures were collected, washed (MBEC Physiology and Genetics Assay, Edmonton, Calif.) with 1x phosphate-buffered saline (PBS), recollected, and were placed in 96-well plates containing overnight cells that resuspended in 150 uL of 1xPBS. Serial dilutions were per were diluted 1:200 in 150 uL LB. Plates were then inserted formed with 1xPBS and sampled on LB agar plates. LBagar into plastic bags to minimize evaporation and inserted in a plates were incubated at 37° C. overnight before counting. Minitron shaker (Infors HT, Bottmingen, Switzerland). After Flow cytometer assay of SOS induction. To monitor 24 hours of growth at 35° C. and 150 rpm, lids were moved M13mp 18-lex.A3s (cp) suppression of the SOS response into new 96-well plates with 200 uLLB with or without 10 (FIG. 10), the inventors used a plasmid containing an SOS PFU/mL of bacteriophage. After 12 hours of treatment at 35° response promoter driving gfp expression in EMG2 cells 10 (PlexO-gfp). After growing 1:500 dilutions of the over C. and 150 rpm, lids were removed, washed three times in 200 night cells for 2 hours and 15 minutes at 37°C. and 300 rpm uL of 1xPBS, inserted into Nunc i262162 microtiter plates (model G25 incubator shaker, New Brunswick Scientific), the with 150 uL 1xPBS, and sonicated in an Ultrasonics 5510 inventors applied ofloxacin and bacteriophage and treated for sonic water bath (Branson, Danbury, Conn.) at 40 kHz for 30 6 hours at 37° C. and 300 rpm. Cells were then analyzed for 15 minutes. Serial dilutions, using the resulting 150 uL 1xPBS, GFP fluorescence using a Becton Dickinson (Franklin Lakes, were performed on LB plates and viable cell counts were N.J.) FACS caliber flow cytometer with a 488-nm argon laser determined. Mean killing (A logo (CFU/mL)) was calculated and a 515-545 nm emission filter (FL1) at low flow rate. The by subtracting mean logo (CFU/mL) after 24 hours of growth following photo-multiplier tube (PMT) settings were used for from mean logo (CFU/mL) after 12 hours of treatment (FIG. analysis: E00 (FSC), 275 (SSC), and 700 (FL1). Becton Dick 17 and FIG. 18). inson CALIBRITE Beads were used for instrument calibra Antibiotic resistance assay. To analyze the effect of subin tion. 200,000 cells were collected for each sample and pro hibitory concentrations of ofloxacin on the development of cessed with MATLAB (Mathworks, Natick, Mass.). antibiotic-resistant mutants, the inventors grew 1:10 dilu Ofloxacinkilling assay. To determine the adjuvant effect of tions of overnight EMG2 in LB media containing either no engineered phage (FIG. 1B, FIG.3A and FIG. 3D), the inven 25 ofloxacin (FIG. 4) or 30 ng/mL ofloxacin (FIG. 7). After 12 tors grew 1:500 dilutions of overnight EMG2 cells for 3 hours hours of growth at 37° C. and 300 rpm, the inventors split the and 30 minutes at 37° C. and 300 rpm to late-exponential cells grown in no ofloxacin into 100 uL aliquots with no phase and determined initial CFUs. Then, the inventors added ofloxacin in 60 wells in 96-well plate format (Costar 3370; 60 ng/mL ofloxacin by itself or in combination with 10 Fisher Scientific, Pittsburgh, Pa.). The inventors also split the PFU/mL bacteriophage (unmodified p, or engineered 30 cells grown in 30 ng/mL ofloxacin into 100 uL aliquots in 60 (PLevas (Pso R: (Pas- (Pomp F: O (Pcs-ompF phage) and treated at wells with either no phage and 30 ng/mL ofloxacin (FIG.7B). 37° C. and 300 rpm. At indicated time points, the inventors (p, phage and 30 ng/mL ofloxacin (FIG. 7C), and (plus determined CFUs as described above. Mean killing (Alogo and 30 ng/mL ofloxacin (FIG. 7D) in 96-well plate format. (CFU/mL)) was determined by Subtracting mean initial logo The inventors placed the 96-well plates in 37° C. and 300 rpm (CFU/mL) from mean logo (CFU/mL) after treatment in 35 with plastic bags to minimize evaporation. After 12 hours of order to compare data from different experiments. This pro treatment, the inventors plated cultures from each well on LB tocol was replicated with E. coli RFS289 to determine the agar-i-100 ng/mL ofloxacinto select for mutants that devel ofloxacin-enhancing effect of engineered (plus phage against oped resistance against ofloxacin. To compare results, the antibiotic-resistant bacteria (FIG. 2). In addition, viable cell inventors plotted histograms of the number of resistant bac counts were obtained for ofloxacin-free EMG2 cultures, 40 teria found in each well in FIGS. 4 and 8. ofloxacin-free EMG2 cultures with (p, phage, and ofloxa Gentamicin and amplicillin killing assays. To determine the cin-free EMG2 cultures with engineered (p phage. antibiotic enhancing or adjuvant effect of engineered bacte Dose response assays. The initial phage inoculation dose riophage forgentamicinandampicillin, the inventors used the response experiments (FIG. 1c and FIG. 15) were handled same protocol as the ofloxacin killing assay except that the using the same protocol as the ofloxacin killing assay except 45 inventors used 10 PFU/mL initial phage inoculations. 5 that 60 ng/mL ofloxacin was added with varying concentra ug/mL gentamicin and 5 lug/mL amplicillin were used in tions of phage. Cultures were treated for 6 hours before FIGS. 1D, 1E, 8A and 8B. obtaining viable cell counts. The ofloxacin dose response Statistical analysis. All CFU data were logo-transformed experiments (FIG. 1C) were also obtained using the same prior to analysis. For all data points in all experiments, in 3 protocol as the ofloxacin killing assay except that 10 PFU/ 50 samples were collected except where noted. Error bars in mL phage were added with varying concentrations of ofloxa figures indicate standard error of the mean. cin and viable cell counts were obtained after 6 hours of treatment. Example 1 Persister killing assay. The inventors performed a persister killing assay to determine whether engineered phage could 55 The inventors have engineered synthetic bacteriophage to help to kill persister cells in a population which survived target genetic networks in order to potentiate bacterial killing initial drug treatment without bacteriophage (FIGS. 11 and in combination therapy with antibiotics. The inventors spe 16). The inventors first grew 1:500 dilutions of overnight cifically targeted genetic networks in E. coli which are not EMG2 for 3 hours and 30 minutes at 37° C. and 300 rpm directly attacked by antibiotics to avoid imposing additional followed by treatment with 200 ng/mL ofloxacin for 3 hours 60 evolutionary pressures for antibiotic resistance. Instead, the to create a population of Surviving bacteria. Then, the inven inventors chose proteins that are responsible for repairing tors added either no phage, 10 PFU/mL control {p, or cellular damage caused by antibiotics, those that control regu 10 PFU/mL engineered p, phage. After 3 hours of addi latory networks, or those that modulate sensitivity to antibi tional treatment, the inventors collected the samples and otics Unlike conventional antibiotics that act by disrupting assayed for viable cell counts as described above. 65 protein activity, the inventors designed an engineered phage Biofilm killing assay. Biofilms were grown using E. coli to overexpress target genes, such as repressors and act as EMG2 cells according to a previously-reported protocol (Lu effective antibiotic adjuvants. US 9,056,899 B2 77 78 Bactericidal antibiotics cause hydroxyl radical formation nation phage and antibiotic treatment up to 12 hours (FIG. which leads to DNA, protein, and lipid damage and ulti 1B) (Hagens et al., (2003) Lett. Appl. Microbiol. 37,318-23: mately, cell death". DNA damage induces the SOS response Hagens et al., (2004) Antimicrob. Agents Chemother. 48, (Miller et al., (2004) Science 305, 1629-1631; Lewin et al., 3817-22: Summers W C (2001) Annu. Rev. Microbiol. 55, (1989) J. Med. Microbiol. 29, 139-144.), which results in 437-451). The inventors confirmed that both p, and DNA repair (FIG.1A). It has been shown that bacterial killing (preplicated significantly during treatment (data not by bactericidal antibiotics can be enhanced by knocking out shown). recA and disabling the SOS response (Kohanski et al., (2007) Cell 130). Here, the inventors used an alternative approach Example 2 and engineered M13mp8 phage to overexpress leXA3, a 10 repressor of the SOS response (Little et al., (1979) Proc Natl To test whether (ps can act as an antibiotic adjuvant in AcadSci USA 76, 6147-51). Overexpression of lexA to sup different situations, the inventors assayed for bacterial killing press the SOS system has been demonstrated to inhibit the with varying initial phage inoculation doses (FIG. 15) and emergence of antibiotic resistance (Cirz et al., (2005) in PLOS varying doses of ofloxacin (FIG. 1C) after 6 hours of treat Biol, p. e17624). The inventors used M13mp 18, a modified 15 ment, respectively. (ps enhanced ofloxacin’s bactericidal version of M13 phage, as the Substrate since it is a non-lytic activity over a wide range of multiplicity-of infections filamentous phage and can accommodate DNA insertions (MOIs), from 1:1000 to 1:1 (FIG. 15). (ps ability to into its genome (Figure S1) (Yanisch-Perron et al., (1985) increase killing by ofloxacin at a low MOI reflects rapid Gene 33, 103-119). replication and infection by M13 phage. For ofloxacin con To repress the SOS response, the inventors placed the centrations of 30 ng/mL and higher, p resulted in much lexA3 gene under the control of the synthetic PLtetO pro greater killing compared with no phage or unmodified phage moter followed by a synthetic ribosome-binding sequence (p (FIG.1C). Thus, the inventors have demonstrated that (RBS) (Kohanski et al., (2007) Cell 130,797-810; Little et al., (p. is a strong adjuvant for ofloxacin at doses below and (1979) Proc Natl AcadSci USA 76, 6147-51; Walker GC above the minimum inhibitory concentration (60 ng/mL, data (1984) Microbiol. Rev. 48, 60-93; Lutz et al., (1997) Nucleic 25 not shown). Acids Res 25, 1203-1210.); The inventors named this phage The inventors next determined whether the engineered “p' (FIG. 1A and Figure S1B) and the unmodified phage could increase killing by classes of antibiotics other M13mp 18 phage (p. PLtetO, which is an inducible pro than quinolones. The inventors tested (ps antibiotic-en moter in the presence of the TetR repressor, is constitutively hancing effect for gentamicin, an aminoglycoside, and ampi on in EMG2 cells, which lack TetR. PLtetO was used for 30 cillin, a f-lactam antibiotic. As demonstrated herein, p. convenience in proof-of-concept experiments as described increased gentamicin's bactericidal action by over 2.5 and 3 herein and would not necessarily be the promoter of choice in orders of magnitude compared with (p, and no phage, real-world situations. Accordingly, one of ordinary skill in the respectively (FIG.1D). (p also improved amplicillin's bac art can readily substitute the PLtetOpromoter with a different tericidal effect by over 2 and 5.5 orders of magnitude com inducible or constitutively active or tissue specific promoter 35 pared with (p and no phage, respectively (FIG. 1E). For of their choice. The inventors confirmed that (ps Sup both gentamicin and ampicillin, (p's strong antibiotic pressed the SOS response induced by ofloxacintreatment by enhancing effect was noticeable after 1 hour of treatment monitoring GFP fluorescence in E. coli K-12 EMG2 cells (FIGS. 1D and 1E). These results are consistent with previous carrying a plasmid with an SOS-responsive promoter driving observations that Areca mutants exhibit increased suscepti gfp expression (Figure S2) (Kohanski et al., (2007) Cell 130, 40 bility to quinolones, aminoglycosides, and B-lactams (Ko 797-810). hanski et al., (2007) Cell 130,797-810), and demonstrate that To test (ps antibiotic-enhancing effect, the inventors engineered phages, such as (ps, can act as general adju obtained time courses for killing of E. coli EMG2 bacteria vants for the three major classes of bactericidal drugs. The with phage and/or ofloxacin treatment. The inventors calcu inventors also found that engineered phage (ps is capable lated viable cell counts by counting colony-forming units 45 of reducing the number of persister cells in populations (CFUs) during treatment with no phage or 10 plaque-form already exposed to antibiotics as well as enhancing antibiotic ing units/mL (PFU/mL) of phage and with no ofloxacin or 60 efficacy against bacteria living in biofilms. For example, ng/mL ofloxacin (FIG. 1B). Bacteria exposed only to ofloxa (ps added to a population previously treated only with cin were reduced by about 1.7 logo (CFU/mL) after 6 hours ofloxacin increased the killing of bacteria that survived the of treatment, reflecting the presence of persisters not killed by 50 initial treatment by approximately 1 and 1.5 orders of mag the drug (FIG. 1B). By 6 hours, (p. improved the bacteri nitude compared with (p, and no phage, respectively cidal effect of ofloxacin by 2.7 orders of magnitude compared (FIG.16). In addition, simultaneous application of p and to unmodified phage (p (~0.99.8% additional killing)and ofloxacin improved killing of biofilm cells by about 1.5 and 2 by over 4.5 orders of magnitude compared to no phage orders of magnitude compared with (p, plus ofloxacin and (~99.998% additional killing) (FIG. 1B). Unmodified phage 55 no phage plus ofloxacin, respectively (FIG. 17). enhanced ofloxacin's bactericidal effect, which is consistent Since the inventors previous experiments all involved with previous observations that unmodified filamentous simultaneous application of bacteriophage and drug, the phage augment antibiotic efficacy against Pseudomonas inventors tested whether later addition of engineered (ps to aeruginosa (Hagens et al., (2006) Microb Drug Resist 12, a previously drug-treated population would also enhance kill 164-168). Other researchers have noted that M13-infected E. 60 ing Late exponential-phase cells were first exposed to 3 hours coli exhibited impaired host stress responses to conditions of treatment by ofloxacinto generate a population of Surviv such as acid stress (Karlsson et al., (2005) Can J Microbiol ing cells and followed by either no phage, 10 PFU/mL (p. 51, 29-35). While wishing not to be bound by theory, the mod, or 10 PFU/mL engineered (ps phage. After 3 hours of mechanism by which unmodified filamentous phage can aug additional treatment, (ps increased killing by 0.94 logo ment antibiotic efficacy is not well characterized but can 65 (CFU/mL) compared with (pandby over 1.3 logo (CFU/ involve membrane disruption or impaired stress responses. mL) compared with no phage (FIG. 11). These results indi No significant bacterial regrowth was apparent with combi cate that engineered (ps bacteriophage increases the killing US 9,056,899 B2 79 80 of bacteria which survive initial antibiotic treatment and sistant cells (median 2.5), resulting in a majority of samples reduce the number of persister cells in a given population. with either no resistant CFUs or <20 resistant CFUs (FIG. 4). Example 3 Example 6 Enhancing Killing of Antibiotic-Resistant Bacteria. In Flexible Targeting of Other Gene Networks. The inventors addition to killing wild-type bacteria with increased efficacy, next demonstrated that the phage platform can be used to the inventors also demonstrate that the engineered phage can target many different gene networks to produce effective enhance killing of bacteria that have already acquired antibi antibiotic adjuvants. To demonstrate this, the inventors engi otic resistance. The inventors applied (p with ofloxacin 10 neered phage to express proteins that regulate non-SOS gene against E. coli RFS289, which carries a mutation (gyra 111) networks (e.g., SOXR and CSrA) or modulate sensitivity to that renders it resistant to quinolone antibiotics (Dwyer et al., antibiotics (e.g., Ompl) (FIG. 5 and FIG.9F) (Lutz et al., (2007) Mol Syst Biol 3,917; Schleif R (1972) Proc Natl Acad (1997) Nucleic Acids Res 25, 1203-10). For example, the Sci USA 69,3479-84). (p. increased the bactericidal action SOXR-SOXS regulon controls a coordinated cellular response of ofloxacin by over 2 and 3.5 orders of magnitude compared 15 to superoxide (Hidalgo et al., (1997) Cell 88, 121-129). SoxR with (p, and no phage, respectively (FIG.2). These results contains a 12Fe-251 cluster that must be oxidized for it to demonstrate that antibiotic-enhancing phage. Such as (p. stimulate SoxS production, which then controls the transcrip can be used to combat antibiotic-resistant bacteria and there tion of downstream genes that respond to oxidative stress fore can have the potential to bring defunct antibiotics back (Hidalgo et al., (1997) Cell 88, 121-129). As quinolones into clinical use. generate Superoxide-based oxidative attack (Dwyer et al., (2007) Mol Syst Biol 3,91: Kohanski et al., (2007) Cell 130, Example 4 797-810), the inventors engineered phage to overexpress wild-type SoxR (cp) to affect this response and improve Increasing Survival of Mice Infected with Bacteria. To ofloxacins bactericidal activity (FIG. 5A). As shown in FIG. determine the clinical relevance of antibiotic-enhancing 25 5B, p enhanced killing by ofloxacin compared with phage in vivo, the inventors applied the engineered phage unmodified phage (p, and no phage (FIG. 5B). The inven (ps with ofloxacinto prevent death in mice infected with tors discovered that the overexpression of SoxR may provide bacteria. Mice were injected with E. coli EMG2 intraperito additional iron-sulfur clusters that could be destabilized to neally 1 hour prior to receiving different intravenous treat increase sensitivity to bactericidal antibiotics (Dwyer et al., ments (FIG. 3A). Eighty percent of mice that received ps 30 (2007) Mol Syst Biol 3,91: Kohanski et al., (2007) Cell 130, with ofloxacin survived, compared with 50% and 20% for 797-810). Alternatively, since SoxR is usually kept at rela mice that received p, plus ofloxacin or ofloxacin alone, tively levels in vivo which are unchanged by oxidative stress respectively (FIG.3B). The inventors have demonstrated that (Hidalgo et al., (1998) EMBO.J. 17, 2629-2636), and the the engineered phage (ps with ofloxacin prevents death in overexpression of large amounts of SoxR may interfere with vivo of mice with a severe bacterial infection, thus demon 35 signal transduction in response to oxidative stress by titrating strating that the in Vivo efficacy of the antibiotic enhancing intracellular iron or oxidizing species or by competing with phages are effective at rescuing infected mice from death, and oxidized SoxR for binding to the soxS promoter (Hidalgo et demonstrates the feasibility of various embodiments of the al., (1998) EMBO.J. 17, 2629-36: Meng Met al., (1999).J invention for clinical use. Bacteriol 181, 4639-4643; Gaudu et al., (1996) Proc Natl 40 AcadSci USA 93, 10094-98). Example 5 CSrA is a global regulator of glycogen synthesis and catabolism, gluconeogenesis, and glycolysis, and has been Reducing the Development of Antibiotic Resistance. shown to represses biofilm formation (Jackson D Wet al., Exposure to subinhibitory concentrations of antibiotics can (2002).J. Bacteriol. 184,290-301). As biofilm formation has lead to initial mutations which confer low-level antibiotic 45 been linked to antibiotic resistance, the inventors assessed if resistance and eventually more mutations that yield high cSrA-expressing phage (cps) would increase Susceptibility level resistance (Martinez et al., (2000) Antimicrob. Agents to antibiotic treatment (Stewart et al., (2001) Lancet 358, Chemother: 44, 1771-77). The inventors assessed if the engi 135-138). In addition, since Ompl is a porin used by quino neered phage, as antibiotic adjuvants, could reduce the num lones to enterbacteria (Hirai et al., (1986) Antimicrob. Agents ber of antibiotic-resistant mutants that result from a bacterial 50 Chemother: 29, 535-538), the inventors also assessed if population exposed to antimicrobial drugs. To test this, the ompF-expressing phage (pr.) would increase killing by inventors grew E. coli EMG2 in media with either no ofloxa ofloxacin (FIG. 5C). After 6 hours, both p and pe. cin for 24 hours, 30 ng/mL ofloxacin for 24 hours, 30 ng/mL increased ofloxacin’s bactericidal effect by approximately 1 ofloxacin for 12 hours followed by p, plus ofloxacin and 3 orders of magnitude compared with p,iific and no treatment for 12 hours, or 30 ng/mL ofloxacin for 12 hours 55 phage, respectively (FIG. 5D). followed by p, plus ofloxacintreatment for 12 hours (FIG. 4). Then, the inventors counted the number of mutants resis Example 7 tant to 100 ng/mL ofloxacin for each of the 60 samples under each growth condition. Growth in the absence of ofloxacin Systems biology analysis often results in the identification yielded very few resistant cells (median=1) (FIG. 4). How 60 of multiple antibacterial targets which are not easily ever, growth with subinhibitory levels of ofloxacin produced addressed by traditional drug compounds. In contrast, engi a high number of antibiotic-resistant bacteria (median=1592) neered phage are well-suited for incorporating multiple tar (FIG. 4). Treatment with unmodified phage (p, decreased gets into a single antibiotic adjuvant. To demonstrate this the number of resistant cells (median=43.5); however, all capability, the inventors designed an M13mp 18 phage to samples contained >1 resistant CFU and over half of the 65 express cSrA and ompF simultaneously (per) to target samples had D20 resistant CFUs (FIG. 4). In contrast, ps cSrA-controlled gene networks and increase drug penetration treatment dramatically suppressed the level of antibiotic-re (FIG.5C). The multi-target phage was constructed by placing US 9,056,899 B2 81 82 RBS and ompl immediately downstream of cSrA in ps resistance and eventually more mutations that yield high (FIG.9F) (Lutz et al., (1997) Nucleic Acids Res 25, 1203 level antibiotic resistance'7. By enhancing ofloxacin's bacte 1210). The inventors demonstrated that per was more ricidal effect, engineered bacteriophage can reduce the num effective at enhancing ofloxacins bactericidal effect com ber of antibiotic-resistant mutants that survive in a bacterial pared with its single-target relatives, p, and ple, in population exposed to antimicrobial drugs. To demonstrate planktonic (FIG. 5D) and biofilm settings (FIG. 18). this effect, the inventors grew E. coli in media with no ofloxa Together, these results demonstrate that engineering phage to cin (FIG. 7A) or 30 ng/mL ofloxacin for 12 hours (FIG. 7B, target non-SOS genetic networks such as networks which FIG. 7C, and FIG. 7D) to produce antibiotic-resistant increase a bacterial cells Susceptibility to an antimicrobial mutants. Then, the inventors divided the cells which grew agent and/or overexpress multiple factors can produce effec 10 under no ofloxacin into 60 individual wells with no ofloxacin tive antibiotic adjuvants. (FIG. 7A). The inventors also divided the cells which grew Example 8 under 30 ng/mL ofloxacin into 60 individual wells for each of the following treatments: no phage and 30 ng/mL ofloxacin To show that other targets can be found to enhance the 15 (FIG.7B), 10 PFU/mL (p, and 30 ng/mL ofloxacin (FIG. efficacy of combination therapy with bacteriophage and anti 7C), and 10 PFU/mL (ps with 30 ng/mL ofloxacin (FIG. biotic, the inventors screened M13mp 18 bacteriophage 7D). After 12 hours of additional growth, the inventors deter which expressed proteins that could modulate sensitivity to mined the number of antibiotic-resistant mutants by plating antibiotics or that control regulatory networks, such as SOXR, and counting the number of cells that grew on LB agar con fur, crp, marr, iccdA, cSrA, and ompl. The inventors did this taining 100 ng/mL ofloxacin. FIG. 7A shows that growth in by obtaining viable cell counts after 6 hours of treatment with the absence of ofloxacin yielded very few resistant cells. ofloxacin. Phage expressing SOXR, cSrA, or omp yielded the However, growth in the presence of a subinhibitory level of greatest improvements in killing by ofloxacin (See FIG. 1). ofloxacin resulted in a very high number of antibiotic-resis Like (ps, these phage expressed their respective proteins tant bacteria (FIG. 7B). Although treatment with (p. under the control of P, tetO and a synthetic RBS (FIGS. 9C, 25 reduced the number of resistant cells, all of the 60 individual 9D, and 9E). Since SoxR regulates a cellular response to wells tested contained at least one resistant CFU and over half Superoxide stress and quinolones stimulate Superoxide-based of the wells had more than 20 resistant CFUs (FIG. 7C). In oxidative attack, the inventors Surmised that overproducing contrast to treatment with no phage or unmodified (p. SoxR could affect this response and improve ofloxacins bac (ps treatment Suppressed the level of resistant cells dra tericidal activity''. As shown in FIG. 6A, soxR-expressing 30 matically, resulting in a majority of wells with either no M13mp 18 (cps) enhanced killing by ofloxacinby about 3.8 observable resistant CFUs or less than 20 CFUs (FIG. 3d). logo (CFU/mL) compared with no phage and by about 1.9 These results demonstrate that engineered (p is effica logo (CFU/mL) compared with unmodified (p, after 6 cious at reducing the number of antibiotic-resistant cells hours of treatment. which can develop in the presence of subinhibitory drug CsrA is a global regulator of glycogen synthesis and 35 concentrations. catabolism, gluconeogenesis, glycolysis, and biofilm forma tion. Since biofilm formation has been linked to antibiotic Example 9 resistance, the inventors assessed if overexpressing cSrA might increase susceptibility to antibiotic treatment. The inventors also sought to determine whether the engi OmpF is a porin which is used by quinolones to enterbacteria 40 neered phage could be applied to different classes of antibi and therefore, the inventors determined that overproducing otics other than the quinolones. Since (ps was the most OmpF would increase killing by ofloxacin". The inventors effective adjuvant for ofloxacin, the inventors tested its adju discovered that cSrA-expressing M13mp 18 (cps) and vant effect for gentamicin, an aminoglycoside, and amplicil ompf-expressing M13mp18 (cp) both increased ofloxa lin, a f-lactam antibiotic. For 5 g/mL gentamicin, (p., cin's bactericidal effect by about 2.7 logo (CFU/mL) com 45 was slightly more effective at enhancing killing of bacterial pared with no phage and 0.8 logo (CFU/mL) compared with cells by ofloxacin compared with no phage (FIG. 8A). (p. unmodified (p, after 6 hours of treatment (FIG. 6B). increased gentamicin's bactericidal action by over 2.5 logo In order to enhance the effectiveness of engineered phage (CFU/mL) compared with (p, and by over 3 logo (CFU/ with cSrA or ompl alone as antibiotic adjuvants, the inventors mL) compared with no phage after 6 hours of treatment (FIG. designed an M13mp 18 phage to express cSrA and ompl 50 8A). For 5ug/mL amplicillin, control (p, alone increased simultaneously (cps) (FIG. 9F). The combination killing by ofloxacin by more than 3 orders of magnitude phage was constructed by modifying (ps to carry an RBS compared to no phage (FIG.4b). (p. improvedampicillin's and ompF immediately downstream of CSrA". (p.A-ompF bactericidal effect by over 2.2 logo (CFU/mL) compared improved killing by ofloxacin by over 0.7 logo (CFU/mL) with unmodified (p and by over 5.5 logo (CFU/mL) compared with {p, and per after 6 hours of treatment 55 compared to no phage (FIG. 8B). For both gentamicin and (FIG. 6B). The dual-target (p-ompF phage performed ampicillin, p’s strong adjuvant effect was noticeable comparably with (ps at various initial phage inoculations after 1 hour of treatment (FIG.8A and FIG.8B). These results with 60 ng/mL ofloxacin (FIG. 6C) and at various concentra are consistent with previous observations that Areca mutants tions of ofloxacin with 10 PFU/mL phage (FIG. 6D). Both exhibit increased Susceptibility to quinolone, aminoglyco phages were more effective than no phage or (p, at 60 side, and B-lactam drugs'. Therefore, engineered bacte increasing killing by ofloxacin. These results demonstrate riophage such as (ps can act as general adjuvants for the that targeting other non-SOS genetic networks and overex three major classes of bactericidal drugs. pressing multiple factors, i.e. multiple repressors can result in Using phage, the inventors have demonstrated that target engineered bacteriophage which are good adjuvants for anti ing genetic networks to potentiate killing by existing antimi biotics. 65 crobial drugs is a highly effective strategy for enhancing the Exposure to subinhibitory concentrations of antibiotics usefulness of antibiotics. The host specificity of phage avoids can lead to initial mutations which confer low-level antibiotic the side effects associated with broad-spectrum antibiotics US 9,056,899 B2 83 84 such as Clostridium difficile overgrowth but requires a library (FIG. 14). These constructs displayed 1.87 and 2.37 logo of phage to be maintained to cover a range of infections. (CFU/mL) less persisters, respectively, compared with wild In some embodiments, libraries of existing phage could be type E. coli EMG2. modified to overexpress other genes, such as for example but not limited to lexA3 to suppress the SOS response in different Example 11 bacterial species''. The inventors have demonstrated herein that combination Example 10 therapy which couples antibiotics with antibiotic-enhancing phage has the potential to be an effective antimicrobial strat A direct method of attacking antibiotic-resistant bacteria is 10 egy. Moreover, the inventors have demonstrated that antibi to express asRNAs to knockdown genes that either confer otic-enhancing phage are effective in Vivo in rescuing bacte antibiotic resistance or promote cell repair and the SOS rially infected mice, and thus have clinical relevance for their response. Thus, the inventors expressed an antisense RNA use in Vivo, in mammalian models of bacterial infections, as (asRNAs) against the cat gene and other antibiotic-resistance well as in human treatment, both for therapeutic and prophy 15 lactic treatment. Thus, the inventors have demonstrated a genes (genes that inactivate antibiotics or pump out antibiot method to modify phage (i.e. bacteriophage) to be engineered ics or genetic circuits that confer persistence or any other to act as effective antibiotic adjuvants in vitro and in vivo and antibiotic resistance phenotype Such as VanA, mecA, and can be used in methods for antimicrobial target identification others) as well as recA, recB, recC., spoT relA, and other as well as for therapeutic use and implementation. The inven genes necessary for cell repair or Survival. These vectors tors have also demonstrated that by targeting non-essential should sensitize cells to antibiotics since they will target gene networks, a diverse set of engineered bacteriophage can genes that inactivate or pump outantibiotics and those that are be developed to Supplement other antimicrobial strategies. necessary for cell repair from damage caused by antibiotics While use of phages in clinical practice is not widely (Dwyer et al., (2007) Mol Syst Biol3:91). Inhibiting the SOS accepted due to a number of issues such as phage immuno response may also reduce the spread of antibiotic resistance 25 genicity, efficacy, target bacteria identification and phage genes (Beaber, et al., (2004) Nature 427: 72-74;Ubeda, et al., selection, host specificity, and toxin release (Merril et al., (2005) Mol Microbiol 56: 836-844). (2003) Nat. Rev. Drug Discov. 2, 489-497; Hagens et al., The designs that have been currently experimented with (2003) Lett. Appl. Microbiol. 37, 318-323; Hagens et al., extend the paired-termini (PTT) design described in (2004) Antimicrob. Agents Chemother: 48, 3817-3822: Nakashima et al., (2006) Nucleic Acids Res 34: e138, which 30 Boratynski et al., (2004) Cell. Mol. Biol. Lett. 9, 253-259; produces an RNA similar to that shown in FIG. 12. The PTT Merrilet al., (1996) Proc Natl AcadSci USA 93,3188-3192), construct produces antisense RNA with longer half-lives in the inventors indicate that one way to reduce the risk of Vivo, allowing for greater antisense effect (Nakashima et al., leaving lysogenic particles in patients after treatment, the (2006) Nucleic Acids Res 34: e138). Using the PT system, we inventors engineered adjuvant phages could be further modi 35 fied to be non-replicative, as has been previously described have constructed antisense RNAS targeting cat, recA, recB. (Hagens et al., (2004) Antimicrob 11). The inventors have and recC (Nakashima et al., (2006) Nucleic Acids Res 34: demonstrated an antibiotic-enhancing phage as a prototype e138). These asRNA constructs have been placed under phage as proofof-conceptantibiotic adjuvants. The inventors inducible control by aTc by cloning into p7E21s1-cat in place indicate that in Some embodiments, a combination of antibi of cat (Lutz et al., (1997) Nucleic Acids Res 25: 1203-1210). 40 otic-enhancing phages or phage cocktails can be used for in The inventors also created all pairwise combinations of asR vivo and in vitrouse, as well as inclinical settings for effective NAS to recA, recB, and recC by placing one asRNA construct efficacy and/or the ability to treat non-F-plasmid containing under the control of PtetO and the other under the control of bacteria. In particular, in Some embodiments phage cocktails PlacO on the same plasmid (Lutzetal. (1997)Nucleic Acids which target different, multiple bacterial receptors can be Res 25: 1203-1210). 45 used, which can have a benefit of reducing the development of All the plasmids described thereafter have been introduced phage resistance by invading bacteria through multiple dif into wild-type E. coli EMG2 cells and have been assayed for ferent means and pathways. Thus, in another embodiment, survival with antibiotic treatment. All cells and suitable con phage cocktails can be used with one or more different anti trols were grown for 8 hours at 37° C. in LB media (with biotics to also enhance bacterial killing as well as reduce appropriate inducers) and challenged with antibiotics such as 50 resistance to both the phages and antibiotics. ofloxacin at 5ug/mL. Cell counts were plated after 8 hours of The inventors have demonstrated use of engineered antibi exposure to antibiotic and counted to assess persistence lev otic-enhancing phages as a phage platform for the develop els. Cells will also be assayed for resistance to specific anti ment of effective antibiotic adjuvants, and is a practical biotics (for example, chloramphenicol in the presence of cat example of how synthetic biology can be applied to important expressing plasmids). 55 real-world biomedical issues. Synthetic biology is focused on the rational and modular engineering of organisms to create The inventors constructed asRNA targeting cat and have novel behaviors. The field has produced many reports of expressed the asRNA in a ColE1-type plasmid. With the synthetic gene circuits and systems with interesting charac cat-askNA vector, the inventors assessed if the chloram teristics (Andrianantoandro et al., (2006) Mol Syst Biol, 2. phenicol MIC of target bacteria is effectively reduced. The 60 2006.0028; Hasty et al., (2002) in Nature, pp. 224-230; inventors constructed vectors with recA-asRNA, recB-as McDaniel et al., (2005) in Curr. Opin. Biotechnol., pp. 476 RNA, recC-asRNA and all pairwise recA, recB, and recC 483.: Chan et al., (2005) in Mol Syst Biol, p. 2005.0018). combinations and assayed for persistence levels with ofloxa More recently, synthetic biologists have begun to address cin (5ug/mL) with 8 hours of growth followed by 8 hours of important industrial and medical problems (Lu et al., (2007) treatment. The vectors which demonstrated the strongest phe 65 Proc Natl Acad Sci USA 104, 11197-216: Anderson et al., notypes were the PtetO-recB-askNA/PlacO-recA-asRNA (2006).J. Mol. Biol. 355,619-627; Loose et al., (2006) Nature and PtetO-recC-asRNA/PlacO-rec3-asRNA plasmids 443, 867-869; Roet al. (2006) Nature 440, 940-943). US 9,056,899 B2 85 86 In some embodiments, the present invention also encom pared with wild-type E. coli EMG2 (FIG. 10). The inventors passes production and use of libraries of natural phage which also made changes in the design of pZE1L-leXA by using have been modified to target gene networks and pathways, non-cleavable leXA variants. such as the SOS response, in different bacterial species (Hick The inventors demonstrated, in lytic phage Such as T7 or man-Brenner et al., (1991) J. Clin. Microbiol. 29, 2817 lysogenic phage Such as M13 and using synthetic biology, 2823). One of ordinary skill in the art could generate and use construction of engineered phage by inserting the vector con Such libraries by using routine methods in the art, such as structs simply into optimal regions in the phage genome to be isolation and genetic modification of natural phage with the expressed during infection (Lu et al., (2007) Proc Natl Acad ability to infect the bacterial species being targeted. With Sci USA 104: 11197-11202). M13 is a filamentous, male current DNA sequencing and synthesis technology, an entire 10 specific phage with a single-stranded, circular DNA genome engineered bacteriophage genome carrying multiple con that infects E. coli. During infection, the genome adopts a double-stranded replicative form (RF) which can be stably structs to target different gene networks could be synthesized maintained in lysogeny. M13 Subsequently replicates and (Baker et al. (2006) Sci. Am. 294, 44-51). Thus, one of ordi secretes mature phage particles into the Surrounding environ nary skill in the art, using Such technologies could carry out 15 ment that can infect other cells. M13 is a commonly used large-scale modifications of phage libraries to produce anti phage for peptide display and DNA sequencing and has been biotic-enhancing phage that can be applied with different modified for genetic manipulation. In some embodiments, antibiotic drugs against a wide range of bacterial infections. M13 and other lysogenic phage can be used as carriers for Targeting clinical bacterial strains with libraries of engi asRNAS or other genetic modules because they allow propa neered phage, which can be carried out by routine testing by gation of the introduced constructs throughout a bacterial one of ordinary skill in the art to identify which engineered population without massive lysis, which can lead to release of phage from the libraries is effective as an antibiotic-enhanc toxic products such as endotoxin or lead to the development ing phage to clinically relevant bacterial strains and has of phage resistant bacteria due to strong evolutionary pres important uses in developing treatments against real-world Sure. As the constructs need to be able to reach a large popu infections. 25 lation of cells, have the desired effects, and then be subse In some embodiments, the engineered phages as described quently killed by antibiotic therapy, lysogenic phages were herein can also be used in industrial, agricultural, and food used by the inventors. For example, the gene constructs could processing settings where bacterial biofilms and other diffi be cloned in place of the lacZ gene in the already modified cult-to-clear bacteria are present (Lu et al., (2007) Proc Natl M13mp18 bacteriophage under the control of a strong bacte AcadSci USA 104, 11 197-216). Accordingly, some embodi 30 rial-species-specific promoter or phage-specific promoter. ments as described herein encompass applying the engi Herein, the inventors have demonstrated that building neered phage as described herein as antibiotic adjuvants in effective bacteriophage adjuvants that target different factors non-medical settings. This could be economically advanta individually or in combination can be achieved in a modular geous, reduce community-acquired antibiotic resistance, and fashion. As the cost of DNA sequencing and synthesis tech be also be useful in testing efficacy of the particular engi 35 nologies continues to be reduced, large-scale modifications of neered phage prior to its use as a treatment and/or in clinical phage libraries should become feasible. With current use (Morens et al., (2004) Nature 430, 242-24949). technology, an entire engineered M13mp 18 genome carrying Another strategy to combat antibiotic resistance is to take multiple constructs to target genetic networks could be syn advantage of the numerous autoregulated repressors inherent thesized for less than $10,000, a price which is sure to in bacteria that regulate resistance genes or cell repair path 40 decrease in the future. Furthermore, systems biology tech ways (Okusu, et al., (1996) J Bacteriol 178: 306–308). For niques can be employed to more rapidly identify new targets example, lexA represses the SOS response until it is cleaved to be used in engineered bacteriophage'''. Antisense RNA by recA in response to DNA damage (Dwyer et al., (2007) could also be delivered by bacteriophage to enhance killing of Mol Syst Biol 3: 91). In addition, marr represses the mar bacteria. Cocktails of engineered phage such as those RAB operon and acrR represses the acrAB operon; both 45 described here could be combined with biofilm-dispersing operons confer resistance to a range of antibiotics (Okusu, et bacteriophage and antibiotics to increase the removal of al., (1996) J Bacteriol 178: 306–308). To increase repression harmful biofilms. of the SOS response orantibiotic-resistance-conferring oper Since the FDA recently approved the use of bacteriophage ons, we propose to overexpress the responsible repressors. against Listeria monocytogenes in food products, it is likely However, simple overexpression may impose a high meta 50 that the engineered phages as disclosed herein can be readily bolic cost on the cells leading to rejection of the introduced adopted for medical, industrial, agricultural, and food pro constructs. Therefore, as an alternative to simple overexpres cessing settings where bacterial biofilms and other difficult Sion, the inventors created an autoregulated negative-feed to-clear bacteria are present'. Potentiating bacterial kill back modules with lexA and other repressors and determine ing in non-medical settings should have economic advantages whether cells are sensitized to antibiotic treatment with these 55 in addition to reducing community-acquired antibiotic resis constructs (FIG. 13). The net effect of this strategy should be tance'. to increase the loop gain of inherent autoregulated negative Conventional drugs typically achieve their therapeutic feedback loops so that any perturbations in the level of repres effect by reducing protein function. In contrast, the bacte sors will be more rapidly restored, hopefully preventing Suc riophage and selective gene targeting approach as described cessful activation of Survival pathways. 60 herein potentiates killing by antibiotics by overexpressing The inventors produced and assessed the pZE1L-lex.A proteins that affect genetic networks, such as leXA3, SOXR, plasmid for persistence levels with ofloxacin (5ug/mL) with and cSrA, or that act on their own to modulate antibiotic 8 hours of growth followed by 8 hours of treatment. The sensitivity, such as ompF. By reducing the SOS response with inventors constructed the pzE1L-lex.A plasmid by utilizing engineered M13mp 18-lex A3 bacteriophage, the inventors the PlexO promoter described in (Dwyer et al., (2007) Mol 65 have potentiated ofloxacins bactericidal effect by over 4.5 Syst Biol 3:91). Cells containing the pZE1L-lex A construct orders of magnitude and reduced the number of persister cells produced about 1.44 logo (CFU/mL) less persisters com (FIG. 1b). The inventors have also demonstrated that other US 9,056,899 B2 87 88 factors such as SOXR, cSrA, and ompl could be targeted for tion therapy with antibiotics and engineered phage resulted in overexpression individually or in combination to enhance no noticeable development of phage resistance. The inventors killing (FIG. 6). The inventors demonstrated that the number demonstrated that targeting genetic networks in bacteria of mutants which acquired antibiotic resistance was signifi which are not primary antibiotic targets yield Substantial cantly decreased by the use of engineered M13mp18-lex.A3 improvements in killing by antimicrobial drugs. Advances in bacteriophage in combination with ofloxacin (FIG. 7). In systems biology and synthetic biology should enable the addition, the inventors confirmed that our engineered bacte practical application of engineered bacteriophage with anti riophage could be used as antibiotic adjuvants for other drugs biotics as a new combination therapy for combating bacterial Such as aminoglycosides and B-lactams (FIG. 8). Combina infections. TABLE 2A Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Ciprofloxacin antimicrobial agent. Code: Accession Number (from world-wide web “ecocyc.org), Categories are as follows: 1-DNA replication, recombination and repair, 1A-functions indirectly affecting category 1,2-transport, efflux, cell wall and cell membrane synthesis, 2A-chaperones and functions related to 2,3-protein synthesis, 4-central metabolic reactions, 5-regulation, 6-prophage encoded genes; cell adhesion, or 7-unassigned genes. Gene knockout(s) from KEIO collection (3) using BW25113 (10) as the starting strain. Table 2A: Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Ciprofloxacin antimicrobial agent MIC (ng/mL)

LOCS E Tag Gene Gene Product Category Test Plating

BW251.13 16 2O b1413 hrp A ATP-dependent helicase 8.75 b2699 recA DNA strand exchange and recombination 2 >8.75 protein with protease and nuclease activity b282O rec DNA helicase, ATP-dependent 7.5 dsDNA ssDNA exonuclease b2822 recC DNA helicase, ATP-dependent 8 >8.75 dsDNA ssDNA exonuclease b3652 recC ATP-dependent DNA helicase, resolution of 6 6 Holliday junctions, branch migrations b2616 recy Recombination and repair protein 10 b1861 ruvA Holliday junction DNA helicase 10 b1863 ruvC Holliday junction nuclease; resolution of 8 >8.75 structures; repair b3813 wrD DNA-dependent ATPase I and helicase II 5 6 b2SO9 xSeA Exodeoxyribonuclease VII large subunit 6 6 bO422 xSeB Exodeoxyribonuclease VII Small subunit 8 b3261 fis DNA-binding protein - chromosome A. 6 >8.75 compaction b1712 ihfA integration host factor alpha-subunit (IHF- A. 7.5 alpha). b0464 acrA AcrAB-TolC Multidrug Efflux Transport 2 7.5 System bO462 acre AcrAB-TolC Multidrug Efflux Transport 2 8 System b3O3S toC AcrAB-TolC Multidrug Efflux Transport 2 4 5 System b0742 ybgF Predicted plasma protein 2 7.5 b0489 qmcA Putative protease 3 >8.75 b0852 rimK Ribosomal protein S6 modification protein. 3 >8.75 b1317 pgmB 3-phosphoglucomutase 4 10 b0736 ybgC Acyl-CoA thioesterase - cytoplasm 4 7.5 b2767 ygcO Predicted 4Fe-4S cluster-containing protein 4 7.5 b1284 deoT DNA-binding transcriptional regulator 5 7.5 b0145 dkSA RNA polymerase-binding transcription factor 5 10 b4172 hf. HF-I, host factor for RNA phage QB 5 7.5 replication b2572 rSeA Sigma-E factor negative regulatory protein. 5 >8.75 b1280 yeiM Putative heat shock protein 5 7.5 b1233 yeh.J Conserved protein Yeh.J 7 7.5 b4402 yijY Predicted protein YY 7 8.75 US 9,056,899 B2 89 TABLE 2B Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Vancomycin antimicrobial agent, or analogue or varient thereof. Table 2B: Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Vancomycin antimicrobial agent, or analogue or varient thereof

LOCS MIC mL Tag Gene Gene Product Category Plating E Test BW251.13 500 b3613 enwic Cytokinesis - murein hydrolase 2 50 b3404 enwz. OSmolarity sensor protein 2 50 b0588 fepc Ferric enterobactin transport ATP-binding 2 50 protein b3201 lptB ATP-binding LiptAB-YrbKABC transporter 2 50 2.0 b1855 msbB Myristoyl-acyl carrier acyltransferase 2 50 b0741 pal Peptidoglycan-associated lipoprotein 2 OO 96 CSO. b2678 proW Glycine betaine:L-proline transport?permease 2 50 b2617 Smp A Outer membrane lipoprotein 2 OO 70 b1252 tonB Cytoplasmic membrane protein; energy 2 25 8SCUCC b2512 yfgL Lipoprotein-outer membrane protein 2 50 assembly b3245 yhdP Transporter activity, membrane protein 2 25 b2S27 scB Hsc20 co-chaperone, with Hsc66 IscU iron- 2A 50 Sulfur cluster b0178 skip Periplasmic chaperone 2A 75 bOO53 SurA Peptidyl-prolyl cis-trans isomerase PPIase 2A 8 4 and chaperone b0939 yebR Predicted periplasmic pilin chaperone 2A 50 b0742 ybgF Predicted periplasmic protein 2 OO b2269 ela) Deubiquitinase 3 50 b0852 rimK Ribosomal protein S6 modification protein. 3 50 b3299 rpm.J. 50S ribosomal protein L36 (Ribosomal 3 50 protein B). b3179 rm 23S rRNA m2U2552 methyltransferase 3 50 b3344 tuSC RNA modification -sulfur transfer protein 3 50 complex b3345 tuSD RNA modification -sulfur transfer protein 3 50 complex b2494 yfgC Predicted peptidase 3 50 b1317 pgmB Putative beta-phosphoglucomutase 4 OO b1773 ydi I Predicted adolase 4 OO b0145 dkSA RNA polymerase-binding transcription factor 5 25 b1237 hns DNA-binding protein H-NS 5 50 b3961 oxyR OxyR transcriptional dual regulator 5 50 b24.05 xapR Xanthosine operon regulatory protein. 5 OO b1280 yeiM Putative heat shock protein 5 OO b1553 ydfP Qin prophage; conserved protein 6 50

TABLE 2C Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Rifampicin antimicrobial agent, or analogue or varient thereof Table 2C: Example of a genes which can be inhibited by an repressor engineered bacteriophage, and in some embodiments, such repressor engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Rifampicin antimicrobial agent, or analogue or varient thereof

MIC Locus (Lig/mL) Tag Gene Gene Product Category Plating

BW251.13 16 b2822 recC DNA helicase, ATP-dependent 1 7.5 dsDNA ssDNA exonuclease b2616 recN Recombination and repair protein 1 7.5 b1652 rnt RibonucleaseT 1 >10 US 9,056,899 B2 91 TABLE 2C-continued Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Rifampicin antimicrobial agent, or analogue or varient thereof Table 2C: Example of a genes which can be inhibited by an repressor engineered bacteriophage, and in some embodiments, such repressor engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Rifampicin antimicrobial agent, or analogue or varient thereof

MIC LOCS (Lig/mL) Tag Gene Gene Product Category Plating b4058 wrA Excision nuclease subunit A 1 7.5 b3781 trix A Thioredoxin electron transfer protein 1A 5 b0464 acrA Acra B-TolC Multidrug Efflux Transport 2 >10 System bO462 acre Acra B-TolC Multidrug Efflux Transport 2 10 System b3613 enwic Cytokinesis - murein hydrolase 2 10 b3404 enwz. OSmolarity sensor protein 2 10 b0588 fepc Ferric enterobactin transport ATP-binding 2 10 protein b1677 pp Major outer membrane lipoprotein precursor 2 5 b3201 lptB ATP-binding LiptAB-YrbKABC transporter 2 10 b1855 msbB Lipid A biosynthesis (KDO)2-(lauroyl)-lipid 2 7.5 VA acyltransferase b0741 pal Peptidoglycan-associated lipoprotein 2 5 CSO. b1090 plsX Fatty acidiphospholipid synthesis protein plsX. 2 10 b0525 ppi B Peptidyl-prolyl cis-trans isomerase B 2 5 b3726 pstA Phosphate transport system permease protein 2 5 b3728 pstS Phosphate-binding periplasmic protein 2 7.5 CSO b3619 rfaD ADP-L-glycero-D-manno-heptose-6- 2 10 epimerase b3OS2 rifa Heptose 1-phosphate adenyltransferase 2 7.5 b3631 rfaC Lipopolysaccharide core biosynthesis protein 2 2 b2617 Smp A Outer membrane lipoprotein 2 5 b3838 tat Sec-independent protein translocase TatB 2 10 b3839 tatC Sec-independent protein translocase TatC 2 10 bO738 toR Colicin import; Tolerance to group A colicins 2 3.5 b1252 tonB Cytoplasmic membrane protein; energy 2 >10 8SCUCC b0742 ybgF Predicted periplasmic protein 2 >10 b2512 yfgL Lipoprotein-outer membrane protein assembly 2 >10 b2807 ygdD Conserved inner membrane protein 2 10 b3245 yhdP Transporter activity, membrane protein 2 10 b0161 degP Periplasmic serine protease and chaperone 2A 10 b0014 dnaK Chaperone protein - chaperone Hsp70; DNA 2A 7.5 biosynthesis b0178 skip Periplasmic chaperone 2A 5 bOO53 SurA Peptidyl-prolyl cis-trans isomerase PPIase and 2A 2 chaperone b0939 yebR Predicted periplasmic pilin chaperone 2A 10 b2269 ela) Deubiquitinase 3 >10 b4375 prf Peptide chain release factor 3 (RF-3). 3 10 b0489 qmcA Putative protease 3 10 b0852 rimK Ribosomal protein S6 modification protein. 3 10 b1269 ruE 23S rRNA pseudouridine synthase 3 10 b3984 rplA 50S ribosomal protein L1. 3 7.5 b3936 rpm. 50S ribosomal protein L31. 3 5 b1089 romF 50S ribosomal protein L32. 3 7.5 b3299 rpm 50S ribosomal protein L36 (Ribosomal protein 3 7.5 B). b2494 yfgC Predicted peptidase 3 5 b1095 fab B-ketoacyl-ACP synthase 4 5 b3058 foB Dihydroneopterin aldolase 4 >10 b4395 gpmB Probable phosphoglycerate mutase gpmB 4 10 B3612 gpmM phosphoglycerate mutase, cofactor 4 >10 independent b0677 nagA N-acetylglucosamine-6-phosphate deacetylase 4 5 b1317 pgmB 3-phosphoglucomutase 4 10 b3386 rpe Ribulose-phosphate 3-epimerase 4 10 b1731 ced A Cell division activator 5 10 b4172 hf. HF-I, host factor for RNA phage Q B 5 10 replication b1237 hns DNA-binding protein H-NS 5 7.5 b3842 rfaEH Transcriptional activator rfaH. 5 7.5 US 9,056,899 B2 93 TABLE 2C-continued Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Rifampicin antimicrobial agent, or analogue or varient thereof Table 2C: Example of a genes which can be inhibited by an repressor engineered bacteriophage, and in some embodiments, such repressor engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Rifampicin antimicrobial agent, or analogue or varient thereof

MIC Locus (Lig/mL) Tag Gene Gene Product Category Plating b2572 rseA Sigma-E factor negative regulatory protein. 5 7.5 b24.05 xapR Xanthosine operon regulatory protein. 5 >10 b1280 yeiM Putative heat shock protein 5 7.5 b0547 ybcN Hypothetical protein in lambdoid DLP12 6 7.5 prophage region b0550.1 ylcG DLP12 prophage; predicted protein 6 5 b0659 ybeY Hypothetical protein 7 10 b1088 yeeD Hypothetical protein 7 5 b1233 yeh.J. Hypothetical protein 7 7.5 b4402 yjY Hypothetical proteinyi.Y. 7 >10

TABLE 2D Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with an Ampicillin antimicrobial agent, or analogue or varient thereof Table 2D: Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in Some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with an Ampicillin antimicrobial agent, or analogue or varient thereof Locus MIC (Lig/mL Tag Gene Gene Description Category E test Plating BW251.13 S.O 6.O b3017 Suf Suppressor of essential cell division protein FtsI 1A, 2 2.0 b0464 acrA Acra B-TolC Multidrug Efflux Transport System 2 .5 bO462 acre Acra B-TolC Multidrug Efflux Transport System 2 2.0 b3O3S toC Acra B-TolC Multidrug Efflux Transport System 2 1.O 2.0 b0632 dacA Penicillin-binding protein 5 precursor 2 1.5 .5 bOO92 dB Subunit of D-alanine:D-alanine ligase B, ADP- 2 O forming b2314 ded) Putative lipoprotein - inner membrane 2 2.0 b1193 entA :ytic murein transglycosylase E 2 2.0 b3613 enwic Cytokinesis - murein hydrolase 2 .5 b3201 lptB ATP-binding LiptAB-YrbKABC transporter 2 2.0 bO149 mrcB Subunit of 5-methylcytosine restriction system 2 2.0 b0741 pal Peptidoglycan-associated lipoprotein precursor. 2 2.0 5 b3838 tat Sec-independent protein translocase TatB 2 1.5 .5 b3839 tatC Sec-independent protein translocase TatC 2 3.0 2.0 bO738 toR Colicin import; Tol-pal system component 2 2.0 b0742 ybgF Hypothetical proteinybgF precursor. 2 .5 b0028 flkpB FKBP-type 16 kDa peptidyl-prolyl cis-trans 2A 2.5 isomerase b2526 hScA Chaperone, member of Hsp70 protein family 2A 2.0 b2S27 scB Hsc20 co-chaperone that acts with Hsc66 in IscU 2A 2.5 iron-sulfur cluster b0178 skip Periplasmic chaperone 2A 2.0 bOO53 SurA Peptidyl-prolyl cis-trans isomerase PPIase and 2A 2.0 chaperone b0489 qmcA Putative protease 3 2.5 b0852 rimK Ribosomal protein S6 modification protein. 3 2.0 b3984 rplA 50S ribosomal protein L1. 3 2.0 2.0 b1089 rpmF 50S ribosomal protein L32. 3 1.5 b4200 rpsF 30S ribosomal protein S6. 3 2.0 b3179 rm 23S rRNA m2U2552 methyltransferase 3 1.5 b2494 yfgC Hypothetical protein yfgC precursor. 3 1.5 b2512 yfgL Lipoprotein component of Outer membrane 3 2.0 protein assembly complex US 9,056,899 B2 95 TABLE 2D-continued Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with an Ampicillin antimicrobial agent, or analogue or varient thereof Table 2D: Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with an Ampicillin antimicrobial agent, or analogue or varient thereof

LOCS MIC (Lig/mL Tag Gene Gene Description Category E test Plating b3734 atpA ATP synthase alpha chain 4 2.5 b3809 dapF Diaminopimelate epimerase 4 2.0 1.O b2065 ded Deoxycytidine triphosphate deaminase (dTP) 4 2.5 b3612 gpmM Phosphoglycerate mutase, cofactor independent 4 1.5 b1317 pgmB 3-phosphoglucomutase 4 1.5 b2232 ubiG 3-demethylubiquinone-9 3-methyltransferase 4 2.0 b2767 ygcO Predicted 4Fe-4S cluster-containing protein 4 2.0 b1284 deoT DNA-binding transcriptional regulator 5 2.0 bO145 kSA RNA polymerase-binding transcription factor 5 2.0 b1130 phoP Transcriptional regulatory protein 5 2.0 b24.05 xapR Xanthosine operon regulatory protein. 5 1.5 b1280 yeiM Putative heat shock proteins 5 1.5 JW 5115 Hypothetical protein 7 2.0 b0631 ybeD conserved protein Ybed 7 2.0 b0659 ybeY conserved protein Ybey 7 2.0 b0762 ybhT Hypothetical protein YbhT precursor 7 2.0 b4402 yjY predicted protein YY 7 1.5

TABLE 2E Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a SulfamethaxaZone antimicrobial agent, or analogue or varient thereof. Table 2E: Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, Such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Sulfamethaxazone antimicrobial agent, or analogue or varient thereof

MIC LOCS (Lig/mL) Tag Gene Gene Product Category Plating

BW251.13 1OOO b1865 nudB dATP pyrophosphohydrolase 1 350 b2699 recA DNA strand exchange and recombination protein 1 400 b282O rec DNA helicase, ATP-dependent dsDNA ssDNA 1 350 exonuclease b2822 recC DNA helicase, ATP-dependent dsDNA ssDNA 1 350 exonuclease b3652 recC ATP-dependent DNA helicase, resolution of 1 500 Holliday junctions b3261 fis DNA-binding protein - chromosome compaction 1A 600 b3613 enwic Cytokinesis - murein hydrolase 2 400 b3201 lptB ATP-binding LiptAB-YrbKABC transporter 2 500 b3726 pstA Phosphate transport system permease 2 550 b3728 pstS Phosphate-binding periplasmic protein 2 550 b3OS2 rifa Heptose 1-phosphate adenyltransferase 2 550 b3O3S toC Acra B-TolC Multidrug Efflux Transport System 2 400 b0742 ybgF Predicted plasma protein 2 >550 b1279 yeiS Conserved inner membrane protein 2 550 b2512 yfgL Lipoprotein component of Outer membrane protein 2 400 assembly complex b1520 yneE Conserved inner membrane protein 2 550 b0161 degP Periplasmic serine protease and chaperone 2A 500 b0014 dnaK Chaperone protein - chaperone Hsp70; DNA 2A 300 biosynthesis b0489 qmcA Putative protease 3 550 b0852 rimK Ribosomal protein S6 modification protein. 3 350 b3984 rplA 50S ribosomal protein L1. 3 500 US 9,056,899 B2 97 TABLE 2E-continued Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a SulfamethaxaZone antimicrobial agent, or analogue or varient thereof. Table 2E: Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, Such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a Sulfamethaxazone antimicrobial agent, or analogue or varient thereof

MIC LOCS (Lig/mL) Tag Gene Gene Product Category Plating b1089 rpmF 50S ribosomal protein L32. 3 550 b3065 rpsU 30S ribosomal protein S21. 3 500 b3809 dapF Diaminopimelate epimerase 4 300 b2065 ded Deoxycytidine triphosphate deaminase (dTP) 4 >550 b3612 gpmM Phosphoglycerate mutase, cofactor independent 4 400 b0116 pdA Dihydrolipoamide dehydrogenase (Glycine 4 400 cleavage) b1317 pgmB 3-phosphoglucomutase 4 500 b1773 ydi I Predicted adolase 4 >550 b2767 ygcO Predicted 4Fe-4S cluster-containing protein 4 550 b1284 deoT DNA-binding transcriptional regulator 5 550 b0145 dkSA Transcription initiation factor 5 550 b1237 hns DNA-binding protein H-NS 5 550 b2572 resA Sigma-E factor negative regulatory protein. 5 >550 b24.05 xapR Xanthosine operon regulatory protein. 5 >550 b1280 yeiM Putative heat shock protein 5 >550 b0550.1 ylcG DLP12 prophage; predicted protein 6 500 b1143 ymfI Prophage genes - e14 prophage; predicted protein 6 500 JW 5115 Hypothetical protein 7 400 JW5474 Hypothetical protein 7 500 b0659 ybeY Hypothetical protein 7 500 b3928 yii U Conserved protein Yii U 7 550 b4402 yjY Predicted protein YY 7 >550

35

TABLE 2F Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a gentamicin antimicrobial agent, or analogue or varient thereof Table 2F: Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, Such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a gentamicin antimicrobial agent, or analogue or varient thereof

MIC LOCS (Lig/mL) Tag Gene Gene Product Category Plating

BW251.13 O.8 b1652 rint RibonucleaseT 1 0.7 b3613 enwic Cytokinesis - murein hydrolase 2 >O.S b3621 rfaC Lipopolysaccharide heptosyltransferase-1 2 0.7 b3791 riff A dTDP-4-oxo-6-deoxy-D-glucose transaminase 2 0.7 b1292 sapC Peptide transport system permease protein 2 O.S b3175 SecG Protein-export membrane - Sec Protein Secretion 2 O.S Complex b3839 tatC Sec-independent protein translocase TatC 2 O.S b3O3S toC Acra B-TolC Multidrug Efflux Transport System 2 O.S b4174 hfK Regulator of FtsH protease 3 O.S b4203 rplI 50S ribosomal protein L9. 3 0.7 b3936 rpmE 50S ribosomal protein L31. 3 O.6 b3344 tuSC RNA modification -sulfur transfer protein 3 O.S complex b3345 tuSD RNA modification -sulfur transfer protein 3 O.S complex US 9,056,899 B2 99 100 TABLE 2F-continued Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a gentamicin antimicrobial agent, or analogue or varient thereof Table 2F: Example of a genes which can be inhibited by an repressor-engineered bacteriophage, and in some embodiments, Such repressor-engineered bacteriophages which inhibit one or more of the following non-SOS defense genes are useful in combination with a gentamicin antimicrobial agent, or analogue or varient thereof

MIC Locus (Lig/mL) Tag Gene Gene Product Category Plating b2494 yfgC Predicted peptidase b3809 dapF Diaminopimelate epimerase 0.7 b3612 gpmM Phosphoglycerate mutase, cofactor independent 0.7 b32O2 RNA polymerase sigma-54 factor. O.S b24OS XapR Xanthosine operon regulatory protein. b1280 yciM Putative heat shock protein JW5360 Hypothetical protein b4557 yidD Predicted protein YidD

TABLE 5 Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. Table 5: Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. organism accession length proteins RNAS genes Acholepiasma phage L2 NC OO1447 11.96S 14 14 Acholeplasma phage MV-L1 NC OO1341 4491 4 4 Acidianus bottle-shaped virus NC OO9452 23814 57 57 Acidiantis filamentous virus 1 NC OO5830 2O869 40 40 Acidiantis filamentous virus 2 NC 009884 31787 52 53 Acidianus filamentous virus 3 NC 01O155 40449 68 68 Acidianus filamentous virus 6 NC 01.0152 39577 66 66 Acidianus filamentous virus 7 NC 01.0153 36895 57 57 Acidianus filamentous virus 8 NC 01 0154 38179 61 61 Acidianus filamentous virus 9 NC 01.0537 41172 73 73 Acidiant is rod-shaped virus 1 NC OO9965 246SS 41 41 Acidianus two-tailed virus NC OO7409 62730 72 72 Acinetobacter phage AP205 NC OO2700 4268 4 4 Actinomyces phage AV-1 NC 009643 17171 22 23 Actinoplanes phage phiAsp2 NC OO5885 S8638 76 76 Acyrthosiphon pisum secondary NC OOO935 36524 S4 S4 endosymbiont phage 1 Aeromonas phage 25 NC OO8208 161475 242 242 Aeromonas phage 31 NC 007022 172963 247 262 Aeromonas phage 44RR2.8t NC OO5135 173591 252 269 Aeromonas phage Aeh1 NC OO5260 233234 352 375 Aeromonas phage phiO18P NC OO9542 33985 45 45 Archaeal BJ1 virus NC OO8695 42271 70 71 Azospirilium phage Cod NC 01 0355 62337 95 95 Bacilius phage 0305phi8-36 NC OO9760 218948 246 246 Bacilius phage AP50 NC 011523 14398 31 31 Bacilius phage B103 NC OO4165 18630 17 17 Bacilius phage BCJA1c NC OO6557 41,092 58 58 Bacilius phage Bam35c NC OO5258 14935 32 32 Bacilius phage Cherry NC 007457 366.15 51 51 Bacilius phage Fah NC 007814 37974 50 50 Bacilius phage GA-1 NC 002649 21129 35 52 Bacilius phage GIL16c NC OO6945 14844 31 31 Bacilius phage Gamma NC 007458 37253 53 53 Bacilius phage IEBH NC 01.1167 53104 86 86 Bacilius phage SPBc2 NC OO1884 134416 185 185 Bacilius phage SPO1 NC O11421 132S62 204 209 Bacilius phage SPP1 NC 004166 44010 101 101 Bacilius phage TP21-L NC 011645 37456 56 56 Bacilius phage WBeta NC OO7734 40867 53 53 Bacilius phage phBC6A51 NC OO4820 61.395 75 75 US 9,056,899 B2 101 102 TABLE 5-continued Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. Table 5: Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. organism accession length proteins RNAS genes Bacilius phage phBC6A52 NC OO4821 38.472 49 49 Bacilius phage phil O5 NC OO4167 39325 51 51 Bacilius phage phi29 NC 01.1048 19282 27 27 Bacilius virus 1 NC 009737 35055 S4 S4 Bacteriophage APSE-2 NC 011551 39867 41 42 Bacteroides phage B40-8 NC 011222 44929 46 46 Bdellovibrio phage philNH2K NC 002643 4594 11 11 Bordeteila phage BIP-1 NC OO5809 42638 48 48 Bordeteila phage BMP-1 NC OO5808 42663 47 47 Bordeteila phage BPP-1 NC 005357 42493 49 49 Burkholderia ambifaria phage BcepF1 NC OO9015 72415 127 127 Burkholderia phage Bcep1 NC OO5263 48.177 71 71 Burkholderia phage Bcep176 NC OO7497 44856 81 81 Burkholderia phage Bcep22 NC OO5262 63879 81 82 Burkholderia phage Bcep43 NC OO5342 48024 65 65 Burkholderia phage Bcep781 NC 004333 48247 66 66 Burkholderia phage BcepB1A NC OO5886 47399 73 73 Burkholderia phage BcepC6B NC OO5887 42415 46 46 Burkholderia phage BcepGomr NC 009447 52414 75 75 Burkholderia phage BcepMu NC OO5882 36748 53 53 Burkholderia phage BcepNY3 NC 009604 47382 70 70 Burkholderia phage BcepNazgul NC 005091 574.55 73 73 Burkholderia phage KS10 NC 011216 37635 49 49 Burkholderia phage phil O26b NC OO5284 S486S 83 83 Burkholderia phage phi52237 NC 0071.45 37639 47 47 Burkholderia phage phió44-2 NC 009235 48674 71 71 Burkholderia phage phiE12-2 NC 009236 36690 50 50 Burkholderia phage phiE125 NC OO3309 53373 71 71 Burkholderia phage phiE202 NC 009234 35741 48 48 Burkholderia phage phiE255 NC 009237 37446 55 55 Chlamydia phage 3 NC 008355 4554 8 8 Chlamydia phage 4 NC OO7461 4530 8 8 Chlamydia phage CPAR39 NC 00218O 4532 7 7 Chlamydia phage Chp1 NC OO1741 4877 12 12 Chlamydia phage Chp2 NC 002194 4563 8 7 Chlamydia phage phiCPG1 NC OO1998 4529 9 9 Clostridium phage 39-O NC O11318 38753 62 62 Clost ridium phage c-st NC 007581 185683 198 198 Clostridium phage phi CD119 NC 007917 53325 79 79 Clostridium phage phi3626 NC OO3524 33507 50 50 Clostridium phage phiC2 NC 009231 56538 82 82 Clostridium phage phiCD27 398 SO930 75 75 Clostridium phage phiSM101 NC OO8265 38092 53 S4 Corynebacterium phage BFK20 NC OO9799 42969 S4 S4 Corynebacterium phage P1201 NC 009816 70579 97 101 Enterobacteria phage 13a 38841 55 55 Enterobacteria phage 933W NC OOO924 61670 8O 84 Enterobacteria phage BA14 39816 52 52 Enterobacteria phage BP-4795 NC 004.813 57930 85 85 Enterobacteria phage BZ13 NC OO 426 34.66 4 4 Enterobacteria phage EPS7 NC 01.0583 111382 170 171 Enterobacteria phage ES18 NC OO6949 46900 79 79 Enterobacteria phage EcoDS1 39252 53 53 Enterobacteria phage FI sensulato NC 004301 4276 4 4 Enterobacteria phage Felix 01 NC OO5282 86155 131 153 Enterobacteria phage Fels-2 NC 010463 33693 47 48 Enterobacteria phage G4 sensulato NC OO 5577 11 13 Enterobacteria phage HK022 NC 002166 4O751 57 57 Enterobacteria phage HK620 NC OO2730 38297 58 58 Enterobacteria phage HK97 NC 002167 39732 61 62 Enterobacteria phage I2-2 NC OO 332 6744 9 9 Enterobacteria phage ID18 sensulato NC 007856 S486 11 11 Enterobacteria phage ID2 NC 007817 S486 11 11 Moscow ID 2001 Enterobacteria phage Ifl NC OO 954 8454 10 10 Enterobacteria phage Ike NC OO2014 6883 10 10 Enterobacteria phage JKO6 NC OO7291 46O72 82 82 Enterobacteria phage JS98 NC 01.01.05 170523 266 269 Enterobacteria phage K1-5 NC OO8152 44385 52 52 Enterobacteria phage K1E NC 007637 452S1 62 62 Enterobacteria hage K1F NC 007456 39704 43 41 US 9,056,899 B2 103 104 TABLE 5-continued Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. Table 5: Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. organism accession length proteins RNAS genes Enterobacteria phage M13 NC OO3287 64O7 10 10 Enterobacteria hage MS2 NC OO1417 3569 4 4 Enterobacteria hage Min27 NC 010237 6339S 83 86 Enterobacteria hage Mu NC OOO929 36717 55 55 Enterobacteria hage N15 NC OO1901 46375 60 60 Enterobacteria hage N4 NC OO872O 7O153 72 72 Enterobacteria NC OO5856 94.800 110 117 Enterobacteria NC OO1895 33593 43 43 Enterobacteria NC OO2371 41724 72 74 Enterobacteria NC OO1609 11624 14 19 Enterobacteria NC OO1421 14927 31 31 Enterobacteria NC OO9821 164270 276 276 Enterobacteria NC OO5340 30636 42 42 Enterobacteria NC OO1890 4215 4 4 Enterobacteria NC OO8515 165890 270 270 Enterobacteria NC 007023 18OSOO 292 292 Enterobacteria NC OO5066 164018 279 279 Enterobacteria NC 004928 167S60 273 275 Enterobacteria NC OO7603 46219 75 75 Enterobacteria NC OO4831 43769 52 52 Enterobacteria phage ST104 NC OO5841 41391 63 63 Enterobacteria NC 004348 4O679 65 65 Enterobacteria NC O05344 39043 66 70 Enterobacteria NC OO3444 37074 53 53 Enterobacteria NC OO5833 48836 78 78 Enterobacteria NC OO3298 38208 55 56 Enterobacteria NC OOO866 168903 278 288 Enterobacteria NC OO5859 121750 162 i 195 Enterobacteria hage T7 NC OO1604 39937 60 60 Enterobacteria hage TLS NC OO9540 499.02 87 87 Enterobacteria hage VT2-Sakai NC OOO902 60942 83 86 Enterobacteria hage WA13 sensulato NC 007821 6068 10 10 Enterobacteria hage YYZ-2008 NC O11356 54896 75 75 Enterobacteria hage alpha,3 NC OO1330 6087 10 10 Enterobacteria hage epsilon15 NC 004775 39671 51 51 Enterobacteria hage lambda NC OO1416 485O2 73 92 Enterobacteria hage phiEco32 NC 010324 77554 128 128 Enterobacteria hage phiEcoM-GJ1 NC 01.01.06 52975 75 76 Enterobacteria hage phiP27 NC OO3356 42575 58 60 Enterobacteria hage phiV10 NC 007804 391.04 55 55 Enterobacteria hage phiX174 sensu NC OO1422 S386 11 11 lato Enterococcus phage phiEF24C NC 009904 142O72 221 226 Erwinia phage Era 103 NC OO9014 45445 53 53 Erwinia phage phiEa21-4 NC 011811 84576 118 144 Escherichia phage rvis NC 011041 137947 233 239 Flavobacterium phage 11b NC OO6356 36012 65 65 Geobacilius phage GBSV1 NC 008376 34683 S4 S4 Geobacilius virus E2 NC OO9552 40863 71 71 Haemophilus phage Aaphi23 NC OO4827 43O33 66 66 Haemophilus phage HP1 NC OO1697 32355 42 42 Haemophilus phage HP2 NC OO3315 31508 37 37 Haloarcula phage SH1 NC 007217 3O889 56 56 Halomonas phage phiHAP-1 NC 010342 392.45 46 46 Haiorubriumv phage HF2 NC OO3345 77670 114 119 Haiovirus HF1 NC 004927 75898 102 106 His1 virus NC 007914 14462 35 35 His2 virus NC 007918 16067 35 35 Iodobacteriophage phiPLPE NC O11142 47453 84 84 Klebsiella phage K11 NC 011043 41181 51 51 Klebsiella phage phiKO2 NC O05857 S1601 64 63 Kluyvera phage Kvp1 NC 011534 39472 47 48 Lactobacillusiohnsonii prophage NC 01.0179 40881 56 56 L771 Lactobacilius phage A2 NC 004112 43411 61 64 Lactobacilius phage KC5a NC 007924 38239 61 61 Lactobacilius phage LL-H NC OO9554 34659 51 51 Lactobacilius phage LP65 NC OO6565 131522 16S 1 179 Lactobacilius phage Lc-Nu NC OO75O1 36466 51 51 Lactobacilius phage Lrm 1 NC O11104 39989 S4 S4 Lactobacilius phage LV-1 NC 011801 38934 47 47 US 9,056,899 B2 105 106 TABLE 5-continued Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. Table 5: Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. organism accession length proteins RNAS genes Lactobacilius phage phiAT3 NC OO5893 39166 55 55 Lactobacilius phage phiL-1 NC OO6936 36674 46 46 Lactobacilius phage philadh NC OOO896 43785 63 63 Lactobacilius phage phigle NC 004305 42259 50 62 Lactobacilius prophage Lj928 NC OO5354 38384 50 50 Lactobacilius prophage Lj965 NC 005355 4O190 46 46 Lactococci is phage 1706 NC 01.0576 55597 76 76 Lactococci is phage 712 NC 008370 3OS10 55 55 Lactococcus phage BK5-T NC OO2796 4OOO3 63 63 Lactococcus phage KSY1 NC 009817 79.232 130 131 Lactococcus phage P008 NC 008363 28S38 58 58 Lactococcus phage P335 sensulato NC 004746 36596 49 49 Lactococci is phage Q54 NC 008364 26537 47 47 Lactococcus phage TP901-1 NC OO2747 37667 56 56 Lactococcus phage Tuc2009 NC OO2703 38347 56 56 Lactococci is phage asccphi28 NC 010363 18762 28 27 Lactococcus phage b|BB29 NC 011046 293.05 S4 S4 Lactococcus phage b|L170 NC OO1909 31754 64 64 Lactococcus phage b|L285 NC 002666 35.538 62 62 Lactococcus phage b|L286 NC 002667 41834 61 61 Lactococcus phage b|L309 NC 002668 36949 56 56 Lactococci is phage bL310 NC 002669 14957 29 29 Lactococci is phage bL311 NC 002670 14S10 22 22 Lactococci is phage bL312 NC 002671 15179 27 27 Lactococci is phage bL67 NC OO1629 22195 37 O Lactococci is phage NC OO1706 22172 39 41 Lactococci is phage o NC 008371 27453 49 49 Lactococcus phage s 3 NC OO5822 32172 51 51 Lactococci is phage NC 004302 33350 50 50 Lactococci is phage NC OO1835 284.51 56 56 Lactococci is phage 6 NC 004O66 36798 61 61 Leticonostoc phage NC 009,534 2435 O O Listeria phage 2389 NC OO3291 37618 59 58 Listeria phage A006 NC 009815 3.8124 62 62 Listeria phage A118 NC OO3216 40834 72 72 Listeria phage A500 NC 009810 38867 63 63 Listeria phage A511 NC 009811 137619 199 215 Listeria phage B025 NC 009812 42653 65 65 Listeria phage B054 NC 009813 481.72 8O 8O Listeria phage P35 NC 009814 35822 56 56 Listeria phage P40 NC O11308 35638 62 62 Listonella phage phiHSIC NC OO6953 37966 47 47 Mannheimia phage phi MHaA1 NC OO82O1 34.525 49 50 Methanobacterium phage psi M2 NC OO1902 26111 32 32 Methanothermobacter phage psi M100 NC OO2628 28798 35 35 Microbacterium phage Min1 NC 009603 46365 77 77 Microcystis phage Ma-LMMO1 NC OO8562 162109 184 186 Morganella phage MmP1 NC 011085 38233 47 47 Mycobacterium phage 244 NC OO8194 74483 142 144 Mycobacterium phage Adjutor NC 01.0763 64S11 86 86 Mycobacterium phage BPs NC 01.0762 41901 63 63 Mycobacterium phage Barnyard NC 004689 70797 109 109 Mycobacterium phage Bethlehem NC OO9878 52250 87 87 Mycobacterium phage Boomer NC 011054 58O37 105 105 Mycobacterium phage Brujita NC 011291 47057 74 74 Mycobacterium phage Butterscotch NC 011286 64562 86 86 Mycobacterium phage Bxb1 NC 002656 50550 86 86 Mycobacterium phage BXZ1 NC 004687 156102 225 2 253 Mycobacterium phage BXZ2 NC 004682 SO913 86 89 Mycobacterium phage Cali NC 011271 155372 222 257 Mycobacterium phage Catera NC OO82O7 153766 218 s 253 Mycobacterium phage Chah NC 011284 6.8450 104 104 Mycobacterium phage Chel2 NC OO82O3 52047 98 101 Mycobacterium phage Che8 NC 004680 594.71 112 112 Mycobacterium phage Che9c NC 004683 57050 84 85 Mycobacterium phage Che9d NC 004686 56276 111 111 Mycobacterium phage Civ1 NC 004681 75931 141 142 Mycobacterium phage Cooper NC OO8195 70654 99 99 Mycobacterium phage Corndog NC 004685 69777 122 122 Mycobacterium phage D29 NC OO1900 49136 79 84 Mycobacterium phage DD5 NC 011022 S1621 87 87 US 9,056,899 B2 107 108 TABLE 5-continued Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. Table 5: Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. organism accession length proteins RNAS genes Mycobacterium hage Fruitloop NC 011288 S8471 in 102 O 102 Mycobacterium hage Giles NC 009993 S4S12 in 79 1 8O Mycobacterium hage Gumball NC 011290 64807 in 88 O 88 Mycobacterium hage Halo NC OO82O2 42289 in 65 O 65 Mycobacterium hage Jasper NC 011020 SO968 in 94 O 94 Mycobacterium hage KBG NC 011019 53572 in 89 O 89 Mycobacterium hage Konstantine NC 011292 68952 in 95 O 95 Mycobacterium hage Kostya NC 011056 75811 in 143 2 145 Mycobacterium hage L5 NC OO1335 S2297 in 85 3 88 Mycobacterium hage Lli NC OO8196 S6852 in 100 O 1OO Mycobacterium hage Lockley NC 011021 S1478 in 90 O 90 Mycobacterium hage Myrna NC 011273 1646O2 in 229 41 270 Mycobacterium hage Nigel NC 011044 69904 in 94 1 95 Mycobacterium hage Omega NC 004688 110865 in 237 2 239 Mycobacterium hage Orion NC OO8197 68427 in 100 O 1OO Mycobacterium hage PBI1 NC OO8198 64494 in 81 O 81 Mycobacterium hage PG1 NC OO5259 68.999 in 100 O 1OO Mycobacterium hage PLot NC OO8200 64787 in 89 O 89 Mycobacterium hage PMC NC OO8205 56692 in 104 O 104 Mycobacterium hage Pacca-0 NC 011287 S8554 in 101 O 101 Mycobacterium hage Phaedrus NC 011057 68090 in 98 O 98 Mycobacterium hage Pipefish NC O08199 69059 in 102 O 102 Mycobacterium hage Porky NC 011055 76312 in 147 2 149 Mycobacterium hage Predator NC 011039 701.10 in 92 O 92 Mycobacterium hage Pukovnik NC 011023 S2892 in 88 1 89 Mycobacterium hage Qyrzula NC OO8204 67188 in 81 O 81 Mycobacterium hage Ramsey NC 011289 S8578 in 108 O 108 Mycobacterium hage Rizal NC 011272 153894 in 220 35 255 Mycobacterium hage Rosebush NC 004684 67480 in 90 O 90 Mycobacterium hage Scott McG NC 011269 154017 in 221 36 257 Mycobacterium hage Solon NC 011267 49487 in 86 O 86 Mycobacterium hage Spud NC 011270 154906 in 222 35 257 Mycobacterium hage TM4 NC OO3387 52797 in 89 O 89 Mycobacterium hage Troll4 NC 011285 64618 in 84 O 84 Mycobacterium hage Tweety NC OO982O S8692 in 109 O 109 Mycobacterium phage U2 NC 009877 51277 in 81 O 81 Mycobacterium phage Wildcat NC OO82O6 78441 in 148 23 171 Mycoplasma phage MAV1 NC OO1942 15644 in 15 O 15 Mycoplasma phage P1 NC OO2515 11660 in 11 O 11 Mycoplasma phage phi MFV1 NC 005964 15141 in 15 O 17 Myxococcus phage Mx8 NC OO3O85 495.34 in 86 O 85 Natrialba phage PhiCh1 NC 004084 58498 in 98 O 98 Pasteurella phage F108 NC OO8193 3OSOS in 44 O 44 Phage Gifsy-1 NC 010392 48491 in 58 1 59 Phage Gifsy-2 NC 010393 45840 in 55 O 56 Phage catI NC OO9514 47021 in 60 O 60 Phage phijL001 NC OO6938 63649 in 90 O 90 Phormidium phage Pf-WMP3 NC OO9551 43249 in 41 O 41 Phormidium phage Pf-WMP4 NC 008367 40938 in 45 O 45 Prochlorococcus phage P-SSM2 NC OO6883 2524O1 in 329 1 330 Prochlorococcus phage P-SSM4 NC OO6884 178249 in 198 O 198 Prochlorococcus phage P-SSP7 NC OO6882 44970 in 53 O 53 Propionibacterium phage B5 NC OO3460 5804 in 10 O 10 Propionibacterium phage PA6 NC OO9541 29739 in 48 O 48 Pseudoalteromonas phage PM2 NC OOO867 10079 in 22 O 22 Pseudomonas phage 119X NC 007807 43365 in 53 O 53 Pseudomonas phage 14-1 NC O11703 6623S in 90 O 90 Pseudomonas phage 201phi2-1 NC 010821 316674 in 461 1 462 Pseudomonas phage 73 NC 007806 42999 in 52 O 52 Pseudomonas phage B3 NC OO6548 384-39 in 59 O 59 Pseudomonas phage D3 NC 002484 S642S in 95 4 99 Pseudomonas phage D3112 NC OO5178 37611 in 55 O 55 Pseudomonas phage DMS3 NC 008717 36415 in 52 O 52 Pseudomonas phage E. NC 007623 211215 in 2O1 O 2O1 Pseudomonas phage F10 NC 007805 391.99 in 63 O 63 Pseudomonas phage F116 NC OO6552 651.9S in 70 O 70 Pseudomonas phage F8 NC 007810 66O15 in 91 O 91 Pseudomonas phage LBL3 NC O11165 64427 in 87 O 87 Pseudomonas phage LKA1 NC 009936 41593 in 56 O 56 Pseudomonas hage LKD16 NC 009935 43200 in 53 O 53 Pseudomonas hage LMA2 NC O11166 66530 in 93 O 93 US 9,056,899 B2 109 110 TABLE 5-continued Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. Table 5: Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. organism accession length proteins RNAS genes Pseudomonas hage LUZ19 NC O 10326 43S48 S4 S4 Pseudomonas hage LUZ24 NC O 10325 45625 68 68 Pseudomonas hage M6 NC O O7809 594.46 85 85 Pseudomonas hage MP22 NC O O9818 36409 51 51 Pseudomonas hage MP29 NC O 11613 36632 51 51 Pseudomonas hage MP38 NC O 11611 3688S 51 51 Pseudomonas hage PA11 NC O O7808 49639 70 70 Pseudomonas hage PAJU2 NC O 11373 46872 79 79 Pseudomonas hage PB1 NC O 11810 65764 93 94 Pseudomonas hage PP7 NC O O1628 3588 4 4 Pseudomonas hage PRR1 NC O O8294 3573 4 4 Pseudomonas hage PT2 NC O 11107 42961 S4 S4 Pseudomonas hage PT5 NC O 111 OS 42954 52 52 Pseudomonas hage PaP2 NC O OS884 43783 58 58 Pseudomonas hage PaP3 NC O O4466 45503 71 75 Pseudomonas hage P NC O O1331 7349 14 14 Pseudomonas hage Pf3 NC O O1418 5833 9 9 Pseudomonas hage SN NC O 11756 66390 92 92 Pseudomonas hage Yu.A NC O 10116 S8663 77 77 Pseudomonas hage gh-1 NC O O4665 37359 4 4 Pseudomonas hage phil2 NC O O4173 6751 Pseudomonas nage NC O O41.75 4100 Pseudomonas nage NC O O4174 2322 Pseudomonas nage NC O O4172 6458 Pseudomonas nage NC O O4171 4213 Pseudomonas nage NC O O4170 2981 Pseudomonas nage NC O O3715 6374 Pseudomonas lage NC O O3716 4O63 Pseudomonas nage phi NC O O3714 2948 Pseudomonas nage NC O O3299 7051 Pseudomonas nage NC O O3300 4741 Pseudomonas hage phi8 NC O O3301 3.192 Pseudomonas phage CTX NC O O3278 3558O Pseudomonas phage phiKMV NC O OSO45 42519 : o : o Pseudomonas phage phiKZ NC O O4629 280334 306 306 Pyrobaculum spherical virus NC O O5872 28337 48 48 Pyrococcus abyssi virus 1 NC O O9597 18098 25 25 Ralstonia phage RSB1 NC O 12O1 43079 47 47 Ralstonia phage RSL1 NC O O811 231256 345 346 Ralstonia phage RSM NC O O8574 8999 15 15 Ralstonia phage RSM3 1399 8929 14 14 Ralstonia phage RSS1 O8575 666.2 12 12 Ralstonia phage p12J OS131 7118 Ralstonia phage phiRSA1 O938.2 3876O 51 51 Rhizobium phage 16-3 1103 6O195 110 109 Rhodothermus phage RM378 O4735 1299.08 146 146 Roseobacter phage SIO1 O2S19 39898 34 34 Salmonella phage E1 O495 45051 51 52 Salmonella phage Fels-1 O391 42723 52 52 Salmonella phage KS7 O6940 4O794 59 59 Salmonella phage SE1 1802 41941 67 67 Salmonella phage SETP3 O9232 42572 53 53 Salmonella phage ST64B O4313 4O149 56 56 Salmonella phage phiSG-JL2 NC O O807 3881S 55 55 Sinorhizobium phage PBC5 NC O O3324 S7416 83 83 Sodalis phage phiSG1 NC O O7902 S2162 47 47 Spiroplasma kuinkei virus NC O O998.7 7870 13 13 SkV1 CR2-3x Spiroplasma phage 1-C74 NC O O3793 7768 13 13 Spiroplasma phage 1-R8A2B NC O O1365 8273 12 12 Spiroplasma phage 4 NC O O3438 4421 Spiroplasma phage SVTS2 NC O O1270 6825 13 13 Sputnik Virophage NC O 11132 18343 21 21 Staphylococcusatiret is phage P68 NC O O46.79 18227 22 22 Staphylococci is phage 11 NC O O4615 43604 53 53 Staphylococci is phage 187 NC O O7047 3962O 77 77 Staphylococcus phage 2638A NC O O7051 41318 57 57 Staphylococci is phage 29 NC O O7061 428O2 67 67 Staphylococci is phage 37 NC O O7055 43681 70 70 Staphylococci is phage 3A NC O O7053 4309S 67 67 Staphylococci is phage 42E NC O O7052 45861 79 79 US 9,056,899 B2 111 112 TABLE 5-continued Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. Table 5: Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. organism accession length proteins RNAS genes Staphylococcits hage 44AHJD NC 004678 16784 21 21 Staphylococcits hage 47 NC 007054 44777 65 65 Staphylococcits hage 52A NC 007062 41690 60 60 Staphylococcits hage 53 NC 007049 43883 74 74 Staphylococcits hage 55 NC 007060 41902 77 77 Staphylococcits hage 66 NC 007046 18199 27 27 Staphylococcits hage 69 NC 007048 42732 69 69 Staphylococcits hage 71 NC 007059 43114 67 67 Staphylococcits hage 77 NC OO5356 41708 69 69 Staphylococcits hage 80alpha NC OO9526 43864 73 73 Staphylococcits hage 85 NC 007050 44283 71 71 Staphylococcits hage 88 NC 007063 43231 66 66 Staphylococcits hage 92 NC 007064 42431 64 64 Staphylococcits hage 96 NC 007057 43576 74 74 Staphylococcits hage CNPH82 NC OO8722 4342O 65 65 Staphylococcits hage EW NC 007056 45286 77 77 Staphylococcits hage G1 NC 007066 138715 214 214 Staphylococcits hage K NC OO5880 127395 115 115 Staphylococcits hage PH15 NC OO8723 44041 68 68 Staphylococcits hage PT1028 NC 007045 15603 22 22 Staphylococcits hage PVL NC OO2321 41401 62 62 Staphylococcits hage ROSA NC 007058 431 SS 74 74 Staphylococcits hage SAP-2 NC OO9875 17938 2O 2O Staphylococcits hage Twort NC 007021 1307O6 195 195 Staphylococcits hage X2 NC 007065 43440 77 77 Staphylococcits hage phi 12 NC 004616 44970 49 49 Staphylococcits hage phil3 NC 004617 42722 49 49 Staphylococcus hage phi2958PVL NC O11344 47342 60 59 Staphylococcits hage phiETA NC OO3288 43O81 66 66 Staphylococcits hage phiETA2 NC OO8798 4326S 69 69 Staphylococcits hage phiETA3 NC OO8799 43282 68 68 Staphylococcits hage philNR11 NC O10147 4301.1 67 67 Staphylococcits hage philNR25 NC 010808 44342 70 70 Staphylococcits hage philN315 NC 004740 44082 65 64 Staphylococcits hage philNM NC OO8583 43128 64 64 Staphylococcits hage philNM3 NC OO8617 44061 65 65 Staphylococcits hage phiPVL108 NC OO8689 44857 59 59 Staphylococcits hage phiSLT NC 002661 42942 61 61 Staphylococcits hage phiSauS NC 011612 45344 62 62 IPLA35 Staphylococcits phage phiSauS NC 011614 42526 l 60 O 61 IPLA88 Staphylococcits phage tp310-1 NC OO9761 41.407 59 59 Staphylococcits phage tp310-2 NC 0097.62 45710 67 67 Staphylococcits phage tp310-3 NC OO9763 41966 58 58 Staphylococcits prophage phiPV83 NC OO2486 45636 65 65 Stenotrophomonas phage S1 NC 011589 40287 48 48 Stenotrophomonas phage phiSMA9 NC 007189 6907 7 7 Streptococcusp hage 2972 NC 007019 34704 44 44 Streptococcusp hage 72.01 NC 002185 3S466 46 46 Streptococcusp hage 858 NC 010353 35543 46 46 Streptococcusp hage C1 NC 004814 16687 2O 2O Streptococcusp hage Cp-1 NC OO1825 19343 25 25 Streptococcusp hage DT1 NC OO2O72 34.815 45 45 Streptococcusp hage E.J-1 NC OO5294 42935 73 73 Streptococcusp hage MM1 NC OO3050 40248 53 53 Streptococcusp hage O1205 NC 004303 43075 57 57 Streptococcusp hage P9 NC 009819 40539 53 53 Streptococcusp hage PH15 NC 010945 391.36 60 60 Streptococcusp hage SM1 NC OO4996 34692 56 56 Streptococcusp hage SMP NC OO8721 36,216 48 48 Streptococcusp hage Sfil1 NC OO2214 398O7 53 53 Streptococcusp hage Sfil 9 NC OOO871 37370 45 45 Streptococcusp hage Sf21 NC OOO872 4O739 50 50 Streptococcusp hage phi3396 NC OO9018 38528 64 64 Streptococci is pyogenes phage 315.1 NC OO4584 39538 56 56 Streptococci is pyogenes phage 315.2 NC OO4585 41072 60 61 Streptococci is pyogenes phage 315.3 NC OO4586 34419 52 52 Streptococci is pyogenes phage 315.4 NC OO4587 41796 64 64 Streptococci is pyogenes phage 315.5 NC OO4588 382O6 55 55 Streptococci is pyogenes phage 315.6 NC OO4589 40014 51 51 US 9,056,899 B2 113 114 TABLE 5-continued Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. Table 5: Examples of bacteriophages which can be engineered to be an inhibitor-engineered bacteriophage, or a repressor-engineered bacteriophage or a Susceptibility-engineered bacteriophage as disclosed herein. organism accession length proteins RNAS genes Streptomyces phage VWB NC OO5345 4922O in 61 O 61 Streptomyces phage mulf6 NC 007967 381.94 in 52 O 52 Streptomyces phage phiBT1 NC 004664 41831 in 55 1 56 Streptomyces phage phiC31 NC OO1978 41491 in 53 1 S4 StX1 converting phage NC OO4913 59866 in 167 O 166 StX2 converting phage I NC OO3525 61765 in 166 O 166 StX2 converting phage II NC 004914 627O6 in 170 O 169 Stx2-converting phage 1717 NC O11357 62147 in 77 O 81 Stx2-converting phage 86 NC 008464 6O238 in 81 3 8O Sulfolobus islandicus filamentous NC OO3214 4O900 in 73 O 73 virus Sulfolobus islandicus rod-shaped virus 1 NC 004087 323O8 in 45 O 45 Sulfolobus islandicus rod-shaped virus 2 NC 004086 3S450 in S4 O S4 Sulfolobus spindle-shaped virus 4 NC 009986 15135 in 34 O 34 Sulfolobus spindle-shaped virus 5 NC 011217 15330 in 34 O 34 Sulfolobus turreted icosahedral virus NC OO5892 17663 in 36 O 36 Sulfolobus virus 1 NC OO1338 15465 in 32 O 33 Sulfolobus virus 2 NC OO5265 14796 in 34 O 34 Sulfolobus virus Kamchatka 1 NC OO5361 17385 in 31 O 31 Sulfolobus virus Ragged Hills NC OO5360 16473 in 37 O 37 Sulfolobus virus STSV1 NC OO6268 75294 in 74 O 74 Synechococcus phage P60 NC OO3390 47872 in 8O O 8O Synechococcus phage S-PM2 NC OO6820 196280 in 236 1 238 Synechococci is phage SynS NC OO9531 46214 in 61 O 61 Synechococcus phage syn9 NC OO8296 177300 in 226 6 232 Temperate phage phiNIH1.1 NC 003157 41796 in 55 O 55 Thaiassomonas phage BA3 NC 009990 37313 in 47 O 47 Thermoproteus tenax spherical virus 1 NC 006556 20933 in 38 O 38 Thermus phage IN93 NC 004.462 19603 in 40 O 32 Thermus phage P23-45 NC 009803 842O1 in 117 O 117 Thermus phage P74-26 NC 009804 83319 in 116 O 116 Thermus phage phiYS40 NC OO8584 152372 in 170 3 170 Vibrio phage K139 NC OO3313 33106 in 44 O 44 Vibrio phage KSF-1 phi NC OO6294 7107 in 12 O 12 Vibrio phage KVP40 NC 005083 244834 in 381 29 415 Vibrio phage VGJphi NC OO4736 7542 in 13 O 13 Vibrio phage VHML NC OO4456 431.98 in 57 O 57 Vibrio phage VP2 NC OO5879 39853 in 47 O 47 Vibrio phage VP5 NC OO5891 39.786 in 48 O 48 Vibrio phage VP882 NC OO9016 38.197 in 71 O 71 Vibrio phage VSK NC OO3327 6882 in 14 O 14 Vibrio phage Vf12 NC OO5949 7965 in 7 O 7 Vibrio phage Vf33 NC OO5948 7965 in 7 O 7 Vibrio phage V fo3K6 NC 002362 8784 in 10 O 10 Vibrio phage V fo4K68 NC 002363 6891 in 8 O 8 Vibrio phage fs NC 004306 6340 in 15 O 15 Vibrio phage fs2 NC OO1956 8651 in 9 O 9 Vibrio phage kappa NC 010275 33134 in 45 O 45 Vibrio phage VP4 NC 007149 39SO3 in 31 O 31 Vibrio phage VpV262 NC OO3907 46O12 in 67 O 67 Xanthomonas phage Cflic NC OO1396 73O8 in 9 O 9 Xanthomonas phage OP1 NC 007709 43785 in 59 O 59 Xanthomonas phage OP2 NC 007710 46643 in 62 O 62 Xanthomonas phage Xopa-11 NC O09543 44520 in 58 O 58 Xanthomonas phage Xp10 NC OO4902 44373 in 60 O 60 Xanthomonas phage Xp15 NC 007024 55770 in 84 O 84 Yersinia pestis phage phi A1122 NC 004777 37555 in 50 O 50 Yersinia phage Berlin NC OO8694 38564 in 45 O 45 Yersinia phage L-413C NC 004745 30728 in 40 O 40 Yersinia phage PY54 NC OO5069 46339 in 67 O 66 Yersinia phage Yepe2 NC 01.1038 38677 in 46 O 46 Yersinia phage phiYeO3-12 NC OO1271 39600 in 59 O 59 US 9,056,899 B2 115 116 TABLE 6 Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Table 6: Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages.

Name Description Length 500 Inducible pBadaraC promoter 1210 3453 Pbad promoter 130 2004 CMV promoter 654 2074 T7 promoter (strong promoter from T7 bacteriophage) 46 4889 OR21 of PR and PRM 101 4924 RecA DlexO DLacO1 862 4927 RecA S WTexO DLacO 862 4929 RecA S WTexO DLacO3 862 4930 RecAD consenLexO lacO1 862 4933 WT SulA Single LexO double LacO1 884 4935 WT SulA Single LexO double LacO2 884 4936 WT SulA Single LexO double LacO3 884 4937 sluA double lexO LacO1 884 4938 sluA double lexO LacO2 884 4939 sluA double lexO LacO3 884 SO38 pLac-RBS-T7 RNA Polymerase 2878 6014 yfbE solo trial 2 3O2 6102 pir (Induces the R6K Origin) 918 I7 90OS T7 Promoter 23 I7 32205 NOT Gate Promoter Family Member (D001O55) 124 J1 30O2 TetR repressed POPS/RIPS generator 74 J1 3O23 3OC6HSL + LuxR dependent POPS/RIPS generator 117 J23100 constitutive promoter family member 35 J23101 constitutive promoter family member 35 J23102 constitutive promoter family member 35 J23103 constitutive promoter family member 35 J23104 constitutive promoter family member 35 J23105 constitutive promoter family member 35 J23106 constitutive promoter family member 35 J23107 constitutive promoter family member 35 J23108 constitutive promoter family member 35 J23109 constitutive promoter family member 35 J23110 constitutive promoter family member 35 J23111 constitutive promoter family member 35 J23112 constitutive promoter family member 35 J23113 constitutive promoter family member 35 J23114 constitutive promoter family member 35 J23115 constitutive promoter family member 35 J23116 constitutive promoter family member 35 J23117 constitutive promoter family member 35 J23118 constitutive promoter family member 35 J44002 pBAD reverse 130 J5 2010 NFkappaB-dependent promoter 814 J5 2O34 CMV promoter 654 J61043 flhF2 Promoter 269 J63005 yeast ADH1 promoter 1445 J63006 yeast GAL1 promoter S49 KO82017 general recombine system 89 K091110 LacI Promoter 56 K091111 LacIQ promoter 56 KO94120 pLacIara-1 103 OOOOO Natural Xylose Regulated Bi-Directional Operator 303 OOOO1 Edited Xylose Regulated Bi-Directional Operator 1 303 OOOO2 Edited Xylose Regulated Bi-Directional Operator 2 303 18O11 PcstA (glucose-repressible promoter) 131 3SOOO pCpXR (CpxR responsive promoter) 55 37029 constitutive promoter with (TA) 10 between -10 and -35 39 elements B Ba K 37030 constitutive promoter with (TA)9 between -10 and -35 37 elements Ba 37046 150 bp inverted tetR promoter 150 Ba 37047 250 bp inverted tetR promoter 250 Ba 37048 350 bp inverted tetR promoter 350 Ba 37049 450 bp inverted tetR promoter 450 Ba 37050 650 bp inverted tetR promoter 6SO Ba 37051 850 bp inverted tetR promoter 8SO Ba ROO10 promoter (lacI regulated) 2OO Ba ROO11 Promoter (lacIregulated, lambda pl. hybrid) 55 Ba ROOS3 Promoter (p22 cII regulated) S4 Ba 1 O10 cI(1) fused to tetR promoter 834 Ba 1 051 Lux cassette right promoter 68 Ba 1 2006 Modified lamdba Prm promoter (repressed by 434 cI) 82 US 9,056,899 B2 117 118 TABLE 6-continued Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Table 6: Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages.

Name Description Length Ba 12036 Modified lamdba Prm promoter (cooperative repression by 434 91 cI) B Ba 12040 Modified lambda P(RM) promoter: -10 region from P(L) and 91 cooperatively repressed by 434 cI I13005 Promoter ROO11 w/YFP (-LVA) TT 920 I13006 Promoter R0040 w/YFP (-LVA) TT 920 I14015 P(Las) TetO 170 I14O16 P(Las) CIO 168 I14017 P(Rhl) 51 I14018 P(Bla) 35 I14033 P(Cat) 38 I14034 P(Kat) 45 I714890 OR321 of PR and PRM 121 I714925 RecA DlexO DLacO2 862 I714926 RecA DlexO DLacO3 862 I714928 RecA S WTexO DLacO2 862 I714931 RecAD consenLexO lacO2 862 I718018 dap Ap promoter 81 I720001 AraBp->rpoN 1632 I720002 glnKp->lacI 1284 I720003 NifHip->cI (lambda) 975 I720005 NifA lacIRFP 3255 I720006 GFP glng cI 291.3 I720007 araBp->rpoN (leucine landing pad) 51 I720008 Ara landing pad (pBBLP 6) 2O I720009 Ara landing pad (pBBLP 7) 23 I720010 Ara landing pad (pBBLP 8) 2O I721 001 Lead Promoter 94 I723020 Pu 32O I728456 MerRT: Mercury-Inducible Promoter + RBS (MerR + part of 635 MerT) B I741018 Right facing promoter (for xylF) controlled by xylRand CRP 221 cAMP I742124 Reverse complement Lac promoter 2O3 I746104 P2 promoter in agroperon from S. aureus 96 I746360 PF promoter from P2 phage 91 I746361 PO promoter from P2 phage 92 I746362 PP promoter from P2 phage 92 I746364 Psid promoter from P4 phage 93 I746365 PLL promoter from P4 phage 92 I748001 Putative Cyanide Nitrilase Promoter 271 I752000 Riboswitch(theophylline) 56 I761011 CinR, CinL and glucose controlled promotor 295 I761014 cinr + cinl (RBS) with double terminator 1661 I764001 Ethanol regulated promoter AOX1 867 I765OOO Fe promoter 104.4 I765OO1 UV promoter 76 I765.007 Fe and UV promoters 1128 J13210 pOmpR dependent POPS producer 245 J22106 rec A (SOS) Promoter 192 J23119 constitutive promoter family member 35 J24669 Tri-Stable Toggle (Arabinose induced component) 31OO J3902 PrFe (PI+ PII rus operon) 272 J58 100 AND-type promoter synergistically activated by cI and CRP 106 J61051 Psal1 1268 KO85005 (lacI)promoter->key3c->Terminator 40S KO88007 GlnRS promoter 38 KO89004 phaC Promoter (-663 from ATG) 663 KO89005 -35 to Tc start site of phaC 49 KO89006 -663 to Tc start site of phaC 361 KO905O1 Gram-Positive IPTG-Inducible Promoter 107 KO90504 Gram-Positive Strong Constitutive Promoter 239 K091 100 pLac lux hybrid promoter 74 K091101 pTet Lac hybrid promoter 83 K091 104 plac/Mnt Hybrid Promoter 87 KO91105 pTet/Mnt Hybrid Promoter 98 K091106 LSrA?cI hybrid promoter 141 KO91107 pLux/cI Hybrid Promoter 57 K091114 LSrARPromoter 248 K091115 LSrRPromoter 1OO K091116 LSrAPromoter 126 K091117 plas promoter 126 K091143 pLasicI Hybrid Promoter 164 US 9,056,899 B2 119 120 TABLE 6-continued Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Table 6: Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages.

Name Description Length Ba KO91146 pLasiLux Hybrid Promoter 126 Ba KO91184 pLux/cI+ RBS + LuxS + RBS + Mint + TT + pLac/Mint + RBS + LuxS + 2616 RBS -- cI - TT KO93000 pRecA with Lex A binding site 48 O1017 MioC Promoter (DNAa-Repressed Promoter) 319 O1018 MioC Promoter (regulating tetR) 969 OSO20 tetR - operator 29 OSO21 cI - operator 27 OSO22 lex A- operator 31 OSO23 lac I - operator 25 OSO24 Gal4 - operator 27 OSO26 Gall promoter S49 05O27 cyc100 minimal promoter 103 OSO28 cyc70 minimal promoter 103 OSO29 cyc43 minimal promoter 103 OSO3O cycl28 minimal promoter 103 OSO31 cyc16 minimal promoter 103 O8O14 PR 234 O8O16 PP 4O6 O8O2S Pl 2OO O92OO AraC and TetR promoter (hybrid) 132 OOOS Alpha-Cell Promoter MF(ALPHA)2 500 OOO6 Alpha-Cell Promoter MF(ALPHA)1 5O1 OO16 A-Cell Promoter STE2 (backwards) 500 2118 rrnBP1 promoter 503 2318 {

Name Description Length Ba 715052 Trp Leader Peptide and anti-terminator? terminator 134 Ba 715053 Trp Leader Peptide and anti-terminator? terminator with hixC 159 insertion I717002 Pr from lambda switch 177 I723011 pontR (estimated promoter for DntR) 26 I723013 ponta (estimated promoter for DntA) 33 I723018 Pr (promoter for XylR) 410 I731004 FecA promoter 90 I732021 Template for Building Primer Family Member 59 I732200 NOT Gate Promoter Family Member (D001Olwt1) 25 I732201 NOT Gate Promoter Family Member (D001O11) 24 I732202 NOT Gate Promoter Family Member (D001O22) 24 I732203 NOT Gate Promoter Family Member (D001O33) 24 I732204 NOT Gate Promoter Family Member (D001O44) 24 I732206 NOT Gate Promoter Family Member (D001O66) 24 I732207 NOT Gate Promoter Family Member (D001O77) 24 I732270 Promoter Family Member with Hybrid Operator (D001O12) 24 I732271 Promoter Family Member with Hybrid Operator (D001O16) 24 I732272 Promoter Family Member with Hybrid Operator (D001O17) 24 I732273 Promoter Family Member with Hybrid Operator (D001O21) 24 I732274 Promoter Family Member with Hybrid Operator (D001O24) 24 I732275 Promoter Family Member with Hybrid Operator (D001O26) 24 I732276 Promoter Family Member with Hybrid Operator (D001O27) 24 I732277 Promoter Family Member with Hybrid Operator (D001O46) 24 I732278 Promoter Family Member with Hybrid Operator (D001O47) 24 I732279 Promoter Family Member with Hybrid Operator (D001O61) 24 I732301 NAND Candidate (U073O26D001O16) 2O I732302 NAND Candidate (UO73O27D001O17) 2O I7323.03 NAND Candidate (U073O22D001O46) 2O I732304 NAND Candidate (U073O22D001O47) 2O I732305 NAND Candidate (U073O22D059O46) 78 I732306 NAND Candidate (U073O11 D002O22) 21 I732351 NOR Candidate (UO37O11 D002O22) 85 I732352 NOR Candidate (UO35O44D001O22) 82 I732400 Promoter Family Member (UO97NUL + D062NUL) 65 I732401 Promoter Family Member (UO97O11 + D062NUL) 85 I732402 Promoter Family Member (UO85O11 + D062NUL) 73 I732403 Promoter Family Member (U073O11 + D062NUL) 61 I732404 Promoter Family Member (U061O11 + D062NUL) 49 I732405 Promoter Family Member (U049011 + D062NUL) 37 I732406 Promoter Family Member (UO37O11 + D062NUL) 25 I732407 Promoter Family Member (UO97NUL + D002O22) 25 I732408 Promoter Family Member (UO97NUL + D014O22) 37 I732409 Promoter Family Member (UO97NUL + D026O22) 49 I732410 Promoter Family Member (UO97NUL + D038O22) 61 I732411 Promoter Family Member (UO97NUL + D050O22) 73 I732412 Promoter Family Member (UO97NUL + D062O22) 85 I732413 Promoter Family Member (UO97O 45 I732414 Promoter Family Member (UO97O 57 I732415 Promoter Family Member (UO97O 69 I732416 Promoter Family Member (UO97O 81 I732417 Promoter Family Member (UO97O 93 I732418 Promoter Family Member (UO97O 205 I732419 Promoter Family Member (UO85O 33 I732420 Promoter Family Member (UO85O 45 I732421 Promoter Family Member (UO85O 57 I732422 Promoter Family Member (UO85O 69 I732423 Promoter Family Member (UO85O 81 I732424 Promoter Family Member (UO85O 93 I732425 Promoter Family Member (U073O 21 I732426 Promoter Family Member (U073O 33 I732427 Promoter Family Member (U073O 45 I732428 Promoter Family Member (U073O 57 I732429 Promoter Family Member (U073O 69 I732430 Promoter Family Member (U073O 81 I732431 Promoter Family Member (U061O 09 I732432 Promoter Family Member (U061O 21 I732433 Promoter Family Member (U061O 33 I732434 Promoter Family Member (U061O 45 I732435 Promoter Family Member (U061O 57 I732436 Promoter Family Member (U061O 69 I732437 Promoter Family Member (U0490 97 I732438 Promoter Family Member (U0490 09 US 9,056,899 B2 125 126 TABLE 6-continued Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Table 6: Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages.

Name Description Length 439 Promoter Family Member (U049011 + D026O22) 121 440 Promoter Family Member (U049011 + D038O22) 133 441 Promoter Family Member (U049011 + D050O22) 145 442 Promoter Family Member (U049011 + D062O22) 157 443 Promoter Family Member (UO37O11 + D002O22) 85 444 Promoter Family Member (UO37O11 + D014O22) 97 445 Promoter Family Member (UO37O11 + D026O22) 109 446 Promoter Family Member (UO37O11 + D038O22) 121 447 Promoter Family Member (UO37O11 + D050O22) 133 448 Promoter Family Member (UO37O11 + D062O22) 145 450 Promoter Family Member (U073O26+ D062NUL) 161 451 Promoter Family Member (U073O27 + D062NUL) 161 452 Promoter Family Member (U073O26+ D062O61) 181 OO8 ORE1X Oleate response element 273 O09 ORE2X oleate response element 332 O10 This promoter encoding for a thiolase involved in beta 8SO oxidation of fatty acids. 101 Double Promoter (constitutive/TetR, negative) 83 102 Double Promoter (cI, negative/TetR, negative) 97 103 Double Promoter (lacI, negative/P22 cII, negative) 87 104 Double Promoter (LuxR/HSL, positive? P22 cII, negative) 101 105 Double Promoter (LuxR/HSL, positive? cI, negative) 99 106 Double Promoter (TetR, negative? P22 cII, negative) 84 107 Double Promoter (cI, negative LacI, negative) 78 O15 two way promoter controlled by XylR and Crp-CAmp 301 O17 dual facing promoter controlled by xylRand CRP-cAMP 3O2 (I741015 reverse complement) O19 Right facing promoter (for xylA) controlled by xylR and CRP 131 cAMP BB promoter to xylF without CRP and several binding sites for 191 XylR B O21 promoter to XylA without CRP and several binding sites for 87 XylR I74 109 Lambda Or operator region 82 I742 126 Reverse lambda cI-regulated promoter 49 I746 363 PV promoter from P2 phage 91 I746 665 Pspac-hy promoter 58 I75 500 pcI (for positive control of pcI-lux hybrid promoter) 77 I75 5O1 plux-cI hybrid promoter 66 I75 502 plux-lachybrid promoter 74 I756 OO2 Kozak Box I756 O14 Lex Aoperator-MajorLatePromoter 229 I756 O15 CMV Promoter with lac operator sites 663 I756 O16 CMV-tet promoter 610 I756 O17 U6 promoter with tet operators 341 I756 O18 Lambda Operator in SV-40 intron 411 I756 O19 Lac Operator in SV40 intron 444 I756 O2O Tet Operator in SV40 intron 391 I756 O21 CMV promoter with Lambda Operator 630 I760 005 Cu-sensitive promoter 16 I761 OOO cinr + cinl (RBS) 1558 I761 OO1 OmpR binding site 62 I766 2OO Ste2 1OOO I766 214 pGal1 10O2 I766 555 pCyc (Medium) Promoter 244 I766 556 pAdh (Strong) Promoter 15O1 I766 557 pSte5 (Weak) Promoter 6O1 I766 558 pFig1 (Inducible) Promoter 1OOO I920 1 ambda cI operatoribinding site 82 JO1005 pspoIIE promoter (spoOAJO1004, positive) 2O6 JO1006 Key Promoter absorbs 3 59 JO3OO7 Maltose specific promotor 2O6 JO31 OO -- No description -- 847 JO4700 Part containing promoter, riboswitch mTCT8-4 theophylline 258 aptamer (JO4705), and RBS B JO4705 Riboswitch designed to turn “ON” a protein 38 JO4800 O4800 (Rev AptRibo) contains a theophylline aptamer 258 upstream of the RBS that should act as a riboswi Ba O4900 Part containing promoter, 8 bp, RBS, and riboswitch mTCT8-4 258 heophylline aptamer (JO4705) Ba OS209 Modifed PrPromoter 49 Ba OS210 Modifed Prm-- Promoter 82 Ba O5215 Regulator for R1-CREBH 41 US 9,056,899 B2 127 128 TABLE 6-continued Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Table 6: Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Description Length JO5216 Regulator for R3-ATF6 41 JO5217 Regulator for R2-YAP7 41 JO5218 Regulator for R4-cMaf 41 JO5221 Tripple Binding Site for R3-ATF6 62 JO5222 ZF-2*e2 Binding Site 37 JO5500 Sensing Device A (cI) 2371 JO55O1 Sensing Device B (cI+ LVA) 2337 J06403 RhIR promoter repressible by CI 51 J07007 ctX promoter 145 JO7010 ToxR inner (aa's 1-198; cytoplasm +TM) 594 JO7019 Feca Promoter (with Fur box) 86 JO7041 POPS/RIPS generator (R0051:B0030) 72 JO7042 POPS/RIPS generator (R0040::B0030) 77 J11003 control loop for PI controller with BBa J1 1002 961 J13211 ROO4.O.BOO32 75 J13212 ROO4.O.BOO33 73 J15301 Pars promoter from Escherichia coli chromosomalars operon. 127 J155O2 copA promoter 287 J16101 BanAp - Banana-induced Promoter 19 J16105 HelPp - “Help' Dependant promoter 26 J16400 ron sensitive promoter (test delete later) 26 J21002 Promoter + LuxR 998 J21003 Promoter - TetR 904 J21004 Promoter + LacL 1372 J21006 LuxR, TetR Generator 1910 J21007 LuxR, TetR, LacL Generator 3290 J22052 Pcya 65 J22086 pX (DnaA binding site) 125 J22126 RecA (SOS) promoter 186 J23150 bp mutant from J23107 35 J23151 bp mutant from J23114 35 J24000 CafAp (Cafeine Dependant promoter) 14 J24001 WigLp (Wiggle-dependent Promotor) 46 J24670 Tri-Stable Toggle (Lactose induced component) 1877 J24671 Tri-Stable Toggle (Tetracycline induced component) 2199 J24.813 ORA3 Promoter from S. cerevisiae 137 J26003 Mushroom Activated Promoter 23 J31013 pLac Backwards (cf. BBa R0010) 2OO J31014 38 J3102 153 J31020 produces taRNA 295 J31022 comK transcription activator from B. subtilis 578 J33100 Arsk and Ars Promoter 472 J34800 Promoter tetracyclin inducible 94 J34806 promoter lac induced 112 J34.809 promoter lac induced 125 J34814 T7 Promoter 28 J45503 hybB Cold Shock Promoter 393 J45504 htpG Heat Shock Promoter 40S J45992 Full-length stationary phase osmy promoter 199 J45993 Minimal stationary phase osmY promoter 57 J45994 Exponential phase transcriptional control device 1109 J48103 ron promoter 140 J48104 NikR promoter, a protein of the ribbon helix-helix family of 40 trancription factors that repress expre J48106 wnfEI 891 J48107 UGT008-3 Promoter/Met32p 588 J48110 Fe Promoter-mRFP1 1009 J48111 E. Coi NikR 926 J48112 wnfH: vanadium promoter 1816 J49000 Roid Rage J49001 Testosterone dependent promoter for species Bicyclus Bicyclus 89 J49006 Nutrition Promoter J4906 WrooHEAD2 (Wayne Rooney's Head dependent promoter) 122 J54015 Protein Binding Site LacI 42 J54O16 promoter lacq S4 J54017 promoter always 98 J54018 promoter always 98 J54101 deltaP-GFP(A) J54102 DeltaP-GFP(A) 813 J54110 MelR regulated promoter 76 J54120 EmrR regulated promoter 46 J54130 BetI regulated promoter 46 US 9,056,899 B2 129 130 TABLE 6-continued Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Table 6: Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Description Length J54200 acq. Promoter 50 J54210 RbsR Binding Site 37 J54220 FadR Binding Site 34 J54230 TetR regulated 38 J54250 LacI Binding Site 42 J56012 nvertible sequence of includes Ptrc promoter 409 J56015 acIQ - promoter sequence 57 J61045 spv spv operon (PoPS out) 1953 J61054 HIP-1 Promoter 53 J61055 HIP-1 fmr. Promoter 53 J64000 rhII promoter 72 J64001 psicA from Saimoneiia 143 J64010 asI promoter 53 J64065 cI repressed promoter 74 J64067 LuxR+ 3OC6HSL independent ROO65 98 J64068 increased strength R0051 49 J64069 ROO65 with luxbox deleted 84 J64700 Trp Operon Promoter 616 J64712 LasR/LasI Inducible & RHLR/RHLI repressible Promoter 157 J64750 SPI-1 TTSS secretion-linked promoter from Salmonella 167 J64800 RHLR/RHILI Inducible & LasR/LasIrepressible Promoter 53 J64804 The promoter region (inclusive of regulator binding sites) of 135 he B. subtilis RocDEF operon J64931 glnKp promoter 147 J64951 E. Coli CreABCD phosphate sensing operon promoter 81 J64979 gln Ap2 151 J64980 Ompr-P strong binding, regulatory region for Team ChallengeO3-2007 B J64981 Ompr-P strong binding, regulatory region for Team 82 Challenge03-2007 B J64982 Ompr-P strong binding, regulatory region for Team Challenge 25 O3-2007 J64983 Strong OmpR Binding Site 2O J64986 LacI Consensus Binding Site 2O J64987 LacI Consensus Binding Site in sigma 70 binding region 32 J64991 TetR 19 J64995 Phage -35 site J64997 T7 consensus -10 and rest 19 J64998 consensus -10 and rest from SP6 19 J70025 Promoter for tetM gene, from pBOT1 plasmid, p AMbeta1 345 J72005 {Ptet: promoter in BBb S4 KO76017 Ubc Promoter 1219 KO78101 aromatic compounds regulatory pcbC promoter 129 KO79017 Lac symmetric - operator library member 2O KO79018 Lac 1 - operator library member 21 KO79019 Lac 2 - operator library member 21 KO79036 Tet O operator library member 15 KO79037 TetO-4C - operator library member 15 KO79038 TetO-wt4C5G - operator library member 15 KO79039 Lex A1 - operaor library member 16 KO79040 Lex A2 - Opeartor library member 16 KO79041 Lambda OR1 - operator library member 17 KO79042 Lambda OR2 - operator library member 17 KO79043 Lambda OR3 - operator library member 17 KO79045 Lac operator library 78 KO79046 Tet operator library 61 KO79047 Lambda operator library 67 KO79048 Lex A operator library 40 KO80000 1582 KO80001 A20 alpha cardiac actin miniPro-BMP4 1402 KO80003 CMV-rtTA 1413 KO80005 TetO (TRE)-nkx2.5-fmdv2A-dsRed 2099 KO80006 TetO (TRE)-gata4-fmdv2A-dsRed 2447 KO80008 TetO (TRE)-nkX-2.5-fmdv2A-gata4-fmdv2A-dsRed 3497 KO85004 riboswitch system with GFP 1345 KO85006 pTet->lock3d->GFP->Ter 932 KO86017 unmodified Lutz-Bujard LacO promoter 55 KO86O18 modified Lutz-Bujard LacO promoter, with alternative sigma 55 factor o24 B KO86019 modified Lutz-Bujard LacO promoter, with alternative sigma 55 factor o24 KO86O20 modified Lutz-Bujard LacO promoter, with alternative sigma 55 factor o24 US 9,056,899 B2 131 132 TABLE 6-continued Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriop nages. Table 6: Examples of promoters which can be opera ively linked to the nucleic acid in the engineered bacteriophages.

Name Description Length

Ba KO86021 modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o24 Ba modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o28 Ba modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o28 Ba KO86024 modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o28 Ba modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o28 Ba modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o2 Ba KO86027 modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o2 Ba modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o2 Ba modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o2 Ba KO86030 modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o8 Ba KO86031 modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o8 Ba KO86032 modified Lu Bui LacO OO er, wi El 8 (8. ive sigma 55 actor o8 B KO86033 modified Lu Bui LacO OO er, wi El 8 (8. ive sigma actor o8 KO90502 Gram-Positive Xy ose-Inducible Promoter KO90503 Gram-Positive Genera Constitutive Promoter K091112 pLacIQ1 promoter KO91156 pLux KO91157 pLux/Las Hybrid Promoter KO93008 reverse BBa ROO 1 KO940O2 plambda P(O-R12) KO94140 pLacIq OOOO3 Edited Xylose Regulated Bi-Directional Operator 3 O1OOO Dual-Repressed Promoter for p22 mint and TetR O1 OO1 Dual-Repressed Promoter for LacI and LambdacI O10O2 Dual-Repressed Promoter for p22 cII and TetR O2909 CA11 gate from synthetic algorithm v1.1 O2910 A12 gate from synthetic algorithm v1.1 O2911 CA13 gate from synthetic algorithm v1.2 O2912 TA12 plus pause sequence O29SO CAOIn null anti-sense input O2951 TA1In anti-sense input to TA1 (BBa K102901) O2952 TA2In anti-sense input to BBa K102952 O2953 TA13n anti-sense input to TA3 (BBa K102903) O2954 TA6In anti-sense input to BBa K102904 O2955 TA7In anti-sense input to BBa K102905 O2956 TA8In anti-sense input to BBa K102906 O2957 TA9In anti-sense input to BBa K102907 O2958 TA10In anti-sense input to BBa K102908 O2959 TA11 In anti-sense input to BBa K102909 O2960 CA12In anti-sense input to anti-terminator BBa K102910 O2961 TA13In anti-sense input to BBa K102911 O2962 TA14In anti-sense input to BBa K102912 O3O21 modified T7 promoter with His-Tag O3O22 Plac with operator and RBS O6673 8xLexAops-Cyclip O668O 8xLexAops-Fig1P O6694 Adh1P (Adh1 Promoter, A end) O6699 Gall Promoter O9584 this is a test part, disregard it 1OOO)4 Alpha-Cell Promoter SteS 1OOO7 A-Cell Promoter MFA2 10008 A-Cell Promoter MFA1 10009 A-Cell Promoter STE2 1 OO14 A-Cell Promoter MFA2 (backwards) 10015 A-Cell Promoter MFA1 (RtL) 12139 oriR6K conditional replication origin 12148 phoPp1 magnesium promoter 12149 PmgtCB Magnesium promoter from Salmonella 12321 { H-NS using MG1655 reverse oligo in BBb format 12701 hns promoter US 9,056,899 B2 133 134 TABLE 6-continued Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Table 6: Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages.

Name Description Length 12706 Pspv2 from Salmonella 474 12707 Pspv from Salmonella 1956 12708 PflA 210 12711 rbs.spvR! 913 12900 Pbad 1225 12904 PconBS 41 1290S PconCS 41 12906 PConC6 41 12907 Pcon 41 13010 overlapping T7 promoter 40 13011 more overlapping T7 promoter 37 13012 weaken overlapping T7 promoter 40 16201 ureD promoter from Pmirabilis 19000 Constitutive weak promoter of lacz 38 19001 Mutated Lacz promoter 38 20010 Triple lexO 114 20023 lexA DBD 249 21011 promoter (lacIregulated) 232 21014 promoter (lambda cI regulated) 90 24OOO CYCYeast Promoter 288 24002 Yeast GPD (TDH3) Promoter 681 25100 nir promoter from Synechocystis sp. PCC6803 88 31017 p qrr4 from Vibrio harveyi 275 37085 optimized (TA) repeat constitutive promoter with 13 bp 31 between -10 and -35 elements B Ba 37086 optimized (TA) repeat constitutive promoter with 15 bp 33 between -10 and -35 elements Ba 37087 optimized (TA) repeat constitutive promoter with 17 bp 35 between -10 and -35 elements Ba 37088 optimized (TA) repeat constitutive promoter with 19 bp 37 between -10 and -35 elements Ba 37089 optimized (TA) repeat constitutive promoter with 21 bp 39 between -10 and -35 elements Ba 37090 optimized (A) repeat constitutive promoter with 17 bp between 35 -10 and -35 elements B Ba K 37091 optimized (A) repeat constitutive promoter with 18 bp between 36 -10 and -35 elements 37124 LacI-repressed promoter A81 103 43010 Promoter ctic for B. subtiis 56 43011 Promoter gsiB for B. subtilis 38 43012 Promoter vega constitutive promoter for B. subtilis 97 43013 Promoter 43 a constitutive promoter for B. subtilis 56 43014 Promoter Xyl for B. subtilis 82 43015 Promoter hyper-spank for B. subtilis 101 45152 Hybrid promoter: P22 c2, LacI NOR gate 142 57042 Eukaryotic CMV promoter 654 6SOOO MET 25 Promoter 387 65015 p.ADH1 yeast constituative promoter 1445 65017 LexA binding sites 393 65037 TEF2 yeast constitutive promoter 403 Ba M13101 M13K07 gene I promoter 47 Ba M13102 M13K07 gene II promoter 48 Ba M13103 M13K07 gene III promoter 48 Ba M13104 M13K07 gene IV promoter 49 Ba M13105 M13K07 gene V promoter 50 Ba M13106 M13K07 gene VI promoter 49 Ba M13108 M13K07 gene VIII promoter 47 Ba M13110 M13110 48 Ba M31201 Yeast CLB1 promoter region, G2/M cell cycle specific 500 Ba M31232 Redesigned M13K07 Gene III Upstream 79 Ba M31252 Redesigned M13K07 Gene V Upstream 72 Ba M31272 Redesigned M13K07 Gene VII Upstream 50 Ba M31282 Redesigned M13K07 Gene VIII Upstream 146 Ba M31292 Redesigned M13K07 Gene IX Upstream 69 Ba M31302 Redesigned M13K07 Gene X Upstream 115 Ba M31370 tacI Promoter 68 Ba M31519 Modified promoter sequence of g3. 60 Ba ROOO1 HMG-CoA Dependent RBS Blocking Segment 53 Ba ROO100 Tet promoter and sRBS 67 Ba R00101 VM1.0 to RiPS converter 36 Ba R0085 T7 Consensus Promoter Sequence 23 Ba RO18O T7 RNAP promoter 23 Ba RO181 T7 RNAP promoter 23 US 9,056,899 B2 135 136 TABLE 6-continued Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Table 6: Examples of promoters which can be operatively linked to the nucleic acid in the engineered bacteriophages. Name Description Length BBa RO182 T7 RNAP promoter 23 BBa RO183 T7 RNAP promoter 23 BBa RO184 T7 promoter (lacI repressible) 44 BBa RO185 T7 promoter (lacI repressible) 44 BBa RO186 T7 promoter (lacI repressible) 44 BBa RO187 T7 promoter (lacI repressible) 44 BBa R1028 Randy Rettberg Standardillator BBa R1074 Constitutive Promoter I 49 BBa R1075 Constitutive Promoter II 49 BBa R2108 Promoter with operator site for C2003 72 BBa R2110 Promoter with operator site for C2003 72 BBa R2111 Promoter with operator site for C2003 72 BBa R2112 Promoter with operator site for C2003 72 BBa R2113 Promoter with operator site for C2003 72 BBa R2182 RiPS generator 44 BBa R22O1 C2006-repressible promoter 45 BBa R6182 RiPS generator 36 BBa SO3331 --Specify Parts List 30 BBa SO3385 Cold-sensing promoter (hybB) BBa ZO251 T7 strong promoter 35 BBa ZO252 T7 weak binding and processivity 35 BBa ZO253 T7 weak binding promoter 35 BBa ZO294 A1, A2, A3, box A 435

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SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 42

<21 Os SEQ ID NO 1 &211s LENGTH: 1434 &212s. TYPE: DNA <213> ORGANISM: Escherichia coli

<4 OOs SEQUENCE: 1 atgtttalaga atgcatttgc talacctgcaa aaggtoggta aatcgctgat gctg.ccggta 60 to cqtactgc ct atcgcagg tattotgctg gg.cgt.cggitt Cogcga attt cagctggctg 12O

ccc.gc.cgttg tatcgcatgt tatggcagaa gCaggcggitt cogt ctittgc aaac atgc.ca 18O

Ctgatttittg cgatcggtgt cqcccticggc tittaccalata acgatggcgt atcc.gc.gctg 24 O

gcc.gcagttgttgcc tatgg catcatggitt aaaaccatgg cc.gtggttgc gccactggta 3 OO

Ctgcatttac Ctgctgaaga aatcgc.ctict aaac acctgg cqgatactgg cqtact cqga 360

gggattatct Coggtgcgat cqcagcgtac atgtttalacc gtttct accg tattaa.gctg 42O

cctgagtatic ttggcttctt td.ccggtaaa cqctttgttgc cqat catttic tdgcctggct 48O

gcc at ctitta citgg.cgttgt gctgtcc titc atttggcc.gc cqattggttctgcaatccag 54 O