US 20100322903A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0322903A1 Collins et al. (43) Pub. Date: Dec. 23, 2010

(54) ENGINEERED AS Related U.S. Application Data ADUVANTS FOR ANTIMICROBAL AGENTS AND COMPOSITIONS AND METHODS OF (60) Provisional application No. 61/020,197, filed on Jan. USE THEREOF 10, 2008. Publication Classification (75) Inventors: James J Collins, Newton, MA (US); Timothy Kuan-Ta Lu, (51) Int. Cl. Boston, MA (US) A6II 35/76 (2006.01) CI2N 7/01 (2006.01) Correspondence Address: A6IP3L/04 (2006.01) RONALDI. EISENSTEIN (52) U.S. Cl...... 424/93.2:435/235.1 100 SUMMER STREET, NIXON PEABODY LLP (57) ABSTRACT BOSTON, MA 02110 (US) The present invention relates to the treatment and prevention (73) Assignees: TRUSTEES OF BOSTON of bacteria and bacterial infections. In particular, the present UNIVERSITY, Boston, MA (US); invention relates to engineered bacteriophages used in com MASSACHUSETTS INSTITUTE bination with antimicrobial agents to potentiate the antimi OF TECHNOLOGY, Cambridge, crobial effect and bacterial killing by the antimicrobial agent. MA (US) The present invention generally relates to methods and com positions comprising engineered bacteriophages and antimi (21) Appl. No.: 12/812,212 crobial agents for the treatment of bacteria, and more particu larly to bacteriophages comprising agents that inhibit (22) PCT Filed: Jan. 12, 2009 resistance genes and/or cell Survival genes, and/or bacteriophages comprising repressors of SOS response genes (86). PCT No.: PCT/USO9/30755 or inhibitors of antimicrobial defense genes and/or express ing an agent which increases the sensitivity of bacteria to an S371 (c)(1), antimicrobial agent in combination with at least one antimi (2), (4) Date: Aug. 31, 2010 crobial agent, and their use thereof. Patent Application Publication Dec. 23, 2010 Sheet 1 of 28 US 2010/0322903A1

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ENGINEERED BACTERIOPHAGES AS co-infecting Enterococcus faecalis, which itself became ADUVANTS FOR ANTMICROBAL AGENTS completely resistant to Vancomycin in nosocomial settings by AND COMPOSITIONS AND METHODS OF 1988'''. Drugs such as that induce the SOS USE THEREOF response can even promote the horizontal dissemination of antibiotic resistance genes by mobilizing genetic elements' CROSS REFERENCE TO RELATED 16. For example, Streptococcus pneumoniae and Neisseria APPLICATIONS gonorrhoeae have also obtained resistance to 0001. This application claims priority under 35 U.S.C. (Morens, et al., (2004) Nature 430: 242-249). Sub-inhibitory 119(e) of U.S. Provisional Patent Application Ser. No. concentrations or incomplete treatment courses can present 61/020,197 filed 10 Jan. 2008, the contents of which are evolutionary pressures for the development of antibiotic incorporated herein by reference in their entirety. resistance'7. Use of antibiotics outside of clinical settings, for example in livestockfor the agricultural industry, has contrib GOVERNMENT SUPPORT uted to the emergence of resistant organisms such as methi 0002 This invention was made with the Government Sup cillin-resistant staphylococci and is unlikely to abate due to port under Contract No. EF-0425719 awarded by the economic reasons and modern farming practices''. Resis National Science Foundation (NSF) and Contract No. tance genes that develop in non-clinical settings may be Sub OD003644 awarded by the National Institutes of Health sequently transmitted to bacterial populations which infect (NIH). The Government has certain rights in the invention. humans, worsening the antibiotic resistance problem'. 0007. In addition to acquiring antibiotic-resistance genes, FIELD OF THE INVENTION a small Subpopulation of cells known as persisters can Survive 0003. The present invention relates to the field of treat antibiotic treatment by entering a metabolically-dormant ment and prevention of bacteria and bacterial infections. In state'''. Persister cells do not typically carry genetic muta particular, the present invention relates to engineered bacte tions but rather exhibit phenotypic resistance to antibiotics'. riophages used in combination with antimicrobial agents to In , the fraction of a population which repre potentiate the antimicrobial effect and bacterial killing of the sents persister cells increases dramatically in late-exponential antimicrobial agent. and stationary phases. Chromosomally-encoded toxins may be important contributors to the persister phenotype but the BACKGROUND underlying mechanisms that control the stochastic persis 0004 Bacteria rapidly develop resistance to antibiotic tence phenomena are not well understood’. Persisters drugs within years of first clinical use". Antibiotic resistance constitute a reservoir of latent cells that can begin to regrow can be acquired by horizontal gene transfer or result from once antibiotic treatment ceases and may be responsible for persistence, in which a small fraction of cells in a population the increased antibiotic tolerance observed in bacterial bio exhibits a non-inherited tolerance to antimicrobials. Since films'. By surviving treatment, persisters may play an antimicrobial drug discovery is increasingly lagging behind important role in the development of mutations or acquisition the evolution of antibiotic resistance, there is a pressing need of genes that confer antibiotic resistance. for new antibacterial therapies. 0008. Several strategies have been proposed for control 0005 Bacterial infections are responsible for significant ling antibiotic resistant infections. New classes of antibiotics morbidity and mortality in clinical settings. Though the would improve the arsenal of drugs available to fight antibi advent of antibiotics has reduced the impact of bacterial dis otic-resistant bacteria but few are in pharmaceutical pipe eases on human health, the constant evolution of antibiotic lines. Surveillance and containment measures have been resistance poses a serious challenge to the usefulness of instituted in government and hospitals so that problematic today’s antibiotic drugs'. Infections that would have been infections are rapidly detected and isolated but do not address easily cured by antibiotics in the past are now able to survive the fundamental evolution of resistance'. Cycling antibiotics to a greater extent, resulting in sicker patients and longer is one method of controlling resistant organisms but is costly hospitalizations. The economic impact of antibiotic-re and may not be efficacious’’’. Reducing the overprescrib sistant infections is estimated to be between USS5 billion and ing of antibiotics has only moderately reduced antibiotic US $24 billion per year in the United States alone". Resis resistance’. Efforts have been also made to lessen the use of tance to antibiotic drugs develops and spreads rapidly, often antibiotics in farming but some use is inevitable'. Using within a few years of first clinical use". However, the drug to kill bacteria has been in practice since the pipelines of pharmaceutical companies have not kept pace early 20" century, particularly in Eastern Europe'''. Bac with the evolution of antibiotic resistance'. teriophage can be chosen to lyse and kill bacteria or can be 0006 Acquired antibiotic resistance results from muta modified to express lethal genes to cause cell death'. tions in antibacterial targets or from genes encoding conju However, bacteriophage which are directly lethal to their gative proteins that pump antibiotics out of cells or inactivate bacterial hosts can also produce phage-resistant bacteria in antibiotics'". Horizontal gene transfer, which can occur via short amounts of time'''''''. In addition to the aforemen transformation, conjugative , or conjugative trans tioned approaches, novel methods for designing antimicro posons, is a major mechanism for the spread of antibiotic bial drugs are becoming more important to extending the resistance genes''. For example, Staphylococcus aureus lifespan of the antibiotic era. Combination therapy with became quickly resistant to Sulpha drugs in the 1940s, peni different antibiotics or antibiotics with phage may enhance cillin in the 1950s, and methicillin in the 1980s'. In 2002, bacterial cell killing and thus reduce the incidence of antibi staphylococci developed resistance to Vancomycin, the only otic resistance, and reduce persisters'. Unmodified fila uniformly effective antibiotic against Staphylococci, by mentous bacteriophage have been shown to augment antibi receiving Vancomycin-resistance genes via conjugation from otic efficacy'. Systems biology analysis can be employed to US 2010/0322903A1 Dec. 23, 2010 identify pathways to target and followed by synthetic biology 0013 Bacteriophage have been used in the past for treat to devise methods to attack those pathways. ment of plant diseases, such as fireblight as described in U.S. 0009 Bacterial biofilms are sources of contamination that Pat. No. 4,678,750. Also, Bacteriophages have been used to destroy biofilms (e.g., U.S. Pat. No. 6,699,701). In addition, are difficult to eliminate in a variety of industrial, environ systems using natural bacteriophages that encode biofilm mental and clinical settings. Biofilms are polymer structures destroying enzymes in general have been described. Art also secreted by bacteria to protect bacteria from various environ provides a number of examples of lytic enzymes encoded by mental attacks, and thus result also in protection of the bac bacteriophages that have been used as enzyme dispersion to teria from disinfectants and antibiotics. Biofilms can be found destroy bacteria (U.S. Pat. No. 6,335,012 and U.S. Patent on any environmental Surface where sufficient moisture and Application Publication No. 2005/0004030). The Eastern nutrients are present. Bacterial biofilms are associated with European research and clinical trials, particularly in treating many human and animal health and environmental problems. human diseases, such as intestinal infections, has apparently For instance, bacteria form biofilms on implanted medical concentrated on use of naturally occurring phages and their devices, e.g., catheters, heart Valves, joint replacements, and combined uses (Lorch, A. (1999), “Bacteriophages: An alter damaged tissue. Such as the lungs of cystic fibrosis patients. native to antibiotics? Biotechnology and Development Bacteria in biofilms are highly resistant to antibiotics and host Monitor, No. 39, p. 14-17). defenses and consequently are persistent sources of infection. 0014 For example, non-engineered bacteriophages have 0010 Biofilms also contaminate surfaces such as water been used as carriers to deliver antibiotics (such as chloroam pipes and the like, and render also other industrial Surfaces phenicol) (Yacoby et al., Antimicrobial agents and chemo hard to disinfect. For example, catheters, in particular central therapy, 2006:50: 2087-2097). Non-engineered bacterioph venous catheters (CVCs), are one of the most frequently used ages have also had aminoglycosides antibiotics, such as tools for the treatment of patients with chronic or critical chloroamphenicol, attached to the outside of filamentous illnesses and are inserted in more than 20 million hospital non-engineered bacteriophage (Yacoby et al., Antimicrobial patients in the USA each year. Their use is often severely agents and chemotherapy, 2007: 51; 2156-2163). M13 non compromised as a result of bacterial biofilm infection which lytic bacteriophages have also been engineered to carry lethal is associated with significant mortality and increased costs. cell death genes Gefand ChpBK. However, these phages have Catheters are associated with infection by many biofilm not been used, or Suggested to be useful in combination with forming organisms such as Staphylococcus epidermidis, Sta antimicrobial or antibiotic agents (Westwater et al., 2003, phylococcus aureus, Pseudomonas aeruginosa, Enterococ Antimicrobial agents and chemotherapy, 47: 1301-1307). cus faecalis and Candida albicans which frequently result in Non-engineered filamentous Pf3 bacteriophages have also generalized blood stream infection. Approximately 250,000 been administered with low concentration of gentamicin, cases of CVC-associated bloodstream infections occur in the where neither the filamentous Pf3 or the gentamicin could US each year with an associated mortality of 12%-25% and eliminate the bacterial infection alone (Hagens et al. Microb. an estimated cost of treatment per episode of approximately Drug resistance, 2006: 12; 164-8). The non-engineered bac S25,000. Treatment of CVC-associated infections with con teriophage and the antibiotic have been admin ventional antimicrobial agents alone is frequently unsuccess istered simultaneously, although the use of the antibiotic was ful due to the extremely high tolerance of biofilms to these more effective than the combination of the antibiotic and agents. Once CVCs become infected the most effective treat bacteriophage (see Table 1 in Huffet al., 2004: Poltry Sci, 83: ment still involves removal of the catheter, where possible, 1994-1947). and the treatment of any Surrounding tissue or systemic infec 0015 Constant evolutionary pressure will ensure that anti tion using antimicrobial agents. This is a costly and risky biotic resistance bacteria will continue to grow in number. procedure and re-infection can quickly occur upon replace The dearth of new antibacterial agents being developed in the ment of the catheter. last 25-30 years certainly bodes poorly for the future of the 0011 Bacteriophages (often known simply as “phages') antibiotic era (Wise, R (2004) J Antimicrob Chemother 54: are that grow within bacteria. The name translates as 306-310). Thus, new methods for combating bacterial infec “eaters of bacteria' and reflects the fact that as they grow, the tions are needed in order to prolong the antibiotic age. For majority of bacteriophages kill the bacterial host in order to example, bacteriophage therapy or synthetic antibacterial release the next generation of bacteriophages. Naturally peptides have been proposed as potential Solutions (Loose et occurring bacteriophages are incapable of infecting anything al., (2006) Nature 443: 867-869; Curtin, et al., (2006) Anti other than specific strains of the target bacteria, undermining microb Agents Chemother 50: 1268-1275). their potential for use as control agents. 0016. Because antibiotic resistance in treating bacterial infections and biofilms poses a significant hurdle to eliminat 0012 Bacteriophages and their therapeutic uses have been ing or controlling or inhibiting bacteria and biofilms with the subject of much interest since they were first recognized conventional antimicrobial drugs, new anti-biofilm strate early in the 20th century. Lytic bacteriophages are viruses that gies, such as phage therapy, should be explored. Novel Syn infect bacteria exclusively, replicate, disrupt bacterial thetic biology technologies are needed to enable the engineer metabolism and destroy the cell upon release of phage prog ing of natural phage with biofilm-degrading enzymes to eny in a process known as lysis. These bacteriophages have produce libraries of enzymatically-active phage, which can very effective antibacterial activity and in theory have several advantages over antibiotics. Most notably they replicate at the complement efforts to screen for new biofilm-degrading bac site of infection and are therefore available in abundance teriophages in the environment. where they are most required; no serious or irreversible side effects of phage therapy have yet been described and selecting SUMMARY alternative phages against resistant bacteria is a relatively 0017. The inventors have discovered a two pronged strat rapid process that can be carried out in days or weeks. egy to significantly reduce or eliminate a bacterial infection. US 2010/0322903A1 Dec. 23, 2010

In particular, the inventors have engineered bacteriophages to ages as disclosed herein which comprise a nucleic acid be used in combination with an antimicrobial agent, such that encoding an agent which inhibits at least one gene involved in the engineered bacteriophage functions as an adjuvant to the bacterial antibiotic resistance and/or cell Survival gene are antimicrobial agent. In particular, the inventors have engi referred to herein as “inhibitor-engineered bacteriophages”. neered bacteriophages to specifically disable (or deactivate) In some embodiments, the agent inhibits the gene expression the bacteria's natural resistance mechanisms to the antimicro and/or protein function of antibiotic resistance genes such as, bial agents and/or phage infection. Accordingly, one aspect of but not limited to cat, VanA or mec). In some embodiments, the present invention generally relates to engineered bacte the agent inhibits the gene expression and/or protein function riophages which have been modified or engineered to (i) of a cell Survival repair gene Such as, but not limited to RecA inhibit at least one bacterial resistance gene, or (ii) to inhibit at least one SOS response gene or bacterial defense gene in RecB. RecC. Spot or RelA. In another embodiment, an bacteria, or (iii) to express a protein which increases the inhibitor-engineered bacteriophages can comprise at least 2, Susceptibility of a bacterial cell to an antimicrobial agent. Any 3, 4, 5 or more, for example 8 different nucleic acids encoding one of these engineered bacteriophages, used alone, or in any inhibitors to antibiotic resistance genes or cell Survival repair combination can be used with an antimicrobial agent. genes, such as at least 2, 3, 4, 5 or more selected from the Accordingly, the inventors have discovered a method to pre group, but not limited to, cat, VanA, mecD, RecA, RecB. vent the development of bacterial resistance to antimicrobial RecC, Spot or RelA and other antibiotic resistance genes or agents and the generation of persistent bacteria by inhibiting cell Survival repair genes. In some embodiments of this aspect the local bacterial synthetic machinery which normally cir and all aspects described herein, an agent encoded by the cumvents the antimicrobial effect, by engineering bacte nucleic acid of an inhibitor-engineered bacteriophage is a riophages to be used in conjunction (or in combination with) protein which inhibits an antibiotic resistance gene and/or an antimicrobial agent, where an engineered bacteriophage cell survival gene or encodes an RNA-inhibitor (RNAi) agent can inhibit an antimicrobial resistance gene, or inhibit a SOS which inhibits the translation and expression of an antibiotic response gene or a non-SOS bacterial defense gene, or resistance gene and/or cell Survival gene. express a protein to increase the Susceptibility of a bacterial 0021. Another aspect of the present invention relates to an cell to an antimicrobial agent. engineered bacteriophage which comprises a nucleic acid 0018. Accordingly, one aspect of the present invention encoding a repressor protein, or fragment thereof of a bacte relates to the engineered bacteriophages as discussed herein rial SOS response gene, oran agent (Such as a protein) which for use in conjunction with (i.e. in combination with) at least inhibits a non-SOS pathway bacterial defense gene and are one antimicrobial agent, and that the engineered bacterioph referred to herein as “repressor-engineered bacteriophages.” ages serve as adjuvants to Such antimicrobial agents. Another In Some embodiments, the repressor of an SOS response gene aspect of the present invention relates to a method for inhib is, for example but not limited to, lex A, or modified version iting bacteria and/or removing bacterial biofilms in environ thereof. In some embodiments, the SOS response gene is, for mental, industrial, and clinical settings by administering a example but is not limited to marr AB, arc AB and lexO. In composition comprising at least one engineered bacterioph Some embodiments of this aspect and all other aspects ages as discussed herein with at least one antimicrobial agent. described herein, an inhibitor of a non-SOS pathway bacterial 0019. One aspect of the present invention relates to meth defense gene is SOXR, or modified version thereof. In some ods of using engineered bacteriophages in combination with embodiments of this aspect and all other aspects described antimicrobial agents to potentiate the antimicrobial effect of herein, an inhibitor of a non-SOS pathway bacterial defense bacterial killing (i.e. eliminating or inhibiting the growth or gene is selected from the group of marr, arc, SOXR, fur, crp, controlling the bacteria) by the antimicrobial agent. Accord iccdA or craA or omp A or modified version thereof. In other ingly, the present invention relates to the discovery of an embodiments of this aspect of the invention, an agent encoded engineered bacteriophage as an antibiotic adjuvant. In some by the nucleic acid of a repressor engineered bacteriophage embodiments, an engineered bacteriophage as discussed which inhibits a non-SOS defense gene can inhibit any gene herein functions as an antibiotic adjuvant for an aminglyco listed in Table 2. In some embodiments, a repressor-engi side antimicrobial agent, such as but not limited to, gentami neered bacteriophage which inhibits a non-SOS defense gene cin, as an antibiotic adjuvant for B-lactam antibiotics, such as can be used in combination with selected antimicrobial but not limited to, amplicillin, and as antibiotic adjuvants for agents, for example, where the repressor-engineered bacte quinolones antimicrobial agents, such as but not limited to, riophage encodes an agent which inhibits a gene listed in . Table 2A, such a repressor-engineered bacteriophage can be 0020. Another aspect of the present invention relates to an used in combination with a ciprofloxacin antimicrobial agent engineered bacteriophage which comprises a nucleic acid or a variant or analogue thereof. Similarly, in other embodi encoding an agent which inhibits at least one gene involved in ments a repressor-engineered bacteriophage which inhibits a antibiotic resistance. In Such and embodiment of this aspect non-SOS defense gene can encode an agent which inhibits a of the invention, an engineered bacteriophage can comprise at gene listed in Table 4B can be used in combination with a least 2, 3, 4, 5 or even more, for example 10 different nucleic Vancomycin antimicrobial agent or a variant or analogue acids which inhibit at least one gene involved in antibiotic thereof. Similarly, in other embodiments a repressor-engi resistance. In an alternative embodiment, an engineered bac neered bacteriophage which inhibits a non-SOS defense gene teriophage can comprise a nucleic acid encoding an agent can encode an agent which inhibits a gene listed in Table 2C. which inhibits at least one gene involved in cell survival 2D, 2E, 2F and 2G can be used in combination with a rifampi repair. In another embodiment, an engineered bacteriophage cin antimicrobial agent, ora amplicillin antimicrobial agent or can comprise at least 2, 3, 4, 5 or even more, for example 10 a SulfmethaxaZone antimicrobial agent or a gentamicin anti different nucleic acids which inhibit at least one gene microbial agent or a antimicrobial agent, involved in cell survival repair. Such engineered bacterioph respectively, or a variant or analogue thereof. US 2010/0322903A1 Dec. 23, 2010

0022. Another aspect of the present invention relates to an agents or phage infection. In some embodiments, the bacte engineered bacteriophage which comprises a nucleic acid riophages can be engineered or modified to express (i) at least encoding an agent, such as but not limited to a protein, which one inhibitor to at least one bacterial resistance gene and/or increases the Susceptibility of a bacteria to an antimicrobial cell Survival gene, or (ii) at least one inhibitor (Such as, but not agent. Such herein engineered bacteriophage which com limited to a repressor) at least one SOS response gene or prises a nucleic acid encoding an agent which increases the bacterial defense gene in bacteria, or (iii) a Susceptibility Susceptibility of a bacteria to an antimicrobial agent can be agent which increases the susceptibility of a bacterial cell to referred to herein as an “susceptibility agent-engineered bac an antimicrobial agent. teriophage' but are also encompassed under the definition of 0026. In some embodiments, any one of these engineered a “repressor-engineered bacteriophage'. In some embodi bacteriophages, used alone, or in any combination can be ments of this aspect, and all other aspects described herein, used with at least one antimicrobial agent. For example, one Such an agent which increases the Susceptibility of a bacteria aspect discussed herein relates to an engineered bacterioph to an antimicrobial agent is referred to as a “susceptibility age which expresses a . Such as an anti agent” and refers to any agent which increases the bacteria's sense nucleic acid inhibitor orantisense RNA (asRNA) which susceptibility to the antimicrobial agent by at least about 10% inhibits at least one, or at least two or at least three antibiotic or at least about 15%, or at least about 20% or at least about genes and/or a cell Survival gene, such as, but not limited to 30% or at least about 50% or more than 50%, or any integer cat, VanA, mecD, RecA, RecB. RecC, Spot or RelA. In between 10% and 50% or more, as compared to the use of the another aspect, an engineered bacteriophage can express an antimicrobial agent alone. In one embodiment, a Susceptibil repressor, or fragment thereof, of at least one, or at least two ity agent is an agent which specifically targets a bacteria cell. or at least three SOS response genes, such as, but not limited In another embodiment, a Susceptibility agent modifies (i.e. to leXA, marr, arc, SOXR, fur, crp, iccdA, craA or omp A. inhibits or activates) a pathway which is specifically 0027. The inventors also demonstrated that a repressor expressed in bacterial cells. In one embodiment, a suscepti engineered bacteriophage and/or an inhibitor-engineered bility agent is an agent which has an additive effect of the bacteriophage and/or a Susceptibility agent-engineered bac efficacy of the antimicrobial agent (i.e. the agent has an addi teriophage can reduce the number of antibiotic-resistant bac tive effect of the killing efficacy or inhibition of growth by the teria in a population and act as a strong adjuvant for a variety antimicrobial agent). In a preferred embodiment, a suscepti of other bactericidal antibiotics, such as for example, but not bility agent is an agent which has a synergistic effect on the limited to gentamicin and amplicillin. efficacy of the antimicrobial agent (i.e. the agent has a syn 0028. In some embodiments of all aspects of the invention, ergistic effect of the killing efficacy or inhibition of growth by any engineered bacteriophage disclosed herein, Such as the antimicrobial agent). repressor-engineered bacteriophage and/or an inhibitor-engi 0023. In one embodiment, a susceptibility agent increases neered bacteriophage and/or a Susceptibility agent-engi the entry of an antimicrobial agent into a bacterial cell, for neered bacteriophage as discussed herein can additionally example, a Susceptibility agent is a porin orporin-like protein, comprise a least one of the degrading enzymes effective at such as but is not limited to, protein OmpF, and Beta barrel degrading bacteria biofilms, such as effective EPS-degrading porins, or other members of the outer membrane porin enzymes specific to the target biofilm, particularly, for (OMP)) functional superfamily which include, but are not example, dispersin B (DspB) which is discussed in PCT limited to those disclosed in worldwide web site: “//biocyc. application PCT/US2005/032365 and U.S. application Ser. org/ECOLI/NEW-IMAGE?object=BC-4.1.B”, or a OMP No. 12/337,677, which are incorporated herein by reference. family member listed in Table 3 as disclosed herein, or a 0029. Also discussed herein is the generation of a diverse variant or fragment thereof. In another embodiment, a sus library of engineered bacteriophages described herein, Such ceptibility agent is an agent, such as but not limited to a as a library of repressor-engineered bacteriophage and/or an protein, which increases iron-sulfur clusters in the bacteria inhibitor-engineered bacteriophage and/or a Susceptibility cell and/or increases oxidative stress or hydroxyl radicals in agent-engineered bacteriophages which are capable of acting the bacteria. Examples of a susceptibility agent which as adjuvants or to enhance antimicrobial agents, which is increases the iron-sulfur clusters include agents which modu advantageous than trying to isolate such bacteriophages that late (i.e. increase or decrease) the Fenton reaction to form function as adjuvants from the environment. By multiplying hydroxyl radicals, as disclosed in Kahanski et al., Cell, 2007, within the bacterial colony or biofilm and hijacking the bac 130; 797-810, which is incorporated herein by reference in its terial machinery, inhibitor engineered bacteriophages entirety. Examples of a Susceptibility agent to be expressed by achieves high local concentrations of both enzyme and lytic a Susceptibility-engineered bacteriophage include, for phage to target multiple biofilm components, even with Small example, those listed in Table 4, or a fragment or variant initial phage inoculations. thereof or described in world-wide-web site “biocyc.org/ 0030) Rapid bacteriophage (also referred to as “phage' ECOLI/NEW-IMAGE?type=COMPOUND&object=CPD herein) replication with Subsequent bacterial lysis and 7: expression of inhibitors of SOS genes renders this a two 0024. In some embodiments, a Susceptibility agent is nota pronged attack strategy for use in combination with antimi chemotherapeutic agent. In another embodiment, a suscepti crobial agents for an efficient, autocatalytic method for inhib bility agent is not a toxin protein, and in another embodiment, iting bacteria and/or removing bacterial biofilms in a Susceptibility agent is not a bacterial toxin protein or mol environmental, industrial, and clinical settings. ecule. 0031. Also disclosed herein is a method for the combined 0025. Accordingly, the inventors have developed a modu use of an inhibitor-engineered bacteriophage and/or a repres lar design strategy in which bacteriophages are engineered to sor-engineered bacteriophage and/or Susceptibility agent-en have enhanced capacity to kill bacteria to disable or deacti gineered bacteriophage with at least one antimicrobial agent. vate the bacteria's natural resistance genes to antimicrobial The inventors have demonstrated that the combined use of an US 2010/0322903A1 Dec. 23, 2010

inhibitor-engineered bacteriophage and/or a repressor-engi 0036) Another aspect of the present invention relates to an neered bacteriophage and/or Susceptibility agent-engineered engineered lysogenic M13 bacteriophage comprising a bacteriophage is at least 4.5 orders of magnitude more effi nucleic acid operatively linked to a M13 promoter, wherein cient than use of the antimicrobial agent alone, and at least the nucleic acid encodes at least one repressor of a SOS two orders of magnitude more efficient at killing or eliminat response gene and/or an inhibitor to a non-SOS bacterial ing the bacteria as compared to use of an antimicrobial agent defense gene. with a non-engineered bacteriophage alone (i.e. an antimicro 0037 Another aspect of the present invention relates to an bial agent in the presence of a bacteriophage which is not an engineered lysogenic M13 bacteriophage comprising a inhibitor-engineered bacteriophage or a repressor-engineered nucleic acid operatively linked to a M13 promoter, wherein bacteriophage or Susceptibility agent-engineered bacterioph the nucleic acid encodes at least one agent that increases the Susceptibility of a bacterial cell to an antimicrobial gene. age). Thus, the inventors have demonstrated a significant and 0038 Another aspect of the present invention relates to an Surprising improvement over the combined use of non-engi engineered lytic T7 bacteriophage comprising a nucleic acid neered bacteriophages and antimicrobial agents as therapies operatively linked to a T7 promoter, wherein the nucleic acid described in prior art. The inventors have also demonstrated encodes at least one agent that inhibits at least one antibiotic that use of Such engineered bacteriophages as disclosed resistance gene and/or at least one cell Survival repair gene. herein, Such as the inhibitor-engineered bacteriophages or 0039. Another aspect of the present invention relates to an repressor-engineered bacteriophages are very effective at engineered lytic T7 bacteriophage comprising a nucleic acid reducing the number of antibiotic resistant bacterial cells operatively linked to a T7 promoter, wherein the nucleic acid which can develop in the presence of sub-inhibitory antimi encodes at least one repressor of a SOS response gene and/or crobial drug concentrations. an inhibitor to a non-SOS bacterial defense gene. 0032. Also, one significant advantage of the present inven 0040 Another aspect of the present invention relates to an tion as compared to methods using non-engineered bacte engineered lytic T7 bacteriophage comprising a nucleic acid riophages in combination with antimicrobial agents is that the operatively linked to a T7 promoter, wherein the nucleic acid use of the engineeredbacteriophages as disclosed herein with encodes at least one agent that increases the Susceptibility of antimicrobial agents allows one to significantly reduce or a bacterial cell to an antimicrobial gene. eliminate a population of persister cells. For example, the 0041. In some embodiments, an antibiotic resistance gene administration or application of an engineered bacteriophage is selected from the group comprising cat, VanA or mecD or as disclosed herein after initial treatment with an antimicro Variants thereof. In some embodiments, a cell Survival gene is bial agent can reduce or eliminate a population of persister selected from the group comprising RecA, RecB. RecC., spot, cells. Furthermore, the inventors have discovered that an RelA or variants thereof. engineered bacteriophage as disclosed herein, such as an 0042. In some embodiments of all aspects described inhibitor-engineered bacteriophage or a repressor-engineered herein, a bacteriophage can comprise an agent which is bacteriophage or Susceptibility agent-engineered bacterioph selected from a group comprising, siRNA, antisense nucleic age can reduce the number of antibiotic resistant mutant acid, asRNA, RNAi, miRNA and variants thereof. In some bacteria that Survive in a bacterial population exposed to one embodiments, the bacteriophage comprises an as RNA agent. or more antimicrobial agents, and therefore the engineered 0043. In some embodiments, the bacteriophage comprises bacteriophages described herein are effective at reducing the a nucleic acid encoding at least two agents that inhibit at least number of antibiotic resistant cells which develop in the pres two different cell survival repair genes, for example but not ence of Sub-inhibitory antimicrobial agent drug concentra limited to, at least two agents that inhibit at least two of RecA tions. RecB or RecC. 0033. Another advantage of the present invention is that it 0044. In some embodiments, the repressor of a SOS allows one to reduce or eliminate multiple applications of the response gene is selected from the group comprising leXA, composition during the treatment of a surface having a bac marr, arck, SOXR, fur, crp, iccdA, craA, ompl or variants or terial biofilm. fragments thereof. In some embodiments, the repressor is 0034. One aspect of the present invention relates to engi LeXA and in some embodiments, the repressor is cSrA or neering or modification of any bacteriophage strain or species omF, and in some embodiments the bacteriophage can com to generate the engineered bacteriophages disclosed herein. prise the nucleic acid encoding a mixture of Lex A, cSrA or For example, an inhibitor-engineered bacteriophage or a omF in any combination. For example, in some embodi repressor-engineered bacteriophage or Susceptibility agent ments, the bacteriophage can comprise the nucleic acid engineered bacteriophage can be any bacteriophage known encoding at least two different repressors of at least one SOS by a skilled artisan. For example, in one embodiment, the response gene. Such as, but not limited to the bacteriophage bacteriophage is a lysogenic bacteriophage, for example but can comprise the repressors cSrA and ompl or variants or not limited to a M13 bacteriophage. In another embodiment, homologues thereof. the bacteriophage is a lytic bacteriophage such as, but not 0045 Another aspect of the present invention relates to a limited to T7 bacteriophage. In another embodiment, the method to inhibit or eliminate a bacterial infection compris bacteriophage is a phage Kora Staphylococcus phage K for ing administering to a surface infected with bacteria; (i) a use against bacterial infections of methicillin-resistant S. bacteriophage comprising a nucleic acid operatively linked to Ca,S. a bacteriophage promoter, wherein the nucleic acid encodes 0035. One aspect of the present invention relates to an at least one agent that inhibits an antibiotic resistance gene engineered lysogenic M13 bacteriophage comprising a and/or a cell Survival repair gene, and (ii) at least one antimi nucleic acid operatively linked to a M13 promoter, wherein crobial agent. the nucleic acid encodes at least one agent that inhibits an 0046. Another aspect of the present invention relates to a antibiotic resistance gene and/or a cell Survival repair gene. method to inhibit or eliminate a bacterial infection compris US 2010/0322903A1 Dec. 23, 2010

ing administering to a surface infected with bacteria: (i) a 0053 Another aspect of the present invention relates to a bacteriophage comprising a nucleic acid operatively linked to composition comprising a lysogenic M13 bacteriophage a bacteriophage promoter, wherein the nucleic acid encodes comprising a nucleic acid operatively linked to a M13 pro at least one repressor of a SOS response gene, and (ii) at least moter, wherein the nucleic acid encodes at least one agent that one antimicrobial agent. inhibits an antibiotic resistance gene and/or a cell Survival 0047 Another aspect of the present invention relates to a repair gene and at least one antimicrobial agent. Another method to inhibit or eliminate a bacterial infection compris aspect of the present invention relates to a composition com ing administering to a surface infected with bacteria: (i) a prising a lysogenic M13 bacteriophage comprising a nucleic bacteriophage comprising a nucleic acid operatively linked to acid operatively linked to a M13 promoter, wherein the a bacteriophage promoter, wherein the nucleic acid encodes nucleic acid encodes at least one repressor of a SOS response at least one agent which increases the Susceptibility of a gene and at least one antimicrobial agent. bacterial cell to a antimicrobial agent, and (ii) at least one 0054 Another aspect of the present invention relates to a antimicrobial agent. composition comprising alytic T7 bacteriophage comprising 0048. In some embodiments of all aspects described a nucleic acid operatively linked to a T7 promoter, wherein herein, a bacteriophage useful in the methods disclosed the nucleic acid encodes at least one agent that inhibits an herein and used to generate an engineered bacteriophage, antibiotic resistance gene and/or a cell Survival repair gene Such as a inhibitor-engineered bacteriophage or a repressor and at least one antimicrobial agent. Another aspect of the engineered bacteriophage or a Susceptibility-engineered bac present invention relates to a composition a lytic T7 bacte teriophage is any bacteriophage know by a skilled artisan. A riophage comprising a nucleic acid operatively linked to a T7 non-limiting list of examples of bacteriophages which can be promoter, wherein the nucleic acid encodes at least one used are disclosed in Table 5 herein. In one embodiment, the repressor of a SOS response gene and at least one antimicro bacteriophage is a lysogenic bacteriophage such as, for bial agent. example a M13 lysogenic bacteriophage. In alternative 0055. In some embodiments, the composition comprises embodiments, a bacteriophage useful in all aspects disclosed antimicrobials agents such as, for example but not limited to, herein is a lytic bacteriophage, for example but not limited to quinolone antimicrobial agents and/or aminoglycoside anti a T7 lytic bacteriophage. In one embodiment, a bacteriophage microbial agents and/or B-lactam antimicrobial agent, for useful in all aspects disclosed herein is a SP6 bacteriophage or example, but not limited to, antimicrobial agents selected a phage K, or a staphylococcus phage K bacteriophage. from a group comprising ciprofloxacin, , and 0049. In some embodiments, administration of any engi ofloxacin, , , , trova neered-bacteriophage as disclosed herein and the antimicro floxacin, , , , paZu bial agent occurs simultaneously, and in alternative embodi floxacin, amikacin, gentamycin, tobramycin, netromycin, ments, the administration of a engineered-bacteriophage streptomycin, kanamycin, paromomycin, neomycin, penicil occurs prior to the administration of the antimicrobial agent. lin, amplicillin, penicillin derivatives, cephalosporins, mono In other embodiments, the administration of an antimicrobial bactams, carbapenems, B-lactamase inhibitors or variants or agent occurs prior to the administration of a engineered analogues thereof. bacteriophage. 0056. In some embodiments, the composition comprises 0050. In some embodiments, antimicrobial agents useful at least one inhibitor-engineered bacteriophage and/or at least in the methods as disclosed herein are quinolone antimicro one repressor-engineered bacteriophage as disclosed herein. bial agents, for example but not limited to, antimicrobial 0057 Another aspect of the present invention relates to a agents selected from a group comprising ciprofloxacin, levo kit comprising a lysogenic M13 bacteriophage comprising floxacin, and ofloxacin, gatifloxacin, norfloxacin, lomefloxa the nucleic acid operatively linked to a M13 promoter, cin, , moxifloxacin, sparfloxacin, gemifloxacin, wherein the nucleic acid encodes at least one agent that inhib or variants or analogues thereof. In some its an antibiotic resistance gene and/or a cell Survival repair embodiments, an antimicrobial agents useful in the methods gene. Another aspect of the present invention relates a kit as disclosed herein is ofloxacin or variants or analogues comprising a lysogenic M13 bacteriophage comprising the thereof. nucleic acid operatively linked to a M13 promoter, wherein 0051. In some embodiments, antimicrobial agents useful the nucleic acid encodes at least one repressor of a SOS in the methods as disclosed herein are aminoglycoside anti response. microbial agents, for example but not limited to, antimicro 0.058 Another aspect of the present invention relates a kit bial agents selected from a group consisting of amikacin, comprising a lytic T7 bacteriophage comprising the nucleic gentamycin, tobramycin, netromycin, streptomycin, kana acid operatively linked to a T7 promoter, wherein the nucleic mycin, paromomycin, neomycin or variants or analogues acid encodes at least one agent that inhibits an antibiotic thereof. In some embodiments, an antimicrobial agent useful resistance gene and/or a cell Survival repair gene. Another in the methods as disclosed herein is gentamicin or variants or aspect of the present invention relates a kit comprising a lytic analogues thereof. T7 bacteriophage comprising the nucleic acid operatively 0052. In some embodiments, antimicrobial agents useful linked to a T7 promoter, wherein the nucleic acid encodes at in the methods as disclosed herein are B-lactam antibiotic least one repressor of a SOS response. antimicrobial agents, such as for example but not limited to, 0059. In some embodiments, the methods and composi antimicrobial agents selected from a group consisting of peni tions as disclosed herein are administered to a subject. In cillin, amplicillin, penicillin derivatives, cephalosporins, some embodiments, the methods to inhibit or eliminate a monobactams, carbapenems, B-lactamase inhibitors or vari bacterial infection comprising administering the composi ants or analogues thereof. In some embodiments, an antimi tions as disclosed herein to a subject, wherein the bacteria are crobial agent useful in the methods as disclosed herein is present in the Subject. In some embodiments, the Subject is a ampicillin or variants or analogues thereof. mammal, for example but not limited to a human. US 2010/0322903A1 Dec. 23, 2010

0060. In some embodiments, any of the bacteriophages as at the end of each day to generate survival curves. FIG. 3B disclosed herein are useful in combination with at least one shows survival curves for infected mice treated with phage antimicrobial agent to reduce the number of bacteria as com and/or ofloxacin demonstrate that engineered phage (ps pared to use of the antimicrobial agent alone. In some plus ofloxacin (closed circles with Solid line) significantly embodiments, any of the bacteriophages as disclosed herein increases Survival of mice compared with unmodified phage are useful in combination with at least one antimicrobial funmod plus ofloxacin (closed squares with Solid line), no agent to inhibit or eliminate a bacterial infection, such as for phage plus ofloxacin (closed diamonds with Solid line), and example inhibit or eliminate a bacteria present a biofilm. no treatment (open diamonds with dashed line). 0061. In some embodiments, the present invention relates 0065 FIGS. 4A-4B show box-and-whisker plot of the to methods to inhibit or eliminate a bacterial infection com total number of E. coli EMG2 cells in 60 observations that prising administering to a Surface infected with bacteria: (i) a were resistant to 100 ng/mL ofloxacin after growth under bacteriophage comprising a nucleic acid operatively linked to various conditions (bars indicate medians, diamonds repre a bacteriophage promoter, wherein the nucleic acid encodes sent outliers). FIG. 4A shows cells grown with no phage and at least one repressor of a SOS response gene, and (ii) at least no ofloxacin for 24 hours had very low numbers of antibiotic one antimicrobial agent. In some embodiments, the bacteria resistant cells. Cells grown with no phage and 30 ng/mL is in a biofilm. ofloxacin for 24 hours had high numbers of resistant cells due to growth in subinhibitory drug concentrations (Martinez et BRIEF DESCRIPTION OF FIGURES al., 2000, Antimicrob. Agents Chemother. 44, 1771-177730). 0062 FIGS. 1A-1E show engineered (p bacteriophage Cells grown with no phage and 30 ng/mL ofloxacin for 12 enhances killing of wild-type E. coli EMG2 bacteria by bac hours followed by 10 PFU/mL unmodified phage funmod tericidal antibiotics. FIG. 1A shows a schematic of combina and 30 ng/mL ofloxacin for 12 hours exhibited a modest level tion therapy with engineered phage and antibiotics. Bacteri of antibiotic-resistant bacteria. Cells grown with no phage cidal antibiotics induce DNA damage via hydroxyl radicals, and 30 ng/mL ofloxacin for 12 hours followed by 10 PFU/ leading to induction of the SOS response. SOS induction mL (p and 30 ng/mL ofloxacin for 12 hours exhibited a low results in DNA repair and can lead to survival (Kohanski et level of antibiotic-resistant bacteria, close to the numbers al., 2007, Cell 130,797-8108). Engineered phage carrying the seen with no ofloxacin and no phage. FIG. 4B shows a leXA3 gene (cps) under the control of the synthetic pro Zoomed-in version of box-and-whisker plot in (a) for moter PLtetO and a ribosome-binding sequence (Lutz et al., increased resolution around low total resistant cell counts 1997, Nucleic Acids Res 25, 1203-121027) acts as an antibi confirms that p with 30 ng/mL ofloxacin treatment otic adjuvant by Suppressing the SOS response and increasing reduced the number of resistant cells to levels similar to that cell death. FIG. 1B shows a killing curves for no phage of no ofloxacin with no phage. (diamonds), unmodified phage (p. (squares), and engi 0.066 FIGS. 5A-5D show engineered bacteriophage tar neered phage (ps (circles) with 60 ng/mL ofloxacinoflox geting single and multiple gene networks (other than the SOS (solid lines, closed symbols). 10 PFU/mL phage was used. A network) as adjuvants for ofloxacin treatment oflox). FIG. growth curve for E. coli EMG2 with no treatment is shown for 5A show Ofloxacin stimulates superoxide generation, which comparison (dotted line, open symbols). p. greatly is normally countered by the oxidative stress response, coor enhanced killing by ofloxacin by 4 hours of treatment. FIG. dinated by SoxR (Kohanski et al., 2007, Cell 130,797-8108). 1C is a ofloxacin dose response showing that (ps (circles Engineered phage producing SoxR (cp) enhances ofloxa with solid line) increases killing even at low levels of drug cin-based killing by disrupting regulation of the oxidative compared with no phage (diamonds with dash-dotted line) stress response. FIG. 5B show killing curves for no phage and punmod (squares with dashed line). 10 PFU/mL phage (diamonds), unmodified phage (p. (squares), and engi was used. FIG. 1D shows killing curves for no phage (dia neered phage (p (downwards-facing triangles) with 60 monds), (p,f(Squares), and (pies (circles) with 5 Lig/mL ng/mL ofloxacin (solid lines, closed symbols). 10, PFU/mL gentamicingent). 10 PFU/mL phage was used. (pus phage phage was used. The killing curve for funmod and a growth greatly increases killing by gentamicin. FIG.1E shows killing curve for E. coli EMG2 with no treatment (dotted line, open curves for no phage (diamonds), p. (squares), and (plus symbols) are reproduced from FIG. 1B for comparison and (circles) with 5ug/mL amplicillin amp). 10 PFU/mL phage show that p, enhances killing by ofloxacin. FIG.5C CSrA was used. (p. phage greatly increases killing by amplicillin. suppresses the biofilm state in which bacterial cells tend to be 0063 FIG. 2 shows that engineered (p bacteriophage more resistant to antibiotics (Jackson et al., 2002, J. Bacteriol. enhances killing of quinolone-resistant E. coli RFS289 bac 184,290-30135). Ompl is a porin used by quinolones to enter teria by ofloxacin. Killing curves for no phage (diamonds), bacterial cells (Hirai K, et al., 1986, Antimicrob. Agents unmodified phage funmod (squares), and engineered phage Chemother. 29, 535-53837). Engineered phage producing (ps (circles) with 1 g/mL ofloxacinoflox (solid lines, both CsrA and OmpF simultaneously (cps) enhances closed symbols). 10 PFU/mL phage was used. (ps greatly antibiotic penetration via OmpF and represses biofilm forma enhanced killing by ofloxacin by 1 hour of treatment. tion and antibiotic tolerance via CSrA to produce an improved 0064 FIGS. 3A-3B show that engineered (p bacte dual targeting adjuvant for ofloxacin. FIG. 5D shows killing riophage increases Survival of mice infected with bacteria. curves for (P. (diamonds), Pir (Squares), and P-4- FIG. 3A shows a schematic of a female Charles River CD-1 (upwards-facing triangles) with 60 ng/mL ofloxacin. 10 mice inoculated with intraperitoneal injections of 8.8x10 PFU/mL phage was used. Phage expressing both csrA and CFU/mouse E. coli EMG2 bacteria. After 1 hour, the mice ompF (cp.se) is a better adjuvant for ofloxacin than received either no treatment or intravenous treatment with no phage expressing cSrA (D) or ompF alone (pr.). phage, unmodified phage (p, or engineered phage (picts 0067 FIGS. 6A-6D show engineered bacteriophage tar with 0.2 mg/kg ofloxacin. 10 PFU/mouse phage was used. geting non-SOS systems in E. coli as adjuvants for ofloxacin The mice were observed for 5 days and deaths were recorded treatment oflox. FIG. 6A shows a killing curves for no US 2010/0322903A1 Dec. 23, 2010 phage (black diamonds), 10 PFU/mL unmodified M13mp18 M13mp18-lex A3 treatment reduced the number of resistant (i.e. p.) (squares), and 10 PFU/mL M13mp18-soxR (i.e. cells under 30 ng/mL ofloxacinto levels similar to that of 0 (psi) (downwards-facing triangles) without ofloxacin (dot ng/mL ofloxacin in FIG. 8A. ted lines, open symbols) or with 60 ng/mL ofloxacin (solid 0069 FIGS. 8A-8B shows engineered M13mp18-lexA3 lines, closed symbols). Killing curves for no phage and bacteriophage enhances killing by other bactericidal drugs. unmodified m13mp 18 phage (p) are reproduced from FIG. 8A shows killing curves for no phage (diamonds), 10 FIG. 1B for comparison and demonstrate that M13mp 18 PFU/mL unmodified M13mp 18 (squares), and 10 PFU/mL soxR (i.e. (p) enhances killing by ofloxacin. 10 PFU/mL M13mp18-lex A3 (circles) with 5 lug/mL gentamicin gent. represents an MOI of approximately 1:10. FIG. 6B shows a Engineered M13mp 18-lex A3 phage greatly improved killing killing curves for 10 PFU/mL M13 mp 18-csrA (cp) (black by gentamicin. 10 PFU/mL represents an MOI of approxi diamonds), 10 PFU/mL M13mp 18-ompF (per) (squares), mately 1:1. FIG. 8B shows a killing curves for no phage and 10 PFU/mL M13mp 18-csrA-ompF (Pas-4-oner) (up (diamonds), 10 PFU/mL unmodified M13mp18 (squares), wards-facing triangles) without ofloxacin (dotted lines, open and 10 PFU/mL M13mp 18-lex A3 (circles) with 5 g/mL symbols) or with 60 ng/mL ofloxacin (solid lines, closed ampicillin amp. Engineered M13mp 18-lex A3 phage symbols). Phage expressing both csrA and ompl (M13mp18 greatly improved killing by amplicillin 10 PFU/mL repre cSrA-ompF or (p) is a better adjuvant for ofloxacin sents an MOI of approximately 1:1. than phage expressing cSrA alone (M13mp18-cSrA. (p.) or (0070 FIGS. 9A-9F show of unmodified ompf alone (M13mp 18-ompF; (P). 10 PFU/mL repre M13mp18 bacteriophage and engineered bacteriophage. sents an MOI of approximately 1:10. FIG. 6C shows a phage Engineered bacteriophage were constructed by inserting dose response which demonstrates that both M13mp18-soxR genetic modules under the control of a synthetic promoter (downwards-facing triangles with solid line) and M13mp18 (P, tetO) and ribosome-binding sequence (RBS) in between cSrA-ompF (upwards-facing triangles with Solid line) are Sad and PVul restriction sites. A terminator (Term) ends effective as adjuvants for ofloxacin (60 ng/mL) over a wide transcription of the respective gene(s). FIG. 9A shows unmodified M13mp 18 (cp) contains lacZ to allow blue range of initial inoculations. Phage dose response curves for white screening of engineered bacteriophage. FIG.9B shows no phage (dash-dotted line) and unmodified M13mp 18 phage engineered M13mp 18 bacteriophage expressing leXA3 (squares with dashed line) are reproduced from FIG. 1c for (cp). FIG.9C shows engineered M13mp 18 bacteriophage comparison. FIG. 6D shows a Ofloxacin dose response with expressing soxR (cp). FIG. 9D shows engineered 10 PFU/mL that shows that both M13mp18-soxR (down M13mp 18 bacteriophage expressing csrA (cp). FIG. 9E wards-facing triangles with solid line) and M13mp 18-csrA ompl (upwards-facing triangles with solid line) improve kill shows engineered M13mp 18 bacteriophage expressing ompl ing throughout a range of drug concentrations. Ofloxacin (cp). FIG.9F shows engineered M13mp18 bacteriophage dose response curves for no phage (diamonds with dash expressing CsrA and ompF (cp.s.l.). (0071 FIGS. 10A-10E show flow cytometry of cells with dotted line) and unmodified M13mp 18 phage (squares with an SOS-responsive GFP exposed to no phage (black dashed line) are reproduced from FIG. 1D for comparison. lines), unmodified phage (p, (red lines), or engineered 0068 FIGS. 7A-7D show histograms of the total number phage (ps (blue lines) for 6 hours with varying doses of of E. coli cells in 60 observations that were resistant to 100 ofloxacin. 10 plaque forming units per mL (PFU/mL) of ng/mL ofloxacin after growth under various conditions. FIG. phage were applied. Cells exposed to no phage or (punmod 7A shows cells grown with no phage and no ofloxacin for 24 showed similar SOS induction profiles, whereas cells with hours had very low numbers of antibiotic-resistant cells. Inset (p exhibited significantly suppressed SOS responses. of FIG. 8A shows the distribution of observations with total FIG. 10A shows 0 ng/mL ofloxacin treatment. FIG. 10B resistant cells between 0 and 50 for increased resolution and shows 20 ng/mL ofloxacin treatment. FIG. 10C show 60 demonstrates that many observations were devoid of antibi ng/mL ofloxacin treatment. FIG. 10D show 100 ng/mL otic-resistant bacteria. FIG. 7B shows cells grown with no phage and 30 ng/mL ofloxacin for 24 hours had high numbers ofloxacin treatment. FIG. 10E shows 200 ng/mL ofloxacin of resistant cells, demonstrating a large increase in antibiotic treatment. resistance due to growth in Subinhibitory drug concentra 0072 FIG. 11 shows persister killing assay demonstrates tions'. No inset is shown because no observations had less that engineered bacteriophage can be applied to a previously than 50 resistant cells. FIG. 7C shows cells grown with no drug-treated population to increase killing of Surviving per phage and 30 ng/mL ofloxacin for 12 hours followed by 10 sister cells. After 3 hours of 200 ng/mL ofloxacin treatment, PFU/mL unmodified M13mp 18 phage and 30 ng/mL ofloxa no phage, 10 PFU/mL control M13mp 18 phage, or 10 PFU/ cin for 12 hours exhibited a modest level of antibiotic-resis mL engineered M13mp 18-lex A3 phage were added to the tant bacteria. Inset of FIG.7C shows the distribution of obser previously drug-treated cultures. Three additional hours later, vations with total resistant cells between 0 and 50 for viable cell counts were obtained and demonstrated that increased resolution and demonstrates that no observations M13mp18-lex A3 was able to reduce persister cell levels bet were devoid of antibiotic-resistant bacteria. FIG. 7D shows ter than no phage or control M13mp8 phage. cells grown with no phage and 30 ng/mL ofloxacin for 12 0073 FIG. 12 shows paired-termini design from hours followed by 10 PFU/mL M13mp 18-lexA3 and 30 Nakashima, etal (2006) Nucleic Acids Res 34: e138, in which ng/mL ofloxacin for 12 hours exhibited a low level of antibi the antisense RNA is cloned between the flanking restriction otic-resistant bacteria compared to no phage and 30 ng/mL sites at the top of the stem. Reprinted from Nakashima, et al ofloxacin in FIG. 7D, and unmodified M13mp 18 and 30 (2006) Nucleic Acids Res 34: e138. ng/mL ofloxacin in FIG. 8C. Inset of FIG. 7D shows the 0074 FIG. 13 shows autoregulated negative-feedback distribution of observations with total resistant cells between module with lexA repressing PlexO from Morens, et al., 0 and 50 for increased resolution and demonstrates that (2004) Nature 430: 242-249, can increase the level of lex A US 2010/0322903A1 Dec. 23, 2010

expression when lex A is cleaved by recA in response to DNA inventors have discovered a method to prevent the develop damage by agents such as ofloxacin. ment of bacterial resistance to antimicrobial agents and the 0075 FIG. 14 shows persistence assay for various con generation of persistent bacteria by inhibiting the local bac structs in wild-type E. coli EMG2 cells after 8 hours of growth terial synthetic machinery which normally circumvents the in the presence of 1 mM IPTG followed by 8 hours of treat antimicrobial effect, by engineering bacteriophages to be ment with 5 lug/mL ofloxacin. Greatly improved cell killing used in conjunction (or in combination with) an antimicrobial was generated by the double knockouts, especially PtetO agent, where an engineered bacteriophage can inhibit an anti recB-asRNA/PlacO-recA-asRNA and PtetO-recC-askNA/ microbial resistance gene, or inhibit a SOS response gene or PlacO-recB-asRNA. pZE1L-lexA also reduced the number a non-SOS bacterial defense gene, or express a protein to of surviving cells compared with wild-type E. coli EMG2. increase the Susceptibility of a bacterial cell to an antimicro 0076 FIG. 15 shows engineered (p bacteriophage bial agent. enhances killing of wild-type E. coli EMG2 bacteria by bac I0082. Accordingly, one aspect of the present invention tericidal antibiotics. Phage dose response shows that (p. relates to the engineered bacteriophages as discussed herein (blue circles with solid line) is a strong adjuvant for ofloxacin for use in conjunction with (i.e. in combination with) at least (60 ng/mL) over a wide range of initial inoculations com one antimicrobial agent, and that the engineered bacterioph pared with no phage (black dash-dotted line) and (p (red ages serve as adjuvants to Such antimicrobial agents. squares with dashed line). The starting concentration of bac I0083. One aspect of the present invention relates to a teria was about 10 CFU/mL (data not shown). method to potentiate the bacterial killing effect of an antimi 0077 FIG. 16 shows persister killing assay demonstrates crobial agent. In particular, one aspect of the present inven that engineered bacteriophage can be applied to a previously tion relates to methods and compositions comprising engi drug-treated population to increase killing of Surviving per neered bacteriophages for use in combination with an sister cells. After 3 hours of 200 ng/mL ofloxacin treatment, antimicrobial agent to potentiate the antimicrobial effect and no phage (black bar), 10 PFU/mL unmodified phage (p, bacterial killing of the antimicrobial agent. Another aspects (red bar), or 10 PFU/mL engineered phage (p3 (blue bar) relates to the use of an engineered bacteriophage as an anti were added to the previously drug-treated cultures. Three biotic adjuvant. In some embodiments of this and all aspects additional hours later, viable cell counts were obtained and described herein, an engineered bacteriophage can be used as demonstrated that (pl. was able to reduce persister cell an antibiotic adjuvant for an aminglycoside antimicrobial levels better than no phage or (p, agent, such as but not limited to, gentamicin, as antibiotic 0078 FIG. 17 shows mean killing with 60 ng/mL ofloxa adjuvants for a f-lactam antibiotic, such as but not limited to, cin after 12 hours of treatment of E. coli EMG2 biofilms ampicillin, and as an antibiotic adjuvant for a quinolone anti pregrown for 24 hours. Where indicated, 10 PFU/mL of (r) microbial agent, such as but not limited to, ofloxacin. In one leXA3 bacteriophage was used. embodiment of this aspect and all aspects described herein, an 0079 FIG. 18 shows the mean killing with 60 ng/mL engineered bacteriophage can function as an antimicrobial ofloxacin after 12 hours of treatment of E. coli EMG2 bio adjuvant orantibiotic adjuvant for at least 2, at least 3, at least films pregrown for 24 hours. Where indicated, 10 PFU/mL 4, at least 5, least 6, at least 7, at least 8, at least 9 or at least 10 of Ps. 1. pner, or (Ps...ar bacteriophage was used. or more different antimicrobial agents at any one time. In 0080 FIG. 19 shows an example of a promoter which can Some embodiments, any of the engineered bacteriophages as be used to express the nucleic acid in the engineered bacte disclosed herein can used in combination with at least one or riophage. FIG. 19 shows a P- (SEQID NO:32), P. more antimicrobial agent, for example an engineered bacte (SEQ ID NO: 33), P (SEQ ID NO. 34) and P riophage as disclosed herein can used in combination with at (SEQ ID NO:35) promoters which can be used. least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different antimicrobial agents. DETAILED DESCRIPTION I0084. In one aspect of the present invention, an engineered 0081. As disclosed herein, the inventors have discovered a bacteriophage as disclosed herein can comprise a nucleic acid two pronged strategy to significantly reduce or eliminate a encoding an agent which inhibits at least one bacterial gene bacterial infection. In particular, the inventors have engi involved in the development of antibiotic resistance. In neered bacteriophages to be used in combination with an another embodiment of this aspect and all aspects described antimicrobial agent, such that the engineered bacteriophage herein, an engineered bacteriophage can comprise a nucleic functions as an adjuvant to the antimicrobial agent. Thus, the acid encoding an agent which inhibits at least one gene inventors have engineered bacteriophages to be used in com involved in bacterial cell survival repair. As discussed previ bination with an antimicrobial agent, such that the engineered ously, Such engineered bacteriophages which comprise a bacteriophage functions as an adjuvant to at least one antimi nucleic acid encoding an agent which inhibits at least one crobial agent. In particular, the inventors have engineered bacterial gene involved in antibiotic resistance and/or at least bacteriophages to specifically disable (or deactivate) the bac one bacterial gene involved in cell survival are referred to teria's natural resistance mechanisms to the antimicrobial herein as “inhibitor-engineered bacteriophages'. In some agents and/or phage infection. Accordingly, one aspect of the embodiments of this aspect and all aspects discussed herein, present invention generally relates to engineered bacterioph an agent which inhibits an antibiotic resistance bacterial gene ages which have been modified or engineered to (i) inhibit at can inhibit the gene expression and/or protein function of least one bacterial resistance gene, or (ii) to inhibit at least one antibiotic resistance genes such as, but not limited to cat, SOS response gene or bacterial defense gene in bacteria, or VanA or mecD. In some embodiments of this aspect and all (iii) to express a protein which increases the Susceptibility of aspects discussed herein, an agent which inhibits a bacterial a bacterial cell to an antimicrobial agent. Any one of these cell Survival gene can inhibit the gene expression and/or engineeredbacteriophages, used alone, or in any combination protein function of a cell Survival repair gene Such as, but not can be used with an antimicrobial agent. Accordingly, the limited to RecA, RecB. RecC, Spot or RelA. US 2010/0322903A1 Dec. 23, 2010

0085. In some embodiments of this aspect and all aspects microbial agent or a metronidazole antimicrobial agent, described herein, an inhibitor-engineered bacteriophage can respectively, or a variant or analogue thereof. comprise a nucleic acid encoding an agent which inhibits at I0087. In some embodiments of this aspect an all other least one gene involved in antibiotic resistance and/or cell aspects discussed herein, a repressor is, for example but not Survival repair. In one embodiment of this aspect and all limited to, leXA, marr, arc, SOXR, fur, crp, iccdA, craA or aspect described herein, an inhibitor-engineered bacterioph ompA or a modified version thereof. In some embodiments, age can comprise at least 2, 3, 4, 5 or even more, for example the SOS response gene is, for example but is not limited to 10 different nucleic acids which inhibit at least one gene, for marFRAB, arcAB and lexO. example, 2, 3, 4, 5 or up to 10 genes involved in antibiotic I0088. In some embodiments of this aspect and all other resistance and/or cell Survival repair. In some embodiment of aspects described herein, a repressor-engineered bacterioph this aspect, an inhibitor-engineered bacteriophage can com age can comprise at least 2, 3, 4, 5 or more, for example 8 prise at least 2, 3, 4, 5 or more, for example 8 different nucleic different nucleic acids encoding different repressors of SOS acids encoding inhibitors to at least one antibiotic resistance response genes, such as at least 2, 3, 4, 5 or more selected from the group, but not limited to, lex A, marr AB, arc AB and lexO gene or to at least one cell Survival repair gene. Such as at least and other repressors of SOS response genes, or least 2, 3, 4, 5 2, 3, 4, 5 or more selected from the group, but not limited to, or more, for example 8 different nucleic acids encoding dif cat, VanA, mec), RecA, RecB. RecC. Spot or RelA and other ferent repressors (i.e. inhibitors) of non-SOS defense genes. antibiotic resistance genes or cell Survival repair genes. In In some embodiments, a repressor engineered bacteriophage some embodiments, any or all different combinations of can comprise any or all different combinations of repressors inhibitors of antibiotic resistance genes and/or cell survival of SOS genes described herein and/or any and all different repair genes can be present in an inhibitor-engineered bacte combinations of inhibitors non-SOS defense genes listed in riophage. Tables 2 and 2A-2G can be present in a repressor-engineered I0086. In another aspect of the present invention, an engi bacteriophage. neered bacteriophage can comprise at least one nucleic acid I0089. In another aspect of the present invention, an engi encoding a repressor protein, or fragment thereof of a bacte neered bacteriophage can comprise at least one nucleic acid rial SOS response gene, oran agent (such as a protein) which encoding an agent, such as but not limited to a protein, which inhibits a non-SOS pathway bacterial defense gene and are increases the Susceptibility of a bacteria to an antimicrobial referred to herein as “repressor-engineered bacteriophages.” agent. Such herein engineered bacteriophage which com In some embodiments, the repressor of an SOS response gene prises a nucleic acid encoding an agent which increases the is, for example but not limited to, lex A, or modified version Susceptibility of a bacteria to an antimicrobial agent can be thereof. In some embodiments, the SOS response gene is, for referred to herein as an “susceptibility agent-engineered bac example but is not limited to marr AB, arcAB and lexO. In teriophage' but are also encompassed under the definition of Some embodiments of this aspect and all other aspects a “repressor-engineered bacteriophage'. In some embodi described herein, an inhibitor of a non-SOS pathway bacterial ments of this aspect, and all other aspects described herein, defense gene can be any agent, such as but not limited to a Such an agent which increases the Susceptibility of a bacteria protein or an RNAi agent. Such as antisense to a non-SOS to an antimicrobial agent is referred to as a “susceptibility gene Such as, for example but not limited to SOXR, or modified agent” and refers to any agent which increases the bacteria's version thereof. In some embodiments of this aspect and all susceptibility to the antimicrobial agent by at least about 10% other aspects described herein, an repressor, Such as an agent or at least about 15%, or at least about 20% or at least about which inhibits a non-SOS pathway bacterial defense gene 30% or at least about 50% or more than 50%, or any integer inhibits, for example genes selected from the group of marr, between 10% and 50% or more, as compared to the use of the arc, SOXR, fur, crp, iccdA or craA or ompA or modified version antimicrobial agent alone. In one embodiment, a Susceptibil thereof. In other embodiments of this aspect of the invention, ity agent is an agent which specifically targets a bacteria cell. a nucleic acid of a repressor engineered bacteriophage is an In another embodiment, a Susceptibility agent modifies (i.e. agent which inhibits a non-SOS defense gene, for example inhibits or activates) a pathway which is specifically the repressor agent can inhibit any gene, or any combination expressed in bacterial cells. In one embodiment, a suscepti of genes listed in Table 2. In some embodiments, a repressor bility agent is an agent which has an additive effect of the engineered bacteriophage which inhibits a non-SOS defense efficacy of the antimicrobial agent (i.e. the agent has an addi gene can be used in combination with selected antimicrobial tive effect of the killing efficacy or inhibition of growth by the agents, for example, where the repressor-engineered bacte antimicrobial agent). In a preferred embodiment, a suscepti riophage encodes an agent which inhibits a gene listed in bility agent is an agent which has a synergistic effect on the Table 2A, such a repressor-engineered bacteriophage can be efficacy of the antimicrobial agent (i.e. the agent has a syn used in combination with a ciprofloxacin antimicrobial agent ergistic effect of the killing efficacy or inhibition of growth by or a variant or analogue thereof. Similarly, in other embodi the antimicrobial agent). ments a repressor-engineered bacteriophage which inhibits a 0090 Accordingly, another aspect of the invention relates non-SOS defense gene can encode an agent which inhibits a to the use of an inhibitor-engineered bacteriophage and/or a gene listed in Table 4B can be used in combination with a repressor-engineered bacteriophage and/or a susceptibility Vancomycin antimicrobial agent or a variant or analogue engineered bacteriophage to potentiate the killing effect of thereof. Similarly, in other embodiments a repressor-engi antimicrobial agents or stated another way, to enhance the neered bacteriophage which inhibits a non-SOS defense gene efficacy of antimicrobial agents. An inhibitor-engineered can encode an agent which inhibits a gene listed in Table 2C. bacteriophages and/or a repressor engineered bacteriophage 2D, 2E, 2F and 2G can be used in combination with a rifampi and/or a susceptibility-engineered bacteriophage is consid cin antimicrobial agent, or a amplicillin antimicrobial agent or ered to potentiate the effectiveness of an antimicrobial agent a sulfmethaxaZone antimicrobial agent or a gentamicin anti if the amount of antimicrobial agent used in combination with US 2010/0322903A1 Dec. 23, 2010

an engineered bacteriophage as disclosed herein is reduced 0094. As used herein, the term “adjuvant” as used herein by at least about 10% without adversely affecting the result, refers to an agent which enhances the pharmaceutical effect for example, without adversely effecting the level of antimi of another agent. As used herein, the bacteriophages as dis crobial activity. In another embodiment, the criteria used to closed herein function as adjuvants to antimicrobial agents, select an inhibitor-engineered bacteriophage and/or a repres Such as, but not limited to antibiotic agents, by enhancing the Sor engineered bacteriophage and/or a susceptibility-engi effect of the antimicrobial agents by at least... 5%, ... at least neered bacteriophage that potentiates the activity of an anti 10%, ... at least 15%, ... at least 20%, ... at least 25%, ... microbial agent is a reduction of at least about 10%, ... or at at least 35%, ... at least 50%, ... at least 60%, ... at least 90% least about 15%, ... or at least about 20%, ... or at least about and all amounts in-between as compared to use of the anti 25%, ... or at least about 35%, ... or at least about 50%, ... microbial agent alone. Accordingly, the engineered bacte or at least about 60%, ... or at least about 90% and all integers riophages as disclosed herein, such as the inhibitor-engi in between 10-90% of the amount of the antimicrobial agent neered bacteriophage and/or repressor engineered without adversely effecting the antimicrobial effect when bacteriophage function as antimicrobial agent adjuvants. compared to the similar amount without the addition of an 0.095 As used herein, the term “inhibitor-engineered bac inhibitor-engineered bacteriophage and/or repressor engi teriophage” refers to a bacteriophage that have been geneti neered bacteriophage and/or a Susceptibility-engineered bac cally engineered to comprise a nucleic acid which encodes an teriophage. Stated another way, an inhibitor-engineered bac agent which inhibits at least one gene involved in antibiotic teriophage and/or repressor engineered bacteriophage and/or resistance and/or cell Survival. Such engineered bacterioph a Susceptibility-engineered bacteriophage is effective as an ages as disclosed herein are termed “inhibitor-engineered adjuvant to an antimicrobial agent when the combination of bacteriophages' as they comprise a nucleic acid which the antimicrobial agent and the engineered bacteriophage encodes at least one inhibitor genes, such as but not limited to results in about the same level (i.e. within about 10%) of antibiotic resistance genes such as, but not limited to cat, antimicrobial effect at reducing the bacterial infection or VanA or mecD, or cell Survival repair gene Such as, but not killing the bacteria with the reduction in the dose (i.e. the limited to RecA, RecB. RecC. Spot or RelA. Naturally, one amount) of the antimicrobial agent. Such a reduction in anti can engineer a bacteriophage to comprise at least one nucleic microbial dose can be, for example by about 10%, or about acid which encodes more than one inhibitor, for example, two 15%, ... or about 20%, ... or about 25%, ... or about 35%, or more inhibitors to the same gene or to at least two different ... or about 50%, ... or about 60%, ... or more than 60% with genes which can be used in the methods and compositions as the same level of antimicrobial efficacy. disclosed herein. 0091. The inventors herein have demonstrated that the 0096. As used herein, the term “repressor-engineered bac engineered bacteriophage can target gene networks that are teriophage” refers to bacteriophages that have been geneti not directly attacked by antibiotics and by doing so, greatly cally engineered to comprise at least one nucleic acid which enhanced the efficacy of antibiotic treatment in bacteria, such encodes a repressor protein, or fragment thereof, where the as Escherichia coli. The inventors demonstrated that Sup repressor protein function to prevent activation of a gene pressing or inhibiting the bacterial SOS response network involved in a SOS response. Alternatively, the term repressor with a repressor-engineered bacteriophage can enhance kill engineeredbacteriophage refers to a bacteriophage which has ing by an antimicrobial agent such as an antibiotic, for been genetically engineered to comprise at least one nucleic example but not limited to, ofloxacin, a quinolone drug, by acid which encodes a repressor protein, such as an inhibitors over 2.7 orders of magnitude as compared with a control (including but not limited to RNAi agents) which inhibits a bacteriophage (i.e. non-engineered bacteriophages) plus non-SOS bacterial defense. Such engineered bacteriophages ofloxacin, and over 4.5 orders of magnitude compared with as disclosed herein are referred to herein as “repressor-engi ofloxacin alone. neered bacteriophages' as they comprise a nucleic acid 0092. The inventors have also demonstrated herein in encoding a repressor protein, for example, but not limited to, Examples 6-8 that a repressor-engineered bacteriophage, leXA, or soxR, or modified version thereof. In some embodi which comprises at least one inhibitor to one or more non ments, a SOS response gene is, for example but is not limited SOS genetic networks are also effective antibiotic adjuvants. to marr AB, arc AB and lexO. One can engineer a repressor The inventors also demonstrated that repressor-engineered engineered bacteriophage to comprise at least one nucleic bacteriophage and/or inhibitor-engineered bacteriophage can acid which encodes more than one repressor, for example at reduce the number of antibiotic-resistant bacteria in a popu least 2, 3, 4 or more repressors to the same or different SOS lation and act as a strong adjuvant for a variety of other response gene, in any combination, can be used in the meth bactericidal antibiotics, such as for example, but not limited ods and compositions as disclosed herein. Similarly, one can to gentamicin and amplicillin Thus, the inventors have dem also engineer a repressor-engineered bacteriophage to com onstrated that by selectively targeting gene networks with prise at least one nucleic acid which encodes more than one bacteriophage, one can enhance killing by antibiotics, thus repressor, for example at least 2, 3, 4 or more repressors, such discovering a highly effective new antimicrobial strategy. as inhibitors which inhibits any number and any combination of non-SOS bacterial defense genes listed in Table 2, and can DEFINITIONS be used in any combination, can be used in the methods and compositions as disclosed herein. The term “repressor-engi 0093. For convenience, certain terms employed in the neered bacteriophage' also encompasses Susceptibility-engi entire application (including the specification, examples, and neered bacteriophages as that term is defined herein. appended claims) are collected here. Unless defined other 0097. As used herein, the term “susceptibility-engineered wise, all technical and scientific terms used herein have the bacteriophage” refers to a bacteriophage that has been geneti same meaning as commonly understood by one of ordinary cally engineered to comprise at least one nucleic acid which skill in the art to which this invention belongs. encodes at least one agent which increases the Susceptibility US 2010/0322903A1 Dec. 23, 2010

of a bacterial cell to an antimicrobial agent. An agent which 0100. The term “synergy' or “synergistically are used increases the Susceptibility of a bacteria to an antimicrobial interchangeably herein, and when used in reference to a Sus agent is referred to herein as a “susceptibility agent” and ceptibility agent, or an engineered bacteriophage such as an includes any agent (Such as a protein or RNAi agent) which Susceptibility-bacteriophage having a synergistic effect of the increases the bacteria's susceptibility to the antimicrobial efficacy of the antimicrobial agent refers to a total increase in agent by at least about 10% or at least about 15%, or at least antimicrobial efficacy (ie killing, or reducing the viability of about 20% or at least about 30% or at least about 50% or more a bacterial population or inhibiting growth of a bacterial than 50%, or any integer between 10% and 50% or more, as population) with the combination of the antimicrobial agent compared to the use of the antimicrobial agent alone. In one and the Susceptibility-engineered bacteriophage components embodiment, a Susceptibility agent is an agent which specifi of the invention, over their single and/or additive efficacy of cally targets a bacteria cell. In another embodiment, a Suscep each component alone. A synergistic effect to increase total tibility agent modifies (i.e. inhibits or activates) a pathway antimicrobial effectiveness can be a result of an increase in antimicrobial effect of both components (i.e. the antimicro which is specifically expressed in bacterial cells. In one bial agent and the Susceptibility-engineered bacteriophage) embodiment, a Susceptibility agent is an agent which has an or alternatively, it can be the result of the increase in activity additive effect of the efficacy of the antimicrobial agent (i.e. of only one of the components (i.e. the antimicrobial agent or the agent has an additive effect of the killing efficacy or the Susceptibility-engineered bacteriophage). For clarifica inhibition of growth by the antimicrobial agent). In a pre tion by way of a non-limiting illustrative example of a syner ferred embodiment, a susceptibility agent is an agent which gistic effect, if an antimicrobial agent is effective at reducing has a synergistic effect on the efficacy of the antimicrobial (ie killing) a bacterial population by 15%, and a susceptibil agent (i.e. the agent has a synergistic effect of the killing ity-engineered bacteriophage was effective at reducing a bac efficacy or inhibition of growth by the antimicrobial agent). terial population by 10%, a synergistic effect of a combina 0098. The term “engineered bacteriophage' as used herein tion of the antimicrobial agent and the Susceptibility refer to any one, or a combination of an inhibitor-engineered engineered bacteriophage could be 50%. Stated another way, bacteriophage or a repressor-engineered bacteriophage or a in this example, any total effect greater than 25% (i.e. greater Susceptibility-engineered bacteriophage as these phrases are than the sum of the antibacterial agent alone (i.e. 15%) and the defined herein. susceptibility agent alone (i.e. 10%) would be indicative of a 0099. The term “additive' when used in reference to a synergistic effect. In some embodiments of the present inven susceptibility agent, or an engineered bacteriophage such as tion, the antimicrobial agent and susceptibility-engineered an Susceptibility-bacteriophage having an additive effect of bacteriophage component show at least Some synergistic anti the efficacy of the antimicrobial agent refers to refers to a total pathogenic activity. A synergistic effect of the combination of increase in antimicrobial efficacy (i e killing, or reducing the an antimicrobial agent with an engineered bacteriophage can viability of a bacterial population or inhibiting growth of a be an increase in at least about 10% or at least about 20% or bacterial population) with the combination of the antimicro at least about 30% or at least about 40% or at least about 50% bial agent and the Susceptibility-engineered bacteriophage or more anti-pathogenic (or antimicrobial) efficacy as com components of the invention, over their single efficacy of each pared to the sum of the antimicrobial effect achieved with use component alone. An additive effect to increase total antimi of the antimicrobial agent alone or the engineered bacterioph crobial effectiveness can be a result of an increase in antimi age alone. crobial effect of both components (i.e. the antimicrobial agent 0101 The term “bidirectional synergy” refers to the and the Susceptibility-engineered bacteriophage) or alterna increase in activity of each component (i.e. the antimicrobial tively, it can be the result of the increase inactivity of only one agent and the engineered bacteriophage) when used in com of the components (i.e. the antimicrobial agent or the Suscep bination with each other, and not merely an increase in activ tibility-engineered bacteriophage). For clarification by way ity of one of the antimicrobial components. In some embodi of a non-limiting illustrative example of a additive effect, if an ments, an antimicrobial agent and engineered bacteriophage antimicrobial agent is effective at reducing a bacterial popu show at least synergistic antimicrobial activity. In some lation by 30%, and a susceptibility-engineered bacteriophage embodiments, an antimicrobial agent and engineered bacte was effective at reducing a bacterial population by 20%, an riophage show bidirectional synergistic antimicrobial activ additive effect of a combination of the antimicrobial agent ity. Stated in other words, for example, bidirectional Synergy and the Susceptibility-engineered bacteriophage could be, for means an engineered bacteriophage enhances the activity of example 35%. Stated another way, in this example, any total an antimicrobial agent and vice versa, an antimicrobial agent effect greater than 30% (i.e. greater than the highest antimi can be used to enhance the activity of the engineered bacte crobial efficacy (i.e. 30% which, in this example is displayed riophage. by the antimicrobial agent) would be indicative of an additive 0102 The term “SOS used in the context of “SOS effect. In some embodiments of the present invention, the response' or "SOS response genes' as used herein refers to an antimicrobial agent and Susceptibility-engineered bacte inducible DNA repair system that allows bacteria to survive riophage component show at least some additive anti-patho Sudden increases in DNA damage. SOS response genes are genic activity. An additive effect of the combination of an repressed to differ rent degrees under normal growth condi antimicrobial agent with an engineered bacteriophage can be tions. Without being bound by theory, the SOS response is a an increase in at least about 10% or at least about 20% or at postreplication DNA repair system that allows DNA replica least about 30% or at least about 40% or at least about 50% or tion to bypass lesions or errors in the DNA. One example is more anti-pathogenic (or antimicrobial) efficacy as compared the SOS repressor RecA protein. The RecA protein, stimu to the highest antimicrobial effect achieved with either the lated by single-stranded DNA, is involved in the inactivation antimicrobial agent alone or the engineered bacteriophage of the Lex A repressor thereby inducing the response. The alone. bacterial SOS response, studied extensively in Escherichia US 2010/0322903A1 Dec. 23, 2010

coli, is a global response to DNA damage in which the cell bial cells, such as bacteria by at least about 30% or at least cycle is arrested and DNA repair and mutagenesis are about 40%, or at least about 50% or at least about 60% or at induced. SOS is the prototypic cell cycle check-point control least about 70% or more than 70%, or any integer between and DNA repair system. A central part of the SOS response is 30% and 70% as compared to in the absence of the antimi the de-repression of more than 20 genes under the direct and crobial agent. In one embodiment, an antimicrobial agent is indirect transcriptional control of the Lex A repressor. The an agent which specifically targets a bacteria cell. In another LeXA regulon includes recombination and repair genes recA, embodiment, an antimicrobial agent modifies (i.e. inhibits or recN, and ruvAB, nucleotide excision repair genes uvraB activates or increases) a pathway which is specifically and uvrD, the error-prone DNA polymerase (pol) genes dinB expressed in bacterial cells. In some embodiments, an anti (encoding pol IV) and umul C (encoding pol V), and DNA microbial agent does not include the following agents; che polymerase II in addition to many other genes functions. In motherapeutic agent, a toxin protein expressed by a bacteria the absence of a functional SOS response (i.e. in the presence or other microorganism (i.e. a bacterial toxin protein) and the of repressors as disclosed herein), cells are sensitive to DNA like. An antimicrobial agent can include any chemical, pep damaging agents. McKenzie et al., PNAS, 2000; 6646–6651: tide (i.e. an antimicrobial peptide), peptidomimetic, entity or Michel, PLoS Biology, 2005; 3: e255, and which are incor porated in their entirety herein by reference. A “non-SOS moiety, or analogues of hybrids thereof, including without gene' also includes a “bacterial defense gene' and refers to limitation synthetic and naturally occurring non-proteina genes expressed by a bacteria or a microorganism which ceous entities. In some embodiments, an antimicrobial agent serve protect the bacteria or microorganism from cell death, is a small molecule having a chemical moiety. For example, for example from being killed or growth Suppressed by an chemical moieties include unsubstituted or substituted alkyl, antimicrobial agent. Typically, inhibition or knocking out aromatic or heterocyclyl moieties including macrollides, lep such non-SOS defense genes increases the susceptibility of a tomycins and related natural products or analogues thereof. microorganism Such as bacteria to an antimicrobial agent. A Antimicrobial agents can be any entity known to have a non-SOS gene' or “bacterial defense gene' is not part of the desired activity and/or property, or can be selected from a SOS-response network, but still serve as protective functions library of diverse compounds. to prevent microorganism cell death. In certain conditions, 0105. The term "agent” as used herein and throughout the Some non-SOS genes and/or bacterial defense genes can be application is intended to refer to any means such as an expressed (i.e. upregulated) on DNA damage or in stressful organic or inorganic molecule, including modified and conditions. Examples of a non-SOS gene is soxS, which is unmodified nucleic acids such as antisense nucleic acids, repressed by SOXR, and examples of defense genes are any RNAi, such as siRNA or shRNA, peptides, peptidomimetics, gene listed in Table 2. receptors, ligands, and antibodies, aptamers, polypeptides, 0103) The term “repressor as used herein, refers to a nucleic acid analogues or variants thereof. protein that binds to an operator of a gene preventing the 0106 The term “antimicrobial peptide' as used herein transcription of the gene. Accordingly, a repressor can effec refers to any peptides with antimicrobial activity, i.e. the tively “suppress' or inhibit the transcription of a gene. The ability to inhibit the growth and/or kill bacterium, for example binding affinity of repressors for the operator can be affected gram positive- and gram negative bacteria. The term antimi by other molecules, such as inducers, which bind to repres crobial peptides encompasses all peptides that have antimi sors and decrease their binding to the operator, while co crobial activity, and are typically, for example but not limited repressors increase the binding. The paradigm of repressor to, short proteins, generally between 12 and 50 amino acids proteins is the lactose repressor protein that acts on the lac long, however larger proteins with Such as, for example operon and for which the inducers are 3-galactosides such as lysozymes are also encompassed as antimicrobial peptides in lactose, it is a polypeptide of 360 amino acids that is active as the present invention. Also included in the term antimicrobial a tetramer. Other examples are the lambda repressor protein peptide are antimicrobial peptidomimetics, and analogues or oflambda bacteriophage that prevents the transcription of the fragments thereof. The term “antimicrobial peptide' also genes required for the lytic cycle leading to lysogeny and the includes all cyclic and non-cyclic antimicrobial peptides, or cro protein, also of lambda, which represses the transcription derivatives or variants thereof, including tautomers, see Li et of the lambda repressor protein establishing the lytic cycle. al. JACS, 2006, 128: 5776-85 and world-wide-web at f/aps. Both of these are active as dimers and have a common struc unmc.edu, at /AP/main.php for examples, which are incorpo tural feature the helix turn helix motif that is thought to bind rated herein in their entirety by reference. In some embodi to DNA with the helices fitting into adjacent major grooves. ments, the antimicrobial peptide is a lipopeptide, and in some Useful repressors according to the present invention include, embodiments the lipopeptide is a cyclic lipopeptide. The but are not limited to leXA, marr, arc, SOXR, fur, crp, iccdA, or lipopeptides include, for example but not limited to, the poly craA or modified version thereof. myxin class of antimicrobial peptides. 0104. The term “antimicrobial agent” as used herein refers 0107 The term “microorganism’ includes any micro to any entity with antimicrobial activity, i.e. the ability to scopic organism or taxonomically related macroscopic inhibit the growth and/or kill bacterium, for example gram organism within the categories algae, bacteria, fungi, yeast positive- and gram negative bacteria. An antimicrobial agent and protozoa or the like. It includes Susceptible and resistant is any agent which results in inhibition of growth or reduction microorganisms, as well as recombinant microorganisms. of viability of a bacteria by at least about 30% or at least about Examples of infections produced by Such microorganisms are 40%, or at least about 50% or at least about 60% or at least provided herein. In one aspect of the invention, the antimi about 70% or more than 70%, or any integer between 30% crobial agents and enhancers thereofare used to target micro and 70% or more, as compared to in the absence of the organisms in order to prevent and/or inhibit their growth, antimicrobial agent. Stated another way, an antimicrobial and/or for their use in the treatment and/or prophylaxis of an agent is any agent which reduces a population of antimicro infection caused by the microorganism, for example multi US 2010/0322903A1 Dec. 23, 2010

drug resistant microorganisms and gram-negative microor implanted medical devices, such as catheters, heart Valves ganisms. In some embodiments, gram-negative microorgan and joint replacements. In particular, catheters are associated isms are also targeted. with infection by many biofilm forming organisms such as 0108. The anti-pathogenic aspects of the invention target Staphylococcus epidermidis, Staphylococcus aureus, the broader class of “microorganism' as defined herein. How Pseudomonas aeruginosa, Enterococcus faecalis and Can ever, given that a multi-drug resistant microorganism is so dida albicans which frequently result in generalized blood difficult to treat, the antimicrobial agent and inhibitor-engi stream infection. In a subject identified to have a catheter neered bacteriophage and/or repressor-engineered bacte infected with bacterial, such as for example, a bacterial riophage in the context of the anti-pathogenic aspect of the infected central venous catheter (CVC), the subject can have invention is Suited to treating all microorganisms, including the infected catheter removed and can be treated by the meth for example multi-drug resistant microorganisms, such as ods and compositions as disclosed herein comprising an engi bacterium and multi-drug resistant bacteria. neeredbacteriophage and antimicrobial agent to eliminate the 0109 Unless stated otherwise, in the context of this speci bacterial infection. Furthermore, on removal of the infected fication, the use of the term “microorganism” alone is not catheter and its replacement with a new catheter, the Subject limited to “multi-drug resistant organism’, and encompasses can also be administered the compositions comprising engi both drug-susceptible and drug-resistant microorganisms. neered bacteriophages and antimicrobial agents as disclosed The term “multi-drug resistant microorganism” refers to herein on a prophylaxis basis to prevent re-infection or the those organisms that are, at the very least, resistant to more re-occurrence of the bacterial infection. Alternatively, a sub than two antimicrobial agents such as antibiotics in different ject can be administered the compositions as disclosed herein antibiotic classes. This includes those microorganisms that comprising engineered bacteriophages and antimicrobial have more resistance than those that are resistant to three or agents on a prophylaxis basis on initial placement of the more antibiotics in a single antibiotic class. This also includes catheter to prevent any antimicrobial infection Such as a bac microorganisms that are resistant to a wider range of antibi terial biofilm infection. The effect can be prophylactic in otics, i.e. microorganisms that are resistant to one or more terms of completely or partially preventing a disease or sign classes of antibiotics. or symptom thereof, and/or can be therapeutic in terms of a 0110. The term “persistent cell” or “persisters” are used partial or complete cure of a disease. interchangeably herein and refer to a metabolically dormant 0114. As used herein, the term “effective amount” is Subpopulation of microorganisms, typically bacteria, which meant an amount of antimicrobial agent and/or inhibitor are not sensitive to antimicrobial agents such as antibiotics. engineered bacteriophages or repressor-engineered bacte Persisters typically are not responsive (i.e. are not killed by riophages effective to yield a desired decrease in bacteria or the antibiotics) as they have non-lethally downregulated the increase to increase the efficacy of antimicrobial agent as pathways on which the antimicrobial agents act i.e. the per compared to the activity of the antimicrobial agent alone (i.e. sister cells have down regulated the pathways which are nor without the engineered bacteriophages as disclosed herein). mally inhibited or corrupted by the antimicrobial agents, such The term “effective amount’ as used herein refers to that as the transcription, translation, DNA replication and cell amount of composition necessary to achieve the indicated wall biosynthesis pathways. Persisters can develop at non effect, i.e. a reduction of the number of viable microorgan lethal (or sub-lethal) concentrations of the antimicrobial isms, such as bacteria, by at reduction of least 5%, at least agent. 10%, by at least 20%, by at least 30%... at least 35%, ... at 0111. The term “analog as used herein refers to a com least 50%, ... at least 60%, ... at least 90% or any reduction position that retains the same structure or function (e.g., bind of viable microorganism in between. As used herein, the ing to a receptor) as a polypeptide or nucleic acid herein. effective amount of the bacteriophage as disclosed herein is Examples of analogs include peptidomimetics, peptide the amount sufficient to enhance the effect of the antimicro nucleic acids, Small and large organic or inorganic com bial agents by at least. .. 5%, at least 10%, ... at least 15%, pounds, as well as derivatives and variants of a polypeptide or ... at least 20%, ... at least 25%, ... at least 35%, ... at least nucleic acid herein. The term “analog as used herein refers to 50%, . . . at least 60%, . . . at least 90% and all amounts a composition that retains the same structure or function (e.g., in-between as compared to use of the antimicrobial agent binding to a receptor) as a polypeptide or nucleic acid herein. alone. Or alternatively result in the same efficacy of the anti 0112. The term “infection' or “microbial infection' which microbial effect with less (i.e. for example by about 10%, or are used interchangeably herein refers to in its broadest sense, about 15%, ... or about 20%, ... or about 25%, ... or about any infection caused by a microorganism and includes bac 35%, ... or about 50%, ... or about 60%, ... or more than 60% terial infections, fungal infections, yeast infections and pro less) amount or dose of the antimicrobial agents as compared toZomal infections. to its use alone to achieve the same efficacy of antimicrobial 0113. The term “treatment and/prophylaxis' refers gener effect. The “effective amount’ or “effective dose' will, obvi ally to afflicting a subject, tissue or cell to obtain a desired ously, vary with Such factors, in particular, the strain of bac pharmacologic arid/or physiologic effect, which in the case of teria being treated, the strain of bacteriophage being used, the the methods of this invention, include reduction or elimina genetic modification of the bacteriophage being used, the tion of microbial infections or prevention of microbial infec antimicrobial agent, as well as the particular condition being tions. The methods as disclosed herein can be used prophy treated, the physical condition of the subject, the type of lactically for example in instances where an individual is subject being treated, the duration of the treatment, the route susceptible for infections or re-infection with a particular of administration, the type of antimicrobial agent and/or bacterial strain or a combination of Such strains. For example, enhancer of antimicrobial agent, the nature of concurrent microbial infections such as bacterial infections such as bio therapy (if any), and the specific formulations employed, the films can occur on any Surface where Sufficient moisture and ratio of the antimicrobial agent and/or enhancers antimicro nutrients are present. One Such surface is the Surface of bial agent components to each other, the structure of each of US 2010/0322903A1 Dec. 23, 2010

these components or their derivatives. The term “effective 0119) As used herein “shRNA” or “small hairpin RNA'. amount' when used in reference to administration of the (also called stem loop) is a type of siRNA. In one embodi compositions comprising an antimicrobial agent and a engi ment, these shRNAs are composed of a short, e.g. about 19 to neered bacteriophage as disclosed hereinto a subject refers to about 25 nucleotide, antisense strand, followed by a nucle the amount of the compositions—to reduce or stop at least otide loop of about 5 to about 9 nucleotides, and the analo one symptom of the disease or disorder, for example a symp gous sense Strand. Alternatively, the sense Strand can precede tom or disorder of the microorganism infection, such as bac the nucleotide loop structure and the antisense strand can terial infection. For example, an effective amount using the follow. methods as disclosed herein would be considered as the 0.120. The terms “microRNA or “miRNA are used inter amount Sufficient to reduce a symptom of the disease or changeably herein are endogenous RNAS, Some of which are disorder of the bacterial infection by at least 10%. An effec known to regulate the expression of protein-coding genes at tive amount as used herein would also include an amount the posttranscriptional level. Endogenous microRNA are sufficient to prevent or delay the development of a symptom small RNAs naturally present in the which are of the disease, alter the course of a symptom disease (for capable of modulating the productive utilization of mRNA. example but not limited to, slow the progression of a symp The term artificial microRNA includes any type of RNA tom of the disease), or reverse a symptom of the disease. sequence, other than endogenous microRNA, which is 0115. As used herein, a “pharmaceutical carrier' is a phar capable of modulating the productive utilization of mRNA. maceutically acceptable solvent, Suspending agent or vehicle MicroRNA sequences have been described in publications for delivering the combination of antimicrobial agent and/or such as Lim, et al., Genes & Development, 17, p. 991-1008 inhibitor-engineered bacteriophages or repressor-engineered (2003), Limetal Science 299, 1540 (2003), Lee and Ambros bacteriophages to the surface infected with bacteria or to a Science, 294, 862 (2001), Lau et al., Science 294, 858-861 subject. The carrier can be liquid or solid and is selected with (2001), Lagos-Quintana et al. Current Biology, 12, 735-739 the planned manner of administration in mind. Each carrier (2002), Lagos Quintana et al. Science 294, 853-857 (2001), must be pharmaceutically “acceptable' in the sense of being and Lagos-Quintana etal, RNA, 9, 175-179 (2003), which are compatible with other ingredients of the composition and non incorporated by reference. Multiple microRNAs can also be injurious to the Subject. incorporated into a precursor molecule. Furthermore, 0116. As used herein, “gene silencing or “gene silenced' miRNA-like stem-loops can be expressed in cells as a vehicle in reference to an activity of in RNAi molecule, for example to deliver artificial miRNAs and short interfering RNAs (siR a siRNA or miRNA refers to a decrease in the mRNA level in NAs) for the purpose of modulating the expression of endog a cell for a target gene by at least about 5%, about 10%, about enous genes through the miRNA and or RNAi pathways. 20%, about 30%, about 40%, about 50%, about 60%, about 0121. As used herein, “double stranded RNA or 70%, about 80%, about 90%, about 95%, about 99%, about “dsRNA refers to RNA molecules that are comprised of two 100% of the mRNA level found in the cell without the pres strands. Double-stranded molecules include those comprised ence of the miRNA or RNA interference molecule. In one of a single RNA molecule that doubles back on itself to form preferred embodiment, the mRNA levels are decreased by at a two-stranded structure. For example, the stem loop structure least about 70%, about 80%, about 90%, about 95%, about of the progenitor molecules from which the single-stranded 99%, about 100%. miRNA is derived, called the pre-miRNA (Bartel et al. 2004. 0117. As used herein, the term “RNAi refers to any type Cell 116:281-297), comprises a dsRNA molecule. of interfering RNA, including but not limited to, siRNAi, I0122) The terms “patient”, “subject” and “individual” are shRNAi, endogenous microRNA and artificial microRNA. used interchangeably herein, and refer to an animal, particu For instance, it includes sequences previously identified as larly a human, to whom treatment including prophylaxis siRNA, regardless of the mechanism of down-stream pro treatment is provided. The term “subject' as used herein cessing of the RNA (i.e. although siRNAs are believed to have refers to human and non-human animals. The term “non a specific method of in vivo processing resulting in the cleav human animals' and “non-human mammals' are used inter age of mRNA, such sequences can be incorporated into the changeably herein includes all vertebrates, e.g., mammals, vectors in the context of the flanking sequences described Such as non-human primates, (particularly higher primates), herein). sheep, dog, rodent (e.g. mouse or rat), guinea pig, goat, pig, 0118. As used herein an “siRNA refers to a nucleic acid cat, rabbits, cows, and non-mammals such as chickens, that forms a double stranded RNA, which double stranded amphibians, reptiles etc. In one embodiment, the Subject is RNA has the ability to reduce or inhibit expression of a gene human. In another embodiment, the Subject is an experimen or target gene when the siRNA is present or expressed in the tal animal or animal Substitute as a disease model. Suitable same cell as the target gene, for example Lp-PLA. The mammals also include members of the orders Primates, double stranded RNA siRNA can be formed by the comple Rodentla, Lagomorpha, Cetacea, Homo sapiens, Carnivora, mentary strands. In one embodiment, a siRNA refers to a Perissodactyla and Artiodactyla. Members of the orders nucleic acid that can form a double stranded siRNA. The Perissodactyla and Artiodactyla are included in the invention sequence of the siRNA can correspond to the full length target because of their similar biology and economic importance, gene, or a Subsequence thereof. Typically, the siRNA is at for example but not limited to many of the economically least about 15-50 nucleotides in length (e.g., each comple important and commercially important animals such as goats, mentary sequence of the double stranded siRNA is about sheep, cattle and pigs have very similar biology and share 15-50 nucleotides in length, and the double stranded siRNA is high degrees of genomic homology. about 15-50 base pairs in length, preferably about 19-30 base I0123. The term “gene' used herein can be a genomic gene nucleotides, preferably about 20-25 nucleotides in length, comprising transcriptional and/or translational regulatory e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in sequences and/or a coding region and/or non-translated length). sequences (e.g., introns, 5'- and 3'-untranslated sequences US 2010/0322903A1 Dec. 23, 2010 and regulatory sequences). The coding region of agene can be complements thereof. As will also be appreciated by those in a nucleotide sequence coding for an amino acid sequence or the art, a single strand provides a probe for a probe that can a functional RNA, such as tRNA, rRNA, catalytic RNA, hybridize to the target sequence under Stringent hybridization siRNA, miRNA and antisense RNA. A gene can also be an conditions. Thus, a nucleic acid also encompasses a probe mRNA or cDNA corresponding to the coding regions (e.g. that hybridizes under stringent hybridization conditions. exons and miRNA) optionally comprising 5'- or 3' untrans I0128 Nucleic acids can be single stranded or double lated sequences linked thereto. A gene can also be an ampli Stranded, or can contain portions of both double Stranded and fied nucleic acid molecule produced in vitro comprising all or single stranded sequence. The nucleic acid can be DNA, both a part of the coding region and/or 5'- or 3'-untranslated genomic and cDNA, RNA, or a hybrid, where the nucleic acid sequences linked thereto. can contain combinations of deoxyribo- and ribo-nucle 0.124. The term “gene product(s) as used herein refers to otides, and combinations of bases including uracil, adenine, include RNA transcribed from a gene, or a polypeptide thymine, cytosine, guanine, inosine, Xanthine hypoxanthine, encoded by a gene or translated from RNA. isocytosine and isoguanine. Nucleic acids can be obtained by 0.125. The term “inhibit or “reduced or “reduce' or chemical synthesis methods or by recombinant methods. “decrease' as used herein generally means to inhibit or I0129. A nucleic acid will generally contain phosphodi decrease the expression of a gene or the biological function of ester bonds, although nucleic acid analogs can be included the protein (i.e. an antibiotic resistance protein) by a statisti that can have at least one different linkage, e.g., phosphora cally significant amount relative to in the absence of an inhibi midate, phosphorothioate, phosphorodithioate, or O-meth tor. The term “inhibition or “inhibit or “reduce when refer ylphosphoroamidite linkages and peptide nucleic acid back ring to the activity of an antimicrobial agent or composition as bones and linkages. Other analog nucleic acids include those disclosed herein refers to prevention of, or reduction in the with positive backbones; non-ionic backbones, and non-ri rate of growth of the bacteria. Inhibition and/or inhibit when bose backbones, including those described in U.S. Pat. Nos. used in the context to refer to an agent that inhibits an antibi 5,235,033 and 5,034,506, which are incorporated by refer otic resistance gene and/or cell Survival refers to the preven ence. Nucleic acids containing one or more non-naturally tion or reduction of activity of a gene or gene product, that occurring or modified nucleotides are also included within when inactivated potentiates the activity of an antimicrobial one definition of nucleic acids. The modified nucleotide ana agent. However, for avoidance of doubt, “inhibit” means sta log can be located for example at the 5'-end and/or the 3'-end tistically significant decrease in activity of the biological of the nucleic acid molecule. Representative examples of function of a protein by at least about 10% as compared to in nucleotide analogs can be selected from Sugar- or backbone the absence of an inhibitor, for example a decrease by at least modified ribonucleotides. It should be noted, however, that about 20%, at least about 30%, at least about 40%, at least also nucleobase-modified ribonucleotides, i.e. ribonucle about 50%, or least about 60%, or least about 70%, or least otides, containing a non naturally occurring nucleobase about 80%, at least about 90% or more, up to and including a instead of a naturally occurring nucleobase such as uridines 100% inhibition (i.e. complete absence of an antibiotic resis or cytidines modified at the 5-position, e.g. 5-(2-amino)pro tance gene protein in the presence of an inhibitor), or any pyl uridine, 5-bromo uridine; adenosines and guanosines decrease in biological activity of the protein (i.e. of an anti modified at the 8-position, e.g. 8-bromo guanosine; deaza biotic resistance gene protein) between 10-100% as com nucleotides, e.g. 7 deaza-adenosine; O- and N-alkylated pared to a in the absence of an inhibitor. nucleotides, e.g. N6-methyl adenosine are suitable. The 0126 The terms “activate” or “increased' or “increase' as 2OH-group can be replaced by a group selected from H.O.R. used in the context of biological activity of a protein (i.e. R. halo, SH, SR, NH, NHR, NR, or CN, wherein R is C-C6 activation of a SOS response gene) herein generally means an alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I. Modifica increase in the biological function of the protein (i.e. SOS tions of the ribose-phosphate backbone can be done for a response protein) by a statically significant amount relative to variety of reasons, e.g., to increase the stability and half-life in a control condition. For the avoidance of doubt, an of such molecules in physiological environments or as probes “increase of activity, or “activation of a protein means a on a biochip. Mixtures of naturally occurring nucleic acids statistically significant increase of at least about 10% as com and analogs can be made; alternatively, mixtures of different pared to the absence of an agonist or activator agent, includ nucleic acid analogs, and mixtures of naturally occurring ing an increase of at least about 20%, at least about 30%, at nucleic acids and analogs can be made. least about 40%, at least about 50%, at least about 60%, at 0.130. As used herein, the terms “administering, and least about 70%, at least about 80%, at least about 90%, at “introducing are used interchangeably and refer to the place least about 100% or more, including, for example at least ment of the bacteriophages and/or antimicrobial agents as 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least disclosed herein onto the surface colonized by bacteria or into 10-fold increase or greater as compared to in a control con a Subject, such as a Subject with a bacterial infection or other dition. microorganism infection, by any method or route which 0127. The term “nucleic acid” or “oligonucleotide' or results in at least partial localization of the engineered-bac "polynucleotide' used herein can mean at least two nucle teriophages and/or antimicrobial agents at a desired site. The otides covalently linked together. As will be appreciated by compositions as disclosed herein can be administered by any those in the art, the depiction of a single strand also defines the appropriate route which results in the effective killing, elimi sequence of the complementary strand. Thus, a nucleic acid nation or control of the growth of the bacteria. also encompasses the complementary Strand of a depicted I0131 The term “vectors' is used interchangeably with single strand. As will also be appreciated by those in the art, “plasmid' to refer to a nucleic acid molecule capable of many variants of a nucleic acid can be used for the same transporting another nucleic acid to which it has been linked purpose as a given nucleic acid. Thus, a nucleic acid also A vector can be a plasmid, bacteriophage, bacterial artificial encompasses Substantially identical nucleic acids and chromosome or yeast artificial chromosome. A vector can be US 2010/0322903A1 Dec. 23, 2010

a DNA or RNA vector. A vector can be either a self replicating required for a given embodiment. The term permits the pres extrachromosomal vector or a vector which integrate into a ence of elements that do not materially affect the basic and host genome. Vectors capable of directing the expression of novel or functional characteristic(s) of that embodiment of genes and/or nucleic acid sequence to which they are opera the invention. tively linked are referred to herein as “expression vectors'. In 0.137 Other than in the operating examples, or where oth general, expression vectors of utility in recombinant DNA erwise indicated, all numbers expressing quantities of ingre techniques are often in the form of "plasmids” which refer to dients or reaction conditions used herein should be under circular double stranded DNA loops which, in their vector stood as modified in all instances by the term “about.” The form are not bound to the chromosome. Other expression term “about when used in connection with percentages can vectors can be used in different embodiments of the invention, meant 1%. for example, but are not limited to, plasmids, episomes, bac 0.138. This invention is further illustrated by the following teriophages or viral vectors, and Such vectors can integrate examples which should not be construed as limiting. The into the host's genome or replicate autonomously in the par contents of all references cited throughout this application, as ticular cell. Otherforms of expression vectors known by those well as the figures and tables are incorporated herein by skilled in the art which serve the equivalent functions can also reference. be used. Expression vectors comprise expression vectors for 0.139. It should be understood that this invention is not stable or transient expression encoding the DNA. limited to the particular methodology, protocols, and 0132) The term “analog as used herein refers to a com reagents, etc., described herein and as Such can vary. The position that retains the same structure or function (e.g., bind terminology used herein is for the purpose of describing ing to a receptor) as a polypeptide or nucleic acid herein. particular embodiments only, and is not intended to limit the Examples of analogs include peptidomimetics, peptide scope of the present invention, which is defined solely by the nucleic acids, Small and large organic or inorganic com claims. Other features and advantages of the invention will be pounds, as well as derivatives and variants of a polypeptide or apparent from the following Detailed Description, the draw nucleic acid herein. The term “analog as used herein refers to ings, and the claims. a composition that retains the same structure or function (e.g., binding to a receptor) as a polypeptide or nucleic acid herein. Inhibitor-Engineered Bacteriophages 0133. The term “derivative' or “variant as used herein refers to a peptide, chemical or nucleic acid that differs from 0140. One aspect of the present invention relates to an the naturally occurring polypeptide or nucleic acid by one or engineered bacteriophage which comprise a nucleic acid more amino acid or nucleic acid deletions, additions, Substi which encodes an agent which inhibits at least one antibiotic tutions or side-chain modifications. Amino acid substitutions resistance gene or at least one cell Survival gene, thereby gene include alterations in which an amino acid is replaced with a silencing Such genes and preventing the development of anti different naturally-occurring or a non-conventional amino biotic resistance and/or increased cell viability of the bacteria acid residue. Such substitutions may be classified as “conser in the presence of the antimicrobial agent. As discussed Vative', in which case an amino acid residue contained in a herein, such engineered bacteriophages which comprise a polypeptide is replaced with another naturally occurring nucleic acid encoding an agent which inhibits at least one amino acid of similar character either in relation to polarity, gene involved in antibiotic resistance and/or at least one cell side chain functionality or size. Survival gene as disclosed herein are referred to herein as 0134) Substitutions encompassed by the present invention “inhibitor-engineered bacteriophages”. may also be “non conservative', in which an amino acid 0.141. In some embodiments, an inhibitor-engineered bac residue which is present in a peptide is substituted with an teriophage can comprise a nucleic acid encoding any type of amino acid having different properties, such as naturally inhibitor, such as a nucleic acid inhibitor. Nucleic acid inhibi occurring amino acid from a different group (e.g., Substitut tors include, for example but are not limited to antisense ing a charged or hydrophobic amino; acid with alanine), or nucleic acid inhibitors, oligonucleosides, RNA interference alternatively, in which a naturally-occurring amino acid is (RNAi) and paired termini (PT) antisense and variants Substituted with a non-conventional amino acid. In some thereof. embodiments amino acid Substitutions are conservative. 0142. In some embodiments of this aspect of the invention, 0135. The articles “a” and “an are used herein to refer to an inhibitor-engineered bacteriophage can encode an agent one or to more than one (i.e., at least one) of the grammatical which inhibits the gene expression and/or protein function of object of the article. By way of example, “an element’ means any bacterial antibiotic resistance genes commonly known by one element or more than one element. Thus, in this specifi persons of ordinary skill in the art, Such as, but not limited to cation and the appended claims, the singular forms 'a'an.” cat (SEQID NO:1), van A (SEQID NO:2) or mecD (SEQID and “the include plural references unless the context clearly NO:3). In alternative embodiments, an agent can inhibit the dictates otherwise. Thus, for example, reference to a pharma gene expression and/or protein function of any bacterial cell ceutical composition comprising “an agent' includes refer Survival repair gene commonly known by persons of ordinary ence to two or more agents. skill in the art such as, but not limited to RecA, RecB, RecC. 0136. As used herein, the term “comprising means that Spot or RelA. other elements can also be present in addition to the defined 0.143 For reference, RecA (recombinase A) can be iden elements presented. The use of "comprising indicates inclu tified by Accession number: P03017 and Gene ID Seq ID sion rather than limitation. The term “consisting of refers to GI: 132224. Table 1 provides the accession numbers and Gene compositions, methods, and respective components thereof ID numbers for examples of antibiotic resistance genes and as described herein, which are exclusive of any element not cell survival genes which can be inhibited in the methods of recited in that description of the embodiment. As used herein the present invention, as well examples of as repressors which the term “consisting essentially of refers to those elements one can use in repressor-engineered bacteriophages. US 2010/0322903A1 Dec. 23, 2010 18

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 M97297 479085 Vancomycin-resistant protein mecA 3 X52593 46610 Penicillin binding protein II recA 4 b2699, NC OOO913.2 947 170 ECK2694, (2820730... 2821791, JW2669, lexB, complement) recH, rnmB, Srf, if, umuB, umuR, Zab rec 5 b2820, NC OOO913.2 947286 exonuclease V (RecBCD ECK2816, (295.0483 ... 2954.025, complex), beta subunit JW2788, ior, complement) ror A recC 6 b2822, NC OOO913.2 947294 exonuclease V (RecBCD ECK2818, (2957082... 296.0450, complex), gamma chain JW 2790 complement) spoT 7 b3650, NC OOO913.2 94.8159 bifunctional (p)ppGpp ECK3640, (38.20423 ... 3822531) synthetase IIguanosine JW3625 3',5'-bis pyrophosphate 3'- pyrophosphohydrolase relA 8 b2784, NC OOO913.2 947244 (p)ppGpp synthetase I/GTP ECK2778, (2909439... 2911673, pyrophosphokinase JW2755, RC complement) lexA 9 b4043, NC OOO913.2 948.544 DNA-binding ECK4035, (4255138... 4255746) transcriptional repressor of JW4003, exra, SOS regulon recA, spr, ts, marR O b1530, NC OOO913.2 94.582S DNA-binding ECK1523, (1617144. ... 1617578) transcriptional repressor of JW5248, cfxB, multiple antibiotic resistance inaR, SOXQ 1 P22gp18 NC OO2371.2 1262.795 Arc; transcriptional (14793... 15022) repressor SoxR 2 baQ63, NC OOO913.2 94.8566 DNA-binding ECK4055, (4275492 ... 4275956) transcriptional dual JW4024, marC regulator, Fe—S center for redox-sensing fur 3 b0683, NC OOO913.2 945295 DNA-binding ECKO671, (709423 ... 709869, transcriptional dual JWO669 complement) regulator of siderophore biosynthesis and transport crp 4 b3357, NC OOO913.2 94.7867 DNA-binding ECK3345, (3484142... 3484774) transcriptional dual JW5702, cap, regulator CS icod 5 b1136, NC OOO913.2 94.5702 e14 prophage; isocitrate ECK1122, (1194346 ... 1195596) dehydrogenase, specific for JW1122, iccdA, NADP iccE cSrA 6 b2696, NC OOO913.2 947176 pleiotropic regulatory (2816983... 2817168, protein for carbon Source complement) metabolism omp A NC OOO913.2 945571 outer membrane protein A (1018236... 1019276, (3a; II*; G: d) complement)

0144) In some embodiments, one can use a modular design or express at least one repressor of a SOS response gene. For strategy in which bacteriophage kill bacteria in a species example, in Some embodiments, the bacteriophage can specific manner are engineered to express at least one inhibi express an nucleic acid inhibitor, Such as an antisense nucleic tor of at least one antibiotic gene and/or a cell Survival gene, acid inhibitor or antisense RNA (asRNA) which inhibits at US 2010/0322903A1 Dec. 23, 2010

least one, or at least two or at least three antibiotic genes use in the inhibitor-engineered bacteriophages disclosed and/or a cell survival gene, such as, but not limited to cat (SEQ herein. In addition to the antibiotic resistance genes discussed ID NO:1), van A (SEQ ID NO:2) mecD (SEQ ID NO:3), herein, other Such antibiotic resistance genes which can be RecA (SEQID NO:4), RecB (SEQID NO:5), RecC (SEQID used include, for example, are katG, rpoB, rpsL, ampC, beta NO:6), Spot (SEQ ID NO:7) or RelA (SEQID NO:8). lactamases, aminoglycoside kinases, meXA, mexB, oprM. 0145 Some aspects of the present invention are directed to ermA, carA, Imra, ereA, VgbA, InVA, mph A. tetA, tetB, use of a inhibitor-engineeredbacteriophage as an adjuvants to van H, van R, VanX, VanY. vanZ, folC, folE, folP, and folk an antimicrobial agent, where an inhibitor-engineered bacte which are disclosed in U.S. Pat. No. 7,125,622, which is riophage encodes at least one inhibitor to an antimicrobial or incorporated herein in its entity by reference. antibacterial resistance gene in the bacteria. Previous uses of Repressor-Engineered Bacteriophages antibiotic resistance genes have been used to increase the susceptibility of bacteria to antimicrobial agents. For 0.147. In another aspect of the present invention, an engi example, US patent application US2002/0076722 discusses a neered bacteriophage can comprise a nucleic acid encoding a method of improving susceptibility of bacteria to antibacte repressor, or fragment thereof, of a SOS response gene or a rial agents by identifying gene loci which decrease the bac non-SOS defense gene and as discussed previously, are terium's Susceptibility to antibacterial agents, and identify referred to herein as “repressor-engineered bacteriophages.” OftX, WbbL, Slt, and Wza as such loci. However, in contrast 0.148. In some embodiments of this aspect and all aspects to the present application, US2002/0076722 does not teach described herein, a repressor-engineered bacteriophage can method to inhibit the loci to increase the bacterial suscepti comprises a nucleic acid encoding a repressor protein, or bility to antibacterial agents. Similarly, U.S. Pat. No. 7,125, fragment thereof of a bacterial SOS response gene, or an 622 discusses a method to identify bacterial antibiotic resis agent (such as a protein) which inhibits a non-SOS pathway tance genes by analyzing pools of bacterial genomic bacterial defense gene. fragments and selecting those fragments which hybridize or 0149. Without wishing to be limited to theory, the SOS have high homology (using computer assisted in silico meth response in bacteria is an inducible DNA repair system which odologies) to numerous known bacterial resistance genes. allows bacteria to Survive Sudden increases in DNA damage. The U.S. Pat. No. 7,125,622 discloses a number of bacterial For instance, when bacteria are exposed to stress they produce resistance genes, including, katG, rpoB, rpsL, ampC, beta can defense proteins from genes which are normally in a lactamases, aminoglycoside kinases, meXA, mexB, oprM. repressed State and allow repair of damaged DNA and reac ermA, carA, Imra, ereA, VgbA, InVA, mph A. tetA, tetB, tivation of DNA synthesis. The SOS response is based upon pp-cat, VanA. VanH. vanR, vanX, vanY. Vanz, folC, folE, folP. the paradigm that bacteria play an active role in the mutation and folk, which are encompassed as targets for the inhibitors of their own genomes by inducing the production of proteins in an inhibitor-engineered bacteriophage as discussed herein. during stressful conditions which facilitate mutations, includ However, in contrast to the present application, U.S. Pat. No. ing Pol II (PolB), Pol IV (dinB) and PolV (umulDandumuC). 7,125,622 does not teach method to inhibit the bacterial resis Inhibition of these proteins, such as Pol II, Pol IV and Pol V tance genes using an inhibitor-engineered bacteriophage of or prevention of their derepression by inhibition of Lex A the present invention, or their inhibition by such an inhibitor cleavage is one strategy to prevent the development of anti engineered bacteriophage in combination with an antimicro biotic-resistant bacteria. The SOS response is commonly trig bial agent. Similarly, International Application WO2008/ gered by single-stranded DNA, which accumulates as a result 110840 discusses the use of six different bacteriophages of either DNA damage or problematic replication or on bac (NCIMB numbers 41174-41179) to increase sensitivity of teriophage infection. In some situations antibiotics trigger the bacteria to antibiotics. However, WO2008/110840 but does SOS response, as some antibiotics, such as fluoroquinolones not teach genetically modifying Such bacteriophages to and B-lactams induce antibiotic-mediated DNA damage. The inhibit bacterial resistance genes or repressing SOS genes. SOS response is discussed in Benedicte Michel, PLoS Biol While there are some reports of modifying bacteriophages to ogy, 2005; 3: 1174-1176; Janion et al., Acta Biochemica increase their effectiveness of killing bacteria, previous stud Polonica, 2001; 48: 599-610 and Smith et al., 2007, 9; 549 ies have mainly focused on optimizing method to degrade 555, and Cirz et al., PLoS Biology, 2005; 6: 1024-1033, and bacteria biofilms, such as, for example introducing a lysase are incorporated herein in their entirety by reference. enzyme Such as alginate lyse (discussed in International 0150. In some embodiments, the repressor of an SOS Application WOO4/062677); or modifying bacteriophages to response gene is, for example but not limited to, lexA (SEQ inhibit the cell which propagates the bacteriophage. Such ID NO:9), or modified version thereof. In other embodiments introducing a KIL gene Such as the Holin gene in the bacte of this aspect of the invention, a SOS response gene is, for riophage (discussed in International Application WO02/ example but is not limited to marRAB (SEQ ID NO:18), 034892 and WO04/046319), or introducing bacterial toxin arcAB (SEQID NO:19) and lexO (SEQID NO:20). genes such as pGef or ChpBK and Toxin A (discussed in U.S. 0151. In some embodiments of this aspect and all other Pat. No. 6,759.229 and Westwater et al., Antimicrobial agents aspects described herein, an inhibitor of a non-SOS pathway and Chemotherapy, 2003., 47: 1301-1307). However, unlike bacterial defense gene is soxR (SEQID NO: 12), or modified the present invention the modified bacteriophages discussed version thereof. In some embodiments of this aspect and all in WOO4/062677, WO02/034892, WOO4/046319, 6,759,229 other aspects described herein, an inhibitor of a non-SOS and Westwater et al., have not been modified to target and pathway bacterial defense gene is selected from the group of disable the bacteria's antimicrobial resistance mechanism by marr (SEQID NO:10), arc (SEQID NO:11), soxR (SEQID inhibiting the bacterial resistance genes or expressing a NO:12), fur (SEQ ID NO:13), crp (SEQ ID NO:14), iccdA repressor to a SOS gene. (SEQ ID NO:15), craA (SEQID NO:16) or ompA (SEQ ID 014.6 An inhibitor to any antimicrobial resistance genes NO:17) or modified version thereof. In some embodiments, a known to one or ordinary skill in the art is encompassed for non-SOS repressor expressed by a repressor-engineered bac US 2010/0322903A1 Dec. 23, 2010

teriophage is soxR (SEQ ID NO: 12) which represses soxS and protects against oxidative stress. TABLE 2-continued 0152. In other embodiments of this aspect of the invention, Examples of non-SOS defense genes which can be inhibited by a a repressor-engineered bacteriophage can express an repres- repressor or an inhibitor expressed by a Sor, or fragment thereof, of at least one, or at least two or at repressor-engineered bacteriophage. least three or more SOS response genes, such as, but not Table 2: Examples of non-SOS defense genes which can limited to leXA, marr, arc, SOXR, fur, crp, iccdA, craA or be inhibitedrepressor-engineered by an repressor or inhibitorbacteriophage expressed by a omp A. Other repressors known by a skilled artisan are also encompassed for use in repressor-engineered bacteriophages. msbB In some embodiments, repressor-engineered bacteriophages N. are used in combination with antimicrobial agents which oxyR trigger the SOS response, or trigger DNA damage. Such as, pal for example fluoroquinolones, ciprofloxacin and B-lactams. pal B 0153. In other embodiments of this aspect of the invention, E. an agent encoded by the nucleic acid of a repressor engi- plsX neered bacteriophage which inhibits a non-SOS defense gene ppiB can inhibit any gene listed in Table 2. prfproW pstA TABLE 2 pstS qmcA Examples of non-SOS defense genes which can be inhibited by a recA repressor or an inhibitor expressed by a rec repressor-engineered bacteriophage. recC Table 2: Examples of non-SOS defense genes which can recC be inhibited by an repressor or inhibitor expressed by a recs repressor-engineered bacteriophage recC) res.A acrA rfaC acrB rfaD atp A rfa bdm rfaG BW251.13 rfa ced A rfA cysB rimlk dacA ruB dapF int dcd e didIB rpiA dedD rp degP rpmE deoT rpmR dinB rpm dkSA rpoN dnaK SF ela) ribsU emtA rrm envC rse A envZ ruvA fab ruvC fepc sapC fis secC fkpB skip follB Smp A gnty suf gor SurA gpmB tat B gpmM tatC gshA tolC gshB toIR hfK tonB hf trxA hins tusC hirpA tus) hscA typA hscE ubiG ihfA uvrA JW 5115 uwrC JW5360 uwrD JW5474 XapR lon XSeA lpdA XseB lipp ybcN lptB ybdN mircB ybeD US 2010/0322903A1 Dec. 23, 2010 21

Susceptibility Agent-Engineered Bacteriophages TABLE 2-continued 0.155. Another aspect of the present invention relates to an Examples of non-SOS defense genes which can be inhibited by a engineered bacteriophage which comprises a nucleic acid repressor or an inhibitor expressed by a repressor-engineered bacteriophage. encoding an agent, such as but not limited to a protein, which Table 2: Examples of non-SOS defense genes which can increases the Susceptibility of a bacteria to an antimicrobial be inhibited by an repressor or inhibitor expressed by a agent. Such herein engineered bacteriophage which com repressor-engineered bacteriophage prises a nucleic acid encoding an agent which increases the Susceptibility of a bacteria to an antimicrobial agent can be ybeY referred to herein as an “susceptibility agent-engineered bac teriophage' or “susceptibility-engineered bacteriophage' but are also encompassed under the definition of a “repressor engineered bacteriophage'. In some embodiments of this aspect, and all other aspects described herein, Such an agent which increases the Susceptibility of a bacteria to an antimi crobial agent is referred to as a 'susceptibility agent' and refers to any agent which increases the bacteria's Susceptibil ity to the antimicrobial agent by about at least 10% or about at 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 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 ym antimicrobial agent (i.e. the agent has an additive effect of the yneE killing efficacy or inhibition of growth by the antimicrobial agent). In a preferred embodiment, a susceptibility agent is an 0154) In some embodiments, a repressor-engineered bac agent which has a synergistic effect on the efficacy of the teriophage which inhibits a non-SOS defense gene can be antimicrobial agent (i.e. the agent has a synergistic effect of used in combination with selected antimicrobial agents, for the killing efficacy or inhibition of growth by the antimicro example, where the repressor-engineered bacteriophage bial agent). encodes an agent which inhibits a gene listed in Table 2A, 0156. In one embodiment, a susceptibility agent increases Such a repressor-engineered bacteriophage can be used in the entry of an antimicrobial agent into a bacterial cell, for combination with a ciprofloxacin antimicrobial agent or a example, a susceptibility agent is a porin orporin-like protein, variant or analogue thereof. Similarly, in other embodiments such as but is not limited to, protein Omp, and Beta barrel a repressor-engineered bacteriophage which inhibits a non porins, or other members of the outer membrane porin (OMP)) functional superfamily which include, but are not SOS defense gene can encode an agent which inhibits a gene limited to those disclosed in worldwide web site: “//biocyc. listed in Table 2B can be used in combination with a Vanco org/ECOLI/NEW-IMAGE?object=BC-4.1.B”, or a OMP mycin antimicrobial agent or a variant or analogue thereof. Similarly, in other embodiments a repressor-engineered bac family member listed in Table 3 as disclosed herein, or a teriophage which inhibits a non-SOS defense gene can variant or fragment thereof. encode an agent which inhibits a gene listed in Table 2C, 2D, 2E, 2F and 2G can be used in combination with a TABLE 3 antimicrobial agent, or a amplicillin antimicrobial agent or a Examples of members of the Outer Membrane Porin (OMP) Superfamily SulfmethaxaZone antimicrobial agent or a gentamicin antimi which can be expressed as a susceptibility agent by a Susceptibility-agent engineered bacteriophage. crobial agent or a metronidazole antimicrobial agent, respec Table 3: Members of The Outer Membrane Porin (OMP) tively, or a variant or analogue thereof. In some embodiments, Functional Superfamily other non-SOS response genes which can be inhibited or repressed in a repressor-engineered bacteriophage includes, bglH (carbohydrate-specific Outer membrane porin, cryptic), btuB (outer membrane receptor for transport of vitamin B12, Ecolicins, for example, but not limited to genes induced by DNA dam and bacteriophage BF23), age, such as DinD, DinF, DinG, Dinl, DinP. OraA, PolB. fadL (long-chain fatty acid outer membrane transporter; sensitivity RecA, RecN, RuvA. RuvB, SbmC, Ssb, SulA, UmuC, to phage T2), fecA (outer membrane receptor; citrate-dependent iron transport, Outer Umul), UvrA, UvrB, and UvrD, as discussed in Dwyer et al., membrane receptor), Mol Systems Biology, 2007: 3: 1-15, which is incorporated fepA (FepA, outer membrane receptor for ferric enterobactin herein in its entirety by reference. In another embodiment, (enterochelin) and colicins B and D), other non-SOS response genes which can be inhibited or fhuA (FhuA outer membrane protein receptor for ferrichrome, repressed in a repressor-engineered bacteriophage includes, colicin M, and phages T1, T5, and phi80), fhuE (outer membrane receptor for ferric iron uptake), for example, but not limited to genes induced by oxidative fiu (putative Outer membrane receptor for iron transport), damage, such as MarA, MarB, MarR, SodA and SoxS, as lamB, discussed in Dwyer et al., Mol Systems Biology, 2007: 3; mdtQ (putative channel/filament protein), 1-15, which is incorporated herein in its entirety by reference. US 2010/0322903A1 Dec. 23, 2010 22

0159. In some embodiments, a susceptibility agent is not a TABLE 3-continued chemotherapeutic agent. In another embodiment, a suscepti bility agent is not a toxin protein, and in another embodiment, Examples of members of the Outer Membrane Porin (OMP) Superfamily which can be expressed as a Susceptibility agent by a Susceptibility-agent a susceptibility agent is not a bacterial toxin protein or mol engineered bacteriophage. ecule. Table 3: Members of The Outer Membrane Porin (OMP) Functional Superfamily Modification of Inhibitor-Engineered Bacteriophages, omp A (outer membrane protein 3a (II*:G:d)), Repressor-Engineered Bacteriophages and Susceptibility ompC, Agent Engineered Bacteriophages ompF, ompG (outer membrane porin OmpG), 0160. In another embodiment, an inhibitor-engineered ompIL (predicted outer membrane porin L.), ompN (Outer membrane pore protein N, non-specific), bacteriophage and/or a repressor-engineered bacteriophage ompW (OmpW, Outer membrane protein), and/or a susceptibility-engineered bacteriophage can be fur pgaA (partially N-deacetylated poly-2-1,6-N-acetyl-D-glucosamine ther be modified to comprise nucleic acids which encode outer membrane porin), phoE phage resistant genes, for example any phage resistant gene toIB known by persons of ordinary skill in the art, such as, but not tolC (TolCouter membrane channel), limited to AbiZ (as disclosed in U.S. Pat. No. 7,169,911 tSX (nucleoside channel; receptor of phage T6 and colicin K), which is incorporated herein by reference), Sie-ooo. Sieroo. yncD (probable TonB-dependent receptor sie, orf2, orf258, orf2(M), olf D, orf304, orfB, orf142, orf203, orf3 up, orf2 gp34. gp33, gp32, gp25, glo, orfl. Sie A, 0157. In another embodiment, a susceptibility agent is an SieB, imm, sim, rexB (McGrathet al., Mol Microbiol, 2002, agent, such as but not limited to a protein, which increases 43:509-520). iron-sulfur clusters in the bacteria cell and/or increases oxi 0.161. In another embodiment, the inhibitor-engineered dative stress or hydroxyl radicals in the bacteria. Examples of bacteriophages and/or repressor-engineered bacteriophages a susceptibility agent which increases the iron-sulfur clusters and/or a susceptibility-engineered bacteriophage can be fur include agents which modultate (i.e. increase or decrease) the ther be modified to comprise nucleic acids which encode Fenton reaction to form hydroxyl radicals, as disclosed in enzymes which assist in breaking down or degrading the Kahanski et al., Cell, 2007, 130; 797-810, which is incorpo biofilm matrix, for example any phage resistant gene known rated herein by reference in its entirety. Examples of a sus as a biofilm degrading enzyme by persons of ordinary skill in ceptibility agent to be expressed by a susceptibility-engi the art, such as, but not limited to Dispersin D aminopepti neered bacteriophage include, for example, those listed in dase, amylase, carbohydrase, carboxypeptidase, catalase, Table 4, or a fragment or variant thereof or described in cellulase, chitinase, cutinase, cyclodextrin glycosyltrans world-wide-web site “biocyc.org/ECOLI/NEW ferase, deoxyribonuclease, esterase, alpha-galactosidase, IMAGE?type=COMPOUND&object=CPD-7”. Examples beta-galactosidase, glucoamylase, alpha-glucosidase, beta of susceptibility agents which increases iron-sulfur clusters in glucosidase, haloperoxidase, invertase, laccase, lipase, man the bacteria cell include, for example but not limited to IscA, nosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, IscR, IscS and IscU. Examples of susceptibility agents which peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, increase iron uptake and utilization and can be used as Sus ribonuclease, transglutaminase, Xylanase or lyase. In other ceptibility agents include, for example but not limited to embodiments, the enzyme is selected from the group consist EntC, ExbB, ExbD, Fecl, Feck, FepB, FepC, Fes, FhuA, ing of cellulases, such as glycosyl hydroxylase family of FhuB, FhuC, FhuF, NrdH, Nrdl, SodA and TonB, as dis cellulases, such as glycosylhydroxylase 5 family of enzymes cussed in Dwyer et al., Mol Systems Biology, 2007: 3: 1-15, also called cellulase A; polyglucosamine (PGA) depoly which is incorporated herein in its entirety by reference. merases; and colonic acid depolymerases, such as 1,4-L- fucodise hydrolase (see, e.g., Verhoef R. et al., Characteriza TABLE 4 tion of a 1,4-beta-fucoside hydrolase degrading collanic acid, Carbohydr Res. 2005 Aug. 15:340(11):1780-8), depolymer Examples of genes which can be expressed as a Susceptibility azing alginase, and DNase I, or combinations thereof, as agent by a Susceptibility-engineered bacteriophage to increase disclosed in the methods as disclosed in U.S. patent applica iron cluster formation in bacteria. tion Ser. No. 1 1/662,551 and International Patent Application Table 4: Example of susceptibility agents which increase iron clusters Wo2006/137847 and provisional patent application 61/014, Cofactor of: serine deaminase, L-serine deaminase, L-serine deaminase, pyruvate formate-lyase activating enzyme, 2,4-dienoyl-CoA reductase 518, which are specifically incorporated herein in their Prosthetic Group of biotin synthase, dihydroxy-acid dehydratase, entirety by reference. dihydroxy-acid dehydratase, lysine 2,3-aminomutase, NADH:ubiquinone 0162. In another embodiment, the inhibitor-engineered oxidoreductase, sulfite reductase-(NADPH), aconitase B, fumarase A, bacteriophages and/or repressor-engineered bacteriophages aconitase, fumarase B, anaerobic coproporphyrinogen III oxidase, Succinate dehydrogenase, nitrate reductase, flavin reductase, and/or a susceptibility-engineered bacteriophage can be fur aconitase B, fumarate reductase ther be modified in a species-specific manner, for example, Cofactor or Prosthetic Group of: quinolinate synthase, ribonucleoside one can modify or select the bacteriophage on the basis for its triphosphate reductase activase, 23S ribosomal RNA 5-methyluridine infectivity of specific bacteria. methyltransferase 0163 A bacteriophage to be engineered or developed into an inhibitor-engineered bacteriophage or repressor-engi 0158. In some embodiments, a susceptibility agent is an neered bacteriophage or a susceptibility-engineered bacte agent such as CsrA, which is described in world-wide web riophage can be any bacteriophage as known by a person of site: “biocyc.org/ECOLI/NEW ordinary skill in the art. In some embodiments, an inhibitor IMAGE?type=ENZYME&object=CPLX0-1041. engineered bacteriophage or a repressor-engineered bacte US 2010/0322903A1 Dec. 23, 2010 riophage or a Susceptibility-engineered bacteriophage is embodiment, the invention provides an engineered T7 select derived from any or a combination of bacteriophages listed in 10-3b phage that expresses both cellulase and 10A capsid Table 5. protein. 0164. In some embodiments, a bacteriophage which is 0167. It is known that wild-type T7 does not productively engineered to become an engineered bacteriophage as dis infect male (F plasmid-containing) E. coli because of inter closed herein is alytic bacteriophage or lysogenic bacterioph actions between the F plasmid protein PifA and T7 genes 1.2 age, or any bacteriophage that infects E. coli, P. aeriginosa, S. or 10 (Garcia, L. R., and Molineux, I.J. 1995. Incomplete aureaus, E. facalis and the like. Such bacteriophages are well entry of bacteriophage T7 DNA into F plasmid-containing known to one skilled in the art and are listed in Table 5, and Escherichia coli. J. Bacteriol. 177:4077-4083.). F plasmid include, but are not limited to, lambda phages, M13. T7, T3, containing E. coli infected by T7 die but do not lyse or release and T-even and T-even like phages, such as T2, and T4, and large numbers of T7 (Garcia, L. R., and Molineux, I.J. 1995. RB69; also phages such as Pfl. Pfa, Bacteroides fragilis phage Incomplete entry of bacteriophage T7 DNA into F plasmid B40-8 and coliphage MS-2 can be used. For example, lambda containing Escherichia coli. J. Bacteriol. 177:4077-4083). phage attacks E. coli by attaching itself to the outside of the Wild-type T3 grows normally on male cells because of T3's bacteria and injecting its DNA into the bacteria. Once injected gene 1.2 product (Garcia, L. R., and Molineux, I.J. 1995, Id.). into its new host, a bacteriophage uses E. coli's genetic When T3 gene 1.2 is expressed in wild-type T7. T7 is able to machinery to transcribe its genes. Any of the known phages productively infect male cells (Garcia, L. R., and Molineux, I. can be engineered to express an agent that inhibits an antibi J. 1995. Id). otic resistance gene or cell Survival gene, or alternatively 0168 Because many biofilm-producing E. coli contain the express a repressor agent oran inhibitor of a non-SOS defense F plasmid (Ghigo, et al., 2001. Natural conjugative plasmids gene for a repressor-engineered bacteriophage, or express a induce bacterial biofilm development. Nature. 412:442-445), Susceptibility agent for a Susceptibility-engineered bacte it is important, although not necessary, for an engineered riophage as described herein. bacteriophage to be able to productively infect also male 0.165. In some embodiments, bacteriophages which have cells. Therefore, in addition to engineering the phage to dis been engineered to be more efficient cloning vectors or natu play a biofilm degrading enzyme on its Surface, one can also rally lack a gene important in infecting all bacteria, Such as engineer it to express the gene necessary for infecting the male and female bacteria can be used to generate engineered male bacteria. For example, one can use the modification bacteriophages as disclosed herein. Typically, bacteriophages described by Garcia and Molineux (Garcia, L. R., and have been engineered to lack genes for infecting all variants Molineux, I.J. 1995. Incomplete entry of bacteriophage T7 and species of bacteria can have reduced capacity to replicate DNA into F plasmid-containing Escherichia coli. J. Bacte in naturally occurring bacteria thus limiting the use of Such riol. 177:4077-4083) to express T3 gene 1.2 in T7. phages in degradation of biofilm produced by the naturally Nucleic Acid Inhibitors of Antibiotic Resistance Genes and/ occurring bacteria. or Cell Survival Genes for Inhibitor-Engineered Bacterioph 0166 For example, the capsid protein of phage T7, gene ages or Nucleic Acid Inhibitors of Non-SOS Defense Genes 10, comes in two forms, the major product 10A (36 kDa) and in Repressor-Engineered Bacteriophages. the minor product 10B (41 kDa) (Condron, B.G., Atkins, J. 0169. In some embodiments of aspects of the invention F., and Gesteland, R. F. 1991. Frameshifting in gene 10 of involving inhibitor-engineered bacteriophages, agents that bacteriophage T7. J. Bacteriol. 173:6998-7003). Capsid pro inhibit an antibiotic resistance gene and/or a cell Survival tein 10B is produced by frameshifting near the end of the gene is a nucleic acid. In another embodiments, repressor coding region of 10A. NOVAGENR modified gene 10 in T7 engineered bacteriophages comprise nucleic acids which to remove the frameshifting site so that only 10B with the inhibit non-SOS defense genes, such as those listed in Table attached user-introduced peptide for Surface display is pro 2, and Tables 2A-2F. An antibiotic resistance gene and/or cell duced (U.S. Pat. No. 5,766,905. 1998. Cytoplasmic bacte survival gene and/or non-SOS defense gene can be inhibited riophage display system, which is incorporated in its entirety by inhibition of the expression of such antibiotic resistance herein by reference). The 10B-enzyme fusion product is too proteins and/or cell survival polypeptide or non-SOS defense large to make up the entire phage capsid because the enzymes gene or by gene silencing methods commonly known by that are typically introduced into phages, such as T7, are large persons of ordinary skill in the art. A nucleic acid inhibitor of (greater thana few hundredamino acids). As a result, T7 select an antibiotic resistance gene and/or a cell Survival gene or 10-3b must be grown in host bacterial strains that produce non-SOS defense gene, includes for example, but is not lim wild-type 10A capsid protein, such as BLT5403 or BLT5615, ited to, RNA interference-inducing (RNAi) molecules, for so that enough 10A is available to be interspersed with the example but are not limited to siRNA, dsRNA, stRNA, 1OB-enzyme fusion product to allow replication of phage shRNA, miRNA and modified versions thereof, where the (U.S. Pat. No. 5,766.905. 1998. Cytoplasmic bacteriophage RNA interference molecule gene silences the expression of display System, which is incorporated in its entirety herein by the antibiotic resistance gene and/or cell Survival gene non reference). However, because most biofilm-forming E. coli SOS-defense gene. In some embodiments, the nucleic acid do not produce wild-type 10A capsid protein, this limits the inhibitor of an antibiotic resistance gene and/or cell survival ability of T7 select 10-3b displaying large enzymes on their gene and/or non-SOS defense gene is an anti-sense oligo Surface to propagate within and lyse some important Strains of nucleic acid, or a nucleic acid analogue, for example but are E. coli. Accordingly, in some embodiments, the present not limited to DNA, RNA, peptide-nucleic acid (PNA), invention provides genetically engineered phages that in pseudo-complementary PNA (pc-PNA), or locked nucleic addition to comprising inhibitors to cell Survival genes or acid (LNA) and the like. In alternative embodiments, the antibiotic resistance genes, or nucleic acids encoding repres nucleic acid is DNA or RNA, and nucleic acid analogues, for Sor proteins, also express all the essential genes for example PNA, pcPNA and LNA. A nucleic acid can be single replication in naturally occurring bacterial strains. In one or double Stranded, and can be selected from a group com US 2010/0322903A1 Dec. 23, 2010 24 prising nucleic acid encoding a protein of interest, oligo or antibiotic resistance protein) encoded by a target gene nucleotides, PNA, etc. Such nucleic acid inhibitors include which has not been targeted and gene silenced by an RNA for example, but are not limited to, a nucleic acid sequence interfering (RNAi) agent. encoding a protein that is a transcriptional repressor, or an (0174 As used herein, the term “short interfering RNA'. antisense molecule, or a ribozyme, or a small inhibitory (siRNA), also referred to herein as “small interfering RNA is nucleic acid sequence such as a RNAi, an shRNAi, an siRNA, defined as an agent which functions to inhibit expression of a a micro RNAi (miRNA), an antisense oligonucleotide etc. target gene, e.g., by RNAi. An siRNA can be chemically synthesized, can be produced by in vitro transcription, or can 0170. In some embodiments, a nucleic acid inhibitor of an be produced within a host cell. In one embodiment, siRNA is antibiotic resistance gene and/or a cell Survival gene and/or a double stranded RNA (dsRNA) molecule of about 15 to non-SOS defense gene can be for example, but not are limited about 40 nucleotides in length, preferably about 15 to about to, paired termini antisense, an example of which is disclosed 28 nucleotides, more preferably about 19 to about 25 nucle in FIG. 8 and disclosed in Nakashima, et al., (2006) Nucleic otides in length, and more preferably about 19, 20, 21, 22, or Acids Res 34: e138, which in incorporated herein in its 23 nucleotides in length, and can contain a 3' and/or 5' over entirety by reference. hang on each Strand having a length of about 0, 1, 2, 3, 4, or 0171 In some embodiments of this aspect and all aspects 5 nucleotides. The length of the overhang is independent described herein, a single-stranded RNA (ssRNA), a form of between the two strands, i.e., the length of the overhang on RNA endogenously found in eukaryotic cells can be used to one strand is not dependent on the length of the overhang on forman RNAi molecule. Cellular ssRNA molecules include the second strand. In some embodiments, the siRNA is messenger RNAS (and the progenitor pre-messenger RNAS), capable of promoting RNA interference through degradation small nuclear RNAs, Small nucleolar RNAs, transfer RNAs or specific post-transcriptional gene silencing (PTGS) of the and ribosomal RNAs. Double-stranded RNA (dsRNA) target messenger RNA (mRNA). induces a size-dependent immune response such that dsRNA 0.175 siRNAs also include small hairpin (also called stem larger than 30 bp activates the interferon response, while loop) RNAs (shRNAs). In one embodiment, these shRNAs shorter dsRNAs feed into the cell's endogenous RNA inter are composed of a short (e.g., about 19 to about 25 nucleotide) ference machinery downstream of the Dicer enzyme. antisense strand, followed by a nucleotide loop of about 5 to (0172 RNA interference (RNAi) provides a powerful about 9 nucleotides, and the analogous sense Strand. Alterna approach for inhibiting the expression of selected target tively, the sense Strand can precede the nucleotide loop struc polypeptides. RNAi uses small interfering RNA (siRNA) ture and the antisense strand can follow. These shRNAS can duplexes that target the messenger RNA encoding the target be contained in plasmids, retroviruses, and lentiviruses and polypeptide for selective degradation. siRNA-dependent expressed from, for example, the pol III U6 promoter, or post-transcriptional silencing of gene expression involves another promoter (see, e.g., Stewart, et al. (2003) RNA Apr; cutting the target messenger RNA moleculeata site guided by 9(4):493-501, incorporated by reference herein in its the siRNA. entirety). (0173 RNA interference (RNAi) is an evolutionally con 0176 Typically a target gene or sequence targeted by gene served process whereby the expression or introduction of silencing by an RNA interfering (RNAi) agent can be a cel RNA of a sequence that is identical or highly similar to a lular gene or genomic sequence encoding an antibiotic resis target gene results in the sequence specific degradation or tant protein or a cell Survival protein. In some embodiments, specific post-transcriptional gene silencing (PTGS) of mes an siRNA can be substantially homologous to the target gene senger RNA (mRNA) transcribed from that targeted gene (see or genomic sequence, or a fragment thereof. As used in this Coburn, G. and Cullen, B. (2002).J. of Virology 76(18): 9225), context, the term "homologous is defined as being Substan thereby inhibiting expression of the target gene. In one tially identical, Sufficiently complementary, or similar to the embodiment, the RNA is double stranded RNA (dsRNA). target mRNA, or a fragment thereof, to effect RNA interfer This process has been described in plants, invertebrates, and ence of the target. In addition to native RNA molecules, RNA mammalian cells. In nature, RNAi is initiated by the dsRNA suitable for inhibiting or interfering with the expression of a specific endonuclease Dicer, which promotes processive target sequence include RNA derivatives and analogs. Pref cleavage of long dsRNA into double-stranded fragments erably, the siRNA is identical to its target. termed siRNAs. siRNAs are incorporated into a protein com 0177. The siRNA preferably targets only one sequence. plex (termed “RNA induced silencing complex,” or “RISC) Each of the RNA interfering agents, such as siRNAs, can be that recognizes and cleaves target mRNAs. RNAi can also be screened for potential off-target effects by, for example, initiated by introducing nucleic acid molecules, e.g., Syn expression profiling. Such methods are known to one skilled thetic siRNAs or RNA interfering agents, to inhibit or silence in the art and are described, for example, in Jackson et al. the expression of a target genes, such an antibiotic resistance Nature Biotechnology 6:635-637, 2003. In addition to gene and/or cell Survival gene and/or non-SOS defense gene. expression profiling, one can also screen the potential target As used herein, “inhibition of target gene expression' sequences for similar sequences in the sequence databases to includes any decrease in expression or proteinactivity or level identify potential sequences which can have off-target of the target gene (i.e. antibiotic resistance gene) or protein effects. For example, according to Jackson et al. (Id.) 15, or encoded by the target gene (i.e. antibiotic resistance protein) perhaps as few as 11 contiguous nucleotides of sequence as compared to the level in the absence of an RNA interfer identity are sufficient to direct silencing of non-targeted tran ence (RNAi) molecule. The decrease in expression or protein Scripts. Therefore, one can initially screen the proposed siR level as result of gene silencing can be of at least 30%, 40%, NAS to avoid potential off-target silencing using the sequence 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as com identity analysis by any known sequence comparison meth pared to the expression of a target gene or the activity or level ods, such as BLAST (Basic Local Alignment Search Tool of the protein (i.e. expression of the antibiotic resistance gene available from or at NIBI). US 2010/0322903A1 Dec. 23, 2010

0178 siRNA molecules need not be limited to those mol Reed, M. W., Cox, T., Virosco, J. S., Adams, A. D., Gall, A., ecules containing only RNA, but, for example, further Scholler, J. K., and Meyer, R. B. (1993) Facile Preparation encompasses chemically modified nucleotides and non and Exonuclease Stability of 3'-Modified Oligodeoxynucle nucleotides, and also include molecules wherein a ribose otides. Nucleic Acids Res. 21 145-150; and Reed, M. W., Sugar molecule is Substituted for another Sugar molecule or a Adams, A. D., Nelson, J. S., and Meyer, R. B., Jr. (1991) molecule which performs a similar function. Moreover, a Acridine and Cholesterol-Derivatized Solid Supports for non-natural linkage between nucleotide residues can be used, Improved Synthesis of 3'-Modified Oligonucleotides. Bio Such as a phosphorothioate linkage. For example, siRNA conjugate Chem. 2217-225 (1993). containing D-arabinofuranosyl Structures in place of the natu 0183. Other siRNAs useful for targeting Lp-PLA expres rally-occurring D-ribonucleosides found in RNA can be used sion can be readily designed and tested. Accordingly, siRNAS in RNAi molecules according to the present invention (U.S. useful for the methods described herein include siRNA mol Pat. No. 5,177,196, which is incorporated herein by refer ecules of about 15 to about 40 or about 15 to about 28 ence). Other examples include RNA molecules containing nucleotides in length. Preferably, the siRNA molecules have the O-linkage between the Sugar and the heterocyclic base of a length of about 19 to about 25 nucleotides. More preferably, the nucleoside, which confers nuclease resistance and tight the siRNA molecules have a length of about 19, 20, 21, or 22 complementary Strand binding to the oligonucleotidesmol nucleotides. The siRNA molecules can also comprise a 3 ecules similar to the oligonucleotides containing 2'-O-methyl hydroxyl group. The siRNA molecules can be single ribose, arabinose and particularly D-arabinose (U.S. Pat. No. stranded or double stranded; such molecules can be blunt 5,177,196, which is incorporated herein in its entirety by ended or comprise overhanging ends (e.g., 5' 3"). In specific reference). embodiments, the RNA molecule is double stranded and 0179 The RNA strand can be derivatized with a reactive either blunt ended or comprises overhanging ends. functional group of a reporter group, such as a fluorophore. 0184. In one embodiment, at least one strand of the RNA Particularly useful derivatives are modified at a terminus or molecule has a 3' overhang from about 0 to about 6 nucle termini of an RNA strand, typically the 3' terminus of the otides (e.g., pyrimidine nucleotides, purine nucleotides) in sense strand. For example, the 2'-hydroxyl at the 3' terminus length. In other embodiments, the 3' overhang is from about 1 can be readily and selectively derivatized with a variety of to about 5 nucleotides, from about 1 to about 3 nucleotides groups. and from about 2 to about 4 nucleotides in length. In one 0180. Other useful RNA derivatives incorporate nucle embodiment the RNA molecule is double stranded—one otides having modified carbohydrate moieties, such as 2"O- strand has a 3' overhang and the other strand can be blunt alkylated residues or 2'-O-methyl ribosyl derivatives and ended or have an overhang. In the embodiment in which the 2'-O-fluoro ribosyl derivatives. The RNA bases can also be RNA molecule is double stranded and both strands comprise modified. Any modified base useful for inhibiting or interfer an overhang, the length of the overhangs can be the same or ing with the expression of a target sequence can be used. For different for each strand. In a particular embodiment, the example, halogenated bases, such as 5-bromouracil and 5-io RNA of the present invention comprises about 19, 20, 21, or douracil can be incorporated. The bases can also be alkylated, 22 nucleotides which are paired and which have overhangs of for example, 7-methylguanosine can be incorporated in place from about 1 to about 3, particularly about 2, nucleotides on of a guanosine residue. Non-natural bases that yield Success both 3' ends of the RNA. In one embodiment, the 3' overhangs ful inhibition can also be incorporated. can be stabilized against degradation. In a preferred embodi 0181. The most preferred siRNA modifications include ment, the RNA is stabilized by including purine nucleotides, 2'-deoxy-2'-fluorouridine or locked nucleic acid (LNA) Such as adenosine or guanosine nucleotides. Alternatively, nucleotides and RNA duplexes containing either phosphodi substitution of pyrimidine nucleotides by modified ana ester or varying numbers of phosphorothioate linkages. Such logues, e.g., Substitution of uridine 2 nucleotide 3' overhangs modifications are known to one skilled in the art and are by 2'-deoxythymidine is tolerated and does not affect the described, for example, in Braasch et al., Biochemistry, 42: efficiency of RNAi. The absence of a 2 hydroxyl significantly 7967-7975, 2003. Most of the useful modifications to the enhances the nuclease resistance of the overhang in tissue siRNA molecules can be introduced using chemistries estab culture medium. lished for antisense oligonucleotide technology. Preferably, 0185. In some embodiments, assessment of the expression the modifications involve minimal 2'-O-methyl modification, and/or knock down of antibiotic resistance gene and/or cell preferably excluding such modification. Modifications also Survival gene protein and/or non-SOS defense genes using preferably exclude modifications of the free 5'-hydroxyl such RNAi agents such as antisense RNA can be determined groups of the siRNA. by a person of ordinary skill in the art determining the viabil 0182 siRNA and miRNA molecules having various ity of a bacteria expressing Such a RNAi agent in the presence “tails' covalently attached to either their 3'- or to their 5'-ends, of an antimicrobial agent. In some embodiments, bacterial or to both, are also known in the art and can be used to cell viability can be determined by using commercially avail stabilize the siRNA and miRNA molecules delivered using able kits. Others can be readily prepared by those of skill in the methods of the present invention. Generally speaking, the art based on the known sequence of the target mRNA. To intercalating groups, various kinds of reporter groups and avoid doubt, the nucleic acid sequence which can be used to lipophilic groups attached to the 3' or 5' ends of the RNA design nucleic acid inhibitors for inhibitor-engineered bacte molecules are well known to one skilled in the art and are riophages as disclosed herein can be based on any antibiotic useful according to the methods of the present invention. resistance gene or any SOS gene or any non-SOS defense Descriptions of syntheses of 3'-cholesterol or 3'-acridine gene listed in Tables 2 or 2A-2F as disclosed herein. modified oligonucleotides applicable to preparation of modi 0186 siRNA sequences are chosen to maximize the fied RNA molecules useful according to the present invention uptake of the antisense (guide) strand of the siRNA into RISC can be found, for example, in the articles: Gamper, H. B., and thereby maximize the ability of the inhibitor to target US 2010/0322903A1 Dec. 23, 2010 26

RISC to target antibiotic resistance gene or cell Survival gene 5,858,988 to Wang, also describe nucleic acid analogs for mRNA for degradation. This can be accomplished by scan enhanced nuclease stability and cellular uptake. ning for sequences that have the lowest free energy of binding 0190. Synthetic siRNA molecules, including shRNA mol at the 5'-terminus of the antisense strand. The lower free ecules, can be obtained using a number of techniques known energy leads to an enhancement of the unwinding of the to those of skill in the art. For example, the siRNA molecule 5'-end of the antisense strand of the siRNA duplex, thereby can be chemically synthesized or recombinantly produced ensuring that the antisense strand will be taken up by RISC using methods known in the art, such as using appropriately and direct the sequence-specific cleavage of the targeted protected ribonucleoside phosphoramidites and a conven mRNA. tional DNA/RNA synthesizer (see, e.g., Elbashir, S. M. et al. 0187 RNA interference molecules and nucleic acid (2001) Nature 411:494-498; Elbashir, S. M., W. Lendeckel inhibitors useful in the methods as disclosed herein can be and T. Tuschl (2001) Genes & Development 15:188-200; produced using any known techniques such as direct chemi Harborth, J. et al. (2001) J. Cell Science 114:4557-4565; cal synthesis, through processing of longer double stranded Masters, J. R. et al. (2001) Proc. Natl. Acad. Sci., USA 98:8012-8017; and Tuschl, T. et al. (1999) Genes & Devel RNAs by exposure to recombinant Dicer protein or Droso opment 13:3191-3197). Alternatively, several commercial phila embryolysates, through an in vitro system derived from RNA synthesis suppliers are available including, but are not S2 cells, using phage RNA polymerase, RNA-dependant limited to, Proligo (Hamburg, Germany), Dharmacon RNA polymerase, and DNA based vectors. Use of cell lysates Research (Lafayette, Colo., USA), Pierce Chemical (part of or in vitro processing can further involve the Subsequent Perbio Science, Rockford, Ill., USA), Glen Research (Ster isolation of the short, for example, about 21-23 nucleotide, ling, Va., USA), ChemGenes (Ashland, Mass., USA), and siRNAs from the lysate, etc. Chemical synthesis usually pro Cruachem (Glasgow, UK). As such, siRNA molecules are not ceeds by making two single stranded RNA-oligomers fol overly difficult to synthesize and are readily provided in a lowed by the annealing of the two single stranded oligomers quality suitable for RNAi. In addition, dsRNAs can be into a double stranded RNA. Other examples include meth expressed as stem loop structures encoded by plasmid vec ods disclosed in WO99/32619 and WO 01/68836, which are tors, retroviruses and lentiviruses (Paddison, P.J. et al. (2002) incorporated herein by reference, teach chemical and enzy Genes Dev. 16:948-958; McManus, M.T. et al. (2002) RNA matic synthesis of siRNA. Moreover, numerous commercial 8:842-850; Paul, C. P. et al. (2002) Nat. Biotechnol. 20:505 services are available for designing and manufacturing spe 508; Miyagishi, M. etal. (2002) Nat. Biotechnol. 20:497-500; cific siRNAs (see, e.g., QIAGEN Inc., Valencia, Calif. and Sui, G. et al. (2002) Proc. Natl. Acad. Sci., USA99:5515 AMBION Inc., Austin,Tex.) 5520; Brummelkamp, T. et al. (2002) Cancer Cell 2:243; Lee, 0188 In one embodiment, the nucleic acid inhibitors of N. S., et al. (2002) Nat. Biotechnol. 20:500-505:Yu, J.Y., et al. antibiotic resistance genes and/or cell Survival genes can be (2002) Proc. Natl. Acad. Sci., USA99:6047-6052; Zeng, Y., obtained synthetically, for example, by chemically synthesiz et al. (2002) Mol. Cell. 9:1327-1333; Rubinson, D. A., et al. ing a nucleic acid by any method of synthesis known to the (2003) Nat. Genet. 33:401–406; Stewart, S.A., et al. (2003) skilled artisan. The synthesized nucleic acid inhibitors of RNA 9:493-501). These vectors generally have a poliII pro antibiotic resistance genes and/or cell Survival genes can then moter upstream of the dsRNA and can express sense and be purified by any method known in the art. Methods for antisense RNA strands separately and/or as a hairpin struc chemical synthesis of nucleic acids include, but are not lim tures. Within cells, Dicer processes the short hairpin RNA ited to, in vitro chemical synthesis using phosphotriester, (shRNA) into effective siRNA. phosphate or phosphoramidite chemistry and Solid phase 0191 The targeted region of the siRNA molecule of the techniques, or via deoxynucleoside H-phosphonate interme present invention can be selected from a given target gene diates (see U.S. Pat. No. 5,705,629 to Bhongle). sequence, e.g., an antibiotic resistance genes and/or cell Sur 0189 In some circumstances, for example, where vival genes coding sequence, beginning from about 25 to 50 increased nuclease stability is desired, nucleic acids having nucleotides, from about 50 to 75 nucleotides, or from about nucleic acid analogs and/or modified internucleoside link 75 to 100 nucleotides downstream of the start codon. Nucle ages can be preferred. Nucleic acids containing modified otide sequences can contain 5' or 3' UTRS and regions nearby internucleoside linkages can also be synthesized using the start codon. One method of designing a siRNA molecule reagents and methods that are well known in the art. For of the present invention involves identifying the 23 nucleotide example, methods of synthesizing nucleic acids containing sequence motif AA(N19)TT (where N can be any nucle phosphonate phosphorothioate, phosphorodithioate, phos otide), and selecting hits with at least 25%, 30%, 35%, 40%, phoramidate methoxyethyl phosphoramidate, formacetal, 45%, 50%, 55%, 60%. 65%, 70% or 75% G/C content. The thioformacetal. diisopropylsilyl acetamidate, carbamate, “TT portion of the sequence is optional. Alternatively, if no dimethylene-sulfide (-CHS CH), dimethylene-sulfox Such sequence is found, the search can be extended using the ide (—CH2—SO CH), dimethylene-sulfone (—CH2— motif NA(N21), where N can be any nucleotide. In this situ SOCH), 2'-O-alkyl, and 2'-deoxy-2'-fluoro' phosphorothio ation, the 3' end of the sense siRNA can be converted to TT to ate internucleoside linkages are well known in the art (see allow for the generation of a symmetric duplex with respect to Uhlmann et al., 1990, Chem. Rev. 90:543-584; Schneider et the sequence composition of the sense and antisense 3' over al., 1990, Tetrahedron Lett. 31:335 and references cited hangs. The antisense siRNA molecule can then be synthe therein). U.S. Pat. Nos. 5.614,617 and 5,223,618 to Cook, et sized as the complement to nucleotide positions 1 to 21 of the al., U.S. Pat. No. 5,714,606 to Acevedo, et al., U.S. Pat. No. 23 nucleotide sequence motif. The use of symmetric 3' TT 5,378,825 to Cook, et al., U.S. Pat. No. 5,672,697 and U.S. overhangs can be advantageous to ensure that the Small inter Pat. No. 5,466,786 to Buhr, et al., U.S. Pat. No. 5,777,092 to fering ribonucleoprotein particles (siRNPs) are formed with Cook, et al., U.S. Pat. No. 5,602,240 to De Mesmacker, et al., approximately equal ratios of sense and antisense target U.S. Pat. No. 5,610,289 to Cook, et al. and U.S. Pat. No. RNA-cleaving siRNPs (Elbashir et al. (2001) supra and US 2010/0322903A1 Dec. 23, 2010 27

Elbashir et al. 2001 supra). Analysis of sequence databases, promoter listed in Table 6 or disclosed in world-wide web site including but are not limited to the NCBI, BLAST, Derwent "partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup other and GenSeq as well as commercially available oligosynthesis regulator&show =1. software such as OLIGOENGINE(R), can also be used to 0.195. In some embodiments, an agent is protein or select siRNA sequences against EST libraries to ensure that polypeptide or RNAi agent that inhibits expression of antibi only one gene is targeted. otic resistance genes and/or cell Survival gene, or a non-SOS 0.192 Accordingly, the RNAi molecules functioning as defense genes. In Such embodiments bacteriophage cells can nucleic acid inhibitors of antibiotic resistance genes and/or be modified (e.g., by homologous recombination) to provide cell Survival genes as disclosed hereinare for example, but are increased expression of such an agent, for example by replac not limited to, unmodified and modified double stranded (ds) ing, in whole or in part, the naturally occurring bacteriophage RNA molecules including short-temporal RNA (stRNA), promoter with all or part of a heterologous promoter so that small interfering RNA (siRNA), short-hairpin RNA the bacteriophage and/or the bacteriophage infected-host cell (shRNA), microRNA (miRNA), double-stranded RNA expresses a high level of the inhibitor agent of antibiotic (dsRNA), (see, e.g. Baulcombe, Science 297:2002-2003, resistance genes and/or cell Survival gene or a repressor oran 2002). The dsRNA molecules, e.g. siRNA, also can contain 3' inhibitor to a non-SOS defense gene or a susceptibility agent. overhangs, preferably 3'UU or 3TT overhangs. In one In some embodiments, a heterologous promoter is inserted in embodiment, the siRNA molecules of the present invention such a manner that it is operatively linked to the desired do not include RNA molecules that comprise ssRNA greater nucleic acid encoding the agent. See, for example, PCT Inter than about 30-40 bases, about 40-50 bases, about 50 bases or national Publication No. WO 94/12650 by Transkaryotic more. In one embodiment, the siRNA molecules of the Therapies, Inc., PCT International Publication No. WO present invention are double stranded for more than about 92/20808 by Cell Genesys, Inc., and PCT International Pub 25%, more than about 50%, more than about 60%, more than lication No. WO 91/09955 by Applied Research Systems, about 70%, more than about 80%, more than about 90% of which are incorporated herein in their entirety by reference. their length. In some embodiments, a nucleic acid inhibitor of 0196. In some embodiments, bacteriophages can be engi antibiotic resistance genes and/or cell Survival genes is any neered as disclosed herein to express an endogenous gene, agent which binds to and inhibits the expression of antibiotic Such as a repressor protein, or a nucleic acid inhibitor of an resistance genes and/or cell Survival gene mRNA, where the antibiotic resistance gene or cell Survival gene comprising the expression of the antibiotic resistance genes and/or cell Sur agent under the control of inducible regulatory elements, in vival mRNA or a product of transcription of nucleic acid which case the regulatory sequences of the endogenous gene encoded by antibiotic resistance genes and/or cell Survival can be replaced by homologous recombination. Gene activa gene is inhibited. tion techniques are described in U.S. Pat. No. 5.272,071 to 0193 In another embodiment of the invention, agents Chappel; U.S. Pat. No. 5,578,461 to Sherwin et al.; PCT/ inhibiting antibiotic resistance genes and/or cell Survival US92/09627 (WO93/09222) by Selden et al.; and PCT/ genes are catalytic nucleic acid constructs. Such as, for US90/06436 (WO91/06667) by Skoultchietal, which are all example ribozymes, which are capable of cleaving RNA tran incorporated herein in their entirety by reference. Scripts and thereby preventing the production of wildtype 0.197 Other exemplary examples of promoter which can protein. Ribozymes are targeted to and anneal with a particu be used include, for example but not limited, Anhydrotetra lar sequence by virtue of two regions of sequence comple cycline(aTc) promoter, PLtetO-1 (Pubmed Nucleotidei mentary to the target flanking the ribozyme catalytic site. U66309), Arabinose promoter (PBAD), IPTG inducible pro After binding, the ribozyme cleaves the targetina site specific moters PTAC (in vectors such as Pubmed Accession manner. The design and testing of ribozymes which specifi #EU546824), PTrc-2. Plac (invectors such as Pubmed Acces cally recognize and cleave sequences of the gene products sion #EU546816), PLlacO-1, PAllacO-1, and Arabinose and described herein, for example for cleavage of antibiotic resis IPTG promoters, such as Placfara-a. Examples of these pro tance genes and/or cell Survival genes or homologues or vari moters are as follows: ants thereof can be achieved by techniques well known to 0198 Anhydrotetracycline (aTc) promoter, such as those skilled in the art (for example Lleber and Strauss, PLtetO-1 (Pubmed Nucleotide# U66309): GCATGCTC (1995) Mol Cell Biol 15:540.551, the disclosure of which is CCTATCAGTGATAGAGATTGACATC incorporated herein by reference). CCTATCAGTGATAGAGATACTGAGCACAT CAGCAG GACGCACTGACCAGGA (SEQ ID NO: 36); Arabinose Promoters of the Engineered Bacteriophages promoter (PBAD): or modified versions which can be found at world-wide web site: partsregistry.org/wiki/index. 0194 In some embodiments of all aspects described php?title=Part:BBa I13453” AAGAAACCAATTGTC herein, an engineered bacteriophage comprises a nucleic acid CATATTGCATCAGACATTGCCGTCACT which expresses an inhibitor to an antibiotic resistance gene GCGTCTTTTACTGGCTCTTCT (such as in inhibitor-engineered bacteriophages) or a repres CGCTAACCAAACCGGTAACCCCGCTTAT sor to a SOS gene or a repressor (or inhibitor) to a non-SOS TAAAAGCATTCTGTAACAAAGCGGGACCAAAGC defense gene (in the case of repressor-engineered bacterioph CATGACAAAAACGCGTAACAAAAGTGTC ages) or a susceptibility agent (in a case of a susceptibility TATAATCACGGCAGAAAAGTCCACATTGATTATTTG agent engineered bacteriophage). In each instance, gene CACGGCGTCACACTTTGCTATGCCATAG expression from the nucleic acid is regulated by a promoter to CATTTTTATCCATAAGATTAGCGGATCCTACC which the nucleic acid is operatively linked to. In some TGACGCTTTTTATCGCAACTCTCTACT embodiments, a promoter is a bacteriophage promoter. One GTTTCTCCATA (SEQ ID NO: 37); IPTG promoters: (i) can use any bacteriophage promoter known by one of ordi PTAC (in vectors such as Pubmed Accession #EU546824, nary skill in the art, for example but not limited to, any which is incorporated herein by reference), (ii) PTrc-2: US 2010/0322903A1 Dec. 23, 2010 28

CCATCGAATGGCTGAAATGAGCTGTTGA at least one antimicrobial agent. In some embodiments of this CAATTAATCATCCGGCTCGTATAATGTGTGGAAT and all aspects described herein, the composition can be TGTGAGCGGATAACAATTTCACACAGGA (SEQ ID administered as a co-formulation with one or more other NO: 38) and temperature sensitive promoters such as non-antimicrobial or therapeutic agents. PLSlcon, GCATGCACAGATAACCATCTGCGGT 0203. In a further embodiment, the invention provides GATAAATTATCTCTGGCGGTGTTGACAT. methods of administration of the compositions and/or phar AAATACCAC TGGCGGTtATAaTGAGCACATCAG maceutical formulations of the invention and include any CAGG/GTATGCAAAGGA (SEQID NO:39) and modified means commonly known by persons skilled in the art. In some variants thereof. embodiments, the Subject is any organism, including for example a mammalian, avian or plant. In some embodiments, Modification of Engineered Bacteriophages. the mammalian is a human, a domesticated animal and/or a 0199. In some embodiments of all aspects described commercial animal. herein, an engineered bacteriophage can also be designed for 0204 While clearance issue is not significant in treatment example, for optimal enzyme activity or to delay cell lysis or of chronic diseases, the problem of phage clearance is an using multiple phage promoters to allow for increased important one that needs to be solved as it can make phage enzyme production, or targeting multiple biofilm EPS com therapy more useful for treating transient infections rather ponents with different proteins. In some embodiments, one than chronic ones. Non-lytic and non-replicative phage have can also target multi-species biofilm with a cocktail of differ been engineered to kill bacteria while minimizing endotoxin ent species-specific engineered enzymatically-active phage, release. Accordingly, the present invention encompasses and combination therapy with other agents other than antimi modification of the inhibitor-engineered and/or repressor crobial agent that are well known to one skilled in the art and engineered bacteriophage and/or Susceptibility engineered phage to improve the efficacy of both types of treatment. bacteriophage with minimal endotoxin release or toxin-free 0200. In some embodiments of all aspects described bacteriophage preparation. herein, an engineeredbacteriophage can also be used together 0205 The specificity of phage for host bacteria is both an with other antibacterial or bacteriofilm degrading agents or advantage and a disadvantage for phage therapy. Specificity chemicals such as EGTA, a calcium-specific chelating agent, allows human cells as well as innocuous bacteria to be spared, effected the immediate and substantial detachment of a P potentially avoiding serious issues such as drug toxicity. Anti aeruginosa biofilm without affecting microbial activity, biotic therapy is believed to alter the microbial flora in the NaCl, CaCl, or MgCl, surfactants and urea. colon due to lack of target specificity, and in Some instances 0201 Phage therapy or bacteriophage therapy has begun allowing resistant C. difficile to proliferate and cause disease to be accepted in industrial and biotechnological settings. For such as diarrhea and colitis. The inhibitor-engineered bacte example, the FDA has previously approved the use of phage riophage and repressor-engineered bacteriophages and/or targeted at Listeria monocytogenes as a food additive. Phage Susceptibility engineered bacteriophage as disclosed herein therapy has been used Successfully for therapeutic purposes are capable of inhibiting the local bacterial synthetic machin in Eastern Europe for over 60 years. The development and use ery which normally circumvent antimicrobial effect to result of phage therapy in clinical settings in Western medicine, in in persistent bacteria. particular for treating mammals such as humans has been 0206 For host specificity (i.e. bacteria specific inhibitor or delayed due to the lack of properly designed clinical trials to repressor-engineered bacteriophages), a well-characterized date as well as concerns with (i) development of phage resis library of phage must be maintained so that an appropriate tance, (ii) phage immunogenicity in the human body and inhibitor-engineered bacteriophage or repressor-engineered clearance by the reticuloendothelial system (RES), (iii) the bacteriophage and/or Susceptibility engineered bacterioph release of toxins upon bacterial lysis, and (iv) phage specific age therapy can be designed for each individual bacterial ity. Many of these concerns are currently being studied and infection. The diversity of bacterial infections implies that it addressed. Such as the isolation and development of long may be difficult for any one particular engineered phage to be circulating phage that can avoid RES clearance for increased an effective therapeutic solution for a wide range of biofilms. in vivo efficacy. Accordingly, in all aspects described herein, Accordingly, in one embodiment, the invention provides use the methods of the present invention are applicable to human of a variety of different engineered bacteriophages in combi treatment as the engineered bacteriophages can be designed nation (i.e. a cocktail of engineered bacteriophages discussed to prevent the development of phage resistance in bacteria. A herein) to cover a range of target bacteria. skilled artisan can also develop and carry out an appropriate 0207. One skilled in the art can generate a collection or a clinical trial for use in clinical applications, such as therapeu library of the inhibitor-engineered bacteriophage and/or tic purposes as well as in human Subjects. In some instances, repressor engineered bacteriophage and/or Susceptibility a skilled artisan could establish and set up a clinical trial to engineered bacteriophage as disclosed herein by new cost establish the specific tolerance of the engineered bacterioph effective, large-scale DNA sequencing and DNA synthesis age in human Subjects. The inventors have already demon technologies. Sequencing technologies allows the character strated herein that inhibitor-engineered bacteriophage and ization of collections of natural phage that have been used in repressor-engineered bacteriophages and Susceptibility-engi phage typing and phage therapy formany years. Accordingly, neered bacteriophages are effective at increasing the efficacy a skilled artisan can use synthesis technologies as described of antimicrobial agents, and are effective in dispersing bio hereinto add different inhibitors to antibiotic resistance genes films, including biofilms present in human organs, such as or cell survival genes, and/or different repressors to different colon or lungs and other organs in a Subject prone to bacterial SOS response genes or non-SOS defense genes or Suscepti infection such as bacterial biofilm infection. bility agents to produce a variety of new inhibitor-engineered 0202 Another aspect relates to a pharmaceutical compo bacteriophage and repressor-engineered bacteriophages and/ sition comprising at least one engineered bacteriophage and or Susceptibility engineered bacteriophage respectively. US 2010/0322903A1 Dec. 23, 2010 29

0208. In particular embodiments, the engineered bacte adversely effecting the level of antimicrobial activity. In riophages as described herein can be engineered to express an another embodiment, the criteria used to select inhibitor endogenous gene, such as a repressor protein, or a nucleic engineered bacteriophages and/or a repressor engineered acid inhibitor of an antibiotic resistance gene or cell survival bacteriophage and/or a susceptibility engineered bacterioph gene comprising the agent under the control of inducible age that can potentiate the activity of an antimicrobial agentis regulatory elements, in which case the regulatory sequences an engineered bacteriophage which enables a reduction of at of the endogenous gene can be replaced by homologous least about 10%, ... or at least about 15%, ... or at least about recombination. Gene activation techniques are described in 20%, ... or at least about 25%, ... or at least about 35%, ... U.S. Pat. No. 5.272,071 to Chappel; U.S. Pat. No. 5,578.461 or at least about 50%, ... or at least about 60%, ... or at least to Sherwin et al.: PCT/US92/09627 (WO93/09222) by about 90% and all integers inbetween 10-90% of the amount Selden et al.; and PCT/US90/06436 (WO91/06667) by (i.e. dose) of the antimicrobial agent without adversely effect Skoultchi et al, which are all incorporated herein in their ing the antimicrobial effect when compared to the similar entirety by reference. amount in the absence of an inhibitor-engineered bacterioph 0209 Furthermore, rational engineering methods with age and/or a repressor engineered bacteriophage and/or a new synthesis technologies can be employed to broaden the Susceptibility engineered bacteriophage. engineered bacteriophage host range. For example, T7 can be 0213. In some embodiments, any antimicrobial agent can modified to express K1-5 endosialidase, allowing it to effec be used which is know by persons of ordinary skill in the art tively replicate in E. coli that produce the K1 polysaccharide can be used in combination with an inhibitor-engineered bac capsule. In some embodiments, the gene 1.2 from phage T3 teriophage or a repressor-engineered bacteriophage and/or a can be used to extend the bacteriophages as disclosed herein Susceptibility engineered bacteriophage. In some embodi to be able to transfect a host range to include E. coli that ments an antimicrobial agent is an antibiotic. Thus, in some contain the Fplasmid, thus demonstrating that multiple modi embodiments, the engineered bacteriophages as disclosed fications of a phage genome can be done without significant herein function as antibiotic adjuvants for aminglycoside impairment of the phage's ability to replicate. Bordetella antimicrobial agents, such as but not limited to, gentamicin, bacteriophage use a reverse-transcriptase-mediated mecha amikacin, gentamycin, tobramycin, netromycin, Streptomy nism to produce diversity in host tropism which can also be cin, kanamycin, paromomycin, neomycin. In some embodi used according to the methods of the present invention to ments, the engineered bacteriophages as disclosed herein create a phage that encodes an agent which inhibits antibiotic function as antibiotic adjuvants for B-lactamantibiotics. Such resistance genes and/or cell Survival genes, or alternatively as but not limited to, ampicillin, penicillin, penicillin deriva encodes repressors of SOS response genes, and is lytic to the tives, cephalosporins, monobactams, carbapenems and 3-lac target bacterium or bacteria. The many biofilm-promoting tamase inhibitors. In some embodiments, the engineered bac factors required by E. coli K-12 to produce a mature biofilm teriophages as disclosed herein function as antibiotic are likely to be shared among different biofilm-forming bac adjuvants for quinolones antimicrobial agents, such as, but terial strains and are thus also targets for engineered enzy not limited to, ofloxacin, ciproflaxacin, levofloxacin, gati matic bacteriophage as disclosed herein. floxacin, norfloxacin, lomefloxacin, trovafloxacin, moxi floxacin, sparfloxacin, gemifloxacin, and paZufloxacin. Antimicrobial Agents 0214. In alternative embodiments, an antimicrobial agent 0210. One aspect of the present invention relates to the can be, for example, but not limited to, a small molecule, a killing or inhibiting the growth of bacteria using a combina peptide, a peptidomimetic, a chemical, a compound and any tion of an inhibitor-engineered bacteriophage and/or a repres entity that inhibits the growth and/or kills a microorganism. In Sor engineered bacteriophage and/or a Susceptibility engi Some embodiments, an antimicrobial agent can include, but is neered bacteriophage with at least one antimicrobial agent. not limited to; antibodies (polyclonal or monoclonal), neu Accordingly, one aspect of the present invention relates to tralizing antibodies, antibody fragments, chimeric antibod methods and compositions comprising engineered bacte ies, humanized antibodies, recombinant antibodies, peptides, riophages for use in combination with antimicrobial agents to proteins, peptide-mimetics, aptamers, oligonucleotides, hor potentiate the antimicrobial effect and bacterial killing func mones, Small molecules, nucleic acids, nucleic acid ana tion or inhibition of growth function of the antimicrobial logues, carbohydrates or variants thereof that function to agent. inactivate the nucleic acid and/or protein of the gene products 0211. Accordingly in some embodiments of this aspect of identified herein, and those as yet unidentified. Nucleic acids the present invention relates to the use of a inhibitor-engi include, for example but not limited to, DNA, RNA, oligo neered bacteriophage and/or a repressor engineered bacte nucleotides, peptide nucleic acid (PNA), pseudo-comple riophage and/or susceptibility engineered bacteriophage to mentary-PNA (pcPNA), locked nucleic acid (LNA), RNAi, potentiate the killing effect of antimicrobial agents. Stated microRNAi, siRNA, shRNA etc. The an antimicrobial agent another way, the inhibitor-engineered or repressor-engi inhibitors can be selected from a group of a chemical, Small neered bacteriophage or Susceptibility engineered bacte molecule, chemical entity, nucleic acid sequences, nucleic riophage can be used to enhance the efficacy of at least one acid analogues or protein or polypeptide or analogue or frag antimicrobial agent. ment thereof. 0212. An inhibitor-engineered bacteriophages and/or a 0215. In some embodiments, an antimicrobial agent is an repressor engineered bacteriophage and/or a Susceptibility antimicrobial peptide, for example but not limited to, meflo engineered bacteriophage is considered to potentiate the quine, Venturicidin A, antimycin, myxothiazol, Stigmatellin, effectiveness of the antimicrobial agent if the amount of anti diuron, iodoacetamide, potassium tellurite hydrate, a L-Vi microbial agent used in combination with the engineered nylglycine, N-ethylmaleimide, L-allyglycine, diarycuino bacteriophages as disclosed herein is reduced by at least 10% line, betaine aldehyde chloride, acivcin, psicofuraine, without adversely affecting the result, for example, without buthionine Sulfoximine, diaminopemelic acid, 4-phospho-D- US 2010/0322903A1 Dec. 23, 2010 30 erythronhydroxamic acid, motexafin gadolinium and/or antibiotics to treat conditions, such as multi-drug resistant Xycitrin or modified versions or analogues thereof. tuberculosis, neutropenic cancer patients with fever, and 0216. In some embodiments, an antimicrobial agent use potentially anthrax. Examples of fluoroquinolones/quinolo ful in combination with an inhibitor-engineered or repressor nes include ciproflaxacin, levofloxacin, and ofloxacin, gati engineered bacteriophage described herein includes, but are floxacin, norfloxacin, lomefloxacin, trovafloxacin, moxi not limited to aminoglycosides, carbapenemes, cephalospor floxacin, sparfloxacin, gemifloxacin, and paZufloxacin. ins, cephems, glycoproteins fluoroquinolones/quinolones, 0222 Glycopeptides and streptogramins representantibi oxazolidinones, penicillins, streptogramins, Sulfonamides otics that are used to treat bacteria that are resistant to other and/or tetracyclines. antibiotics, such as methicillin-resistant staphylococcus 0217 Aminoglycosides are a group of antibiotics found to aureus (MRSA). They are also be used for patients who are be effective against gram-negative. Aminoglycosides are allergic to penicillin Examples of glycopeptides include van used to treat complicated urinary tract infections, septicemia, comycin, teicoplanin, and daptomycin. peritonitis and other severe intra-abdominal infections, severe pelvic inflammatory disease, endocarditis, mycobac 0223 B-lactam antibiotics are a broad class of antibiotics terium infections, neonatal sepsis, and various ocular infec which include penicillin derivatives, cephalosporins, mono tions. They are also frequently used in combination with bactams, carbapenems and B-lactamase inhibitors; basically, penicillins and cephalosporins to treat both gram-positive and any antibioticor agent or antimicrobial agent which contains gram-negative bacteria. Examples of aminoglycosides a B-lactam nucleus in its molecular structure. Without being include amikacin, gentamycin, tobramycin, netromycin, bound by theory, B-Lactam antibiotics are bactericidal, and streptomycin, kanamycin, paromomycin, and neomycin. act by inhibiting the synthesis of the peptidoglycan layer of 0218 Carbapenems are a class of broad spectrum antibi bacterial cell walls. The peptidoglycan layer is important for otics that are used to fight gram-positive, gram-negative, and cell wall structural integrity, especially in Gram-positive anaerobic microorganisms. Carbapenems are available for organisms. The final transpeptidation step in the synthesis of intravenous administration, and as such are used for serious the peptidoglycan is facilitated by transpeptidases known as infections which oral drugs are unable to adequately address. penicillin binding proteins (PBPs). B-lactam antibiotics are For example, carbapenems are often used to treat serious analogues of D-alanyl-D-alanine—the terminal amino acid single or mixed bacterial infections, such as lower respiratory residues on the precursor NAM/NAG-peptide subunits of the tract infections, urinary tract infections, intra-abdominal nascent peptidoglycan layer. The structural similarity infections, gynecological and postpartum infections, septice between B-lactam antibiotics and D-alanyl-D-alanine facili mia, bone and joint infections, skin and skin structure infec tates their binding to the active site of penicillin binding tions, and meningitis. Examples of carbapenems include imi proteins (PBPs). The B-lactam nucleus of the molecule irre penem/cilastatin Sodium, meropenem, ertapenem, and versibly binds to (acylates) the Ser403 residue of the PBP active site. This irreversible inhibition of the PBPs prevents panipenem/betamipron. the final crosslinking (transpeptidation) of the nascent pepti 0219 Cephalosporins and cephems are broad spectrum doglycan layer, disrupting cell wall synthesis. Under normal antibiotics used to treat gram-positive, gram-negative, and circumstances peptidoglycan precursors signal a reorganiza spirochaetal infections. Cephems are considered the next tion of the bacterial cell wall and consequently trigger the generation Cephalosporins with newer drugs being stronger activation of autolytic cell wall hydrolyses. Inhibition of against gram negative and older drugs better against gram cross-linkage by B-lactams causes a build-up of peptidogly positive. Cephalosporins and cephems are commonly Substi can precursors which triggers the digestion of existing pepti tuted for penicillin allergies and can be used to treat common doglycan by autolytic hydrolases without the production of urinary tract infections and upper respiratory infections (e.g., new peptidoglycan. This as a result further enhances the pharyugitis and tonsillitis). bactericidal action of B-lactam antibiotics. 0220 Cephalosporins and cephems are also used to treat otitis media, Some skin infections, bronchitis, lower respira 0224 Carbapenems are used to treatgram-positive, gram tory infections (pneumonia), and bone infection (certain; negative, and/or anaerobes. members), and are a preferred antibiotic for Surgical prophy 0225 Oxazolidinones are commonly administered to treat laxis. Examples of Cephalosporins include cefixime, cefpo gram-positive infections. Oxazolidinones are commonly doxime, ceftibuten, cefdinir, cefaclor, cefprozil, loracarbef, used as an alternative to other antibiotic classes for bacteria cefadroxil, cephalexin, and cephradineze. Examples of ceph that have developed resistance. Examples of oxazolidinones ems include cefepime, cefpirome, cefataxidime pentahy include linezolid. drate, ceftazidime, ceftriaxone, ceftazidime, cefotaxime, 0226 Penicillins are broad spectrum used to treat gram cefteram, cefotiam, cefuroxime, cefamandole, cefuroxime positive, gram-negative, and spirochaetal infections. Condi axetil, cefotetan, cefazolin Sodium, cefazolin, cefalexin. tions that are often treated with penicillins include pneumo 0221 Fluoroquinolones/quinolones are antibiotics used to coccal and meningococcal meningitis, dermatological treat gram-negative infections, though some newer agents infections, ear infections, respiratory infections, urinary tract have activity against gram-positive bacteria and anaerobes. infections, acute sinusitis, pneumonia, and Lyme disease. Fluoroquinolones/quinolones are often used to treat condi Examples of penicillins include penicillin, amoxicillin, tions such as urinary tract infections, sexually transmitted amoxicillin-clavulanate, amplicillin, ticarcillin, piperacillin diseases (e.g., gonorrhea, chlamydial urethritisficerviciitis, taZobactam, carbenicillin, piperacillin, meZocillin, benzathin pelvic inflammatory disease), gram-negative gastrointestinal penicillin G penicillin V potassium, methicillin, nafcillin, infections, Soft tissue infections, pphthalmic infections, der oxacillin, cloxacillin, and dicloxacillin. matological infections, sinusitis, and respiratory tract infec 0227 Streptogramins are antibiotics developed in tions (e.g., bronchitis, pneumonia, and tuberculosis). Fluoro response to bacterial resistance that diminished effectiveness quinolones/quinolones are used in combination with other of existing antibiotics. Streptogramins are a very Small class US 2010/0322903A1 Dec. 23, 2010 of drugs and are currently very expensive. Examples of strep cycline; tobramycin; ; ; trimetrex togramins include quinupristin/dafopristin and pristinamy ate; trovafloxacin; Vancomycin; Verdamicin; azithromycin; cin. and linezolid. 0228 Sulphonamides are broad spectrum antibiotics that have had reduced usage due to increase in bacterial resistance Uses of the Engineered Bacteriophages to them. Sulphonamides are commonly used to treat recurrent 0231. Accordingly, the inventors have demonstrated that attacks of rheumatic fever, urinary tract infections, prevention an antimicrobial agent when used in combination with an of infections of the throat and chest, traveler's diarrhea, inhibitor-engineered bacteriophage (which expresses an whooping cough, meningococcal disease, sexually transmit inhibitor to an antibiotic resistance gene or a cell Survival ted diseases, toxoplasmosis, and rhinitis. Examples of Sul gene) and/or in combination with a repressor-engineered bac fonamides include co-trimoxazole, trime teriophage (which expresses at least one repressor to a SOS thoprim, , , and trimethoprim. response gene, or at least one inhibitor or repressor to a 0229 Tetracyclines are broad spectrum antibiotics that are non-SOS defense gene) and/or in combination with a suscep often used to treat gram-positive, gram-negative, and/or spi tibility engineered bacteriophage is effective at killing bacte rochaetal infections. Tetracyclines are often used to treat ria, Such as a bacterial infection or a bacteria biofilm than use mixed infections, such as chronic bronchitis and peritonitis, of the antimicrobial alone or the use of the antimicrobial agent urinary tract infections, rickets, chlamydia, gonorrhea, Lyme used in combination with a non-engineered bacteriophage. disease, and periodontal disease. Tetracyclines are an alter The inventors have also discovered that engineered bacte native therapy to penicillin in syphilis treatment and are also riophages can be adapted to work with a variety of different used to treat acne and anthrax. Examples of tetracyclines antimicrobial agents as well as be modified to express other include tetracycline, demeclocycline, minocycline, and biofilm-degrading enzymes to target a wide range of bacteria doxycycline. and bacteria biofilms. In some embodiments, an antimicro 0230. Other antimicrobial agents and antibiotics contem bial agent is used in combination with at least one engineered plated herein useful in combination with the engineered bac bacteriophage as disclosed herein, and optionally an addition teriophages as disclosed herein according to the present bacteriophage which is not an inhibitor-engineered or repres invention (some of which can be redundant with the list sor-engineered bacteriophage or a susceptibility engineered above) include, but are not limited to; abrifam; acrofloxacin: bacteriophage, but a bacteriophage which is modified to aptecin, amoxicillin plus clavulonic acid; apalcillin, apramy express atherapeutic gene oratoxin gene or a biofilm degrad cin; astromicin; arbekacin; aspoxicillin; azidozillin; azlocil ing gene. Such bacteriophages are well known in the art and lin; aztreonam; bacitracin; benzathine penicillin; benzylpeni are encompassed for use in the methods and compositions as cillin: clarithromycin, carbencillin; cefaclor, cefadroxil; disclosed herein. cefalexin, cefamandole; cefaparin, cefatrizine; cefazolin; cef buperaZone; cefcapene; cefdinir, cefditoren, cefepime, cefe Bacterial Infections tamet, cefixime; cefinetazole; cefiminox, cefoperaZone; cefo ranide; cefotaxime; cefotetan, cefotiam, cefoxitin; 0232. One aspect of the present invention relates to the use cefpimizole; ce?piramide; cefpodoxime, cefprozil, cefradine; of the methods and compositions comprising an inhibitor cefroxadine, cefsulodin; ceftazidime; ceftriaxone; engineered and/or repressor-engineered bacteriophage and/ cefuroxime; cephalexin; chloramphenicol; chlortetracycline; or a susceptibility engineered bacteriophage in combination ciclacillin: ; clemizole penicillin; cleocin, cleocin with an antimicrobial agent to inhibit the growth and/or kill T, cloxacillin; corifam; daptomycin; daptomycin; demeclo (or reduce the cell viability) of a microorganism, such as a cycline; descquinolone; dibekacin; dicloxacillin; dirithromy bacteria. In some embodiments of this aspect and all aspects cin, doxycycline; ; epicillin; ethambutol; described herein, a microorganism is a bacterium. In some gemifloxacin; fenampicin; finamicina; ; flomoxef. embodiments, the bacteria are gram positive and gram nega flucloxacillin; ; flurithromycin; fosfomycin; fos tive bacteria. In some embodiments, the bacteria are multi midomycin; fusidic acid; gatifloxacin; gemifloxaxin, isepa drug resistant bacterium. In further embodiments, the bacte micin; isoniazid; josamycin; kanamycin; kasugamycin; ria are polymyxin-resistant bacterium. In some embodiments, kitasamycin; kalrifam, latamoxef levofloxacin, levofloxacin; the bacterium is a persister bacteria. Examples of gram-nega lincomycin; lineZolid; lomefloxacin; loracarbaf, lymecy tive bacteria are for example, but not limited to Paeruginosa, cline; mecillinam; methacycline; methicillin; metronidazole; A. bumannii, Salmonella spp., Klebsiella pneumonia, Shigeila meZlocillin; midecamycin; minocycline; miokamycin; moxi spp. and/or Stenotrophomonas maltophilia. In one embodi floxacin, nafcillin, nafcilling ; neomycin; ment, the bacteria to be targeted using the phage of the inven netilmicin; norfloxacin; , oflaxacin; oleandomy tion include E. coli, S. epidermidis, Yersina pestis and cin, oxacillin, ; oxytetracycline; paromycin; Pseudomonas fluorescens. paZufloxacin; ; penicilling; penicillin V; phenethi 0233. In some embodiments, the methods and composi cillin; phenoxymethyl penicillin; ; piperacillin tions as disclosed herein can be used to kill or reduce the and taZobactam combination; ; procaine peni viability of a bacterium, for example a bacterium such as, but cillin; propicillin; pyrimethamine; rifadin: : rifa not limited to: Bacillus cereus, Bacillus anbhracis, Bacillus mide; rifampin: rifapentene; rifomycin; rimactane, rofact, cereus, Bacillus anthracia, Clostridium botulinum, rokitamycin; rollitetracycline; roXithromycin; ; Clostridium difficle, Clostridium tetani, Clostridium perfrin ; sparfloxacin; spectinomycin; spiramycin; Sulfa gens, Corynebacteria diptheriae, Enterococcus (Streptococ diazine, Sulfadoxine; Sulfamethoxazole; sisomicin; Strepto cus D), Lieteria monocytogenes, Pneumoccoccal infections mycin; Sulfamethoxazole; sulfisoxazole; quinupristan-dalfo (Streptococcus pneumoniae), Staphylococcal infections and pristan; teicoplanin; temocillin; gatifloxacin, tetracycline; Streptococcal infections; Gram-negative bacteria including tetroxoprim; tellithromycin; thiamphenicol; ticarcillin; tige Bacteroides, Bordetella pertussis, Brucella, Campylobacter US 2010/0322903A1 Dec. 23, 2010 32 infections, enterohaemorrhagic Escherichia coli (EHEC/E. lens; 6. Skin infections such as nail infections in people coli O157:17), enteroinvasive Escherichia coli (EIEC), whose hands are frequently exposed to water, 7. Gastrointes enterotoxigenic Escherichia coli (ETEC), Haemophilus tinal tract infections; 8. Muscoskeletal system infections. influenzae, Helicobacter pylori, Klebsiella pneumoniae, 0237 Examples of infections caused by A. baumannii Legionella spp., Moraxella catarrhalis, Neisseria gonnor include: A) Nosoconial infections 1. Bacteraemia and sepsis, rhoeae, Neisseria meningitidis, Proteus spp., Pseudomonas 2. respiratory tract infections in mechanically ventilated aeruginosa, Salmonella spp., Shigella spp., Vibrio cholera patients; 3. Post-Surgery infections on invasive devices; 4. and Yersinia; acid fast bacteria including Mycobacterium wound infectious, particularly in burn wound patients; 5. tuberculosis, Mycobacterium avium-intracellulars, Myobac infection in patients with acquired immunodeficiency syn terium johnei, Mycobacterium leprae, atypical bacteria, drome, cancer chemotherapy, steroid therapy, hematological Chlamydia, Myoplasma, Rickettsia, Spirochetes, Treponema malignancies, organ transplantation, renal replacement pallidum, Borrelia recurrentis, Borrelia burgdorfii and Lep therapy, and other situations with severe neutropenia; 6. uri to spira icterohemorrhagiae, Actinomyces, Nocardia, P nary tract infections; 7. Endocarditis by intravenous admin aeruginosa, A. bumannii, Salmonella spp., Klebsiella pneu istration of contaminated drug solutions; 8. Cellulitis. B) monia, Shigeila spp. and/or Stenotrophomonas maltophilia Community-acquired infections; a. community-acquired and other miscellaneous bacteria. pulmonary infections; 2. Meningitis; Cheratitis associated 0234 Bacterial infections include, but are not limited to, with contaminated contact lens; 4. War-Zone community infections caused by Bacillus cereus, Bacillus anbhracis, acquired infections. C) Atypical infections: 1. Chronic gas Bacillus cereus, Bacillus anthracia, Clostridium botulinum, tritis. Clostridium difficle, Clostridium tetani, Clostridium perfrin 0238 Examples of infections caused by Stenotrophomo gens, Corynebacteria diptheriae, Enterococcus (Streptococ nas maltophilia include Bacteremia, pneumonia, meningitis, cus D), Lieteria monocytogenes, Pneumoccoccal infections wound infections and urinary tract infections. Some hospital (Streptococcus pneumoniae), Staphylococcal infections and breaks are caused by contaminated disinfectant Solutions, Streptococcal infections/Gram-negative bacteria including respiratory devices, monitoring instruments and ice Bacteroides, Bordetella pertussis, Brucella, Campylobacter machines. Infections usually occur in debilitated patients infections, enterohaemorrhagic Escherichia coli (EHEC/E. with impaired host defense mechanisms. coli O157:17) enteroinvasive Escherichia coli (EIEC), entero 0239 Examples of infections caused by Klebsiella pneu toxigenic Escherichia coli (ETEC), Haemophilus influenzae, moniae include community-acquired primary lobar pneumo Helicobacter pylori, Klebsiella pneumoniae, Legionella spp., nia, particularly in people with compromised pulmonary Moraxella catarrhalis, Neisseria gonnorrhoeae, Neisseria function and alcoholics. It also caused wound infections, soft meningitidis, Proteus spp., Pseudomonas aeruginosa, Sal tissue infections and urinary tract infections. monella spp., Shigella spp., Vibrio cholera and Yersinia; acid 0240 Examples of infections caused by Salmonella app. fast bacteria including Mycobacterium tuberculosis, Myco are acquired by eating contaminated food products. Infec bacterium avium-intracellulars, Myobacterium johnei, tions include enteric fever, enteritis and bacteremia. Mycobacterium leprae, atypical bacteria, Chlamydia, Myo 0241 Examples of infections caused by Shigella spp. plasma, Rickettsia, Spirochetes, Treponema pallidum, Borre include gastroenteritis (shigellosis). lia recurrentis, Borrelia burgdorfi and Leptospira icterohe 0242. The methods and compositions as disclosed herein morrhagiae and other miscellaneous bacteria, including comprising an inhibitor-engineered or repressor-engineered Actinomyces and Nocardia. bacteriophage and at least one antimicrobial agent can also be 0235. In some embodiments, the microbial infection is used in various fields as where antiseptic treatment or disin caused by gram-negative bacterium, for example, P aerugi fection of materials it required, for example, Surface disinfec nosa, A. bumannii, Salmonella spp., Klebsiella pneumonia, tion. Shigeila spp. and/or Stenotrophomonas maltophilia. 0243 The methods and compositions as disclosed herein Examples of microbial infections include bacterial wound comprising an inhibitor-engineered or repressor-engineered infections, mucosal infections, enteric infections, septic con bacteriophage and at least one antimicrobial agent can be ditions, pneumonia, trachoma, onithosis, trichomoniasis and used to treat microorganisms infecting a cell, group of cells, salmonellosis, especially in Veterinary practice. or a multi-cellular organism. 0236 Examples of infections caused by P. aeruginosa 0244. In one embodiment, an antimicrobial agent and an include: A) Nosoconial infections, 1. Respiratory tract infec engineered bacteriophage as described herein can be used to tions in cystic fibrosis patients and mechanically-ventilated reduce the rate of proliferation and/or growth of microorgan patients; 2. Bacteraemia and sepsis; 3. Wound infections, isms. In some embodiments, the microorganism are either or particularly in burn wound patients; 4. Urinary tract infec both gram-positive or gram-negative bacteria, whether Such tions: 5. Post-surgery infections on invasive devises 5. bacteria are cocci (spherical), rods, vibrio (comma shaped), Endocarditis by intravenous administration of contaminated or spiral. drug Solutions; 7, Infections in patients with acquired immu 0245. Of the cocci bacteria, micrococcusandstaphylococ nodeficiency syndrome, cancer chemotherapy, steroid cus species are commonly associated with the skin, and Strep therapy, hematological malignancies, organ transplantation, tococcus species are commonly associated with tooth enamel renal replacement therapy, and other situations with severe and contribute to tooth decay. Of the rods family, bacteria neutropenia. B) Community-acquired infections: 1. Commu Bacillus species produce endospores seen in various stages of nity-acquired respiratory tract infections; 2. Meningitis; 3. development in the photograph and B. cereus cause a rela Folliculitis and infections of the ear canal caused by contami tively mild food poisoning, especially due to reheated fried nated waters; 4. Malignant otitis externa in the elderly and food. Of the vibrio species, V. cholerae is the most common diabetics; 5. Osteomyelitis of the caleaneus in children: Eye bacteria and causes cholera, a severe diarrhea disease result infections commonly associated with contaminated contact ing from a toxin produced by bacterial growth in the gut. Of US 2010/0322903A1 Dec. 23, 2010 the spiral bacteria, rhodospirillum and Treponema pallidum are the common species to cause infection (e.g., Treponema TABLE 7-continued pallidum causes syphilis). Spiral bacteria typically grow in shallow anaerobic conditions and can photosynthesize to Examples of bacteria. obtain energy from Sunlight. Table 7: Examples of Bacteria 0246 Moreover, the present invention relates to use of or Bacilius cereus Vibrio choierae Borrelia burgdorferi methods comprising an antimicrobial agent and an engi Bacilius subtilis Escherichia Coi K12 Ehrichia chafeensis neered bacteriophage as disclosed herein can be used to Streptococci is phenonia Bartonelia henselae Treponema reduce the rate of growth and/or kill either gram positive, pallidiin gram negative, or mixed flora bacteria or other microorgan Streptococci is pyogenes Haemophilus Chlamydia influenzae trachomatis isms. In one embodiment, the composition consists essen Cliostridium tetani Salmonella typhi tially of at least one antimicrobial agent and at least one Listeria monocytogenes Shigella dysentriae engineered bacteriophage, such as an inhibitor-engineered Mycobacterium Yerinisa pestis bacteriophage or repressor-engineered bacteriophage or a tuberculosis Susceptibility engineered bacteriophage as disclosed herein Staphyloccoctis Pseudomona for the use to reduce the rate of growth and/or kill either gram epidermidis aeruginosa positive, gram negative, or mixed flora bacteria or other microorganisms. In another embodiment, the composition 0248. In some embodiments, antimicrobial agent and contains at least one antimicrobial agent and at least one engineered bacteriophages described herein can be used to engineered bacteriophage, such as an inhibitor-engineered treat an already drug resistant bacterial Strain such as Methi bacteriophage or repressor-engineered bacteriophage or a cillin-resistant Staphylococcus aureus (MRSA) or Vancomy Susceptibility engineered bacteriophage as disclosed herein cin-resistant enterococcus (VRE) of variant strains thereof. for the use to reduce the rate of growth and/or kill either gram 0249. In some embodiments, the present invention also positive, gram negative, or mixed flora bacteria or other contemplates the use and methods of use of an antimicrobial microorganisms. agent and an engineered bacteriophage as described herein in all combinations with other antimicrobial agents and/or anti 0247 Such bacteria are for example, but are not limited to, biotics to fightgram-positive bacteria that maintain resistance listed in Table 7. Further examples of bacteria are, for to certain drugs. example but not limited to Baciccis Antracis: Enterococcus 0250 In some embodiments, an antimicrobial agents and faecalis, Corynebacterium, diphtheriae, Escherichia coli, an engineered bacteriophage as disclosed herein can be used Streptococcus coelicolor, Streptococcus pyogenes, Strepto to treat infections, for example bacterial infections and other bacillus moniliformis, Streptococcus agalactiae, Streptococ conditions such as urinary tract infections, ear infections, cus pneurmoniae, Salmonella typhi; Salmonella paratyphi; sinus infections, bacterial infections of the skin, bacterial Salmonella schottmulleri, Salmonella hirshieldii; Staphylo infections of the lungs, sexually transmitted diseases, tuber coccus epidermidis, Staphylococcus aureus, Klebsiella culosis, pneumonia, Lyme disease, and Legionnaire's dis pneumoniae, Legionella pneumophila, Helicobacter pylori; ease. Thus any of the above conditions and other conditions Mycoplasma pneumonia, Mycobacterium tuberculosis, resulting from a microorganism infection, for example a bac Mycobacterium leprae, Yersinia enterocolitica, Yersinia pes terial infection or a biofilm can be prevented or treated by the tis, Vibrio cholerae, Vibrio parahaemolyticus, Rickettsia pro compositions of the invention herein. wOzekii, Rickettsia rickettsii, Rickettsia akari. Clostridium Biofilms difficile, Clostridium tetani; Clostridium perfingens, 0251 Another aspect of the present invention relates to the Clostridianz novyi, Clostridianz Septicum, Clostridium use of an inhibitor-engineered bacteriophage and/or a repres botulinum, Legionella pneumophila, Hemophilus influenzue; sor-engineered bacteriophage and/or a susceptibility engi Hemophilus parainfluenzue, Hemophilus aegyptus, Chlamy neered bacteriophage in combination with any antimicrobial dia psittaci, Chlamydia trachonzatis, Bordetella pertcsis, agent to eliminate or reduce a bacterial biofilm, for example a Shigella spp., Campylobacter jejuni; Proteus spp., Citro bacterial biofilm in a medical, or industrial, or biotechnologi bacter spp.; Enterobacter spp., Pseudomonas aeruginosa, cal setting. Propionibacterium spp.; Bacillus anthracia; Pseudomonas 0252 For instance, some bacteria, including P. aerugi syringae; Spirrilum minus; Neisseria meningitidis, Listeria nosa, actively form tightly arranged multi-cell structures in monocytogenes, Neisseria gonorrheae, Treponema palli vivo known as biofilm. The production of biofilm is important dum, Francisella tularensis, Brucella spp.; Borrelia recur for the persistence of infectious processes such as seen in rentis, Borrelia hennsii; Borrelia turicatue, Borrelia burg pseudomonal lung-infections in patients with cystic fibrosis dorferi; Mycobacterium avium, Mycobacterium Smegmatis; and diffuse panbronchiolitis and many other diseases. A bio Methicillin-resistant Staphyloccus aureus; Vanomycin-resis film is typically resistant to phagocytosis by host immune tant enterococcus; and multi-drug resistant bacteria (e.g., cells and the effectiveness of antibiotics at killing bacteria in bacteria that are resistant to more than 1, more than 2, more biofilm structures can be reduced by 10 to 1000 fold. Biofilm than 3, or more than 4 different drugs). production and arrangement is governed by quorum sensing systems. The disruption of the quorum sensing system in TABLE 7 bacteria Such as Paeruginosa is an importantanti-pathogenic activity as it disrupts the biofilm formation and also inhibits Examples of bacteria. alginate production Table 7: Examples of Bacteria Selection of Subjects Administered a Composition Compris Staphyloccoctisatiretts Nisseria menigintidis Helicbacter pylori Bacilius anthracis Nisseria gonerrhoeae Legioneiia ing an Engineered Bacteriophage pnemophia 0253) In some embodiments, a subject amenable for the method described herein or for the administration with a US 2010/0322903A1 Dec. 23, 2010 34 composition comprising at least one antimicrobial agent and agent can be administered prior to the administration of the an inhibitor-engineered bacteriophage and/or a repressor-en engineered bacteriophage, and the time at which the antimi gineered bacteriophage and/or a Susceptibility engineered crobial agent is released from the time-release capsule coin bacteriophage is selected based on the desired treatment cides with the time of the administration of the engineered regime. For instance, a subject is selected for treatment if the bacteriophage. subject has a bacterial infection where the bacteria form a 0257. In some embodiments, an antimicrobial agent can biofilm, or where the subject has been non-responsive to prior be a pro-drug, where it is activated by a second agent. Accord therapy or administration with an antimicrobial agent. ingly, in Such embodiments, an antimicrobial pro-drug agent 0254 Accordingly, in some embodiments, a Subjects is can be administered to a subject at the same time, concurrent administered a combination of at least one antimicrobial with, or prior to, or after the administration of an inhibitor agent and at least one inhibitor-engineered bacteriophage engineeredbacteriophage and/or repressor-engineered bacte and/or a repressor-engineered bacteriophage and/or a suscep riophage and/or Susceptibility engineered bacteriophage, and tibility engineered bacteriophage to potentiate the effect of administration of an agent which activates the pro-drug into the antimicrobial agent. its active form can be administered the same time, concurrent 0255. In some embodiments, a subject can be adminis with, or prior to, or after the administration of the inhibitor tered a composition comprising at least one antimicrobial engineeredbacteriophage and/or repressor-engineered bacte agent, for example at least 2, 3, or 4 or as many of 10 different riophage and/or Susceptibility engineered bacteriophage. antimicrobial agents and at least one engineered bacterioph 0258. In some embodiments, a subject is selected for the age as disclosed herein, for example, for example at least 2, 3, administration with the compositions as disclosed herein by or 4 or as many of 10 different engineered bacteriophages as identifying a Subject that needs a specific treatment regimen disclosed herein. In some embodiments, the composition can of an antimicrobial agent, and is administered an antimicro comprise an antimicrobial agent and at least one or a variety bial agent concurrently with, or prior to, or after administra of different repressor-engineered bacteriophages with at least tion with an inhibitor-engineered bacteriophage and/or a one or a variety of different inhibitor-engineered bacterioph repressor-engineered bacteriophage and/or Susceptibility ages and/or with at least one or a variety of Susceptibility engineered bacteriophage as disclosed herein. engineered bacteriophages. In alternative embodiments, the 0259. Using a subject with cystic fibrosis as an exemplary composition can comprise at least two, or at least 3, 4, 5 or as example, a Subject could be administered an antimicrobial many of 10 different inhibitor-engineered bacteriophages, agent to avoid chronic endobronchial infections, such as wherein each of the inhibitor-engineered bacteriophages those caused by pseudomonas aeruginosis or Steintrophomo comprise a nucleic acid which encodes at least one inhibitor nas maltophilia. One such antimicrobial agent which can be to a different antibiotic resistance gene and/or cell survival used is colistin, however, administration of colistin at the repair gene. In alternative embodiments, the composition can doses and the duration required to efficiently prevent Such comprise at least two, or at least 3, 4, 5 or as many of 10 endobronchial infections in Subjects is highly toxic and in different repressor-engineered bacteriophages, wherein each Some instances fatal. Accordingly, in some embodiments, of the repressor-engineered bacteriophages comprise a Such a subject selected for a treatment regimen would be nucleic acid which encodes at least one repressor to a differ administered compositions as disclosed herein comprising an ent SOS response gene and/or at least one repressor or inhibi antimicrobial agent and an inhibitor-engineered bacterioph torto a non-SOS defense gene. Any combination and mixture age and/or a repressor-engineered bacteriophage and/or Sus of antimicrobial agents and mixture of inhibitor-engineered ceptibility engineered bacteriophage. Thus in Such embodi bacteriophages and/or repressor-engineered bacteriophages ments, an antimicrobial agent can be used at a lower dose and/or Susceptibility engineered bacteriophages are useful in when used in combination with an inhibitor-engineered bac teriophage and/or repressor-engineered bacteriophage and/or the compositions and methods of the present invention. Susceptibility engineered bacteriophage as compared to the 0256 In some embodiments, an antimicrobial agent is administered to a Subject at the same time, prior to, or after the use of Such an antimicrobial agent alone. Thus one aspect of administration of an inhibitor-engineered bacteriophage and/ the invention relates to methods to reduce or decrease the dose or a repressor-engineered bacteriophage and/or Susceptibility of an antimicrobial agent while maintaining efficacy of Such engineered bacteriophage. In some embodiments, an antimi an antimicrobial agent, and thus reduce toxic side affects crobial agent can be formulated to a specific time-release for associated with higher doses. activity, Such as the antimicrobial agent is present in a time release capsule. In Such embodiments, an antimicrobial agent Pharmaceutical Formulations and Compositions that is formulated for time-release can be administered to a 0260 The inhibitor-engineered bacteriophage and repres Subject at the same time, concurrent with, or prior to, or after sor-engineered bacteriophages as disclosed herein can befor the administration of an inhibitor-engineered bacteriophage mulated in combination with one or more pharmaceutically and/or a repressor-engineered bacteriophage and/or Suscep acceptable anti-microbial agents. In some embodiments, tibility engineered bacteriophage. Methods of formulation of combinations of different antimicrobial agents can be tailored an antimicrobial agent for release in a time-dependent man to be combined with a specific inhibitor-engineered bacte ner are disclosed herein as “sustained release pharmaceutical riophage and a repressor-engineered bacteriophage and/or compositions' in the section entitled “pharmaceutical formu Susceptibility engineered bacteriophage, where the inhibitor lations and compositions.” Accordingly, in Such embodi engineeredbacteriophage and/or repressor-engineered bacte ments, a time-release antimicrobial agent can be adminis riophages and/or Susceptibility engineered bacteriophage are tered to a subject at the same time (i.e. concurrent with), prior designed to target different (or the same) microorganisms or to or after the administration of an engineered bacteriophage bacteria, which contribute towards morbidity and mortality. A independent to the time to which the antimicrobial agent pharmaceutically acceptable composition comprising an becomes active. In some embodiments, an antimicrobial inhibitor-engineered bacteriophage and/or a repressor-engi US 2010/0322903A1 Dec. 23, 2010

neered bacteriophage and/or susceptibility engineered bacte disclosed herein which comprise a combination of at least one riophage and an antimicrobial agent as disclosed herein, are antimicrobial agents and an engineered bacteriophage can Suitable for internal administration to an animal, for example further comprise a pharmaceutically acceptable carrier. The human. composition can further comprise other components or 0261. In some embodiments, an inhibitor-engineered bac agents useful for delivering the composition to a Subject are teriophage and/or a repressor-engineered bacteriophage and/ known in the art. Addition of Such carriers and other compo or Susceptibility engineered bacteriophage as disclosed nents to the agents as disclosed herein is well within the level herein can be used for industrial sterilizing, sterilizing chemi of skill in this art. cals such as detergents, disinfectants, and ammonium-based chemicals (e.g. quaternary ammonium compounds such as 0266. In some embodiments, the composition is a compo QUATAL, which contains 10.5% N-alkyldimethyl-benzlam sition for sterilization of a physical object, that is infected monium HCl and 5.5% gluteraldehyde as active ingredients, with bacteria, Such as Sterilization of hospital equipment, Ecochimie Ltée, Quebec, Canada), and can be used in con industrial equipment, medical devices and food products. In currently with, or prior to or after the treatment or adminis another embodiment, the compositions are a pharmaceutical tration of an antimicrobial agent. Such sterilizing chemicals composition useful to treat a bacterial infection in a Subject, are typically used in the art for sterilizing industrial work for example a human or animal Subject. Surfaces (e.g. in food processing, or hospital environments), 0267 In some embodiments, a pharmaceutical composi and are not suitable for administration to an animal. tion as disclosed herein can be administered as a formulation 0262. In another aspect of the present invention relates to adapted for passage through the blood-brain barrier or direct a pharmaceutical composition comprising an inhibitor-engi contact with the endothelium. In some embodiments, the neered bacteriophage and/or repressor-engineered bacte pharmaceutical compositions can be administered as a for riophage and/or Susceptibility engineered bacteriophage and mulation adapted for systemic delivery. In some embodi an antimicrobial agent and a pharmaceutically acceptable ments, the compositions can be administered as a formulation excipient. Suitable carriers for the engineered bacteriophages adapted for delivery to specific organs, for example but not of the invention, and their formulations, are described in Remington's Pharmaceutical Sciences, 16" ed., 1980, Mack limited to the liver, bone marrow, or systemic delivery. Publishing Co., edited by Oslo et al. Typically an appropriate 0268 Alternatively, pharmaceutical compositions can be amount of a pharmaceutically acceptable salt is used in the added to the culture medium of cells ex vivo. In addition to the formulation to render the formulation isotonic. Examples of antimicrobial agent and engineered bacteriophages. Such the carrier include buffers such as saline, Ringer's solution compositions can contain pharmaceutically-acceptable carri and dextrose solution. The pH of the solution is preferably ers and other ingredients or agents known to facilitate admin from about 5 to about 8, and more preferably from about 7.4 istration and/or enhance uptake (e.g., Saline, dimethylsulfox to about 7.8. Further carriers include sustained release prepa ide, lipid, polymer, affinity-based cell specific-targeting rations such as semipermeable matrices of solid hydrophobic systems). In some embodiments, a pharmaceutical composi polymers, which matrices are in the form of shaped articles, tion can be incorporated in a gel, Sponge, or other permeable e.g. liposomes, films or microparticles. It will be apparent to matrix (e.g., formed as pellets or a disk) and placed in proX those of skill in the art that certain carriers can be more imity to the endothelium for sustained, local release. The preferable depending upon for instance the route of adminis composition can be administered in a single dose or in mul tration and concentration of the an engineered bacteriophage tiple doses which are administered at different times. being administered. 0269 Pharmaceutical compositions can be administered 0263 Administration to human can be accomplished by to a subject by any known route. By way of example, the means determined by the underlying condition. For example, composition can be administered by a mucosal, pulmonary, if the engineeredbacteriophage is to be delivered into lungs of topical, or other localized or systemic route (e.g., enteral and an individual, inhalers can be used. If the composition is to be parenteral). The phrases “parenteral administration' and delivered into any part of the gut or colon, coated tablets, “administered parenterally as used herein means modes of Suppositories or orally administered liquids, tablets, caplets administration other than enteral and topical administration, and so forth can be used. A skilled artisan will be able to usually by injection, and includes, without limitation, intra determine the appropriate way of administering the phages of venous, intramuscular, intraarterial, intrathecal, intraven the invention in view of the general knowledge and skill in the tricular, intracapsular, intraorbital, intracardiac, intradermal, art intraperitoneal, transtracheal, Subcutaneous, Subcuticular, 0264 Compounds as disclosed herein, can be used as a intraarticular, Sub capsular, Subarachnoid, intraspinal, intrac medicament or used to formulate a pharmaceutical composi erebro spinal, and intrasternal injection, infusion and other tion with one or more of the utilities disclosed herein. They injection or infusion techniques, without limitation. The can be administered in vitro to cells in culture, in vivo to cells phrases “systemic administration.” “administered systemi in the body, or ex vivo to cells outside of a subject that can cally”, “peripheral administration' and “administered later be returned to the body of the same subject or another peripherally as used herein mean the administration of the Subject. Such cells can be disaggregated or provided as Solid agents as disclosed herein Such that it enters the animal's tissue in tissue transplantation procedures. system and, thus, is subject to metabolism and other like 0265 Compositions comprising at least one antimicrobial processes, for example, Subcutaneous administration. agent and at least one engineered bacteriophage (i.e. an 0270. The phrase “pharmaceutically acceptable' is inhibitor engineered and/or repressor-engineered bacterioph employed herein to refer to those compounds, materials, age and/or Susceptibility engineered bacteriophage) as dis compositions, and/or dosage forms which are, within the closed herein can be used to produce a medicament or other Scope of sound medical judgment, Suitable for use in contact pharmaceutical compositions. Use of the compositions as with the tissues of human beings and animals without exces US 2010/0322903A1 Dec. 23, 2010 36 sive toxicity, irritation, allergic response, or other problem or in a subject who is free therefrom as well as slowing or complication, commensurate with a reasonable benefit/risk reducing progression of existing disease. ratio. 0276. In some embodiments, efficacy of treatment can be 0271 The phrase “pharmaceutically acceptable carrier' measured as an improvement in morbidity or mortality (e.g., as used herein means a pharmaceutically acceptable material, lengthening of Survival curve for a selected population). Pro composition or vehicle. Such as a liquid or Solid filler, diluent, phylactic methods (e.g., preventing or reducing the incidence excipient, solvent or encapsulating material, involved in car of relapse) are also considered treatment. rying or transporting the Subject agents from one organ, or 0277 Dosages, formulations, dosage Volumes, regimens, portion of the body, to another organ, or portion of the body. and methods for analyzing results aimed at reducing the num Each carrier must be “acceptable' in the sense of being com ber of viable bacteria and/or activity can vary. Thus, mini patible with the other ingredients of the formulation, for mum and maximum effective dosages vary depending on the example the carrier does not decrease the impact of the agent method of administration. Suppression of the clinical on the treatment. In other words, a carrier is pharmaceutically changes associated with bacterial infections or infection with inert. a microorganism can occur within a specific dosage range, 0272 Suitable choices in amounts and timing of doses, which, however, varies depending on the organism receiving formulation, and routes of administration can be made with the dosage, the route of administration, whether the antimi the goals of achieving a favorable response in the Subject with crobial agents are administered in conjunction with the engi a bacterial infection or infection with a microorganism, for neered bacteriophages as disclosed herein, and in some example, a favorable response is killing or elimination of the embodiments with other co-stimulatory molecules, and the microorganism or bacteria, or control of or inhibition of specific regimen administration. For example, in general, growth of the bacterial infection in the subject or a subject at nasal administration requires a Smaller dosage than oral, risk thereof (i.e., efficacy), and avoiding undue toxicity or enteral, rectal, or vaginal administration. other harm thereto (i.e., safety). Therefore, “effective' refers 0278 For oral or enteral formulations for use with the to Such choices that involve routine manipulation of condi present invention, tablets can be formulated in accordance tions to achieve a desired effect or favorable response. with conventional procedures employing Solid carriers well 0273 A bolus of the pharmaceutical composition can be known in the art. Capsules employed for oral formulations to administered to a subject over a short time, Such as once a day be used with the methods of the present invention can be made is a convenient dosing schedule. Alternatively, the effective from any pharmaceutically acceptable material. Such as gela daily dose can be divided into multiple doses for purposes of tin or cellulose derivatives. Sustained release oral delivery administration, for example, two to twelve doses per day. systems and/or enteric coatings for orally administered dos Dosage levels of active ingredients in a pharmaceutical com age forms are also contemplated. Such as those described in position can also be varied so as to achieve a transient or U.S. Pat. No. 4,704,295, “Enteric Film-Coating Composi Sustained concentration of the composition in the Subject, tions issued Nov. 3, 1987; U.S. Pat. No. 4,556,552, “Enteric especially in and around the area of the bacterial infection or Film-Coating Compositions, issued Dec. 3, 1985; U.S. Pat. infection with a microorganism, and to result in the desired No. 4,309.404, “Sustained Release Pharmaceutical Compo therapeutic response or protection. It is also within the skill of sitions issued Jan. 5, 1982; and U.S. Pat. No. 4,309,406, the art to start doses at levels lower than required to achieve “Sustained Release Pharmaceutical Compositions, issued the desired therapeutic effect and to gradually increase the Jan.5, 1982, which are incorporated herein in their entirety by dosage until the desired effect is achieved. reference. 0274 The amount of the pharmaceutical compositions to 0279. Examples of solid carriers include starch, sugar, be administered to a Subject is dependent upon factors known bentonite, silica, and other commonly used carriers. Further to a persons of ordinary skill in the art such as bioactivity and non-limiting examples of carriers and diluents which can be bioavailability of the antimicrobial agent (e.g., half-life in the used in the formulations of the present invention include body, stability, and metabolism of the engineered bacterioph saline, syrup, dextrose, and water. age); chemical properties of the antimicrobial agent (e.g., 0280 Practice of the present invention will employ, unless molecular weight, hydrophobicity, and solubility); route and indicated otherwise, conventional techniques of cell biology, scheduling of administration, and the like. It will also be cell culture, molecular biology, microbiology, recombinant understood that the specific dose level of the composition DNA, protein chemistry, and immunology, which are within comprising antimicrobial agents and engineered bacterioph the skill of the art. Such techniques are described in the ages as disclosed herein to be achieved for any particular literature. See, for example, Molecular Cloning: A Labora Subject can depend on a variety of factors, including age, tory Manual, 2nd edition. (Sambrook, Fritsch and Maniatis, gender, health, medical history, weight, combination with one eds.), Cold Spring Harbor Laboratory Press, 1989; DNA or more other drugs, and severity of disease, and bacterial Cloning, Volumes I and II (D. N. Glover, ed), 1985; Oligo strain or microorganism the Subject is infected with, such as nucleotide Synthesis, (M. J. Gait, ed.), 1984; U.S. Pat. No. infection with multi-resistant bacterial strains. 4,683,195 (Mullis et al.); Nucleic Acid Hybridization (B. D. (0275. The term “treatment”, with respect to treatment of a Hames and S. J. Higgins, eds.), 1984; Transcription and bacterial infection or bacterial colonization, inter alia, pre Translation (B. D. Hames and S. J. Higgins, eds.), 1984; venting the development of the disease, or altering the course Culture of Animal Cells (R. I. Freshney, ed). Alan R. Liss, of the disease (for example, but not limited to, slowing the Inc., 1987: Immobilized Cells and Enzymes, IRL Press, progression of the disease), or reversing a symptom of the 1986: A Practical Guide to Molecular Cloning (B. Perbal), disease or reducing one or more symptoms and/or one or 1984; Methods in Enzymology, Volumes 154 and 155 (Wu et more biochemical markers in a Subject, preventing one or al., eds), Academic Press, New York; Gene Transfer Vectors more symptoms from worsening or progressing, promoting for Mammalian Cells (J. H. Miller and M. P. Calos, eds.), recovery or improving prognosis, and/or preventing disease 1987, Cold Spring Harbor Laboratory: Immunochemical US 2010/0322903A1 Dec. 23, 2010 37

Methods in Cell and Molecular Biology (Mayer and Walker, 19. The bacteriophage of any of paragraphs 14 to 15, wherein eds.), Academic Press, London, 1987; Handbook of Experi the agent which increases the susceptibility of a bacteria cell ment Immunology, Volumes I-IV (D. M. Weir and C. C. to an antimicrobial agent is craA or variants or fragments Blackwell, eds.), 1986; Manipulating the Mouse Embryo, thereof. Cold Spring Harbor Laboratory Press, 1986. 20. The bacteriophage of any of paragraphs 14 to 15, wherein In some embodiments of the present invention may be defined the agent which increases the susceptibility of a bacteria cell in any of the following numbered paragraphs: to an antimicrobial agent is craA or variants or fragments 1. An engineered bacteriophage comprising a nucleic acid thereof. operatively linked to a promoter, wherein the nucleic acid 21. The bacteriophage of any of paragraphs 14 to 15, wherein encodes at least one agent that inhibits an antibiotic resistance the agent which increases the susceptibility of a bacteria cell gene and/or a cell Survival repair gene. to an antimicrobial agent modifies a pathway specifically 2. The bacteriophage of any of paragraph 1, wherein the expressed in a bacterial cell. antibiotic resistance gene is selected from the group compris 22. The bacteriophage of any of paragraphs 14 to 15 or 21, ing cat, VanA or mecD or variants thereof. wherein modification is inhibition or activation of a pathway 3. The bacteriophage of any of paragraphs 1 or 2, wherein the specifically expressed in a bacterial cell. cell Survival gene is selected from the group comprising 23. The bacteriophage of any of paragraphs 14 to 15, wherein RecA, RecB. RecC., spot, RelA or variants thereof. the agent which increases iron-sulfur clusters in the bacterial 4. The bacteriophage of any of paragraphs 1 to 3, wherein the cell. agent is selected from a group comprising, siRNA, antisense nucleic acid, asRNA, RNAi, miRNA and variants thereof. 24. The bacteriophage of any of paragraphs 14 to 15, wherein 5. The bacteriophage of any of paragraphs 1 to 4, wherein the the agent which increases oxidative stress in a bacterial cellor agent is an antisense RNA (asRNA). increases hydrozyl radicals in a bacterial cell. 6. The bacteriophage of any of paragraphs 1 to 5, wherein the 25. The bacteriophage of any of paragraphs 14 to 24, wherein bacteriophage comprises a nucleic acid encoding at least two the agent is not substantially toxic a bacterial cell in the agents that inhibit at least two different cell survival repair absence of an antimicrobial agent. genes. 26. The bacteriophage of any of paragraphs 14 to 25, wherein 7. The bacteriophage of any of paragraphs 1 to 6, wherein the the agent is not a chemotherapeutic agent oran protein toxin. bacteriophage comprises a nucleic acid encoding at least two 27. The bacteriophage of any of paragraphs 14 to 26, wherein agents that inhibit at least two of RecA, RecB or RecC. the bacteriophage comprises a nucleic acid encoding at least 8. An engineered bacteriophage comprising a nucleic acid two different proteins which increase the susceptibility of a operatively linked to a promoter, wherein the nucleic acid bacteria cell to an antimicrobial agent. encodes at least one repressor of a SOS response gene and/or 28. The bacteriophage of any of paragraphs 14 to 27, wherein bacterial defense gene. the proteins are csrA and ompl or variants or fragments 9. The bacteriophage of any of paragraphs 8, wherein the thereof. repressor of a SOS response gene is lexA. 29. The bacteriophage of any of paragraphs 1 to 28, wherein 10. The bacteriophage of any of paragraphs 8 or 9, wherein the bacteriophage is a lysogenic bacteriophage. the repressor of a defense gene is SoxR. 30. The bacteriophage of any of paragraphs 1 to 29, wherein 11. The bacteriophage of any of paragraphs 8 to 10, wherein the lysogenic bacteriophage is a M13 bacteriophage. the repressor is selected from the group consisting of marr, 31. The bacteriophage of any of paragraphs 1 to 29, wherein arcR, fur, crp, iccdA or variants or fragments thereof. the bacteriophage is a lytic bacteriophage. 12. The bacteriophage any of paragraphs 8 to 11, wherein the 32. The bacteriophage of any of paragraphs 1 to 29, or 31 bacteriophage comprises a nucleic acid encoding at least two wherein the lytic bacteriophage is a T7 bacteriophage. different repressors of at least one SOS response gene. 33. A method to inhibit or eliminate a bacterial infection 13. The bacteriophage any of paragraphs 8 to 12, wherein the comprising administering to a surface infected with bacteria; bacteriophage comprises a nucleic acid encoding at least two (a) a bacteriophage comprising a nucleic acid operatively different repressors of at least one bacterial defense gene. linked to a bacteriophage promoter, wherein the nucleic acid 14. An engineered bacteriophage comprising a nucleic acid encodes at least one agent that inhibits an antibiotic resistance operatively linked to a promoter, wherein the nucleic acid gene and/or a cell Survival repair gene, and (b) at least one encodes at least one agent which increases the Susceptibility antimicrobial agent. of a bacteria cell to an antimicrobial agent. 34. A method to inhibit or eliminate a bacterial infection 15. The bacteriophage of paragraph 14, wherein the agent comprising administering to a surface infected with bacteria; which increases the susceptibility of a bacteria cell to an (a) a bacteriophage comprising a nucleic acid operatively antimicrobial agent increases the efficacy of the antimicrobial linked to a bacteriophage promoter, wherein the nucleic acid effect of the antimicrobial agent by at least 10%. encodes at least one repressor of a SOS response gene or a 16. The bacteriophage any of paragraphs 14 or 15, wherein bacterial-defense gene, and (b) at least one antimicrobial the agent which increases the susceptibility of a bacteria cell agent. to an antimicrobial agent increases the entry of an antimicro 35. A method to inhibit or eliminate a bacterial infection bial agent to a bacterial cell. comprising administering to a surface infected with bacteria; 17. The bacteriophage of any of paragraphs 14 to 16, wherein (a) a bacteriophage comprising nucleic acid operatively the agent which increases the entry of an antimicrobial agent linked to a bacteriophage promoter, wherein the nucleic acid to a bacterial cell is a porin. a encodes at least one agent which increases the Susceptibility 18. The bacteriophage of any of paragraphs 14 to 17, wherein ofa bacteria cell to an antimicrobial agent, and (b) at least one the porin is ompl or variants or fragments thereof. antimicrobial agent. US 2010/0322903A1 Dec. 23, 2010

36. The method of paragraph 33, wherein the bacteriophage is 56. A composition comprising a bacteriophage comprising a a bacteriophage according to any of paragraphs 1 to 7 or nucleic acid operatively linked to a promoter, wherein the 29-32. nucleic acid encodes at least one repressor of a SOS response 37. The method of paragraph34, wherein the bacteriophage is gene or a antimicrobial defense gene and at least one antimi a bacteriophage according to any of paragraphs 8 to 13 or crobial agent. 29-32. 57. A composition comprising a bacteriophage comprising a 38. The method of paragraph 35, wherein the bacteriophage is nucleic acid operatively linked to a promoter, wherein the a bacteriophage according to any of paragraphs 14 to 32. nucleic acid encodes at least one protein which increases the 39. The method of any of paragraphs 33 to 38, wherein the Susceptibility of a bacteria cell to an antimicrobial agent and administration of the bacteriophage and the antimicrobial at least one antimicrobial agent. agent occurs simultaneously. 58. The composition of any of paragraphs 55 to 57, wherein 40. The method of any of paragraphs 33 to 38, wherein the the antimicrobial agent is a quinolone antimicrobial agent, or administration of the bacteriophage occurs prior to the aminoglycoside antimicrobial agent or B-lactam antimicro administration of the antimicrobial agent. bial agent. 41. The method of any of paragraphs 33 to 38, wherein the 59. The composition of any of paragraphs 55 or 58, wherein administration of the antimicrobial agent occurs prior to the the bacteriophage is according to any paragraphs 1-7 or administration of the bacteriophage. 29-32. 42. The method of any of paragraphs of any of paragraphs 33 60. The composition of paragraphs 56 or 58, wherein the to 38, wherein the antimicrobial agent is a quinolone antimi bacteriophage is according to any paragraphs 8 to 13 or crobial agent. 29-32. 43. The method of paragraph 33 to 42, wherein the antimi 61. The composition of paragraphs 57 or 58, wherein the crobial agent is selected from a group consisting of bacteriophage is according to any paragraphs 14 to 32. ciproflaxacin, levofloxacin, and ofloxacin, gatifloxacin, nor 62. A kit comprising a bacteriophage comprising the nucleic floxacin, lomefloxacin, trovafloxacin, moxifloxacin, spar acid operatively linked to a promoter, wherein the nucleic floxacin, gemifloxacin, paZufloxacin or variants or analogues acid encodes at least one agent that inhibits an antibiotic thereof. resistance gene and/or a cell Survival repair gene. 44. The method of any of paragraphs 33 to 38, wherein the 63. A kit comprising a bacteriophage comprising the nucleic antimicrobial agent is ofloxacin or variants or analogues acid operatively linked to a promoter, wherein the nucleic thereof. acid encodes at least one repressor of a SOS response or an 45. The method of any of paragraphs 33 to 38, wherein the antimicrobial defense gene. antimicrobial agent is an aminoglycoside antimicrobial 64. A kit comprising a bacteriophage comprising the nucleic agent. acid operatively linked to a promoter, wherein the nucleic 46. The method of paragraph 45, wherein the antimicrobial acid encodes at least one protein which increases the Suscep agent is selected from a group consisting of amikacin, genta tibility of a bacteria cell to an antimicrobial agent and at least mycin, tobramycin, metromycin, Streptomycin, kanamycin, one antimicrobial agent. paromomycin, neomycin or variants or analogues thereof. 65. The use of a bacteriophage according to any of paragraphs 47. The method of any of paragraphs 33 to 38, wherein the 1 to 23 in combination with an antimicrobial agent to reduce antimicrobial agent is gentamicin or variants or analogues the number of bacteria as compared to use of the antimicro thereof. bial agent alone. 48. 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 an B-lactam antibiotic antimicrobial bacteria is in a biofilm. agent. 67. A combination of at least two bacteriophages of any of 49. The method of any of paragraphs 33 to 38, wherein the paragraphs 1 to 23 with at least one antimicrobial agent. antimicrobial agent is selected from a group consisting of 68. The combination of paragraph 67, wherein the antimicro penicillin, amplicillin, penicillin derivatives, cephalosporins, bial agent is a quinolone antimicrobial agent. monobactams, carbapenems, B-lactamase inhibitors or vari 69. The combination of paragraph 67, wherein the antimicro ants or analogues thereof. bial agent is selected from a group consisting of ciproflaxacin, 50. The method of any of paragraphs 33 to 38, wherein the levofloxacin, and ofloxacin, gatifloxacin, norfloxacin, lom antimicrobial agent is amplicillin or variants or analogues efloxacin, trovafloxacin, moxifloxacin, Sparfloxacin, gemi thereof. floxacin, paZufloxacin or variants or analogues thereof. 51. The method of any of paragraphs 33 to 38, wherein the 70. The combination of paragraph 67, wherein the antimicro bacteria is present in a Subject. bial agent is ofloxacin or variants or analogues thereof. 52. The method of any of paragraphs 33 to 51, wherein the 71. The combination of paragraph 67, wherein the antimicro Subject is a mammal. bial agent is an aminoglycoside antimicrobial agent. 53. The method of any of paragraph 33 to 52, wherein the 72. The combination of paragraph 67, wherein the antimicro mammal is a human. bial agent is selected from a group consisting of amikacin, 54. The method of any of paragraphs 33 to 53, wherein the gentamycin, tobramycin, netromycin, streptomycin, kana bacteria is in a biofilm. mycin, paromomycin, neomycin or variants or analogues 55. A composition comprising a bacteriophage comprising a thereof. nucleic acid operatively linked to a promoter, wherein the 73. The combination of paragraph 67, wherein the antimicro nucleic acid encodes at least one agent that inhibits an anti bial agent is gentamicin or variants or analogues thereof. biotic resistance gene and/or a cell Survival repair gene and at 74. The combination of paragraph 67, wherein the antimicro least one antimicrobial agent. bial agent is an B-lactam antibiotic antimicrobial agent. US 2010/0322903A1 Dec. 23, 2010 39

75. The combination of paragraph 67, wherein the antimicro Inc. (Ipswich, Mass.). E. coli XL-10 cells used for cloning, bial agent is selected from a group consisting of penicillin, amplifying phage, and plating phage were obtained from ampicillin, penicillin derivatives, cephalosporins, monobac Stratagene (La Jolla, Calif.). tams, carbapenems, B-lactamase inhibitors or variants orana 0285 T4 DNA ligase and all restriction enzymes were logues thereof. purchased from New England Biolabs, Inc. (Ipswich, Mass.). 76. The combination of paragraph 67, wherein the antimicro PCR reactions were carried out using PCR SUPERMIX bial agent is amplicillin or variants or analogues thereof. HIGH FIDELITY from INVITROGEN (Carlsbad, Calif.) or 77. The combination of paragraph 67, wherein the composi PHUSION HIGH FIDELITY from New England Biolabs, tion comprises a combination of any of the antimicrobial Inc. (Ipswich, Mass.). Purification of PCR reactions and agents according to paragraphs 68-76. restriction digests was carried out with the QIAQUICKGEL 78. Use of a bacteriophage of any of claims 1 to 32 with at Extraction or PCR Purification kits (QIAGEN, Valencia, least one antimicrobial agent. Calif.). Plasmid DNA was isolated using the QIAPREPSPIN 79. Use of a combination of at least two of any the bacterioph Miniprep kit (QIAGEN, Valencia, Calif.). All other chemicals ages of claims 1 to 32 with at least one antimicrobial agent. and materials were purchased from Fisher Scientific, Inc. 80. The use of a bacteriophage of claim 78 or 79 or any to (Hampton, N.H.). claims 1 to 32 to inhibit or eliminate a bacterial infection. 0286 Engineering M13mp 18 bacteriophage to target 81. The use of a bacteriophage of claim 78 or 79, wherein the genetic networks. To construct engineered phage, leXA3, bacteria is present in a Subject. SOXR, cSrA, and ompl genes were first placed under the 82. The use of a bacteriophage of claim 81, wherein the control of the PtetO promoter in the pZE11G vector''. Subject is a mammal. Using PCR with primers 5' ttatca gg.tacc atgAAAGCGT 83. The use of a bacteriophage of claim 82, wherein the TAACGGCC 3' (SEQID NO: 18) and 5' atacataagctt TTA mammal is a human. CAGCCA GTCGCCG 3' (SEQ ID NO: 19), lexA3 was cloned between the KpnI and HindIII sites of pZE11G to form 84. The use of a bacteriophage of claim 78 or 79, wherein the pZE11-lex.A3. Since SoxR has an internal KpnI site, the bacteria is in a biofilm. inventors built a synthetic RBS by sequential PCR using 5' 85. Use of a composition of any of claims 55 to 57 to inhibit agaggagaaaggtaccatgGAAAAGAAATTACCCCG 3' (SEQ or eliminate a bacterial infection. ID NO: 20) and 5'atacataagctt TTAGTTTTGTTCATCTTC 86. The use of the composition of claim 85, wherein the CAG3' (SEQID NO: 21) followed by 5' agtaga gaattic attaaa bacteria is present in a subject. gaggagaaaggtaccatg3' (SEQID NO: 22) and 5' atacataagctt 87. The use of the composition of claim 86, wherein the TTAGTTTTGTTCATCTTCCAG3' (SEQID NO. 23). The Subject is a mammal. resulting EcoRI-RBS-soxR-HindIII DNA was ligated to an 88. The use of the composition of claim 87, wherein the XhoI-P, tetO-EcoRI fragment excised from pZE11G and the mammal is a human. entire DNA fragment was ligated into pZE11G between XhoI 89. The use of the composition of claim 85, wherein the and HindIII to form pZE11-soxR. Primers for csrA for bacteria is in a biofilm. cloning into pZE11G in between KpnI and HindIII to form 0281. The following Examples are provided to illustrate pZE1 1-cSrA were 5' agaggagaaa gg.tacc atgCTGATTC the present invention, and should not be construed as limiting TGACTCGT 3' (SEQ ID NO: 24) and 5' atacat aagctt thereof. TTAGTAACTGGACTG CTGG 3' (SEQID NO:25); and for ompl to form pZE 11-ompF, 5' agaggagaaaggtacc atgAT EXAMPLES GAAG C GCAATATTCT3' (SEQ ID NO: 26) and 5' atacat aagctt TTAGAACTG GTAAACGATA CC 3' (SEQ ID NO: 0282. The examples presented herein relate to the methods 27). To express csrA and ompF simultaneously under the and compositions comprising inhibitor-engineered bacte control of P, tetO, we PCR amplified RBS-ompF DNA from riophages, repressor-engineered bacteriophages or Suscepti p7E11-ompF using 5'ccagtic aagctt attaaagaggagaaaggtacc bility-agent engineered bacteriophages and antimicrobial 3' (SEQID NO: 28) and 5' atacat GGATCCTTAGAACTG agents. Throughout this application, various publications are GTAAACGATA CC 3' (SEQ ID NO: 29) and cloned the referenced. The disclosures of all of the publications and product in between HindIII and BamHI in pZE1 1-csrA to those references cited within those publications in their form p7E1 1-csrA-ompF. The resulting plasmids were trans entireties are hereby incorporated by reference into this appli formed into E. coli XL-10 cells. cation in order to more fully describe the state of the art to 0287 All PtetO-gene constructs followed by terminator which this invention pertains. The following examples are not T1 of the rrnB operon and preceded by a stop codon were intended to limit the scope of the claims to the invention, but PCR amplified from the respective pZE 11 plasmids with are rather intended to be exemplary of certain embodiments. primers 5' aataca GAGCTC cTAA tecctatcagtgatagagattg 3' Any variations in the exemplified methods which occur to the (SEQID NO:30) and 5' taatct CGATCG totaggg.cgg.cggat 3" skilled artisan are intended to fall within the scope of the (SEQ ID NO: 31) and cloned into the Sad and Pvul sites of present invention. M13mp18 (FIG. 5)'''. Resulting phage genomes were 0283 Methods transformed into XL-10 cells, mixed with 200 uL overnight 0284. Bacterial strains, bacteriophage, and chemicals. E. XL-10 cells in 3 mL top agar, 1 mM IPTG, and 40 uL of 20 coli K-12 EMG2 cells, which lack 0 antigens, were obtained mg/mL X-gal, and poured onto LB agar-chloramphenicol from the Yale Coli Genetic Stock Center (CGSC #4401). E. (30 ug/mL) plates for plaque formation and blue-white coli RFS289 cells, which contain a gyra 111 mutation ren screening. After overnight incubation of plates at 3TC, white dering them resistant to quinolones, were obtained from the plaques were scraped and placed into 1:10 dilutions of over Yale Coli Genetic Stock Center (CGSC #5742). M13mp 18 night XL-10 cells and grown for 5 hours. Replicative form bacteriophage was purchased from New England Biolabs, (RF) M13mp18 DNA was collected by DNA minipreps of the US 2010/0322903A1 Dec. 23, 2010 40 bacterial cultures. All insertions into M13mp 18 were verified protocol as the ofloxacin killing assay except that 10 PFU/ by PCR and restriction digests of RF DNA. Infective bacte mL phage were added with varying concentrations of ofloxa riophage solutions were obtained by centrifuging infected cin and viable cell counts were obtained after 6 hours of cultures for 5 minutes at 16,100xg and collecting Superna treatment. tants followed by filtration through Nalgene #190-2520 0.2 0293 Persister killing assay. The inventors performed a um filters (Nalge Nunc International, Rochester, N.Y.). persister killing assay to determine whether engineered phage 0288 Determination of plaque forming units. To obtain could help to kill persister cells in a population which sur plaque forming units, we added serial dilutions of bacterioph vived initial drug treatment without bacteriophage (FIGS. 11 age performed in 1xRBS to 200 uL of overnight XL-10 cells and 16). The inventors first grew 1:500 dilutions of overnight in 3 mL top agar, 1 mMIPTG, and 40 uL of 20 mg/mL X-gal, EMG2 for 3 hours and 30 minutes at 3TC and 300 rpm and poured the mixture onto LB agar--chloramphenicol (30 followed by treatment with 200 ng/mL ofloxacin for 3 hours ug/mL) plates. After overnight incubation at 3TC. plaques to create a population of Surviving bacteria. Then, the inven were counted. tors added either no phage, 10 PFU/mL control {p, or 0289 Determination of colony forming units. To obtain 10 PFU/mL engineered p, phage. After 3 hours of addi CFU counts, 150 uL of relevant cultures were collected, tional treatment, the inventors collected the samples and washed with 1x phosphate-buffered saline (PBS), recol assayed for viable cell counts as described above. lected, and resuspended in 150 uL of 1xPBS. Serial dilutions 0294 Biofilm killing assay. Biofilms were grown using E. were performed with 1xPBS and sampled on LBagar plates. coli EMG2 cells according to a previously-reported protocol LBagar plates were incubated at 3TC overnight before count (Lu and Collins, 2007). Briefly, lids containing plastic pegs 1ng. (MBEC Physiology and Genetics Assay, Edmonton, Calif.) 0290 Flow cytometer assay of SOS induction. To monitor were placed in 96-well plates containing overnight cells that M13mp 18-lex A3's (cp) suppression of the SOS response were diluted 1:200 in 150 uL LB. Plates were then inserted (FIG. 10), the inventors used a plasmid containing an SOS into plastic bags to minimize evaporation and inserted in a response promoter driving gfp expression in EMG2 cells Minitron shaker (Infors HT, Bottmingen, Switzerland). After (PlexO-gfp). After growing 1:500 dilutions of the over 24 hours of growth at 35° C. and 150 rpm, lids were moved night cells for 2 hours and 15 minutes at 3TC and 300 rpm into new 96-well plates with 200 uLLB with or without 10 (model G25 incubator shaker, New Brunswick Scientific), the PFU/mL of bacteriophage. After 12 hours of treatment at 35° inventors applied ofloxacin and bacteriophage and treated for C. and 150 rpm, lids were removed, washed three times in 200 6 hours at 3TC and 300 rpm. Cells were then analyzed for uL of 1xPBS, inserted into Nunc #262162 microtiter plates GFP fluorescence using a Becton Dickinson (Franklin Lakes, with 150 uL 1xPBS, and sonicated in an Ultrasonics 5510 N.J.) FACS caliber flow cytometer with a 488-nm argon laser sonic water bath (Branson, Danbury, Conn.) at 40 kHz for 30 and a 515-545 nm emission filter (FL1) at low flow rate. The minutes. Serial dilutions, using the resulting 150 uL 1xPBS, following photo-multiplier tube (PMT) settings were used for were performed on LB plates and viable cell counts were analysis: E00 (FSC), 275 (SSC), and 700 (FL1). Becton Dick determined. Mean killing (A logo (CFU/mL)) was calculated inson CALIBRITE Beads were used for instrument calibra by Subtracting mean logo (CFU/mL) after 24hours of growth tion. 200,000 cells were collected for each sample and pro from mean logo (CFU/mL) after 12 hours of treatment (FIG. cessed with MATLAB (Mathworks, Natick, Mass.). 17 and FIG. 18). 0291. Ofloxacin killing assay. To determine the adjuvant 0295) Antibiotic resistance assay. To analyze the effect of effect of engineered phage (FIG. 1B, FIG. 3A and FIG. 3D), subinhibitory concentrations of ofloxacin on the development the inventors grew 1:500 dilutions of overnight EMG2 cells of antibiotic-resistant mutants, the inventors grew 1:10 dilu for 3 hours and 30 minutes at 3TC and 300 rpm to late tions of overnight EMG2 in LB media containing either no exponential phase and determined initial CFUs. Then, the ofloxacin (FIG. 4) or 30 ng/mL ofloxacin (FIG. 7). After 12 inventors added 60 ng/mL ofloxacin by itself or in combina hours of growth at 3TC and 300 rpm, the inventors split the tion with 10 PFU/mL bacteriophage (unmodified (p, or cells grown in no ofloxacin into 100 uL aliquots with no engineered (PLevas (Pso R: (Pas- (Pomp F: O (Pcs-ompF phage) and ofloxacin in 60 wells in 96-well plate format (Costar 3370; treated at 37° C. and 300 rpm. At indicated time points, the Fisher Scientific, Pittsburgh, Pa.). The inventors also split the inventors determined CFUs as described above. Mean killing cells grown in 30 ng/mL ofloxacin into 100 uL aliquots in 60 (A logo (CFU/mL)) was determined by subtracting mean wells with either no phage and 30 ng/mL ofloxacin (FIG.7B). initial logo (CFU/mL) from mean logo (CFU/mL) after (p., phage and 30 ng/mL ofloxacin (FIG. 7C), and ps treatment in order to compare data from different experi and 30 ng/mL ofloxacin (FIG. 7D) in 96-well plate format. ments. This protocol was replicated with E. coli RFS289 to The inventors placed the 96-well plates in 37° C. and 300 rpm determine the ofloxacin-enhancing effect of engineered with plastic bags to minimize evaporation. After 12 hours of (p phage against antibiotic-resistant bacteria (FIG. 2). In treatment, the inventors plated cultures from each well on LB addition, viable cell counts were obtained for ofloxacin-free agar-i-100 ng/mL ofloxacinto select for mutants that devel EMG2 cultures, ofloxacin-free EMG2 cultures with (p. oped resistance against ofloxacin. To compare results, the phage, and ofloxacin-free EMG2 cultures with engineered inventors plotted histograms of the number of resistant bac (Peas phage. teria found in each well in FIGS. 4 and 8. 0292 Dose response assays. The initial phage inoculation 0296 Gentamicin and amplicillin killing assays. To deter dose response experiments (FIG. 1c and FIG. 15) were mine the antibiotic enhancing or adjuvant effect of engi handled using the same protocolas the ofloxacinkilling assay neered bacteriophage for gentamicin and amplicillin, the except that 60 ng/mL ofloxacin was added with varying con inventors used the same protocol as the ofloxacin killing centrations of phage. Cultures were treated for 6 hours before assay except that the inventors used 10 PFU/mL initial phage obtaining viable cell counts. The ofloxacin dose response inoculations. 5 g/mL gentamicin and 5 g/mL amplicillin experiments (FIG. 1C) were also obtained using the same were used in FIGS. 1D, 1E, 8A and 8B. US 2010/0322903A1 Dec. 23, 2010

0297 Statistical analysis. All CFU data were logo-trans units (CFUs) during treatment with no phage or 10 plaque formed prior to analysis. For all data points in all experiments, forming units/mL (PFU/mL) of phage and with no ofloxacin n=3 samples were collected except where noted. Error bars in or 60 ng/mL ofloxacin (FIG. 1B). Bacteria exposed only to figures indicate standard error of the mean. ofloxacin were reduced by about 1.7 logo (CFU/mL) after 6 hours of treatment, reflecting the presence of persisters not Example 1 killed by the drug (FIG. 1B). By 6 hours, cp improved the 0298. The inventors have engineered synthetic bacte bactericidal effect of ofloxacin by 2.7 orders of magnitude riophage to target genetic networks in order to potentiate compared to unmodified phage (p (~0.99.8% additional bacterial killing in combination therapy with antibiotics. The killing) and by over 4.5 orders of magnitude compared to no inventors specifically targeted genetic networks in E. coli phage (~99.998% additional killing) (FIG. 1B). Unmodified which are not directly attacked by antibiotics to avoid impos phage enhanced ofloxacin's bactericidal effect, which is con ing additional evolutionary pressures for antibiotic resis sistent with previous observations that unmodified filamen tance. Instead, the inventors chose proteins that are respon tous phage augment antibiotic efficacy against Pseudomonas sible for repairing cellular damage caused by antibiotics, aeruginosa (Hagens et al., (2006) Microb Drug Resist 12, those that control regulatory networks, or those that modulate 164-168). Other researchers have noted that M13-infected E. sensitivity to antibiotics Unlike conventional antibiotics that coli exhibited impaired host stress responses to conditions act by disrupting protein activity, the inventors designed an such as acid stress (Karlsson et al., (2005) Can J Microbiol engineered phage to overexpress target genes. Such as repres 51, 29-35). While wishing not to be bound by theory, the mechanism by which unmodified filamentous phage can aug sors and act as effective antibiotic adjuvants. ment antibiotic efficacy is not well characterized but can 0299 Bactericidal antibiotics cause hydroxyl radical for involve membrane disruption or impaired stress responses. mation which leads to DNA, protein, and lipid damage and No significant bacterial regrowth was apparent with combi ultimately, cell death. DNA damage induces the SOS nation phage and antibiotic treatment up to 12 hours (FIG. response (Miller et al., (2004) Science 305, 1629-1631; 1B) (Hagens et al., (2003) Lett. Appl. Microbiol. 37,318-23: Lewin et al., (1989).J. Med. Microbiol. 29, 139-144.), which Hagens et al., (2004) Antimicrob. Agents Chemother. 48, results in DNA repair (FIG. 1A). It has been shown that 3817-22: Summers W C (2001) Annu. Rev. Microbiol. 55, bacterial killing by bactericidal antibiotics can be enhanced 437-451). The inventors confirmed that both (p, and by knocking out recA and disabling the SOS response (Ko (preplicated significantly during treatment (data not hanski et al., (2007) Cell 130). Here, the inventors used an alternative approach and engineered M13mp8 phage to over shown). express lexA3, a repressor of the SOS response (Little et al., Example 2 (1979) Proc Natl AcadSci USA 76,6147-51). Overexpression oflex A to suppress the SOS system has been demonstrated to 0302) To test whether p can act as an antibiotic adju inhibit the emergence of antibiotic resistance (Cirz et al., vant in different situations, the inventors assayed for bacterial (2005) in PLoS Biol, p. e17624). The inventors used killing with varying initial phage inoculation doses (FIG. 15) M13mp 18, a modified version of M13 phage, as the substrate and varying doses of ofloxacin (FIG. 1C) after 6 hours of since it is a non-lytic filamentous phage and can accommo treatment, respectively. (ps enhanced ofloxacin's bacteri date DNA insertions into its genome (Figure S1) (Yanisch cidal activity over a wide range of multiplicity-of infections Perronet al., (1985) Gene 33, 103-119). (MOIs), from 1:1000 to 1:1 (FIG. 15). (p.'s ability to 0300. To repress the SOS response, the inventors placed increase killing by ofloxacin at a low MOI reflects rapid the lex A3 gene under the control of the synthetic PLtetO replication and infection by M13 phage. For ofloxacin con promoter followed by a synthetic ribosome-binding sequence centrations of 30 ng/mL and higher, p resulted in much (RBS) (Kohanski et al., (2007) Cell 130,797-810; Little et al., greater killing compared with no phage or unmodified phage (1979) Proc Natl AcadSci USA 76, 6147-51; Walker GC (p (FIG.1C). Thus, the inventors have demonstrated that (1984) Microbiol. Rev. 48, 60-93; Lutz et al., (1997) Nucleic (p. is a strong adjuvant for ofloxacin at doses below and Acids Res 25, 1203-1210.); The inventors named this phage above the minimum inhibitory concentration (60 ng/mL, data "p' (FIG. 1A and Figure S1B) and the unmodified not shown). M13mp 18 phage (p. PLtetO, which is an inducible pro 0303. The inventors next determined whether the engi moter in the presence of the TetR repressor, is constitutively neered phage could increase killing by classes of antibiotics on in EMG2 cells, which lack TetR. PLtetO was used for other than quinolones. The inventors tested (ps antibi convenience in proof-of-concept experiments as described otic-enhancing effect forgentamicin, an aminoglycoside, and herein and would not necessarily be the promoter of choice in ampicillin, a B-lactam antibiotic. As demonstrated herein, real-world situations. Accordingly, one of ordinary skill in the (ps increased gentamicin's bactericidal action by over 2.5 art can readily substitute the PLtetOpromoter with a different and 3 orders of magnitude compared with p, and no inducible or constitutively active or tissue specific promoter phage, respectively (FIG. 1D). (ps also improved amplicil of their choice. The inventors confirmed that (p. Sup lin's bactericidal effect by over 2 and 5.5 orders of magnitude pressed the SOS response induced by ofloxacintreatment by compared with p, and no phage, respectively (FIG. 1E). monitoring GFP fluorescence in E. coli K-12 EMG2 cells For both gentamicin and amplicillin, p’s strong antibiotic carrying a plasmid with an SOS-responsive promoter driving enhancing effect was noticeable after 1 hour of treatment gfp expression (Figure S2) (Kohanski et al., (2007) Cell 130, (FIGS. 1D and 1E). These results are consistent with previous 797-810). observations that Areca mutants exhibit increased suscepti 0301 To test (ps antibiotic-enhancing effect, the bility to quinolones, aminoglycosides, and B-lactams (Ko inventors obtained time courses for killing of E. coli EMG2 hanski et al., (2007) Cell 130,797-810), and demonstrate that bacteria with phage and/or ofloxacintreatment. The inventors engineered phages, such as (pes, can act as general adju calculated viable cell counts by counting colony-forming vants for the three major classes of bactericidal drugs. The US 2010/0322903A1 Dec. 23, 2010 42 inventors also found that engineered phage (ps is capable phages are effective at rescuing infected mice from death, and of reducing the number of persister cells in populations demonstrates the feasibility of various embodiments of the already exposed to antibiotics as well as enhancing antibiotic invention for clinical use. efficacy against bacteria living in biofilms. For example, (p added to a population previously treated only with Example 5 ofloxacin increased the killing of bacteria that survived the 0307 Reducing the Development of Antibiotic Resis initial treatment by approximately 1 and 1.5 orders of mag tance. Exposure to Subinhibitory concentrations of antibiotics nitude compared with (p, and no phage, respectively can lead to initial mutations which confer low-level antibiotic (FIG.16). In addition, simultaneous application of p and resistance and eventually more mutations that yield high ofloxacin improved killing of biofilm cells by about 1.5 and 2 level resistance (Martinez et al., (2000) Antimicrob. Agents orders of magnitude compared with (p, plus ofloxacin and Chemother: 44, 1771-77). The inventors assessed if the engi no phage plus ofloxacin, respectively (FIG. 17). neered phage, as antibiotic adjuvants, could reduce the num 0304 Since the inventors previous experiments all ber of antibiotic-resistant mutants that result from a bacterial involved simultaneous application of bacteriophage and population exposed to antimicrobial drugs. To test this, the drug, the inventors tested whether later addition of engineered inventors grew E. coli EMG2 in media with either no ofloxa (ps to a previously drug-treated population would also cin for 24 hours, 30 ng/mL ofloxacin for 24 hours, 30 ng/mL enhance killing Late exponential-phase cells were first ofloxacin for 12 hours followed by p, plus ofloxacin exposed to 3 hours of treatment by ofloxacinto generate a treatment for 12 hours, or 30 ng/mL ofloxacin for 12 hours population of surviving cells and followed by either no phage, followed by p, plus ofloxacintreatment for 12 hours (FIG. 10 PFU/mL (p, or 10 PFU/mL engineered (ps phage. 4). Then, the inventors counted the number of mutants resis tant to 100 ng/mL ofloxacin for each of the 60 samples under After 3 hours of additional treatment, (ps increased killing each growth condition. Growth in the absence of ofloxacin by 0.94 logo (CFU/mL) compared with (p, and by over yielded very few resistant cells (median=1) (FIG. 4). How 1.3 logo (CFU/mL) compared with no phage (FIG. 11). ever, growth with subinhibitory levels of ofloxacin produced These results indicate that engineered (p bacteriophage a high number of antibiotic-resistant bacteria (median=1592) increases the killing of bacteria which survive initial antibi (FIG. 4). Treatment with unmodified phage (p, decreased otic treatment and reduce the number of persister cells in a the number of resistant cells (median=43.5); however, all given population. samples contained >1 resistant CFU and over half of the samples had >20 resistant CFUs (FIG. 4). In contrast, p. Example 3 treatment dramatically suppressed the level of antibiotic-re sistant cells (median 2.5), resulting in a majority of samples 0305 Enhancing Killing of Antibiotic-Resistant Bacteria. with either no resistant CFUs or <20 resistant CFUs (FIG. 4). In addition to killing wild-type bacteria with increased effi cacy, the inventors also demonstrate that the engineered Example 6 phage can enhance killing of bacteria that have already (0308 Flexible Targeting of Other Gene Networks. The acquired antibiotic resistance. The inventors applied ps inventors next demonstrated that the phage platform can be with ofloxacin against E. coli RFS289, which carries a muta used to target many different gene networks to produce effec tion (gyrA111) that renders it resistant to quinolone antibiot tive antibiotic adjuvants. To demonstrate this, the inventors ics (Dwyer et al., (2007) Mol Syst Biol 3,917; Schleif R engineered phage to express proteins that regulate non-SOS (1972) Proc Natl AcadSci USA 69,3479-84). (p. increased gene networks (e.g., SOXR and CSrA) or modulate sensitivity the bactericidal action of ofloxacinby over 2 and 3.5 orders of to antibiotics (e.g., Ompl) (FIG. 5 and FIG.9F) (Lutz et al., magnitude compared with (p., and no phage, respectively (1997) Nucleic Acids Res 25, 1203-10). For example, the (FIG. 2). These results demonstrate that antibiotic-enhancing SOXR-SOXS regulon controls a coordinated cellular response phage, such as (ps can be used to combat antibiotic-resis to superoxide (Hidalgo et al., (1997) Cell 88, 121-129). SoxR tant bacteria and therefore can have the potential to bring contains a 12Fe-251 cluster that must be oxidized for it to defunct antibiotics back into clinical use. stimulate SoxS production, which then controls the transcrip tion of downstream genes that respond to oxidative stress (Hidalgo et al., (1997) Cell 88, 121-129). As quinolones Example 4 generate Superoxide-based oxidative attack (Dwyer et al., (2007) Mol Syst Biol 3,91: Kohanski et al., (2007) Cell 130, 0306 Increasing Survival of Mice Infected with Bacteria. 797-810), the inventors engineered phage to overexpress To determine the clinical relevance of antibiotic-enhancing wild-type SoxR (cps) to affect this response and improve phage in vivo, the inventors applied the engineered phage ofloxacin's bactericidal activity (FIG. 5A). As shown in FIG. (ps with ofloxacinto prevent death in mice infected with 5B, p enhanced killing by ofloxacin compared with bacteria. Mice were injected with E. coli EMG2 intraperito unmodified phage (p, and no phage (FIG. 5B). The inven neally 1 hour prior to receiving different intravenous treat tors discovered that the overexpression of SoxR may provide ments (FIG. 3A). Eighty percent of mice that received ps additional iron-sulfur clusters that could be destabilized to with ofloxacin survived, compared with 50% and 20% for increase sensitivity to bactericidal antibiotics (Dwyer et al., mice that received p, plus ofloxacin or ofloxacin alone, (2007) Mol Syst Biol 3,91: Kohanski et al., (2007) Cell 130, respectively (FIG.3B). The inventors have demonstrated that 797-810). Alternatively, since SoxR is usually kept at rela the engineered phage (ps with ofloxacin prevents death in tively levels in vivo which are unchanged by oxidative stress vivo of mice with a severe bacterial infection, thus demon (Hidalgo et al., (1998) EMBO.J. 17, 2629-2636), and the strating that the in Vivo efficacy of the antibiotic enhancing overexpression of large amounts of SoxR may interfere with US 2010/0322903A1 Dec. 23, 2010 signal transduction in response to oxidative stress by titrating logo (CFU/mL) compared with no phage and by about 1.9 intracellular iron or oxidizing species or by competing with logo (CFU/mL) compared with unmodified (p after 6 oxidized SoxR for binding to the SoxS promoter (Hidalgo et hours of treatment. al., (1998) EMBO.J. 17, 2629-36: Meng Met al., (1999) J 0312 CSrA is a global regulator of glycogen synthesis and Bacteriol 181, 4639-4643; Gaudu et al., (1996) Proc Natl catabolism, gluconeogenesis, glycolysis, and biofilm forma AcadSci USA 93, 10094-98). tion. Since biofilm formation has been linked to antibiotic 0309 CsrA is a global regulator of glycogen synthesis and resistance, the inventors assessed if overexpressing cSrA catabolism, gluconeogenesis, and glycolysis, and has been might increase susceptibility to antibiotic treatment. shown to represses biofilm formation (Jackson D Wet al., OmpF is a porin which is used by quinolones to enterbacteria (2002).J. Bacteriol. 184,290-301). As biofilm formation has and therefore, the inventors determined that overproducing been linked to antibiotic resistance, the inventors assessed if OmpF would increase killing by ofloxacin". The inventors cSrA-expressing phage (cps) would increase Susceptibility discovered that cSrA-expressing M13mp 18 (cps) and to antibiotic treatment (Stewart et al., (2001) Lancet 358, ompf-expressing M13mp 18 (cp) both increased ofloxa 135-138). In addition, since Ompl is a porin used by quino cin's bactericidal effect by about 2.7 logo (CFU/mL) com lones to enterbacteria (Hirai et al., (1986) Antimicrob. Agents pared with no phage and 0.8 logo (CFU/mL) compared with Chemother: 29, 535-538), the inventors also assessed if unmodified (p, after 6 hours of treatment (FIG. 6B). ompF-expressing phage (p) would increase killing by 0313. In order to enhance the effectiveness of engineered ofloxacin (FIG. 5C). After 6 hours, both (ps and pe phage with cSrA or ompl alone as antibiotic adjuvants, the increased ofloxacin's bactericidal effect by approximately 1 inventors designed an M13mp 18 phage to express cSrA and and 3 orders of magnitude compared with p, and no ompf simultaneously (p.) (FIG.9F). The combina phage, respectively (FIG. 5D). tion phage was constructed by modifying (ps to carry an RBS and ompF immediately downstream of csrA. (p. Example 7 one F improved killing by ofloxacin by over 0.7 logo (CFU/ mL) compared with p and prafter 6 hours of treatment 0310 Systems biology analysis often results in the iden (FIG. 6B). The dual-target (p-ompF phage performed tification of multiple antibacterial targets which are not easily comparably with (ps at various initial phage inoculations addressed by traditional drug compounds. In contrast, engi with 60 ng/mL ofloxacin (FIG. 6C) and at various concentra neered phage are well-suited for incorporating multiple tar tions of ofloxacin with 10 PFU/mL phage (FIG. 6D). Both gets into a single antibiotic adjuvant. To demonstrate this phages were more effective than no phage or (p, at capability, the inventors designed an M13mp 18 phage to increasing killing by ofloxacin. These results demonstrate express csrA and omple simultaneously (cps) to target that targeting other non-SOS genetic networks and overex cSrA-controlled gene networks and increase drug penetration pressing multiple factors, i.e. multiple repressors can result in (FIG.5C). The multi-target phage was constructed by placing engineered bacteriophage which are good adjuvants for anti RBS and ompl immediately downstream of cSrA in ps biotics. (FIG.9F) (Lutz et al., (1997) Nucleic Acids Res 25, 1203 0314 Exposure to subinhibitory concentrations of antibi 1210). The inventors demonstrated that p. was more otics can lead to initial mutations which confer low-level effective at enhancing ofloxacin's bactericidal effect com antibiotic resistance and eventually more mutations that yield pared with its single-target relatives, p, and ple, in high-level antibiotic resistance'. By enhancing ofloxacin's planktonic (FIG. 5D) and biofilm settings (FIG. 18). bactericidal effect, engineered bacteriophage can reduce the Together, these results demonstrate that engineering phage to number of antibiotic-resistant mutants that Survive in a bac target non-SOS genetic networks such as networks which terial population exposed to antimicrobial drugs. To demon increase a bacterial cells Susceptibility to an antimicrobial strate this effect, the inventors grew E. coli in media with no agent and/or overexpress multiple factors can produce effec ofloxacin (FIG. 7A) or 30 ng/mL ofloxacin for 12 hours (FIG. tive antibiotic adjuvants. 7B, FIG. 7C, and FIG. 7D) to produce antibiotic-resistant mutants. Then, the inventors divided the cells which grew Example 8 under no ofloxacin into 60 individual wells with no ofloxacin (FIG. 7A). The inventors also divided the cells which grew 0311. To show that other targets can be found to enhance under 30 ng/mL ofloxacin into 60 individual wells for each of the efficacy of combination therapy with bacteriophage and the following treatments: no phage and 30 ng/mL ofloxacin antibiotic, the inventors screened M13mp18 bacteriophage (FIG.7B), 10 PFU/mL (p, and 30 ng/mL ofloxacin (FIG. which expressed proteins that could modulate sensitivity to 7C), and 10 PFU/mL (p, with 30 ng/mL ofloxacin (FIG. antibiotics or that control regulatory networks, such as SOXR, 7D). After 12 hours of additional growth, the inventors deter fur, crp, marr, iccdA, cSrA, and ompl. The inventors did this mined the number of antibiotic-resistant mutants by plating by obtaining viable cell counts after 6 hours of treatment with and counting the number of cells that grew on LB agar con ofloxacin. Phage expressing SOXR, cSrA, or omp yielded the taining 100 ng/mL ofloxacin. FIG. 7A shows that growth in greatest improvements in killing by ofloxacin (See FIG. 1). the absence of ofloxacin yielded very few resistant cells. Like (ps, these phage expressed their respective proteins However, growth in the presence of a subinhibitory level of under the control of P, tetO and a synthetic RBS (FIGS. 9C, ofloxacin resulted in a very high number of antibiotic-resis 9D, and 9E)'. Since SoxR regulates a cellular response to tant bacteria (FIG. 7B). Although treatment with (p. Superoxide stress and quinolones stimulate Superoxide-based reduced the number of resistant cells, all of the 60 individual oxidative attack, the inventors Surmised that overproducing wells tested contained at least one resistant CFU and over half SoxR could affect this response and improve ofloxacin's bac of the wells had more than 20 resistant CFUs (FIG. 7C). In tericidal activity''. As shown in FIG. 6A, soxR-expressing contrast to treatment with no phage or unmodified (p. M13mp 18 (cps) enhanced killing by ofloxacinby about 3.8 (ps treatment Suppressed the level of resistant cells dra US 2010/0322903A1 Dec. 23, 2010 44 matically, resulting in a majority of wells with either no 0319. The designs that have been currently experimented observable resistant CFUs or less than 20 CFUs (FIG. 3d). with extend the paired-termini (PT7) design described in These results demonstrate that engineered (ps is effica Nakashima et al., (2006) Nucleic Acids Res 34: e138, which cious at reducing the number of antibiotic-resistant cells produces an RNA similar to that shown in FIG. 12. The PTT which can develop in the presence of subinhibitory drug construct produces antisense RNA with longer half-lives in concentrations. Vivo, allowing for greater antisense effect (Nakashima et al., (2006) Nucleic Acids Res 34: e138). Using the PT system, we Example 9 have constructed antisense RNAS targeting cat, recA, recB. and recC (Nakashima et al., (2006) Nucleic Acids Res 34: 0315. The inventors also sought to determine whether the e138). These asRNA constructs have been placed under engineered phage could be applied to different classes of inducible control by aTc by cloning into p7E21s1-cat in place antibiotics other than the quinolones. Since (p, was the of cat (Lutz et al., (1997) Nucleic Acids Res 25: 1203-1210). most effective adjuvant for ofloxacin, the inventors tested its The inventors also created all pairwise combinations of asR adjuvant effect forgentamicin, an aminoglycoside, and ampi NAS to recA, recB, and recC by placing one asRNA construct cillin, a f-lactam antibiotic. For 5 Lig/mL gentamicin, (p., under the control of PtetO and the other under the control of was slightly more effective at enhancing killing of bacterial PlacO on the same plasmid (Lutzetal. (1997)Nucleic Acids cells by ofloxacin compared with no phage (FIG. 8A). (p. Res 25: 1203-1210). increased gentamicin's bactericidal action by over 2.5 logo 0320 All the plasmids described thereafter have been (CFU/mL) compared with p, and by over 3 logo (CFU/ introduced into wild-type E. coli EMG2 cells and have been mL) compared with no phage after 6 hours of treatment (FIG. assayed for survival with antibiotic treatment. All cells and 8A). For 5ug/mL amplicillin, control (p, alone increased suitable controls were grown for 8 hours at 37° C. in LB killing by ofloxacin by more than 3 orders of magnitude media (with appropriate inducers) and challenged with anti compared to no phage (FIG.4b). (p. improved amplicillin's biotics Such as ofloxacin at 5 g/mL. Cell counts were plated bactericidal effect by over 2.2 logo (CFU/mL) compared after 8 hours of exposure to antibiotic and counted to assess with unmodified (p and by over 5.5 logo (CFU/mL) persistence levels. Cells will also be assayed for resistance to compared to no phage (FIG. 8B). For both gentamicin and specific antibiotics (for example, chloramphenicol in the ampicillin, ps's strong adjuvant effect was noticeable after presence of cat-expressing plasmids). 1 hour of treatment (FIG. 8A and FIG. 8B). These results are 0321. The inventors constructed asRNA targeting cat and consistent with previous observations that Areca mutants have expressed the asRNA in a ColE1-type plasmid. With the exhibit increased susceptibility to quinolone, aminoglyco cat-askNA vector, the inventors assessed if the chloram side, and B-lactam drugs. Therefore, engineered bacte phenicol MIC of target bacteria is effectively reduced. The riophage such as (ps can act as general adjuvants for the inventors constructed vectors with recA-asRNA, recB-as three major classes of bactericidal drugs. RNA, recC-asRNA and all pairwise recA, recB, and recC 0316. Using phage, the inventors have demonstrated that combinations and assayed for persistence levels with ofloxa targeting genetic networks to potentiate killing by existing cin (5ug/mL) with 8 hours of growth followed by 8 hours of antimicrobial drugs is a highly effective strategy for enhanc treatment. The vectors which demonstrated the strongest phe ing the usefulness of antibiotics. The host specificity of phage notypes were the PtetO-recB-asRNA/PlacO-recA-asRNA avoids the side effects associated with broad-spectrum anti and PtetO-recC-asRNA/PlacO-rec3-asRNA plasmids biotics such as Clostridium difficile overgrowth but requires a (FIG. 14). These constructs displayed 1.87 and 2.37 logo library of phage to be maintained to cover a range of infec (CFU/mL) less persisters, respectively, compared with wild tions: type E. coli EMG2. 0317. In some embodiments, libraries of existing phage could be modified to overexpress other genes, such as for Example 11 example but not limited to lexA3 to suppress the SOS response in different bacterial species'. 0322 The inventors have demonstrated herein that com bination therapy which couples antibiotics with antibiotic Example 10 enhancing phage has the potential to be an effective antimi crobial strategy. Moreover, the inventors have demonstrated 0318. A direct method of attacking antibiotic-resistant that antibiotic-enhancing phage are effective in vivo in res bacteria is to express asRNAs to knockdown genes that either cuing bacterially infected mice, and thus have clinical rel confer antibiotic resistance or promote cell repair and the evance for their use in vivo, in mammalian models of bacterial SOS response. Thus, the inventors expressed an antisense infections, as well as in human treatment, both for therapeutic RNA (asRNAs) against the cat gene and other antibiotic and prophylactic treatment. Thus, the inventors have demon resistance genes (genes that inactivate antibiotics or pump out strated a method to modify phage (i.e. bacteriophage) to be antibiotics or genetic circuits that confer persistence or any engineered to act as effective antibiotic adjuvants in vitro and other antibiotic resistance phenotype such as VanA, mecA, in vivo and can be used in methods for antimicrobial target and others) as well as recA, recB, recC, spoT relA, and other identification as well as for therapeutic use and implementa genes necessary for cell repair or Survival. These vectors tion. The inventors have also demonstrated that by targeting should sensitize cells to antibiotics since they will target non-essential gene networks, a diverse set of engineered bac genes that inactivate or pump outantibiotics and those that are teriophage can be developed to Supplement other antimicro necessary for cell repair from damage caused by antibiotics bial strategies. (Dwyer et al., (2007) Mol Syst Biol3:91). Inhibiting the SOS 0323 While use of phages in clinical practice is not widely response may also reduce the spread of antibiotic resistance accepted due to a number of issues such as phage immuno genes (Beaber, et al., (2004) Nature 427: 72-74;Ubeda, et al., genicity, efficacy, target bacteria identification and phage (2005) Mol Microbiol 56: 836-844). selection, host specificity, and toxin release (Merril et al., US 2010/0322903A1 Dec. 23, 2010

(2003) Nat. Rev. Drug Discov. 2, 489-497; Hagens et al., ing phage to clinically relevant bacterial strains and has (2003) Lett. Appl. Microbiol. 37, 318-323; Hagens et al., important uses in developing treatments against real-world (2004) Antimicrob. Agents Chemother: 48, 3817-3822: infections. Boratynski et al., (2004) Cell. Mol. Biol. Lett. 9, 253-259; 0326 In some embodiments, the engineered phages as Merrilet al., (1996) Proc Natl AcadSci USA 93,3188-3192), described herein can also be used in industrial, agricultural, the inventors indicate that one way to reduce the risk of and food processing settings where bacterial biofilms and leaving lysogenic particles in patients after treatment, the other difficult-to-clear bacteria are present (Lu et al., (2007) inventors engineered adjuvant phages could be further modi Proc Natl AcadSci USA 104, 11 197-216). Accordingly, some fied to be non-replicative, as has been previously described embodiments as described herein encompass applying the (Hagens et al., (2004) Antimicrob 11). The inventors have engineered phage as described herein as antibiotic adjuvants demonstrated an antibiotic-enhancing phage as a prototype in non-medical settings. This could be economically advan phage as proofof-conceptantibiotic adjuvants. The inventors tageous, reduce community-acquired antibiotic resistance, and be also be useful in testing efficacy of the particular indicate that in Some embodiments, a combination of antibi engineered phage prior to its use as a treatment and/or in otic-enhancing phages or phage cocktails can be used for in clinical use (Morens et al., (2004) Nature 430, 242-24949). vivo and in vitrouse, as well as in clinical settings for effective 0327. Another strategy to combat antibiotic resistance is efficacy and/or the ability to treat non-F-plasmid containing to take advantage of the numerous autoregulated repressors bacteria. In particular, in Some embodiments phage cocktails inherent in bacteria that regulate resistance genes or cell which target different, multiple bacterial receptors can be repair pathways (Okusu, et al., (1996) J Bacteriol 178: 306 used, which can have a benefit of reducing the development of 308). For example, lexA represses the SOS response until it is phage resistance by invading bacteria through multiple dif cleaved by recA in response to DNA damage (Dwyer et al., ferent means and pathways. Thus, in another embodiment, (2007) Mol Syst Biol 3:91). In addition, marr represses the phage cocktails can be used with one or more different anti marr AB operon and acrR represses the acrAB operon; both biotics to also enhance bacterial killing as well as reduce operons confer resistance to a range of antibiotics (Okusu, et resistance to both the phages and antibiotics. al., (1996) J Bacteriol 178: 306–308). To increase repression 0324. The inventors have demonstrated use of engineered of the SOS response orantibiotic-resistance-conferring oper antibiotic-enhancing phages as a phage platform for the ons, we propose to overexpress the responsible repressors. development of effective antibiotic adjuvants, and is a prac However, simple overexpression may impose a high meta tical example of how synthetic biology can be applied to bolic cost on the cells leading to rejection of the introduced important real-world biomedical issues. Synthetic biology is constructs. Therefore, as an alternative to simple overexpres focused on the rational and modular engineering of organisms Sion, the inventors created an autoregulated negative-feed to create novel behaviors. The field has produced many back modules with lexA and other repressors and determine reports of synthetic gene circuits and systems with interesting whether cells are sensitized to antibiotic treatment with these constructs (FIG. 13). The net effect of this strategy should be characteristics (Andrianantoandro et al., (2006) Mol Syst to increase the loop gain of inherent autoregulated negative Biol. 2, 2006.0028; Hasty et al., (2002) in Nature, pp. 224 feedback loops so that any perturbations in the level of repres 230; McDaniel et al., (2005) in Curr: Opin. Biotechnol., pp. sors will be more rapidly restored, hopefully preventing Suc 476-483. Chanet al., (2005) in Mol Syst Biol, p. 2005.0018). cessful activation of Survival pathways. More recently, synthetic biologists have begun to address 0328. The inventors produced and assessed the pZE1L important industrial and medical problems (Lu et al., (2007) lexA plasmid for persistence levels with ofloxacin (5ug/mL) Proc Natl Acad Sci USA 104, 11 197-216: Anderson et al., with 8 hours of growth followed by 8 hours of treatment. The (2006).J. Mol. Biol. 355,619-627; Loose et al., (2006) Nature inventors constructed the pzE1L-lex.A plasmid by utilizing 443, 867-869; Roet al. (2006) Nature 440, 940-943). the PlexO promoter described in (Dwyer et al., (2007) Mol 0325 In some embodiments, the present invention also Syst Biol 3:91). Cells containing the pZE1L-lex A construct encompasses production and use of libraries of natural phage produced about 1.44 logo (CFU/mL) less persisters com which have been modified to target gene networks and path pared with wild-type E. coli EMG2 (FIG. 10). The inventors ways, such as the SOS response, in different bacterial species also made changes in the design of pZE1L-leXA by using (Hickman-Brenneret al., (1991).J. Clin. Microbiol. 29, 2817 non-cleavable leXA variants. 2823). One of ordinary skill in the art could generate and use 0329. The inventors demonstrated, in lytic phage such as Such libraries by using routine methods in the art, such as T7 or lysogenic phage such as M13 and using synthetic biol isolation and genetic modification of natural phage with the ogy, construction of engineered phage by inserting the vector ability to infect the bacterial species being targeted. With constructs simply into optimal regions in the phage genome to current DNA sequencing and synthesis technology, an entire be expressed during infection (Lu et al., (2007) Proc Natl engineered bacteriophage genome carrying multiple con Acad Sci USA 104: 11197-11202). M13 is a filamentous, structs to target different gene networks could be synthesized male-specific phage with a single-stranded, circular DNA (Baker et al. (2006) Sci. Am. 294, 44-51). Thus, one of ordi genome that infects E. coli. During infection, the genome nary skill in the art, using Such technologies could carry out adopts a double-stranded replicative form (RF) which can be large-scale modifications of phage libraries to produce anti stably maintained in lysogeny. M13 Subsequently replicates biotic-enhancing phage that can be applied with different and secretes mature phage particles into the Surrounding envi antibiotic drugs against a wide range of bacterial infections. ronment that can infect other cells. M13 is a commonly used Targeting clinical bacterial strains with libraries of engi phage for peptide display and DNA sequencing and has been neered phage, which can be carried out by routine testing by modified for genetic manipulation. In some embodiments, one of ordinary skill in the art to identify which engineered M13 and other lysogenic phage can be used as carriers for phage from the libraries is effective as an antibiotic-enhanc asRNAS or other genetic modules because they allow propa US 2010/0322903A1 Dec. 23, 2010 46 gation of the introduced constructs throughout a bacterial cult-to-clear bacteria are present'. Potentiating bacterial population without massive lysis, which can lead to release of killing in non-medical settings should have economic advan toxic products such as endotoxin or lead to the development tages in addition to reducing community-acquired antibiotic of phage resistant bacteria due to strong evolutionary pres resistance'. Sure. As the constructs need to be able to reach a large popu 0332 Conventional drugs typically achieve their thera lation of cells, have the desired effects, and then be subse peutic effect by reducing protein function. In contrast, the quently killed by antibiotic therapy, lysogenic phages were bacteriophage and selective gene targeting approach as used by the inventors. For example, the gene constructs could described herein potentiates killing by antibiotics by overex be cloned in place of the lacZ gene in the already modified pressing proteins that affect genetic networks, such as leXA3, M13mp 18 bacteriophage under the control of a strong bacte SOXR, and cSrA, or that act on their own to modulate antibiotic rial-species-specific promoter or phage-specific promoter. sensitivity, such as ompF. By reducing the SOS response with 0330 Herein, the inventors have demonstrated that build engineered M13mp 18-lex A3 bacteriophage, the inventors ing effective bacteriophage adjuvants that target different have potentiated ofloxacin's bactericidal effect by over 4.5 factors individually or in combination can be achieved in a orders of magnitude and reduced the number of persister cells modular fashion. As the cost of DNA sequencing and synthe (FIG. 1b). The inventors have also demonstrated that other sis technologies continues to be reduced, large-scale modifi factors such as SOXR, cSrA, and ompl could be targeted for cations of phage libraries should become feasible'''. With overexpression individually or in combination to enhance current technology, an entire engineered M13mp18 genome killing (FIG. 6). The inventors demonstrated that the number carrying multiple constructs to target genetic networks could of mutants which acquired antibiotic resistance was signifi be synthesized for less than S10,000, a price which is sure to cantly decreased by the use of engineered M13mp18-lex.A3 decrease in the future". Furthermore, systems biology tech bacteriophage in combination with ofloxacin (FIG. 7). In niques can be employed to more rapidly identify new targets addition, the inventors confirmed that our engineered bacte to be used in engineered bacteriophage'''. Antisense RNA riophage could be used as antibiotic adjuvants for other drugs could also be delivered by bacteriophage to enhance killing of Such as aminoglycosides and B-lactams (FIG. 8). Combina bacteria. Cocktails of engineered phage such as those tion therapy with antibiotics and engineered phage resulted in described here could be combined with biofilm-dispersing no noticeable development of phage resistance. The inventors bacteriophage and antibiotics to increase the removal of demonstrated that targeting genetic networks in bacteria harmful biofilms. which are not primary antibiotic targets yield Substantial 0331 Since the FDA recently approved the use of bacte improvements in killing by antimicrobial drugs. Advances in riophage against Listeria monocytogenes in food products, it systems biology and synthetic biology should enable the is likely that the engineered phages as disclosed herein can be practical application of engineered bacteriophage with anti readily adopted for medical, industrial, agricultural, and food biotics as a new combination therapy for combating bacterial processing settings where bacterial biofilms and other diffi 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 1 8.75 b2699 recA DNA strand exchange and recombination 1 2 >8.75 protein with protease and nuclease activity b282O rec DNA helicase, ATP-dependent 1 7.5 dsDNA ssDNA exonuclease b2822 recC DNA helicase, ATP-dependent 1 8 >8.75 dsDNA ssDNA exonuclease b3652 recC ATP-dependent DNA helicase, resolution of 1 6 6 Holliday junctions, branch migrations b2616 recy Recombination and repair protein 1 10 b1861 ruvA Holliday junction DNA helicase 1 10 b1863 ruvC Holliday junction nuclease; resolution of 1 8 >8.75 structures; repair b3813 wrD DNA-dependent ATPase I and helicase II 1 5 6 US 2010/0322903A1 Dec. 23, 2010 47

TABLE 2A-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 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 b2SO9 xSeA Exodeoxyribonuclease VII large subunit 1 6 6 bO422 xSeB Exodeoxyribonuclease VII Small subunit 1 8 b3261 fis DNA-binding protein - chromosome 1A 6 >8.75 compaction b1712 ihfA Integration host factor alpha-subunit (IHF- 1A 7.5 alpha). b0464 acrA Acra B-TolC Multidrug Efflux Transport 2 7.5 System bO462 acre Acra B-TolC Multidrug Efflux Transport 2 8 System b3O3S toC Acra B-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 yjY Predicted protein YijY 7 8.75

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

Locus MIC mL Tag Gene Gene Product Category Plating E Test

BW251.13 500 b3613 envC Cytokinesis - murein hydrolase 2 150 b3404 envZ OSmolarity sensor protein 2 150 b0588 fepc Ferric enterobactin transport ATP-binding 2 150 protein b3201 lptB ATP-binding LiptAB-YrbKABC transporter 2 150 2.0 b1855 InsbB Myristoyl-acyl carrier acyltransferase 2 150 b0741 pal Peptidoglycan-associated lipoprotein 2 1OO 96 precursor. b2678 proW Glycine betaine:L-proline transport?permease 2 150 b2617 Smp A Outer membrane lipoprotein 2 1OO 70 US 2010/0322903A1 Dec. 23, 2010 48

TABLE 2B-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 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 b1252 tonB Cytoplasmic membrane protein; energy 2 125 transducer b2512 yfgL Lipoprotein-outer membrane protein 2 150 assembly b3245 yhdP Transporter activity, membrane protein 2 125 b2S27 scB Hsc20 co-chaperone, with Hsc66 IscU iron- 2A 150 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 b4058 uvra Excision nuclease subunit A 1 7.5 b3781 trx A Thioredoxin electron transfer protein 1A 5 b0464 acrA Acra B-TolC Multidrug Efflux Transport 2 >10 System b0462 acrE3 Acra B-TolC Multidrug Efflux Transport 2 10 System b3613 envC Cytokinesis - murein hydrolase 2 10 US 2010/0322903A1 Dec. 23, 2010 49

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 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 QB 5 10 replication b1237 hns DNA-binding protein H-NS 5 7.5 b3842 rfaEH Transcriptional activator rfaH. 5 7.5 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 US 2010/0322903A1 Dec. 23, 2010 50

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 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 2010/0322903A1 Dec. 23, 2010 51

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

Tag Gene Gene Description Category E test Plating b3734 atpA ATP synthase alpha chain 2.5 b3809 dapF Diaminopimelate epimerase 1.O b206S dco Deoxycytidine triphosphate deaminase (dTP) 2.5 b3612 Phosphoglycerate mutase, cofactor independent 1.5 b1317 pgmB 3-phosphoglucomutase 1.5 b2232 ubiG 3-demethylubiquinone-9 3-methyltransferase 2.0 b2767 ygcO Predicted 4Fe-4S cluster-containing protein 2.0 b1284 deoT DNA-binding transcriptional regulator 2.0 bO145 dkSA RNA polymerase-binding transcription factor 2.0 b1130 phoP Transcriptional regulatory protein 2.0 b24OS XapR Xanthosine operon regulatory protein. 1.5 b1280 yciM Putative heat shock proteins 1.5 JW 5115 Hypothetical protein 2.0 ybeD conserved protein Ybed 2.0 ybeY conserved protein Ybey 2.0 ybhT Hypothetical protein YbhT precursor 2.0 predicted protein YY 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 fis DNA-binding protein - chromosome compaction A. 600 envC Cytokinesis - murein hydrolase 400 lptB ATP-binding LiptAB-YrbKABC transporter 500 pstA Phosphate transport system permease 550 pstS Phosphate-binding periplasmic protein 550 rfaE Heptose 1-phosphate adenyltransferase 550 tolC Acra B-TolC Multidrug Efflux Transport System 400 ybgF Predicted plasma protein >550 yciS Conserved inner membrane protein 550 yfgL Lipoprotein component of Outer membrane protein 400 assembly complex yneE Conserved inner membrane protein 2 550 degP Periplasmic serine protease and chaperone 2A 500 US 2010/0322903A1 Dec. 23, 2010 52

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 bOO14 naK Chaperone protein - chaperone Hsp70; DNA 2A 300 biosynthesis bO489 amcA Putative protease 3 550 bO852 rimlk Ribosomal protein S6 modification protein. 3 350 b3984 rplA 50S ribosomal protein L1. 3 500 b1089 rpm E 50S ribosomal protein L32. 3 550 b306S ribsU 30S ribosomal protein S21. 3 500 b3809 apF Diaminopimelate epimerase 4 300 b206S cod Deoxycytidine triphosphate deaminase (dTP) 4 >550 b3612 gpmM Phosphoglycerate mutase, cofactor independent 4 400 bO116 pdA Dihydrolipoamide dehydrogenase (Glycine 4 400 cleavage) b1317 pgmB 3-phosphoglucomutase 4 500 b1773 yd I Predicted adolase 4 >550 b2767 ygcO Predicted 4Fe-4S cluster-containing protein 4 550 b1284 eoT DNA-binding transcriptional regulator 5 550 bO145 kSA Transcription initiation factor 5 550 b1237 hns DNA-binding protein H-NS 5 550 b2572 res.A Sigma-E factor negative regulatory protein. 5 >550 b24OS XapR Xanthosine operon regulatory protein. 5 >550 b1280 yciM Putative heat shock protein 5 >550 bOSSO.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 ybeY Hypothetical protein 7 500 yii U Conserved protein Yii U 7 550 yjY Predicted protein YY 7 >550

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

BW251.13 O.8 b1652 int RibonucleaseT 1 0.7 b3613 envC Cytokinesis - murein hydrolase 2 >O.S b3621 rfaC Lipopolysaccharide heptosyltransferase-1 2 0.7 b3791 rff A dTDP-4-oxo-6-deoxy-D-glucose transaminase 2 0.7 b1292 SapC Peptide transport system permease protein 2 O.S b3175 secC Protein-export membrane - Sec Protein Secretion 2 O.S Complex tatC Sec-independent protein translocase TatC 2 O.S tolC Acra B-TolC Multidrug Efflux Transport System 2 O.S US 2010/0322903A1 Dec. 23, 2010 53

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 LOCS (Lig/mL) Tag Gene Gene Product Category Plating 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 b2494 yfgC Predicted peptidase 3 >O.S b3809 dapF Diaminopimelate epimerase 4 0.7 b3612 gpmM Phosphoglycerate mutase, cofactor independent 4 0.7 b3202 rpoN RNA polymerase sigma-54 factor. 5 O.S b24.05 xapR Xanthosine operon regulatory protein. 5 >O.S b1280 yeiM Putative heat shock protein 5 >0.7 JW5360 Hypothetical protein 7 >0.8 b4557 yidD Predicted protein YidD 7 O.S

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 Acholeplasma phage L2 NC OO1447 11965 in 14 O 14 Acholeplasma phage MV-L1 NC 001341 4491 in 4 O 4 Acidianus bottle-shaped virus NC OO9452 23814 in 57 O 57 Acidianus filamentous virus 1 NC O0583.0 2O869 in 40 O 40 Acidianus filamentous virus 2 NC 009884 31787 in 52 1 53 Acidianus filamentous virus 3 NC 01.0155 40449 in 68 O 68 Acidianus filamentous virus 6 NC 01.0152 39577 in 66 O 66 Acidianus filamentous virus 7 NC 01.0153 36895 in 57 O 57 Acidianus filamentous virus 8 NC 01 0154 38179 in 61 O 61 Acidianus filamentous virus 9 NC 01.0537 41172 in 73 O 73 Acidianus rod-shaped virus 1 NC OO9965 246SS in 41 O 41 Acidianus two-tailed virus NC OO7409 62730 in 72 O 72 Acinetobacter phage AP205 NC OO2700 4268 in 4 O 4 Actinomyces phage AV-1 NC 009643 17171 in 22 1 23 Actinoplanes phage phiAsp2 NC O05885 S8638 in 76 O 76 Acyrthosiphon pisum secondary NC OOO935 36524 in S4 O S4 endosymbiont phage 1 Aeromonas phage 25 NC OO8208 161475 in 242 13 242 Aeromonas phage 31 NC 007022 172963 in 247 15 262 Aeromonas phage 44RR2.8t NC O05135 173591 in 252 17 269 Aeromonas phage Aehl NC OO5260 233234 in 352 23 375 Aeromonas phage phiO18P NC OO9542 33985 in 45 O 45 Archaeal BJ1 virus NC OO8695 42271 in 70 1 71 AZOspirillum phage Cod NC 01 0355 62337 in 95 O 95 Bacillus phage 0305phi8-36 NC OO9760 218948 in 246 O 246 Bacillus phage AP50 NC 011523 14398 in 31 O 31 Bacillus phage B103 NC 004165 1863.On 17 O 17 Bacillus phage BCJA1c NC OO6557 41,092 in 58 O 58 Bacillus phage Bam35c NC O05258 14935 in 32 O 32 Bacillus phage Cherry NC 007457 36615 in 51 O 51 US 2010/0322903A1 Dec. 23, 2010 54

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 Bacillus phage Fah NC 007814 37974 in 50 O 50 Bacillus phage GA-1 NC 002649 21129 in 35 1 52 Bacillus phage GIL16c NC OO6945 14844 in 31 O 31 Bacillus phage Gamma NC 007458 37253 in 53 O 53 Bacillus phage IEBH NC 01.1167 53104 in 86 O 86 Bacillus phage SPBc2 NC OO1884 134416 in 185 O 185 Bacillus phage SPO1 NC 01.1421 132S62 in 204 5 209 Bacillus phage SPP1 NC 004166 44010 in 101 O 101 Bacillus phage TP21-L NC 011645 37456 in 56 O 56 Bacillus phage WBeta NC OO7734 40867 in 53 O 53 Bacillus phage phBC6A51 NC 004.820 61395 in 75 O 75 Bacillus phage phBC6A52 NC 004.821 38472 in 49 O 49 Bacillus phage phil O5 NC 004167 3932S in 51 O 51 Bacillus phage phi29 NC 01.1048 19282 in 27 O 27 Bacillus virus 1 NC 009.737 35055 in S4 O S4 Bacteriophage APSE-2 NC 011551 39867 in 41 1 42 Bacteroides phage B40-8 NC 011222 44929 in 46 O 46 Bdellovibrio phage phi MH2K NC 002643 4594 in 11 O 11 Bordetella phage BIP-1 NC OO5809 42638 in 48 O 48 Bordetella phage BMP-1 NC OO5808 42663 in 47 O 47 Bordetella phage BPP-1 NC OO5357 42493 in 49 O 49 Burkholderia ambifaria phage BcepF1 NC 009015 72415 in 127 O 127 Burkholderia phage Bcep1 NC OO5263 48.177 in 71 O 71 Burkholderia phage Bcep176 NC OO7497 44856 in 81 O 81 Burkholderia phage Bcep22 NC OO5262 63879 in 81 1 82 Burkholderia phage Bcep43 NC OO5342 48024 in 65 O 65 Burkholderia phage Bcep781 NC 004333 48247 in 66 O 66 Burkholderia phage BcepB1A NC O05886 47399 in 73 O 73 Burkholderia phage BcepC6B NC O05887 42415 in 46 O 46 Burkholderia phage BcepGomr NC 009447 52414 in 75 O 75 Burkholderia phage BcepMu NC O05882 36748 in 53 O 53 Burkholderia phage BcepNY3 NC 009604 47382 in 70 1 70 Burkholderia phage BcepNazgul NC 005091 57455 in 73 O 73 Burkholderia phage KS10 NC 011216 3763S in 49 O 49 Burkholderia phage phil O26b NC O05284 S486S in 83 O 83 Burkholderia phage phi52237 NC 007145 37639 in 47 O 47 Burkholderia phage phió44-2 NC 009235 48674 in 71 O 71 Burkholderia phage phiE12-2 NC 009236 36690 in 50 O 50 Burkholderia phage phiE125 NC 003.309 S3373 in 71 O 71 Burkholderia phage phiE202 NC 009234 35741 in 48 O 48 Burkholderia phage phiE255 NC 009237 37446 in 55 O 55 Chlamydia phage 3 NC 008355 4554 in 8 O 8 Chlamydia phage 4 NC 007461 4530 in 8 O 8 Chlamydia phage CPAR39 NC 00218O 4532 in 7 O 7 Chlamydia phage Chp1 NC OO1741 4877 in 12 O 12 Chlamydia phage Chp2 NC 002194 4563 in 8 O 7 Chlamydia phage phiCPG1 NC OO1998 4529 in 9 O 9 Clostridium phage 39-O NC 011318 38753 in 62 O 62 Clostridium phage c-st NC OO7581 185683 in 198 O 198 Clostridium phage phi CD119 NC 007917 S332S in 79 O 79 Clostridium phage phi3626 NC 003524 33507 in 50 O 50 Clostridium phage phiC2 NC 009231 56538 in 82 O 82 Clostridium phage phiCD27 NC O11398 SO930 in 75 O 75 Clostridium phage phiSM101 NC OO8265 38092 in 53 1 S4 Corynebacteriumphage BFK20 NC OO9799 42969 in S4 O S4 Corynebacteriumphage P1201 NC 009816 7.0579 in 97 4 101 Enterobacteria phage 13a NC 011045 38841 in 55 O 55 Enterobacteria phage 933W NC 000924 61670 in 8O 4 84 Enterobacteria phage BA14 NC 011040 39816 in 52 O 52 Enterobacteria phage BP-4795 NC 004.813 S7930 in 85 O 85 Enterobacteria phage BZ13 NC 001426 3466 in 4 O 4 Enterobacteria phage EPS7 NC 01.0583 111382 in 170 O 171 Enterobacteria phage ES18 NC OO6949 46900 in 79 O 79 Enterobacteria phage EcoDS1 NC 011042 39252 m 53 O 53 Enterobacteria phage FI sensulato NC 004301 4276 in 4 O 4 Enterobacteria phage Felix 01 NC O05282 861 SS in 131 22 153 Enterobacteria phage Fels-2 NC 010463 33693 in 47 O 48 US 2010/0322903A1 Dec. 23, 2010 55

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 G4 sensulato NC 001420 5577 in 11 O 13 Enterobacteria phage HKO22 NC 002166 4O751 in 57 O 57 Enterobacteria phage HK620 NC OO2730 38297 in 58 O 58 Enterobacteria phage HK97 NC 002167 39732 in 61 O 62 Enterobacteria phage I2-2 NC OO1332 6744 in 9 O 9 Enterobacteria phage ID18 sensulato NC 007856 5486 in 11 O 11 Enterobacteria phage ID2 NC 007817 5486 in 11 O 11 Moscow ID 200 Enterobacteria phage Ifl NC OO1954 8454 in 10 O 10 Enterobacteria phage Ike NC OO2014 6883 in 10 O 10 Enterobacteria phage JK06 NC 007291 46072 in 82 O 82 Enterobacteria phage JS98 NC 01.01.05 170523 in 266 3 269 Enterobacteria phage K1-5 NC OO8152 44385 in 52 O 52 Enterobacteria phage K1E NC OO7637 45251 in 62 O 62 Enterobacteria phage K1F NC 007456 39704 in 43 O 41 Enterobacteria phage M13 NC OO3287 6407 in 10 O 10 Enterobacteria phage MS2 NC OO1417 3569 in 4 O 4 Enterobacteria phage Min27 NC 010237 6339S in 83 3 86 Enterobacteria phage Mu NC 000929 36717 in 55 O 55 Enterobacteria phage N15 NC OO1901 46.375 in 60 O 60 Enterobacteria phage N4 NC 00872O 701S3 72 O 72 Enterobacteria phage P NC OO5856 948OO in 110 4 117 Enterobacteria phage P2 NC OO1895 33593 in 43 O 43 Enterobacteria phage P22 NC OO2371 41724 in 72 2 74 Enterobacteria phage P4 NC OO1609 11624 in 14 5 19 Enterobacteria phage PRD1 NC OO1421 14927 in 31 O 31 Enterobacteria phage Phil NC 0098.21 16427O in 276 O 276 Enterobacteria phage PsP3 NC OO5340 30636 in 42 O 42 Enterobacteria phage Qbeta NC OO1890 4215 in 4 O 4 Enterobacteria phage RB32 NC OO8515 165890 in 270 8 270 Enterobacteria phage RB43 NC 007023 18OSOO in 292 1 292 Enterobacteria phage RB49 NC 005066 164018 in 279 O 279 Enterobacteria phage RB69 NC 004928 167S60 in 273 2 275 Enterobacteria phage RTP NC 007603 46219 in 75 O 75 Enterobacteria phage SP6 NC 004831 43769 in 52 O 52 Enterobacteria phage ST104 NC 005841 41391 in 63 O 63 Enterobacteria phage ST64T NC 004348 4O679 in 65 O 65 Enterobacteria phage Sfö NC O05344 39043 in 66 2 70 Enterobacteria phage SfV NC 003444 37074 in 53 O 53 Enterobacteria phage T NC OO5833 48836 in 78 O 78 Enterobacteria phage T3 NC OO3298 382O8 in 55 O 56 Enterobacteria phage T4 NC OOO866 168903 in 278 10 288 Enterobacteria phage T5 NC O05859 1217SO in 162 33 195 Enterobacteria phage T7 NC 001604 399.37 in 60 O 60 Enterobacteria phage TLS NC O09540 499O2 in 87 O 87 Enterobacteria phage VT2-Sakai NC OOO902 60942 in 83 3 86 Enterobacteria phage WA13 sensulato NC 007821 6068 in 10 O 10 Enterobacteria phage YYZ-2008 NC O11356 54896 in 75 O 75 Enterobacteria phage alpha3 NC OO1330 6087 in 10 O 10 Enterobacteria phage epsilon15 NC 004775 39671 in 51 O 51 Enterobacteria phage lambda NC 001416 485O2 in 73 O 92 Enterobacteria phage phiEco32 NC 010324 77554 in 128 1 128 Enterobacteria phage phiEcoM-GJ1 NC 01.01.06 52975 in 75 1 76 Enterobacteria phage phiP27 NC 003356 42575 in 58 2 60 Enterobacteria phage phiV10 NC 007804 391.04 in 55 O 55 Enterobacteria phage phiX174 sensu NC OO1422 S386 in 11 O 11 atO Enterococcus phage phiEF24C NC 009904 142O72 in 221 5 226 rwinia phage Era 103 NC 009014 45445 in 53 O 53 Erwinia phage phiEa21-4 NC 011811 84576 in 118 26 144 Escherichia phage rv5 NC 011041 137947 in 233 6 239 Flavobacterium phage 11b NC OO6356 36O12 in 65 O 65 Geobacillus phage GBSV1 NC 008376 34683 in S4 O S4 Geobacillus virus E2 NC O09552 408.63 in 71 O 71 Haemophilus phage Aaphi23 NC 004.827 43O33 in 66 O 66 Haemophilus phage HP1 NC OO1697 323SS in 42 O 42 Haemophilus phage HP2 NC OO3315 31508 in 37 O 37 US 2010/0322903A1 Dec. 23, 2010 56

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 Haloarcula phage SH1 NC 007217 3O889 in 56 O 56 Halomonas phage phiHAP-1 NC 010342 392.45 in 46 O 46 Halorubrum phage HF2 NC OO3345 77670 in 114 5 119 Halovirus HF NC 004927 75898 in 102 4 106 His1 virus NC 007914 14462 in 35 O 35 His2 virus NC 007918 16067 in 35 O 35 Odobacteriophage phiPLPE NC 01.1142 47453 in 84 O 84 Klebsiella phage K1 NC 011043 41181 in 51 O 51 Klebsiella phage phiKO2 NC OO5857 516O1 in 64 O 63 Kluyvera phage KVp1 NC 011534 39472 in 47 1 48 Lactobacillus johnsonii prophage NC 01.0179 40881 in 56 O 56 L771 Lactobacillus phage A2 NC 004112 43411 in 61 O 64 Lactobacillus phage KC5a NC 007924 38239 in 61 O 61 Lactobacillus phage LL-H NC OO9554 34659 in 51 O 51 Lactobacillus phage LP65 NC OO6565 131522 in 16S 14 179 Lactobacillus phage Lc-Nu NC 0075O1 36466 in 51 O 51 Lactobacillus phage Lirm1 NC O11104 39989 in S4 O S4 Lactobacillus phage LV-1 NC 011801 38934 in 47 O 47 Lactobacillus phage phiAT3 NC O05893 391.66 in 55 O 55 Lactobacillus phage phiL-1 NC OO6936 36674 in 46 O 46 Lactobacillus phage philadh NC OOO896 43785 in 63 O 63 Lactobacillus phage phigle NC 004305 42259 in 50 O 62 Lactobacillus prophage Lj928 NC OO5354 38384 in 50 1 50 Lactobacillus prophage Lj965 NC OO5355 4O190 in 46 4 46 Lactococcus phage 1706 NC 01.0576 55597 in 76 O 76 Lactococcus phage 712 NC 008370 3OS10 in 55 O 55 Lactococcus phage BK5-T NC OO2796 400O3 in 63 O 63 Lactococcus phage KSY1 NC 009817 79232 in 130 3 131 Lactococcus phage P008 NC 008363 28S38 in 58 O 58 Lactococcus phage P335 sensulato NC 004746 36596 in 49 O 49 Lactococcus phage Q54 NC 008364 26S37 in 47 O 47 Lactococcus phage TP901-1 NC OO2747 37667 in 56 O 56 Lactococcus phage Tuc2009 NC OO2703 38347 in 56 O 56 Lactococcus phage asccphi28 NC 010363 18762 in 28 O 27 Lactococcus phage b|BB29 NC 011046 293 OS in S4 O S4 Lactococcus phage bL170 NC OO1909 31754 in 64 O 64 Lactococcus phage bL.285 NC 002666 3SS38 in 62 O 62 Lactococcus phage bL286 NC 002667 41834 in 61 O 61 Lactococcus phage bL309 NC 002668 36949 in 56 O 56 Lactococcus phage bL310 NC 002669 14957 in 29 O 29 Lactococcus phage bL311 NC 002670 14510 in 22 O 22 Lactococcus phage bL312 NC 002671 15179 in 27 O 27 Lactococcus phage bL67 NC OO1629 22195 in 37 O O LactococcuS phage c2 NC OO1706 22172 in 39 2 41 Lactococcus phage 50 NC 008371 27453 in 49 O 49 Lactococcus phage phiLC3 NC O05822 32172 in 51 O 51 Lactococcus phagerlt NC 004302 333 SO 50 O 50 Lactococcus phage sk1 NC OO1835 284.51 in 56 O 56 Lactococcus phage ul36 NC 004.066 36798 in 61 O 61 Leuconostoc phage L5 NC 009534 2435 in O O O Listeria phage 2389 NC OO3291 37618 in 59 1 58 Listeria phage A006 NC 009815 3.8124 in 62 O 62 Listeria phage A118 NC OO3216 40834 in 72 O 72 Listeria phage A500 NC 009810 38867 in 63 O 63 Listeria phage A511 NC 009811 137619 in 199 16 215 Listeria phage B025 NC 009812 42653 in 65 O 65 Listeria phage B054 NC 009813 481.72 in 8O O 8O Listeria phage P35 NC 009814 35822 in 56 O 56 Listeria phage P40 NC O11308 35638 in 62 O 62 Listonella phage phiHSIC NC OO6953 37966 in 47 O 47 Mannheimia phage phi MHaA1 NC OO82O1 3452S in 49 O 50 Methanobacterium phage psi M2 NC OO1902 26111 in 32 O 32 Methanothermobacter phage psi M100 NC 002628 28798 in 35 O 35 Microbacterium phage Min1 NC 009603 46365 in 77 O 77 Microcystis phage Ma-LMMO1 NC OO8562 162109 in 184 2 186 Morganella phage MmP1 NC 011085 38.233 in 47 O 47 US 2010/0322903A1 Dec. 23, 2010 57

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 Myco bacterium hage 244 NC 008.194 74483 142 144 Myco bacterium hage Adjutor NC 01.0763 64S11 86 86 Myco bacterium hage BPs NC 01.0762 41901 63 63 Myco bacterium hage Barnyard NC 004689 70797 109 109 Myco bacterium hage Bethlehem NC OO9878 52250 87 87 Myco bacterium hage Boomer NC 011054 58O37 105 105 Myco bacterium hage Brujita NC 011291 47057 74 74 Myco bacterium hage Butterscotch NC 011286 64562 86 86 Myco bacterium hage Bxb NC 002656 50550 86 86 Myco bacterium hage BXZ NC 004687 156102 225 2 253 Myco bacterium hage BXZ2 NC 004682 SO913 86 89 Myco bacterium hage Cali NC 011271 155372 222 257 Myco bacterium hage Catera NC OO82O7 153766 218 s 253 Myco bacterium hage Chah NC 011284 6.8450 104 104 Myco bacterium hage Chel2 NC OO82O3 52047 98 101 Myco bacterium hage Che8 NC 004680 594.71 112 112 Myco bacterium hage Che9c NC 004683 57050 84 85 Myco bacterium hage Che9d NC 004686 56276 111 111 Myco bacterium hage Cw NC 004681 75931 141 142 Myco bacterium hage Cooper NC OO8195 70654 99 99 Myco bacterium hage Corndog NC 004685 69777 122 122 Myco bacterium hage D29 NC OO1900 49136 79 84 Myco bacterium hage DD5 NC 011022 S1621 Myco bacterium hage Fruitloop NCO11288 S8471 Myco bacterium hage Giles NC 009993 S4S12 Myco bacterium hage Gumball NC 011290 64807 Myco bacterium hage Halo NC OO82O2 42289 Myco bacterium hage Jasper NC 011020 SO968 Myco bacterium hage KBG NC 011019 53.572 Myco bacterium hage Konstantine NC 011292 68952 Myco bacterium hage Kostya NC 011056 75811 Myco bacterium hage L5 NC OO1335 52.297 Myco bacterium hage Lli NC O08196 56852 Myco bacterium hage Lockley NC 011021 S1478 Myco bacterium hage Myrna NC 011273 1646O2 Myco bacterium hage Nigel NC 011044 69904 Myco bacterium hage Omega NC 004688 11086S Myco bacterium hage Orion NC OO8197 68427 Myco bacterium hage PBI1 NC OO8198 64494 Myco bacterium hage PG1 NC O05259 68.999 Myco bacterium hage PLot NC O08200 64787 Myco bacterium hage PMC NC OO8205 56692 Myco bacterium hage Pacca.0 NC 011287 58554 Myco bacterium hage Phaedrus NC 011057 68090 Myco bacterium hage Pipefish NC O08199 69059 Myco bacterium hage Porky NC 011055 76312 Myco bacterium hage Predator NC 011039 701 10 Myco bacterium hage Pukovnik NC 011023 S2892 Myco bacterium hage QyrZula NC O08204 67188 Myco bacterium hage Ramsey NC 011289 58578 Myco bacterium hage Rizal NC 011272 153894 Myco bacterium hage Rosebush NC 004684 67480 Myco bacterium hage Scott McG NC 011269 154017 33 Myco bacterium hage Solon NC 011267 494.87 Myco bacterium hage Spud NC 011270 154906 222 3 257 Myco bacterium hage TM4 NC OO3387 52.797 Myco bacterium hage Troll4 NC 011285 64618 Myco bacterium hage Tweety NC OO982O S8692 109 109 Myco bacteriumphage U2 NC OO9877 51277 81 81 Myco bacterium phage Wildcat NC O08206 78441 148 171 Myco plasma phage MAV1 NC OO1942 15644 15 15 Myco plasma phage P1 NC OO2515 11660 11 11 Myco plasma phage phi MFV1 NC 005964 15141 15 17 Myxococcus phage Mx8 NC OO3O85 49534 86 85 Natrialba phage PhiCh1 NC 004084 S8498 98 98 Pasteurella phage F108 NC OO8193 30505 44 44 Phage Gifsy-1 NC 010392 484.91 58 59 US 2010/0322903A1 Dec. 23, 2010 58

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 Phage Gifsy-2 NC 010393 45840 in 55 O 56 Phage catI NC 009514 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 O09541 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 O05178 37611 in 55 O 55 Pseudomonas phage DMS3 NC 008717 36415 in 52 O 52 Pseudomonas phage EL 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 NCO11165 64427 in 87 O 87 Pseudomonas phage LKA1 NC 009936 41593 in 56 O 56 Pseudomonas phage LKD16 NC 009.935 43200 in 53 O 53 Pseudomonas phage LMA2 NC 01.1166 66530 in 93 O 93 Pseudomonas phage LUZ19 NC 010326 43548 in S4 O S4 Pseudomonas phage LUZ24 NC 010325 45625 in 68 O 68 Pseudomonas phage M6 NC 007809 59446 in 85 O 85 Pseudomonas phage MP22 NC 009818 36409 in 51 O 51 Pseudomonas phage MP29 NC 011613 36632 in 51 O 51 Pseudomonas phage MP38 NC 011611 36885 in 51 O 51 Pseudomonas phage PA11 NC 007808 49639 in 70 O 70 Pseudomonas phage PAJU2 NC 01.1373 46872 in 79 O 79 Pseudomonas phage PB1 NC 011810 65764 in 93 O 94 Pseudomonas phage PP7 NC OO1628 3588 in 4 O 4 Pseudomonas phage PRR1 NC OO8294 3573 in 4 O 4 Pseudomonas phage PT2 NC 01.1107 42961 in S4 O S4 Pseudomonas phage PT5 NC 01.1105 42954 in 52 O 52 Pseudomonas phage PaP2 NC OO5884 43783 in 58 O 58 Pseudomonas phage PaP3 NC 004.466 45SO3 in 71 4 75 Pseudomonas phage P NC OO1331 7349 in 14 O 14 Pseudomonas phage Pf3 NC 001418 5833 in 9 O 9 Pseudomonas phage SN NC O11756 66390 in 92 O 92 Pseudomonas phage Yu.A. NC 01 0116 S8663 in 77 O 77 Pseudomonas phage gh-1 NC 004665 37359 in 42 O 42 Pseudomonas phage phil2 NC 004173 6751 in 6 O 6 Pseudomonas phage phil2 NC OO4175 4100 in 5 O 5 Pseudomonas phage phil2 NC 004174 2322 in 4 O 4 Pseudomonas phage phil3 NC 004172 6458 in 4 O 4 Pseudomonas phage phil3 NC 004171 4213 in 5 O 5 Pseudomonas phage phil3 NC OO4170 2981 in 4 O 4 Pseudomonas phage phió NC 003715 6374 in 4 O 4 Pseudomonas phage phió NC 003716 4O63 in 4 O 4 Pseudomonas phage phió NC 003714 2948 in 5 O 5 Pseudomonas phage phi8 NC OO3299 7051 in 7 O 7 Pseudomonas phage phi8 NC OO3300 4741 in 6 O 6 Pseudomonas phage phi8 NC OO3301 3.192 m 6 O 6 Pseudomonas phage phiCTX NC OO3278 3SS80 in 47 O 47 Pseudomonas phage phiKMV NC 005045 42519 in 49 O 49 Pseudomonas phage phiKZ NC 004629 28O334 in 306 O 306 Pyrobaculum spherical virus NC OO5872 28337 in 48 O 48 Pyrococcus abyssi virus 1 NC O09597 18098 in 25 O 25 Ralstonia phage RSB1 NC 011201 43079 in 47 O 47 Ralstonia phage RSL1 NC 010811 231256 in 345 2 346 Ralstonia phage RSM NC OO8574 8999 in 15 O 15 US 2010/0322903A1 Dec. 23, 2010 59

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 Ralstonia phage RSM3 NC O11399 8929 14 14 Ralstonia phage RSS1 NC OO8575 666.2 12 12 Ralstonia phage p12J NC O05131 7118 9 9 Ralstonia phage phiRSA1 NC 0093.82 3876O 51 51 Rhizobium phage 16-3 NC 01.1103 6O195 110 109 Rhodothermus phage RM378 NC 004.735 1299.08 146 146 Roseobacter phage SIO1 NC OO2519 39898 34 34 Salmonella phage E1 NC 0104.95 45051 51 52 Salmonella phage Fels-1 NC 010391 42723 52 52 Salmonella phage KS7 NC OO6940 4O794 59 59 Salmonella phage SE1 NC 011802 41941 67 67 Salmonella phage SETP3 NC 009232 42572 53 53 Salmonella phage ST64B NC 0043.13 4O149 56 56 Salmonella phage phiSG-JL2 NC 010807 3881S 55 55 Sinorhizobium phage PBC5 NC OO3324 S7416 83 83 Sodalis phage phiSG1 NC 007902 S2162 47 47 piroplasma kunkelii virus NC 009.987 7870 13 13 kV1 CR2-3x piroplasma phage 1-C74 NC 003793 7768 13 13 piroplasma phage 1-R8A2B NC OO1365 8273 12 12 piroplasma phage 4 NC OO3438 4421 9 9 piroplasma phage SVTS2 NC OO1270 6825 13 13 putnik virophage NC 01.1132 18343 21 21 aphylococcus aureus phage P68 NCOO4679 18227 22 22 hylococcus phage 11 NC 004615 43604 53 53 hylococcus phage 187 NC 007047 3962O 77 77 hylococcus phage 2638A NC 007051 41318 57 57 hylococcus phage 29 NC 007061 428O2 67 67 hylococcus phage 37 NC 007055 43681 70 70 hylococcus phage 3A NC 007053 4309S 67 67 hylococcus phage 42E NC 007052 45861 79 79 hylococcus phage 44AHJD NC 004678 16784 21 21 hylococcus phage 47 NC 007054 44777 65 65 hylococcus phage 52A NC 007062 41690 60 60 hylococcus phage 53 NC 007049 43883 74 74 hylococcus phage 55 NC 007060 41902 77 77 hylococcus phage 66 NC 007046 18199 27 27 hylococcus phage 69 NC 007048 42732 69 69 hylococcus phage 71 NC 007059 43114 67 67 hylococcus phage 77 NC OO5356 41708 69 69 hylococcus phage 80alpha NC O09526 43864 73 73 hylococcus phage 85 NC 007050 44283 71 71 hylococcus phage 88 NC 007063 43231 66 66 hylococcus phage 92 NC 007064 42431 64 64 hylococcus phage 96 NC 007057 43576 74 74 hylococcus phage CNPH82 NC 008722 4342O 65 65 hylococcus phage EW NC 007056 45286 77 77 hylococcus phage G1 NC 007066 138715 214 214 hylococcus phage K NC OO5880 127395 115 115 hylococcus phage PH15 NC 008723 44041 68 68 hylococcus phage PT1028 NC 007045 15603 22 22 hylococcus phage PVL NC 002321 41401 62 62 hylococcus phage ROSA NC 007058 431 SS 74 74 hylococcus phage SAP-2 NC OO9875 17938 2O 2O hylococcus phage Twort NC 007021 1307O6 195 195 hylococcus phage X2 NC 007065 43440 77 77 hylococcus phage phi 12 NC 004616 44970 49 49 hylococcus phage phil3 NC 004617 42722 49 49 hylococcus phage phi2958PVL NC 011344 47342 60 59 hylococcus phage phiETA NC OO3288 43O81 66 66 hylococcus phage phiETA2 NC 008798 4326S 69 69 hylococcus phage phiETA3 NC 008799 43282 68 68 hylococcus phage phiMR11 NC 01 0147 4301.1 67 67 hylococcus phage phiMR25 NC 010808 44342 70 70 hylococcus phage phiN315 NC 004740 44082 65 64 hylococcus phage phiNM NC OO8583 43128 64 64 hylococcus phage phiNM3 NC OO8617 44061 65 65 US 2010/0322903A1 Dec. 23, 2010 60

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 Staphylococcus phage phiPVL108 NC OO8689 44857 59 O 59 Staphylococcus phage phiSLT NC 002661 42942 61 O 61 Staphylococcus phage phiSauS NC 011612 45344 62 O 62 IPLA35 Staphylococcus phage phiSauS NC 011614 42526 l 60 O 61 IPLA88 aphylococcus phage tp310-1 NC OO9761 41.407 59 59 aphylococcus phage tp310-2 NC OO9762 45710 67 67 aphylococcus phage tp310-3 NC OO9763 41966 58 58 aphylococcus prophage phiPV83 NC OO2486 45636 65 65 enotrophomonas phage S1 NC 011589 40287 48 48 enotrophomonas phage phiSMA9 NC 007189 6907 7 7 re OCOCCS phage 2972 NC 007019 34704 44 44 re OCOCCS hage 72.01 NC 002185 3S466 46 46 re OCOCCS hage 858 NC 010353 35543 46 46 re OCOCCS hage C1 NC 004814 16687 2O 2O re OCOCCS hage Cp-1 NC OO1825 19343 25 25 re OCOCCS hage DT1 NC OO2O72 34.815 45 45 re OCOCCS hage E.J-1 NC 005294 42935 73 73 re OCOCCS hage MM1 NC OO3050 40248 53 53 re OCOCCS hage O1205 NC 004303 43075 57 57 re OCOCCS hage P9 NC 009819 40539 53 53 re OCOCCS hage PH15 NC 010945 391.36 60 60 re OCOCCS hage SM1 NCOO4996 34692 56 56 re OCOCCS hage SMP NC 008721 36,216 48 48 re OCOCCS hage Sfil1 NC OO2214 398O7 53 53 re OCOCCS hage Sfil 9 NC OOO871 37370 45 45 re OCOCCS hage Sf21 NC OOO872 4O739 50 50 re OCOCCS hage phi3396 NC OO9018 38528 64 64 re OCOCCS pyogenes phage 315.1 NC OO4584 39538 56 56 re OCOCCS pyogenes phage 315.2 NC OO4585 41072 60 61 re OCOCCS pyogenes phage 315.3 NC OO4586 34419 52 52 re OCOCCS pyogenes phage 315.4 NC OO4587 41796 64 64 re OCOCCS pyogenes phage 315.5 NC OO4588 382O6 55 55 re OCOCCS pyogenes phage 315.6 NC OO4589 40014 51 51 re omyces phage VWB NC 005.345 4922O 61 61 re omyces phage mu1/6 NC 007967 38.194 52 52 re omyces phage phiBT1 NC 004664 41831 55 56 re omyces phage phiC31 NC OO1978 41491 53 S4 tX1 converting phage NC 004913 S9866 167 166 bx2 converting phage I NC OO3525 61765 166 166 bx2 converting phage II NC 004.914 627O6 170 169 bx2-converting phage 1717 NC O11357 621.47 77 81 Stx2-converting phage 86 NC 008464 6O238 81 8O Sulfolobus islandicus filamentous NC OO3214 4O900 73 73 virus Sulfolobus islandicus rod-shaped virus 1 NC 004087 323O8 45 45 Sulfolobus islandicus rod-shaped virus 2 NC 004086 35450 S4 S4 Sulfolobus spindle-shaped virus 4 NC 009986 15135 34 34 Sulfolobus spindle-shaped virus 5 NC 011217 15330 34 34 Sulfolobus turreted icosahedral virus NC O05892 17663 36 36 Sulfolobus virus 1 NC OO1338 1546S 32 33 Sulfolobus virus 2 NC OO5265 14796 34 34 Sulfolobus virus Kamchatka 1 NC OO5361 17385 31 31 Sulfolobus virus Ragged Hills NC OO5360 16473 37 37 Sulfolobus virus STSV1 NC OO6268 75294 74 74 Synechococcus phage P60 NC OO3390 47872 8O 8O Synechococcus phage S-PM2 NC OO6820 19628O 236 238 Synechococcus phage Syn5 NC 009531 46214 61 61 Synechococcus phage syn9 NC O08296 177300 226 232 Temperate phage phiNIH1.1 NC OO3157 41796 55 55 Thalassomonas phage BA3 NC 009990 373.13 47 47 Thermoproteus tenax spherical virus 1 NC OO6556 20933 38 38 Thermus phage IN93 NC 004.462 19603 40 32 Thermus phage P23-45 NC 0098O3 842O1 117 117 Thermus phage P74-26 NC 009804 83319 116 116 Thermus phage phiYS40 NC OO8584 152372 170 170 US 2010/0322903A1 Dec. 23, 2010 61

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 Vibrio phage K139 NC OO3313 33106 in 44 44 Vibrio phage KSF-1 phi NC OO6294 7107 in 12 12 Vibrio phage KVP40 NC 005083 244834 in 381 415 Vibrio phage VGJphi NC 004.736 7542 in 13 13 Vibrio phage VHML NC 004.456 431.98 in 57 57 Vibrio phage VP2 NC OO5879 39853 in 47 47 Vibrio phage VP5 NC O05891 39.786 in 48 48 Vibrio phage VP882 NC OO9016 38.197 in 71 71 Vibrio phage VSK NC OO3327 6882 in 14 14 Vibrio phage Vf12 NC OO5949 7965 in 7 7 Vibrio phage Vf33 NC 005.948 7965 in 7 7 Vibrio phage V fo3K6 NC 002362 8784 in 10 10 Vibrio phage V fo4K68 NC 002363 6891 in 8 8 Vibrio phage fs NC 004306 6340 in 15 15 Vibrio phage fs2 NC OO1956 8651 in 9 9 Vibrio phage kappa NC 010275 33134 in 45 45 Vibriophage VP4 NC 007149 39SO3 in 31 31 Vibriophage VpV262 NC OO3907 46O12 in 67 67 Xanthomonas phage Cflic NC OO1396 73O8 in 9 9 Xanthomonas phage OP1 NC 007709 43785 in 59 59 Xanthomonas phage OP2 NC OO7710 46643 in 62 62 Xanthomonas phage Xopa-11 NC 009543 44520 in 58 58 Xanthomonas phage Xp10 NC 004902 44373 in 60 60 Xanthomonas phage Xp15 NC 007024 55770 in 84 84 Yersinia pestis phage phi A1122 NC OO4777 37555 in 50 50 Yersinia phage Berlin NC 008694 38564 in 45 45 Yersinia phage L-413C NC 004745 30728 in 40 40 Yersinia phage PY54 NC 005069 46339 in 67 66 Yersinia phage Yepe2 NC 01.1038 38677 in 46 46 Yersinia phage phiYeO3-12 NC OO1271 39600 in 59 59

TABLE 6

Exam ples 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 IO500 inducible pBadaraC promoter 1210 I13453 Pbad promoter 130 7 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 RecA D 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 LacC1 884 4938 sluA double lexO LacC2 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 7 900S T7 Promoter 23 I732205 NOT Gate Promoter Family Member (D001O55) 124 J13002 TetR repressed POPS/RIPS generator 74 J13023 3OC6HSL + LuxR dependent POPS/RIPS generator 117 J23100 constitutive promoter family member 35 J23101 constitutive promoter family member 35 US 2010/0322903A1 Dec. 23, 2010 62

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 J23102 constitutive promoter family member 35 BBa J23103 constitutive promoter family member 35 BBa J23104 constitutive promoter family member 35 BBa J23105 constitutive promoter family member 35 BBa J23106 constitutive promoter family member 35 BBa J23107 constitutive promoter family member 35 BBa J23108 constitutive promoter family member 35 BBa J23109 constitutive promoter family member 35 BBa J23110 constitutive promoter family member 35 BBa J23111 constitutive promoter family member 35 BBa J23112 constitutive promoter family member 35 BBa J23113 constitutive promoter family member 35 BBa J23114 constitutive promoter family member 35 BBa J23115 constitutive promoter family member 35 BBa J23116 constitutive promoter family member 35 BBa J23117 constitutive promoter family member 35 BBa J23118 constitutive promoter family member 35 BBa J44002 pBAD reverse 130 BBa J52010 NFkappaB-dependent promoter 814 BBa JS2O34 CMV promoter 654 BBa J61043 flhF2 Promoter 269 BBa Jó3005 yeast ADH1 promoter 1445 BBa Jó3006 yeast GAL1 promoter S49 BBa K082017 general recombine system 89 BBa K091110 LacI Promoter 56 BBa K091111 LacIQ promoter 56 BBa K094120 placI?ara-1 103 BBa K100000 Natural Xylose Regulated Bi-Directional Operator 303 BBa K100001 Edited Xylose Regulated Bi-Directional Operator 1 303 BBa K100002 Edited Xylose Regulated Bi-Directional Operator 2 303 BBa K118011 Pest A (glucose-repressible promoter) 131 BBa K135000 pCpXR (CpxR responsive promoter) 55 BBa K137029 constitutive promoter with (TA) 10 between-10 and -35 39 elements BBa K137030 constitutive promoter with (TA)9 between-10 and -35 37 elements BBa K137046 150 bp inverted tetR promoter 150 BBa K137047 250 bp inverted tetR promoter 250 BBa K137048 350 bp inverted tetR promoter 350 BBa K137049 450 bp inverted tetR promoter 450 BBa K137050 650 bp inverted tetR promoter 6SO BBa K137051 850 bp inverted tetR promoter 8SO BBa ROO10 promoter (lacI regulated) 2OO BBa ROO11 Promoter (lacIregulated, lambda pl. hybrid) 55 BBa R0053 Promoter (p22 cII regulated) S4 BBa I1010 cI(1) fused to tetR promoter 834 BBa. I1051 Lux cassette right promoter 68 BBa I12006 Modified lamdba Prm promoter (repressed by 434 cI) 82 BBa I12036 Modified lamdba Prm promoter (cooperative repression by 434 91 cI) BBa I12040 Modified lambda P(RM) promoter: -10 region from P(L) and 91 cooperatively repressed by 434 cI BBa I13005 Promoter ROO11 w/YFP (-LVA) TT 920 BBa I13006 Promoter R0040 w/YFP (-LVA) TT 920 BBa I14015 P(Las) TetO 170 BBa I14016 P(Las) CIO 168 BBa I14017 P(Rhl) 51 BBa I14018 P(Bla) 35 BBa I14033 P(Cat) 38 BBa I14034 P(Kat) 45 BBa I714890 OR321 of PR and PRM 121 BBa. I714925 RecA DlexO DLacO2 862 BBa I714926 RecA DlexO DLacO3 862 BBa I714928 RecA S WTexO DLacO2 862 BBa I714931 RecA D consenLexO lacO2 862 BBa I718018 dap Ap promoter 81 BBa I720001 AraBp->rpoN 1632 BBa I720002 glnKp->lacI 1284 BBa I720003 NifHip->cI (lambda) 975 US 2010/0322903A1 Dec. 23, 2010 63

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. I720005 NifA lacIRFP 3255 BBa I720006 GFP glng cI 291.3 BBa I720007 araBp->rpoN (leucine landing pad) 51 BBa I720008 Ara landing pad (pBBLP 6) 2O BBa I720009 Ara landing pad (pBBLP 7) 23 BBa I720010 Ara landing pad (pBBLP 8) 2O BBa I721001 Lead Promoter 94 BBa. I723020 Pu 32O BBa I728456 MerRT: Mercury-Inducible Promoter + RBS (MerR + part of 635 MerT) BBa I741018 Right facing promoter (for xylF) controlled by xylR and CRP- 221 cAMP BBa I742124 Reverse complement Lac promoter 2O3 BBa I746104 P2 promoter in agroperon from S. aureus 96 BBa I746360 PF promoter from P2 phage 91 BBa I746361 PO promoter from P2 phage 92 BBa I746362 PP promoter from P2 phage 92 BBa I746364 Psid promoter from P4 phage 93 BBa I746365 PLL promoter from P4 phage 92 BBa I748001 Putative Cyanide Nitrilase Promoter 271 BBa I752000 Riboswitch(theophylline) 56 BBa I761011 CinR, CinL and glucose controlled promotor 295 BBa I761014 cinr + cinl (RBS) with double terminator 1661 BBa I764001 Ethanol regulated promoter AOX1 867 BBa I765000 Fe promoter 104.4 BBa I765.001 UV promoter 76 BBa I765.007 Fe and UV promoters 1128 BBa J13210 pompR dependent POPS producer 245 BBa J22106 recA (SOS) Promoter 192 BBa J23119 constitutive promoter family member 35 BBa J24669 Tri-Stable Toggle (Arabinose induced component) 31OO BBa J3902 PrFe (PI+ PII rus operon) 272 BBa J58100 AND-type promoter synergistically activated by cI and CRP 106 BBa J61051 Psal1 1268 BBa K085005 (lacI)promoter->key3c->Terminator 40S BBa KO88007 GlnRS promoter 38 BBa KO89004 phaC Promoter (-663 from ATG) 663 BBa KO89005 -35 to Tc start site of phaC 49 BBa KO89006 -663 to Tc start site of phaC 361 BBa KO90501 Gram-Positive IPTG-Inducible Promoter 107 BBa KO90504 Gram-Positive Strong Constitutive Promoter 239 BBa K091 100 pLac lux hybrid promoter 74 BBa K091101 pTet Lac hybrid promoter 83 BBa KO91104 plac/Mnt Hybrid Promoter 87 BBa KO91105 pTet/Mnt Hybrid Promoter 98 BBa K091106 LSrA?cI hybrid promoter 141 BBa KO91107 pLux/cI Hybrid Promoter 57 BBa K091114 LSrARPromoter 248 BBa K091115 LSrRPromoter 1OO BBa K091116 LSrA Promoter 126 BBa K091117 plas promoter 126 BBa K091143 pLasicI Hybrid Promoter 164 BBa K091146 plasiLux Hybrid Promoter 126 BBa K091184 pLux/cI+ RBS + LuxS + RBS + Mint + TT + pLac/Mint + RBS + LuxS + 2616 RBS -- cI - TT BBa K093000 pRecA with Lex A binding site 48 BBa K101017 MioC Promoter (DNAa-Repressed Promoter) 319 BBa K101018 MioC Promoter (regulating tetR) 969 BBa K105020 tetR- operator 29 BBa K105021 cI - operator 27 BBa K105022 lex A- operator 31 BBa K105023 lac I - operator 25 BBa K105024 GalA - operator 27 BBa K105026 Gall promoter S49 BBa K105027 cyclOO minimal promoter 103 BBa K105028 cyc70 minimal promoter 103 BBa K105029 cyc43 minimal promoter 103 BBa K105030 cyc28 minimal promoter 103 BBa K105031 cyclé minimal promoter 103 US 2010/0322903A1 Dec. 23, 2010 64

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 K108014 PR 234 BBa K108016 PP 4O6 BBa K108025 Pu 2OO BBa K109200 AraC and TetR promoter (hybrid) 132 BBa K110005 Alpha-Cell Promoter MF(ALPHA)2 500 BBa K110006 Alpha-Cell Promoter MF(ALPHA)1 5O1 BBa K110016 A-Cell Promoter STE2 (backwards) 500 BBa K112118 rrnBP1 promoter 503 BBa K112318 {

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 ROO61 Promoter (HSL-mediated luxR repressor) 30 BBa ROO63 Promoter (luxR & HSL regulated -- lux pl.) 151 BBa R0065 Promoter (lambda cI and luxR regulated -- hybrid) 97 BBa R0071 Promoter (RhlR & C4-HSL regulated) 53 BBa ROO73 Promoter (Mnt regulated) 67 BBa ROO74 Promoter (PenIregulated) 77 BBa ROO75 Promoter (TP901 cI regulated) 117 BBa ROOT7 Promoter (cinRand HSL regulated, RBS--) 231 BBa R0078 Promoter (cinRand HSL regulated) 225 BBa R0081 inhibitor (AraC loop attachment with O2 site) 183 BBa R0082 Promoter (OmpR, positive) 108 BBa ROO83 Promoter (OmpR, positive) 78 BBa R0084 Promoter (OmpR, positive) 108 BBa R1050 Promoter, Standard (HK022 cI regulated) 56 BBa R1051 Promoter, Standard (lambda cI regulated) 49 BBa R1052 Promoter, Standard (434 cI regulated) 46 BBa R1053 Promoter, Standard (p22 cII regulated) 55 BBa R1062 Promoter, Standard (luxR and HSL regulated -- lux pR) 56 BBa R2000 Promoter, Zif23 regulated, test: between 45 BBa R2001 Promoter, Zif23 regulated, test: after 52 BBa R2002 Promoter, Zif23 regulated, test: between and after 52 BBa R2109 Promoter with operator site for C2003 72 BBa R2114 Promoter with operator site for C2003 72 BBa. I10498 Oct-4 promoter 1417 BBa I12001 Promoter (PRM+) 96 BBa. I12003 Lambda Prm Promoter 88 BBa I12005 ambda Prm Inverted Antisense (No start codon) 85 BBa. I12008 Barkai-Leibler design experiment part A (p22cII) 1154 BBa I12010 Modified lamdba Prm promoter (repressed by p22 cII) 78 BBa I12014 Repressor, 434 cI (RBS-LVA-) 636 BBa I12021 inducible Lambda cI Repressor Generator (Controlled by IPTG 2370 and LacI) BBa I12031 Barkai-Leibler design experiment Part A (Lambda cI) with 1159 cooperativity BBa I12032 Modified lamdba Prm promoter (repressed by p22 cI with 106 cooperativity) RBS-- BBa I12034 Modified lamdba Prm promoter (repressed by 434 cI with 102 cooperativity) RBS-- BBa I12035 Modified lamdba Prm promoter (repressed by p22 cI without 106 cooperativity) RBS-- BBa I12037 Reporter 3 for Barkai-Leibler oscillator 1291 BBa I12044 Activator for BL oscillator with reporter protein, 2112 (cooperativity) BBa 12045 BL oscillator, cooperativity, reporter protein, kickstart 4139 BBa I12046 Activator for BL oscillator with reporter protein, (cooperativity 2112 and L-strain -10 region) BBa 12047 BL oscillator, cooperativity + replaced -10 region (Lilac), 4139 reporter protein, kickstart BBa I12210 plac Or2-62 (positive) 70 BBa I12212 TetR-TetR-4C heterodimer promoter (negative) 61 BBa I12219 Wild-type TetR(B) promoter (negative) 71 BBa I13062 LuxRQPI 822 BBa I13267 intermediate part from assembly 317 1769 BBa I13406 Pbadi AraC with extra REN sites 1226 BBa. I14021 plTetO1.RBS.Cin 810 BBa I2O255 Promoter-RBS 57 BBa I2O256 Promoter-RBS 56 BBa. I2O258 Promoter-RBS 56 BBa I714932 RecA D consenLexO lacO3 862 BBa I715003 hybrid pLac with UV5 mutation 55 BBa I715052 Trp Leader Peptide and anti-terminator?terminator 134 BBa I715053 Trp Leader Peptide and anti-terminator?terminator with hixC 159 insertion BBa I717002 Pr from lambda Switch 177 BBa I723011 pontR (estimated promoter for DntR) 26 BBa I723013 ponta (estimated promoter for DntA) 33 BBa I723018 Pr (promoter for XylR) 410 BBa I731004 FecA promoter 90 BBa I732021 Template for Building Primer Family Member 159 US 2010/0322903A1 Dec. 23, 2010 66

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 I732200 NOT Gate Promoter Family Member (D001Olwt1) 25 Ba I732201 NOT Gate Promoter Family Member (D001O11) 24 Ba I732202 NOT Gate Promoter Family Member (D001O22) 24 Ba I732203 NOT Gate Promoter Family Member (D001O33) 24 Ba I732204 NOT Gate Promoter Family Member (D001O44) 24 Ba I732206 NOT Gate Promoter Family Member (D001O66) 24 Ba I732207 NOT Gate Promoter Family Member (D001O77) 24 Ba I732270 Promoter Family Member with Hybrid Operator (D001O12) 24 Ba I732271 Promoter Family Member with Hybrid Operator (D001O16) 24 Ba I732272 Promoter Family Member with Hybrid Operator (D001O17) 24 Ba I732273 Promoter Family Member with Hybrid Operator (D001O21) 24 Ba I732274 Promoter Family Member with Hybrid Operator (D001O24) 24 Ba I732275 Promoter Family Member with Hybrid Operator (D001O26) 24 Ba I732276 Promoter Family Member with Hybrid Operator (D001O27) 24 Ba I732277 Promoter Family Member with Hybrid Operator (D001O46) 24 Ba I732278 Promoter Family Member with Hybrid Operator (D001O47) 24 Ba I732279 Promoter Family Member with Hybrid Operator (D001O61) 24 Ba I732301 NAND Candidate (U073O26D001O16) 2O Ba I732302 NAND Candidate (U073O27 D001O17) 2O Ba I732303 NAND Candidate (U073O22D001O46) 2O Ba I732304 NAND Candidate (U073O22D001O47) 2O Ba I732305 NAND Candidate (U073O22D059O46) 78 Ba I732306 NAND Candidate (U073O11 D002O22) 21 Ba I732351 NOR Candidate (UO37O11 D002O22) 85 Ba I732352 NOR Candidate (UO35O44D001O22) 82 Ba I732400 Promoter Family Member (U097NUL + D062NUL) 65 Ba I732401 Promoter Family Member (UO97O11 + D062NUL) 85 Ba I732402 Promoter Family Member (UO85O11 + D062NUL) 73 Ba I732403 Promoter Family Member (U073O11 + D062NUL) 61 Ba I732404 Promoter Family Member (U061O11 + D062NUL) 49 Ba I732405 Promoter Family Member (U049011 + D062NUL) 37 Ba I732406 Promoter Family Member (UO37O11 + D062NUL) 25 Ba I732407 Promoter Family Member (UO97NUL + D002O22) 25 Ba I732408 Promoter Family Member (UO97NUL + D014O22) 37 I732409 Promoter Family Member (UO97NUL + D026O22) 49 Ba I732410 Promoter Family Member (UO97NUL + D038O22) 61 Ba I732411 Promoter Family Member (UO97NUL + D050O22) 73 Ba I732412 Promoter Family Member (UO97NUL + D062O22) 85 Ba I732413 Promoter Family Member (UO97O D002O22) 45 Ba I732414 Promoter Family Member (UO97O D014O22) 57 Ba I732415 Promoter Family Member (UO97O D026O22) 69 Ba I732416 Promoter Family Member (UO97O D038O22) 81 Ba I732417 Promoter Family Member (UO97O D050O22) 93 Ba I732418 Promoter Family Member (UO97O D062O22) 205 Ba I732419 Promoter Family Member (UO85O D002O22) 33 Ba I732420 Promoter Family Member (UO85O D014O22) 45 Ba I732421 Promoter Family Member (UO85O D026O22) 57 Ba I732422 Promoter Family Member (UO85O D038O22) 69 Ba I732423 Promoter Family Member (UO85O D050O22) 81 Ba I732424. Promoter Family Member (UO85O D062O22) 93 Ba I732425 Promoter Family Member (U073O D002O22) 21 Ba I732426 Promoter Family Member (U073O D014O22) 33 Ba I732427 Promoter Family Member (U073O D026O22) 45 Ba I732428 Promoter Family Member (U073O D038O22) 57 Ba I732429 Promoter Family Member (U073O D050O22) 69 Ba I732430 Promoter Family Member (U073O D062O22) 81 Ba I732431 Promoter Family Member (U061O D002O22) 09 Ba I732432 Promoter Family Member (U061O D014O22) 21 Ba I732433 Promoter Family Member (U061O D026O22) 33 Ba I732434 Promoter Family Member (U061O D038O22) 45 Ba I732435 Promoter Family Member (U061O D050O22) 57 Ba I732436 Promoter Family Member (U061O D062O22) 69 Ba I732437 Promoter Family Member (U0490 D002O22) 97 Ba I732438 Promoter Family Member (U0490 D014O22) 09 Ba I732439 Promoter Family Member (U0490 D026O22) 21 Ba I732440 Promoter Family Member (U0490 D038O22) 33 Ba I732441 Promoter Family Member (U0490 D050O22) 45 Ba I732442 Promoter Family Member (U0490 D062O22) 57 Ba I732443 Promoter Family Member (UO37O D002O22) 85 US 2010/0322903A1 Dec. 23, 2010 67

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 I732444 Promoter Family Member (UO37O11 + D014O22) 97 BBa I732445 Promoter Family Member (UO37O11 + D026O22) 109 BBa I732446 Promoter Family Member (UO37O11 + D038O22) 121 BBa I732447 Promoter Family Member (UO37O11 + D050O22) 133 BBa I732448 Promoter Family Member (UO37O11 + D062O22) 145 BBa I732450 Promoter Family Member (U073O26+ D062NUL) 161 BBa I732451 Promoter Family Member (U073O27 + D062NUL) 161 BBa I732452 Promoter Family Member (U073O26+ D062O61) 181 BBa I735008 ORE1X Oleate response element 273 BBa I735009 ORE2X oleate response element 332 BBa I735.010 This promoter encoding for a thiolase involved in beta- 8SO oxidation of fatty acids. BBa I739101 Double Promoter (constitutive?TetR, negative) 83 BBa I739102 Double Promoter (cI, negative/TetR, negative) 97 BBa I739103 Double Promoter (lacI, negative/P22 cII, negative) 87 BBa I739104 Double Promoter (LuxRHSL, positive? P22 cII, negative) 101 BBa I739105 Double Promoter (LuxRHSL, positive/cI, negative) 99 BBa I739106 Double Promoter (TetR, negative/P22 cII, negative) 84 BBa I739107 Double Promoter (cI, negative/LacI, negative) 78 BBa I741015 two way promoter controlled by XylR and Crp-CAmp 301 BBa I741017 dual facing promoter controlled by xylRand CRP-cAMP 3O2 (I741015 reverse complement) BBa I741019 Right facing promoter (for xylA) controlled by xylR and CRP- 131 cAMP BBa I741020 promoter to XylF without CRP and several binding sites for 191 xylR BBa I741021 promoter to XylA without CRP and several binding sites for 87 XylR BBa I741109 Lambda Or operator region 82 BBa I742126 Reverse lambda cI-regulated promoter 49 BBa I746363 PV promoter from P2 phage 91 BBa I746665 Pspac-hy promoter 58 BBa I751500 pcI (for positive control of pcI-lux hybrid promoter) 77 BBa I751501 plux-cI hybrid promoter 66 BBa I751502 plux-lachybrid promoter 74 BBa I756002 Kozak Box 7 BBa I756014 Lex Aoperator-MajorLatePromoter 229 BBa I756015 CMV Promoter with lac operator sites 663 BBa I756016 CMV-tet promoter 610 BBa I756O17 U6 promoter with tet operators 341 BBa I756O18 Lambda Operator in SV-40 intron 411 BBa I756O19 Lac Operator in SV40 intron 444 BBa I756020 Tet Operator in SV40 intron 391 BBa I756021 CMV promoter with Lambda Operator 630 BBa I760005 Cu-sensitive promoter 16 BBa I761000 cinr + cinl (RBS) 1558 BBa I761001 OmpR binding site 62 BBa I766200 pSte2 1OOO BBa I766214 pCal1 10O2 BBa I766555 pCyc (Medium) Promoter 244 BBa I766556 p.Adh (Strong) Promoter 15O1 BBa I766557 pSte5 (Weak) Promoter 6O1 BBa I766558 pFig1 (Inducible) Promoter 1OOO BBa I9201 ambda cI operatoribinding site 82 BBa J01005 pspoIIE promoter (spoOAJO1004, positive) 2O6 BBa JO1006 Key Promoter absorbs 3 59 BBa J03007 Maltose specific promotor 2O6 BBa JO3100 -- No description -- 847 BBa JO4700 Part containing promoter, riboswitch mTCT8-4 theophylline 258 aptamer (JO4705), and RBS BBa JO4705 Riboswitch designed to turn “ON” a protein 38 BBa JO4800 O4800 (Rev AptRibo) contains a theophylline aptamer 258 upstream of the RBS that should act as a riboswi BBa JO4900 Part containing promoter, 8 bp, RBS, and riboswitch mTCT8-4 258 heophylline aptamer (JO4705) BBa JO5209 Modifed PrPromoter 49 BBa J05210 Modifed Prm-- Promoter 82 BBa JO5215 Regulator for R1-CREBH 41 BBa J05216 Regulator for R3-ATF6 41 US 2010/0322903A1 Dec. 23, 2010 68

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 JO5217 Regulator for R2-YAP7 41 BBa J05218 Regulator for R4-cMaf 41 BBa J05221 Tripple Binding Site for R3-ATF6 62 BBa J05222 ZF-2*e2 Binding Site 37 BBa J05500 Sensing Device A (cI) 2371 BBa J05501 Sensing Device B (cI+ LVA) 2337 BBa J06403 RhIR promoter repressible by CI 51 BBa JO7007 ctX promoter 145 BBa J07010 ToxR inner (aa's 1-198; cytoplasm +TM) 594 BBa J07019 Feca Promoter (with Fur box) 86 BBa JO7041 POPS/RIPS generator (R0051:B0030) 72 BBa J07042 POPS/RIPS generator (R0040::B0030) 77 BBa J1 1003 control loop for PI controller with BBa J1 1002 961 BBa J13211 ROO4.O.BOO32 75 BBa J13212 R0040.B0033 73 BBa J15301 Pars promoter from Escherichia coli chromosomalars operon. 127 BBa J15502 copA promoter 287 BBa J16101 BanAp - Banana-induced Promoter 19 BBa J16105 HelPp - “Help' Dependant promoter 26 BBa J16400 ron sensitive promoter (test delete later) 26 BBa J21002 Promoter + LuxR 998 BBa J21003 Promoter - TetR 904 BBa J21004 Promoter + LacL 1372 BBa J21006 LuxR, TetR Generator 1910 BBa J21007 LuxR, TetR, LacL Generator 3290 BBa J22052 Pcya 65 BBa J22086 pX (DnaA binding site) 125 BBa J22126 RecA (SOS) promoter 186 BBa J23150 bp mutant from J23107 35 BBa J23151 bp mutant from J23114 35 BBa J24000 CafAp (Cafeine Dependant promoter) 14 BBa J24001 WigLp (Wiggle-dependent Promotor) 46 BBa J24670 Tri-Stable Toggle (Lactose induced component) 1877 BBa J24671 Tri-Stable Toggle (Tetracycline induced component) 2199 BBa J24813 ORA3 Promoter from S. cerevisiae 137 BBa J26003 Mushroom Activated Promoter 23 BBa J31013 plac Backwards (cf. BBa R0010 2OO BBa J31014 crRNA 38 BBa J3102 bBad:RBS 153 BBa J31020 produces taRNA 295 BBa J31022 comK transcription activator from B. subtilis 578 BBa J33100 Arsk and Ars Promoter 472 BBa J34800 Promoter tetracyclin inducible 94 BBa J34.806 promoter lac induced 112 BBa J34809 promoter lac induced 125 BBa J34814 T7 Promoter 28 BBa J45503 hybB Cold Shock Promoter 393 BBa J45504 htpG Heat Shock Promoter 40S BBa J45992 Full-length stationary phase osmy promoter 199 BBa J45993 Minimal stationary phase osmY promoter 57 BBa J45994 Exponential phase transcriptional control device 1109 BBa J48103 ron promoter 140 BBa J48104 NikR promoter, a protein of the ribbon helix-helix family of 40 trancription factors that repress expre BBa J48106 winfh 891 BBa J48107 UGT008-3 Promoter/Met32p 588 BBa J48110 Fe Promoter-mRFP1 1009 BBa J48111 E. Coi NikR 926 BBa J48112 VnfH: vanadium promoter 1816 BBa J49000 Roid Rage 4 BBa J49001 Testosterone dependent promoter for species Bicyclus Bicyclus 89 BBa J49006 Nutrition Promoter 3 BBa J4906 WrooHEAD2 (Wayne Rooney's Head dependent promoter) 122 BBa J54015 Protein Binding Site LacI 42 BBa J54016 promoter lacq S4 BBa J54017 promoter always 98 BBa JS4018 promoter always 98 BBa J54101 deltaP-GFP(A) BBa J54102 DeltaP-GFP(A) 813 US 2010/0322903A1 Dec. 23, 2010 69

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 J54110 MelR regulated promoter 76 BBa J54120 EmrR regulated promoter 46 BBa J54130 BetI regulated promoter 46 BBa J54200 acq Promoter 50 BBa J54210 RbsR Binding Site 37 BBa J54220 FadR Binding Site 34 BBa J54230 TetR regulated 38 BBa J54250 LacI Binding Site 42 BBa J56012 nvertible sequence of dna includes Ptrc promoter 409 BBa J56015 acIQ - promoter sequence 57 BBa J61045 spv spv operon (PoPS out) 1953 BBa J61054 HIP-1 Promoter 53 BBa J61055 HIP-1 fmr. Promoter 53 BBa Jó4000 rhill promoter 72 BBa J64001 psicA from Saimoneiia 143 BBa J64010 asI promoter 53 BBa Jó4065 cI repressed promoter 74 BBa J64067 LuxR+ 3OC6HSL independent ROO65 98 BBa Jó4068 increased strength R0051 49 BBa J64069 ROO65 with luxbox deleted 84 BBa J64700 Trp Operon Promoter 616 BBa J64712 LasR/LasI Inducible & RHLR/RHLI repressible Promoter 157 BBa Jó4750 SPI-1 TTSS secretion-linked promoter from Salmonella 167 BBa J64800 RHLR/RHILI Inducible & LasR/LasIrepressible Promoter 53 BBa Jó4804 The promoter region (inclusive of regulator binding sites) of 135 he B. subtilis RocDEF operon BBa Jó4931 glnKp promoter 147 BBa Jó4951 E. Coli CreABCD phosphate sensing operon promoter 81 BBa Jó4979 gln Ap2 151 BBa Jó4980 OmpR-P strong binding, regulatory region for Team ChallengeO3-2007 BBa Jó4981 OmpR-P strong binding, regulatory region for Team 82 ChallengeO3-2007 BBa Jó4982 OmpR-P strong binding, regulatory region for Team Challenge 25 O3-2007 BBa Jó4983 Strong OmpR Binding Site 2O BBa J64986 LacI Consensus Binding Site 2O BBa J64987 LacI Consensus Binding Site in sigma 70 binding region 32 BBa J64991 TetR 19 BBa J64995 Phage-35 site 6 BBa J64997 T7 consensus-10 and rest 19 BBa J64998 consensus-10 and rest from SP6 19 BBa J70025 Promoter for tetM gene, from pBOT1 plasmid, p AMbeta1 345 BBa J72005 Ptet: promoter in BBb S4 BBa KO76O17 Ubc Promoter 1219 BBa K078101 aromatic compounds regulatory pcbC promoter 129 BBa K079017 Lac Symmetric - operator library member 2O BBa K079018 Lac 1 - operator library member 21 BBa K079019 Lac 2 - operator library member 21 BBa KO79036 Tet O operator library member 15 BBa KO79037 TetO-4C - operator library member 15 BBa KO79038 TetO-wt/4C5G - operator library member 15 BBa K079039 LexA 1 - operaor library member 16 BBa K079040 LexA2 - Opeartor library member 16 BBa K079041 Lambda OR1 - operator library member 17 BBa K079042 Lambda OR2 - operator library member 17 BBa K079043 Lambda OR3 - operator library member 17 BBa K079045 Lac operator library 78 BBa KO79046 Tet operator library 61 BBa K079047 Lambda operator library 67 BBa K079048 LexA operator library 40 BBa KO.80000 TCFbs-BMP4 1582 BBa KO80001 A20 alpha cardiac actin miniPro-BMP4 1402 BBa KO80003 CMV-rtTA 1413 BBa K08.0005 TetO (TRE)-nkx2.5-fmdv2A-dsRed 2099 BBa K08.0006 TetO (TRE)-gata4-fmdv2A-dsRed 2447 BBa K080008 TetO (TRE)-nkX-2.5-fmdv2A-gata4-fmdv2A-dsRed 3497 BBa KO85004 riboswitch system with GFP 1345 BBa KO85006 pTet->lock3d->GFP->Ter 932 US 2010/0322903A1 Dec. 23, 2010 70

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 K086017 unmodified Lutz-Bujard LacO promoter 55 BBa K086018 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o24 BBa K086019 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o24 BBa K086020 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o24 BBa K086021 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o24 BBa K086022 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o28 BBa K086023 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o28 BBa K086024 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o28 BBa K086025 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o28 BBa K086026 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o2 BBa K086027 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o2 BBa K086028 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o2 BBa K086029 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o2 BBa KO86030 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o8 BBa K086031 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o8 BBa K086032 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o8 BBa K086033 modified Lutz-Bujard LacO promoter, with alternative sigma 55 actor o8 BBa KO90502 Gram-Positive Xylose-Inducible Promoter 126 BBa KO90503 Gram-Positive General Constitutive Promoter 91 BBa K091112 placIQ1 promoter 56 BBa KO91156 pLux 55 BBa KO91157 plux/Las Hybrid Promoter 55 BBa KO93008 reverse BBa ROO11 55 BBa K094002 planbda P(O-R12) OO BBa K094140 pLacIq. 8O BBa K100003 Edited Xylose Regulated Bi-Directional Operator 3 303 BBa K101000 Dual-Repressed Promoter for p22 mint and TetR 61 BBa K101001 Dual-Repressed Promoter for LacI and LambdacI 16 BBa K101002 Dual-Repressed Promoter for p22 cII and TetR 66 BBa K102909 TA11 gate from synthetic algorithm v1.1 34 BBa K102910 TA12 gate from synthetic algorithm v1.1 O7 BBa K102911 TA13 gate from synthetic algorithm v1.2 90 BBa K102912 TA12 plus pause sequence O8 BBa K102950 TAOIn null anti-sense input 75 BBa K102951 TA1In anti-sense input to TA1 (BBa K102901) 57 BBa K102952 TA2In anti-sense input to BBa K102952 68 BBa K102953 TA13n anti-sense input to TA3 (BBa K102903) 68 BBa K102954 TA6In anti-sense input to BBa K102904 69 BBa K102955 TA7In anti-sense input to BBa K102.905 68 BBa K102956 TA8In anti-sense input to BBa K102906 68 BBa K102957 TA9In anti-sense input to BBa K102907 73 BBa K102958 TA10In anti-sense input to BBa K102908 83 BBa K102959 TA11 In anti-sense input to BBa K102909 78 BBa K102960 TA12In anti-sense input to anti-terminator BBa K102910 73 BBa K102961 TA13In anti-sense input to BBa K102911 71 BBa K102962 TA14In anti-sense input to BBa K102912 8O BBa K103021 modified T7 promoter with His-Tag 66 BBa K103022 Plac with operator and RBS 279 BBa K106673 8xLexAops-Cyclp 418 BBa K106680 8xLexAops-Fig1P 1169 BBa K106694. Adh1P (Adh1 Promoter, A! end) 1511 BBa K106699 Gal1 Promoter 686 BBa K109584 this is a test part, disregard it US 2010/0322903A1 Dec. 23, 2010 71

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 K110004 Alpha-Cell Promoter Ste3 5O1 BBa K110007 A-Cell Promoter MFA2 5O1 BBa K110008 A-Cell Promoter MFA1 5O1 BBa K110009 A-Cell Promoter STE2 5O1 BBa K110014 A-Cell Promoter MFA2 (backwards) 550 BBa K110015 A-Cell Promoter MFA1 (RtL) 436 BBa K112139 oriR6K conditional replication origin 4.08 BBa K112148 phoPp1 magnesium promoter 81 BBa K112149 PmgtCB Magnesium promoter from Salmonella 28O BBa K112321 H-NS!} using MG1655 reverse oligo in BBb format 414 BBa K112701 hns promoter 669 BBa K112706 Pspv2 from Salmonella 474 BBa K112707 Pspv from Salmonella 1956 BBa K112708 PfhuA 210 BBa K112711 rbs.spvR. 913 BBa K112900 Pbad 1225 BBa K112904 PconB5 41 BBa K112905 PconCS 41 BBa K112906 PconG6 41 BBa K112907 Pcon 41 BBa K113010 overlapping T7 promoter 40 BBa K113011 more overlapping T7 promoter 37 BBa K113012 weaken overlapping T7 promoter 40 BBa K116201 ureD promoter from Pmirabilis BBa K119000 Constitutive weak promoter of lacz 38 BBa K1 19001 Mutated LacZ promoter 38 BBa K120010 Triple lexO 114 BBa K120023 lexA DBD 249 BBa K121011 promoter (lacIregulated) 232 BBa K121014 promoter (lambda cI regulated) 90 BBa K124000 pCYCYeast Promoter 288 BBa K124002 Yeast GPD (TDH3) Promoter 681 BBa K125100 nir promoter from Synechocystis sp. PCC6803 88 BBa K131017 p qrr4 from Vibrio harveyi 275 BBa K137085 optimized (TA) repeat constitutive promoter with 13 bp 31 between-10 and -35 elements BBa K137086 optimized (TA) repeat constitutive promoter with 15 bp 33 between-10 and -35 elements BBa K137087 optimized (TA) repeat constitutive promoter with 17 bp 35 between-10 and -35 elements BBa K137088 optimized (TA) repeat constitutive promoter with 19 bp 37 between-10 and -35 elements BBa K137089 optimized (TA) repeat constitutive promoter with 21 bp 39 between-10 and -35 elements BBa K137090 optimized (A) repeat constitutive promoter with 17 bp between- 35 10 and -35 elements BBa K137091 optimized (A) repeat constitutive promoter with 18 bp between- 36 10 and -35 elements BBa K137124 LacI-repressed promoter A81 103 BBa K143010 Promoter ctic for B. subtiis 56 BBa K143011 Promoter gsiB for B. subtilis 38 BBa K143012 Promoter vega constitutive promoter for B. subtilis 97 BBa K143013 Promoter 43 a constitutive promoter for B. subtilis 56 BBa K143014 Promoter Xyl for B. subtilis 82 BBa K143015 Promoter hyper-spank for B. subtilis 101 BBa K145152 Hybrid promoter: P22 c2, LacI NOR gate 142 BBa K157042 Eukaryotic CMV promoter 654 BBa K165OOO MET 25 Promoter 387 BBa K165015 p.ADH1 yeast constituative promoter 1445 BBa K165017 LexA binding sites 393 BBa K165037 TEF2 yeast constitutive promoter 403 BBa M13101 M13K07 gene I promoter 47 BBa M13102 M13K07 gene II promoter 48 BBa M13103 M13K07 gene III promoter 48 BBa M13104 M13K07 gene IV promoter 49 BBa M13105 M13K07 gene V promoter 50 BBa M13106 M13K07 gene VI promoter 49 BBa M13108 M13K07 gene VIII promoter 47 BBa M13110 M13110 48