REVIEW ARTICLE published: 30 April 2013 doi: 10.3389/fmicb.2013.00103 The intrinsic resistome of bacterial pathogens

Jorge Olivares, Alejandra Bernardini, Guillermo Garcia-Leon, Fernando Corona, Maria B. Sanchez and Jose L. Martinez* Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain

Edited by: Intrinsically resistant bacteria have emerged as a relevant health problem in the last years. Marcelo Tolmasky, California state Those bacterial species, several of them with an environmental origin, present naturally low- University Fullerton, USA level susceptibility to several drugs. It has been proposed that intrinsic resistance is mainly Reviewed by: the consequence of the impermeability of cellular envelopes, the activity of multidrug efflux Morten Otto Alexander Sommer, Technical University of Denmark, pumps or the lack of appropriate targets for a given family of drugs. However, recently Denmark published articles indicate that the characteristic phenotype of susceptibility to antibiotics Alfonso J. Soler-Bistue, Institut of a given bacterial species depends on the concerted activity of several elements, what Pasteur, France has been named as intrinsic resistome. These determinants comprise not just classical *Correspondence: resistance genes. Other elements, several of them involved in basic bacterial metabolic Jose L. Martinez, Centro Nacional de Biotecnología, Consejo Superior de processes, are of relevance for the intrinsic resistance of bacterial pathogens. In the present Investigaciones Científicas, Darwin 3, review we analyze recent publications on the intrinsic resistomes of Escherichia coli and 28049 Madrid, Spain. Pseudomonas aeruginosa. We present as well information on the role that global regulators e-mail: [email protected] of bacterial metabolism, as Crc from P. aeruginosa, may have on modulating bacterial susceptibility to antibiotics. Finally, we discuss the possibility of searching inhibitors of the intrinsic resistome in the aim of improving the activity of drugs currently in use for clinical practice.

Keywords: intrinsic resistance, MDR efflux pump, biofilms, swarming, phenotypic resistance, persistence

Clinical definition of antibiotic resistance is mainly based on the purposes. Consequently, for any bacterial species an intrinsic resis- View metadata, citation and similar papers at core.ac.uk brought to you by CORE bacterial response to treatment. One microorganism is considered tome can be defined, irrespective on whether or not this species as resistant if there exists a high likelihood of therapeutic fail- is classified as intrinsically resistant in the basis of clinical break- provided by Frontiers - Publisher Connector ure upon antibiotic treatment. Consequently, resistance has been points. From the studies on the intrinsic resistome, two categories operationally defined in the basis of breakpoints of the minimal of genes have emerged (Fajardo et al., 2008). Those which inac- inhibitory concentrations (MICs). More recently, an ecological tivation make bacteria more resistant to antibiotics and those definition of antibiotic resistance is emerging, which is based in which inactivation make bacteria more susceptible. The first ones the MIC value identifying the upper limit of the wild-type popu- define elements that are relevant for the acquisition of resistance. lation (Turnidge et al., 2006). This is defined as the ecological cut For instance, the mutation in a transcriptional repressor of a off (ECOFF) value and all strains presenting MICs above ECOFF multidrug (MDR) efflux pump turns the microorganism more are considered resistant from an ecological point of view, even if resistant to antibiotics (Alonso and Martinez, 1997, 2000, 2001). they are classified as susceptible in the basis of clinical breakpoints. Mapping these elements is important to define the capability of an When defining the intrinsic resistome, we are facing the same sit- organism to evolve toward resistance by mutation (Martinez and uation. Bacteria are classically considered intrinsically resistant in Baquero, 2000) and is thus relevant for predicting the evolution the basis of the clinical definition, in other words, if their infections of resistance (Martinez et al., 2007, 2011a). The other group of cannot be treated with a given antibiotic. Three are the most rel- elements define the determinants that contribute to the natural evant causes of this intrinsic resistance: lack of the target, activity phenotype of susceptibility to antibiotics of a given species, and of chromosomally encoded antibiotic-inactivating enzymes and constitute the bona fide intrinsic resistome. Inactivation of these reduced uptake of the antibiotic, the later includes reduced per- elements make bacteria more susceptible to antibiotics, which may meability of the cellular envelopes and activity of efflux pumps be useful for improving efficacy of current drugs (Martinez, 2012). (Nikaido, 1989, 1994; Li et al., 1994; Fernandez and Hancock, This has been the situation of inhibitors of plasmid-encoded β- 2012). More recently, in the same line of the ecological defini- lactamases, which have been demonstrated to be efficient drugs tion of resistance, the “intrinsic resistome” has been defined as the to be used in combination with β-lactams (Reading and Cole, set of elements that contributes directly or indirectly to antibiotic 1977). Similarly, the inhibition of a MDR efflux pump (or another resistance, and whose presence is independent of previous antibi- mechanism of intrinsic resistance) might also improve the effi- otic exposure and is not due to horizontal gene transfer (HGT; cacy of antibiotics currently in use or allow the utilization of Fajardo et al., 2008; Wright, 2010). others (Lomovskaya et al., 1999; Renau et al., 1999; Lomovskaya This definition encompasses all the chromosomally encoded and Watkins, 2001). For instance, macrolides are not used for elements that have not been recently acquired as the consequence treatment of Gram-negative infections because these organisms of the recent human use of antibiotics for therapy and farming are intrinsically resistant to this family of antibiotics. However,

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the major Escherichia coli efflux pump AcrAB extrudes macrolides present in natural ecosystems (Davies, 2006; Linares et al., 2006; and its inactivation might increase the susceptibility of Escherichia Yim et al., 2006, 2007; Fajardo and Martinez, 2008), this situation coli to these antibiotics (Chollet et al., 2004). This evidence indi- allows a complementary view to the traditional weapons/shields cates that macrolides might be useful for treating Gram-negative roles that antibiotics and their resistance genes may have at natu- infections if they are used in combination with an inhibitor of ral ecosystems (Martinez, 2008; Aminov, 2009, 2010; Fajardo et al., MDR efflux pumps. 2009; Martinez et al., 2009a; Allen et al., 2010; Davies and Davies, As stated above, the main causes of intrinsic resistance from 2010; Sengupta et al., 2013). a clinical viewpoint are lack of the target and the inactiva- Studying the intrinsic resistome is of relevance for predict- tion, low uptake and efflux of the antibiotic. However, all ing evolution of resistance (Martinez et al., 2007, 2011a), for bacterial species harbor in their genomes genes encoding MDR understanding the linkage between resistance and other bacte- efflux pumps, and several present also chromosomally encoded rial processes as virulence (Martinez and Baquero, 2002; Martinez antibiotic-inactivating enzymes, even though they are not classi- et al., 2009a,b) or metabolism (Martinez and Rojo, 2011), and for fied as intrinsically resistant from the clinical point of view (Saier defining novel targets which inactivation make bacteria more sus- et al.,1998; Webber and Piddock,2003; Piddock,2006; Poole,2007; ceptible to antibiotics (Martinez et al., 2011b; Martinez, 2012). In Vila and Martinez, 2008; Nikaido, 2009). The study of the intrin- this article we present information of those organisms (Escherichia sic resistome of bacterial pathogens has shown that in addition coli and Pseudomonas aeruginosa) for which more information to these elements, several others contribute to the phenotype of on their intrinsic resistome is available. We discuss as well some resistance. Among them, there are the aforementioned classical issues concerning transient phenotypic resistance, which also resistance genes, but there exist also several other elements belong- depends on the intrinsic capabilities of the bacteria for evad- ing to all functional categories, including elements of the bacterial ing antibiotics action (Figure 1). Finally, we present updated general metabolism (Fajardo et al., 2008). These results indicate information on studies on the development of inhibitors of that the specific phenotype of susceptibility to antibiotics of a resistance. given bacterial species is an emergent property consequence of the concerted action of several elements (Girgis et al., 2009). The METHODS FOR ANALYZING THE INTRINSIC RESISTOME large functional diversity of the elements of the intrinsic resistome Genome-wide analysis of the intrinsic resistome of a given indicates this has not evolved to specifically counteract the activ- microorganism requires using high-throughput technologies. ity of the antibiotics. Together with the proposal that antibiotics Among them, the use of insertion or deletion libraries, allows might be molecular signals at the low concentrations they are likely determining the contribution of each single gene to the

FIGURE 1 |The different elements in bacterial resistance to antibiotics. some others increased resistance is acquired. Nevertheless, acquisition All bacteria have a repertoire of elements that contribute to their of a phenotype of increased resistance to antibiotics not always implies a characteristic phenotype of susceptibility to antibiotics, what has been genetic change, either because of mutation or as the consequence of the dubbed as intrinsic resistome. Some of the elements of this resistome are acquisition of a resistance gene by horizontal gene transfer. Phenotypic, classical resistance elements, as antibiotic-inactivating enzymes, whereas non-inheritable resistance can be achieved by different processes that include, others belong to all functional categories. The mutation of some of these among others, growth in biofilms, swarming adaptation, and development of elements makes bacteria more susceptible to antibiotics, whereas for persistence.

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characteristic phenotype of a given bacteria (Liu et al., 2010). that might be of relevance for its susceptibility to antibiotics as This method is the best suited to determine if the inactivation those involved in the repair of DNA damage or cell membrane of a gene changes bacterial susceptibility. However, it does not synthesis and integrity. In addition to these comprehensive analy- allow analyzing the effect of mutations that do not fully inacti- sis, other studies based on the whole-genome sequence of strains vate a given determinant but changes its activity. Alternatively, the evolving in the presence of antibiotics (Toprak et al., 2012)oron use of plasmid libraries containing each open reading frame of the enrichment of specific mutants of transposon-tagged libraries a given genome allows to establish the contribution to resistance grown with antibiotics have been used to track genes relevant for of each gene when is overexpressed or when it is transferred to the development of resistance (Zhang et al., 2012). However, in a heterologous host (Soo et al., 2011; Spindler et al., 2013). This this type of analysis, mutants are competing one to each other and method is only useful for analyzing acquired resistance, but not only those with the highest fitness will be selected. for studying genes which inactivation alters the bacterial suscep- Escherichia coli as a Gram-negative bacterium presents two tibility to antimicrobials. The use of high-throughput sequencing cell membranes, and both are non-specific barriers preventing and microarray technologies is also useful for studying intrin- drug influx into the cell. The lipopolysaccharide (LPS) of the sic resistance. These methodologies can be used for comparing outer membrane protects the cell against hydrophobic antibi- populations grown in the absence and in the presence of antibi- otics and polycationic compounds such as aminoglycosides and otics. These populations can be formed by the type of libraries polymyxins. LPS presents anionic groups and it is the first bar- above mentioned (transposon-tagged of expression libraries), in rier where some antibiotics bind (Hancock, 1984). Because of which case the enrichment of mutants or clones in the presence this, changes in the LPS can alter the susceptibility to antibi- of antibiotics allow defining genes contributing to resistance (Gir- otics. For instance, the loss of rffA, a gene encoding an enzyme gis et al., 2009; Zhang et al., 2012; Spindler et al., 2013). While implicated in the LPS synthesis, increases susceptibility to gentam- these methods are faster and cheaper than methods based in ana- icin (Tamae et al., 2008). The Enterobacteriaceae common antigen lyzing the susceptibility of each single mutant/clone, their main (ECA) is also an intrinsic resistance element that provides pro- drawback is that among all potential determinants that contribute tection against organic acids (Barua et al., 2002). In a similar way to resistance, only those with lower fitness costs will be enriched as happens with the LPS, changes in ECA may alter the suscep- and several of the elements that may be detected by classical sus- tibility to antibiotics. In line with this reasoning is the finding ceptibility tests would not be detected using enrichment-based that inactivation of the ECA biosynthesis protein WzxE increases technologies. This drawback may be a benefit when the anal- Escherichia coli susceptibility to nalidixic acid and amikacin (Gir- ysis is not made on a library, but using a wild-type strain. In gis et al., 2009). Mechanisms that control the negative charge of these circumstances, sequencing the evolving population allows the outer membrane, as theYrb system and the Pmr regulon, mod- in a single step defining the mutations that produce resistance ulate the bacterial susceptibility to neutral or negatively charged and present the smallest fitness costs among those analyzed, as compounds, such as nalidixic acid, lomefloxacin, and doxycycline well as to determine potential compensatory mutations (Gull- (Girgis et al., 2009). berg et al., 2011; Toprak et al., 2012). These mutations are the The gates of entry of several nutrients through the outer mem- most likely found in clinical isolates (Martinez et al., 2007, 2011a; brane are the porins, which are also used by antibiotics for entering Shcherbakov et al., 2010). into the cell (Nikaido, 1994). Many Escherichia coli strains have developed resistance to antibiotics as β-lactams and quinolones THE Escherichia coli INTRINSIC RESISTOME by mutations in the genes that encode porins or regulate their Although Escherichia coli is traditionally considered a suscepti- expression (Hirai et al., 1986; Adler et al., 2013). In addition, tran- ble organism, acquired resistance to antibiotics was first detected sient down-regulation of the expression of the porins can also in enteric bacteria, Escherichia coli, Shigella, and Salmonella, trigger phenotypic resistance (Fernandez and Hancock, 2012). in the late 1950s to early 1960s (Watanabe, 1963). Nowadays, A general mechanism that prevents the cellular accumulation of Escherichia coli accounts for 17.3% of clinical infections requir- drugs is their active extrusion from the cell or from the cytoplas- ing hospitalization and is the second most common cause of mic membrane through MDR efflux pumps (Nikaido, 1998a,b). infection behind Staphylococcus aureus (18.8%). Among outpa- The AcrAB–TolC system of Escherichia coli is one of the best- tient infections, Escherichia coli is the most common organism characterized MDR transporters (Ma et al., 1995). This system (38.6%). consists of AcrA, a membrane fusion protein; AcrB, a trans- The antibiotic intrinsic resistome of Escherichia coli has been porter of the RND family; and TolC, an outer membrane protein. studied by testing the susceptibility to several antibiotics of AcrAB–TolC is a major determinant for drug intrinsic resistance mutants from gene knockout collections (Tamae et al., 2008; Liu in Escherichia coli (Sulavik et al., 2001). Furthermore, its expres- et al., 2010) or transposon-tagged mutant libraries (Girgis et al., sion is increased in the presence of bile salts, which are present 2009). The results from these screenings showed that several genes in the habitat of Escherichia coli, the gut (Thanassi et al., 1997; participate in the phenotype of susceptibility to antibiotics in this Rosenberg et al., 2003). This means that Escherichia coli might species. Among them, some are classical resistance genes. Indeed, present a lower susceptibility to antibiotics when is growing inside this bacterium has different known resistance mechanisms; such as its host as the consequence of the overexpression of the MDR the AmpC β-lactamase and MDR efflux systems like AcrAB–TolC efflux pump AcrAB–TolC. Several other MDR transporters pro- (Lindberg and Normark,1986; Ma et al.,1993; Lubelski et al.,2007; vide resistance to a narrow range of compounds. Escherichia coli Jacoby, 2009). In addition, Escherichia coli harbors several genes contains five putative ABC-type MDR-like transporters. However,

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none of these systems provides an appreciable drug resistance to hfq, hns, mfd, nusB, rseA, xapR, yciT; Liu et al., 2010). These genes Escherichia coli, excepting YbjYZ (macrolide-specific ABC-type act in different ways, some of them involved in the stress response. efflux carrier), which confers resistance to macrolides composed For instance the DksA transcription factor regulates genes involved of 14- and 15-membered lactones, as erythromycin (Kobayashi in double-strand break repair (Meddows et al., 2005). However, et al., 2001; Nishino and Yamaguchi, 2001). The EmrAB is other determinants are global regulators of basic processes of the another MDR pump that protects the cell from several chem- bacterial physiology. This is the case of Fur, that is the master regu- ically unrelated antimicrobial agents, e.g., the protonophores lator in the response to iron availability (Bagg and Neilands, 1987), carbonyl cyanide m-chlorophenylhydrazone and tetrachlorosali- an environmental cue with relevance for infection (de Lorenzo cyl anilide and the antibiotics nalidixic acid and thiolactomycin and Martinez, 1988; Martinez et al., 1990). This indicates that (Lomovskaya et al., 1995). bacterial physiology and the bacterial response to environmen- In addition to MDR efflux pumps, Escherichia coli harbors tal inputs are cornerstones for the phenotype of susceptibility to antibiotic-inactivating enzymes. It is worth mentioning that whilst antibiotics. In addition to genes with known functions, there MDR efflux pumps usually confer low-level resistance to a wide are several other determinants of the Escherichia coli intrinsic range of compounds, antibiotic-inactivating enzymes confer fre- resistome which functional role is not known. This is the case quently high-level resistance to antibiotics belonging to a single of yecR and yfgC. Removal of yfgC decreases the MIC of van- structural family. All Escherichia coli strains have the chromo- comycin, rifampicin, and ampicillin (Tamae et al., 2008). yfgC somally encoded β-lactamase AmpC. Although it contributes to has homology to peptidases, and apparently it is located in the resistance to antibiotics, it is difficult to assume that this is its orig- bacterial inner membrane (Gardy et al., 2005). The existence of inal functional role if we take into consideration that the natural elements of unknown function conferring resistance to several habitat of Escherichia coli is the gut, which microbiota does not antibiotics bear witness regarding the complexity and the variety include β-lactam producers. AmpC β-lactamases hydrolyze broad of intrinsic resistance mechanisms encoded in the Escherichia coli and extended-spectrum cephalosporins but are not inhibited by genome. β-lactamases inhibitors such as clavulanic acid (Jacoby, 2009). Considering that β-lactams inhibit peptidoglycan transpeptida- THE INTRINSIC RESISTOME OF Pseudomonas aeruginosa tion, determinants involved in this process might be important for Pseudomonas aeruginosa is one of the most metabolically versatile the susceptibility to these antibiotics. This is the case for mltB and bacteria described so far (Lister et al.,2009). This particular feature slt, which encode membrane-bound lytic murein transglycosylases allows it to colonize multiple environments, being isolated from (von Rechenberg et al., 1996) and of ampG, which encodes a trans- seawater (Levin and Cabelli, 1972), soil (Green et al., 1974), inter- porter involved in the recycling of murein (Jacobs et al., 1994). acting mutualistically with plant roots (Green et al., 1974; Walker These genes are not only relevant for β-lactam susceptibility. They et al.,2004), as a plant pathogen (Walker et al.,2004; Records,2011) are also important for the regular physiology of Escherichia coli, and infecting animals (Petersen et al., 2002), including humans since the loss of mltB, slt, ampG, and ampC is deleterious (Girgis (Stover et al., 2000). In addition of its role in infections, P. aerugi- et al., 2009). nosa has been isolated at hospitals in different inorganic surfaces, Among those determinants involved in the intrinsic resistance from ventilation and intubation equipments, contact lens and even of Escherichia coli, some deal with the general response to stress, in hydrotherapy pools (Morales et al., 2012). This versatility and including the repair of damaged DNA. It is known that quinolones ability to survive on minimum nutritional requirements (Favero produce DNA damage and induce the SOS repair system (Howard et al., 1971) have made this bacterium one of the most successful et al., 1993). However, other antibiotics with a different mech- nosocomial opportunistic pathogens (Lister et al., 2009). anism of action as nitrofurantoin and metronidazol can also Not only is this ability to survive anywhere the cause of the damage DNA, and the RecF pathway of DNA repair of single- ecological success of this organism. This bacterium also shows strand breaks and gaps (recF, recO, recR, recQ, and recJ; Amundsen high levels of antibiotic resistance which often makes impossible and Smith, 2003) is important to prevent damages caused by these its eradication. Evolutionary forces have built its high resistance, drugs. It has been reported that the generation of hydroxyl radicals and countless elements contribute to intrinsic and acquired resis- leading to double-strands breaks contributes to cell death caused tance of this bacterium, considered a model organism to study by bactericidal antibiotics (Kohanski et al., 2008). In this sense mechanisms of resistance (Poole et al., 1993). DNA repair mechanisms involved in the maintenance of DNA Although the recent acquisition of antibiotic resistance genes integrity as the RecBCD system or genes involved in the response has been extensively described in P. aeruginosa (Woodford et al., of the cell to damaging agents (dinG, xseA, xseB, and gshB) consti- 2011), these acquired elements are not the unique cause of antibi- tute a valuable battery of defense mechanisms against antibiotics otic resistance in P. aeruginosa; multiple elements contribute (Liu et al., 2010). A similar response to stress might be in the basis substantially to the resistance of this bacterium. Many works have of the role of ribosomal proteins on intrinsic resistance. Indeed, been performed in order to unravel the intrinsic resistome of P. several genes that encode ribosomal proteins (rplA, rpmE, rpmJ) aeruginosa. Transposon-tagged libraries have demonstrated that or proteins involved in their modification (rimK) have a role in the the inactivation of several genes cause changes in antibiotic sus- susceptibility to a set of antibiotics belonging to different structural ceptibility. The majority of these genes are not related with the cell families (Liu et al., 2010). envelope or efflux pumps and many of them are involved in bac- Resistance is also provided by some genes that encode elements terial metabolism (Breidenstein et al., 2008; Fajardo et al., 2008; that are involved in the regulation of gene expression (dksA, fur, Schurek et al., 2008; Alvarez-Ortega et al., 2010; Fernandez et al.,

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2013). Mutants with changes in susceptibility to ciprofloxacin, the importance of the cellular membrane as a barrier to avoid the aminoglycosides, β-lactams, and polymyxin B have been identified entrance of antibiotics inside the cell (Schurek et al., 2008). using this method. Efflux pumps contribute considerably to antibiotic resistance Ciprofloxacin is a broad-spectrum fluoroquinolone that target in P. aeruginosa. They are involved in the extrusion of toxic the bacterial enzymes DNA gyrase and topoisomerase IV (Drlica substances, including antibacterial compounds, from inside the and Zhao, 1997). Thirty-five and 79 mutants with increased and cell to the external environment (Webber and Piddock, 2003). decreased susceptibilities to this antibiotic, respectively (Breiden- In this bacterium, 12 different RND-type efflux systems (Blair stein et al., 2008), were identified in a screening of a PA14 mutant and Piddock, 2009) that can eventually contribute to antibiotic transposon library (Liberati et al., 2006). The majority of these resistance have been described. However, only MexAB-OprM mutants demonstrated only twofold changes in susceptibility com- has shown to play a relevant role on the intrinsic resistance of pared with the respective isogenic strain; just the mutant in ftsK P. aeruginosa to antibiotics (Kohler et al., 1997). Expression of was eightfold more susceptible. Mutants in four genes involved the other efflux pumps under standard growing conditions is in DNA replication and repair (Holliday junction helicase ruvA, too low to achieve resistance, an issue that is also frequently the ATP-dependent RNA helicase recG, recombinase xerD, and described for other bacteria (Grkovic et al., 2001, 2002). However, the site specific recombinase sss) present increased susceptibility MDR efflux pumps can be overexpressed, and hence render resis- to ciprofloxacin. Additionally, ruvA, xerD, and fstK grow slower tance, either because of mutations in their regulatory elements or compared with the wild-type. On the contrary, mutants in genes because of the presence of effectors that trigger their expression nuoBDGHIJLN that encode a dehydrogenase are less susceptible to (Grkovic et al., 2001, 2002; Blair and Piddock, 2009; Hernandez ciprofloxacin. These mutants in the nuo operon present decreased et al., 2009, 2011). susceptibility to tobramycin also (Schurek et al., 2008) supporting Since intrinsic resistance is a multifactorial phenomenon, it the idea that some of the elements involved in intrinsic resistance might be modulated by global regulators. This is the case of have not evolved to counteract the activity of a specific antibiotic the P. aeruginosa “catabolite repression control” regulator Crc (Fajardo et al., 2008; Martinez and Rojo, 2011). (MacGregor et al., 1991). Crc is a post-transcriptional repressor Despite this apparent lack of specificity, there is not a common that regulates the use of preferred carbon sources in nutrient- response to all antibiotics, even for those belonging to the same complex ecosystems (Morales et al., 2004; Moreno et al., 2009). structural family. In a screening using imipenem, meropenem and Recent work has shown that inhibition of Crc makes P. aerug- ceftazidime just one mutant (PA0908) presented reduced suscep- inosa less virulent and more susceptible to different antibiotics tibility to all three antibiotics and two (glnK and ftsK) showed (Linares et al., 2010), the latter being a consequence of increased increased susceptibility to all three antibiotics (Alvarez-Ortega expression of transporters and changes in the LPS composition. et al., 2010). Even more, the mutant in ftsK, which encodes a pro- This shows the existence of networks connecting antibiotic resis- tein involved in cell division (Lewenza et al., 2005), presents an tance, bacterial virulence and metabolism. The hubs of these increased susceptibility to ciprofloxacin too (Breidenstein et al., networks may be good targets in the search of novel antimicro- 2008). In the same way, mutants in many genes that were more bials targeting simultaneously resistance, virulence, and bacterial susceptible to β-lactams (PA0401, pyrB and pyrD; Alvarez-Ortega physiology. et al., 2010) presented decreased susceptibility to polymyxin B (Fernandez et al., 2013). However, this is not a general trend, PHENOTYPIC RESISTANCE since mutants in galU, ampR, lptc, aroB, wapR, and ssg showed Most studies on antibiotic resistance are based in the analysis of decreased susceptibility to β-lactams and they are more suscep- resistant organisms that have acquired the phenotype of resis- tible to polymyxin B also (Fernandez et al., 2013). The majority tance as the consequence of genetic, inheritable, changes, which of these genes participate actively in the central metabolism of can be mutations (including gene rearrangements) of the acqui- P. aeruginosa (Stover et al., 2000) reinforcing the idea that genes sition, through HGT, of resistance genes (Walsh, 2000). However, involved in metabolism can play an important role in the intrinsic there are other situations, grouped under the name of phenotypic resistance to antibiotics of the microorganisms. resistance, in which bacteria present a non-inheritable situation The cell membrane is considered one of the principal contribu- of resistance to the antibiotics (Levin, 2004; Levin and Rozen, tors to intrinsic resistance (Nikaido, 1989). In this way mutations 2006). Phenotypic resistance is thus defined as a transient sit- in many genes that take part in the LPS synthesis in P. aerug- uation in which a bacterial population, usually susceptible to inosa produce changes of the susceptibility against β-lactams antibiotics, is transiently resistant (Figure 1). The elements con- and tobramycin. A mutant in wapR, which encodes a protein tributing to this phenotype are a part of the intrinsic resistome involved in the biosynthesis of the LPS core (Poon et al., 2008), that are only unveiled under specific growing conditions (Martinez is less susceptible to ceftazidime and meropenem; mutations in et al., 1994). Below, some examples of phenotypic resistance are adjacent genes (PA5001, PA5002, PA5003, and PA5005) which described. participate in the LPS synthesis also present reduced suscepti- bility to ceftazidime and some mutants present cross resistance PERSISTENCE with meropenem (Alvarez-Ortega et al., 2010). A similar effect Persistence is defined as a situation in which a bacterial subpopula- was observed in mutants of genes involved in the O-antigen tion is not killed by a given antibiotic under conditions in which the synthesis (wbpZ, wbpY, wzt, wzm, and wbpW). These mutants bulk of the population is inhibited. Once antibiotic is removed and presented decreased susceptibility to tobramycin, demonstrating growth resumes, persistent cells behave as antibiotic susceptible as

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the original population, which means that persistence is not the a characteristic extended time lag. An example of genes involved consequence of a genetic change. The existence of persister cells in this phenotype is hipA7 (Moyed and Bertrand, 1983). Type II into a population is known since 1944, when this phenomenon was persisters constitute a subpopulation of slowly growing cells. An first reported for staphylococcal infections treated with penicillin example of genes involved in this phenotype is hipQ (Wolfson (Bigger, 1944). Currently, persistence has been described for many et al., 1990). other bacterial species and antibiotics. As stated, persisters are phe- In agreement with the potential role that both bacterial notypic variants that present increased resistance to antibiotics and metabolism and TA systems may have on the establishment of are genetically identical to the wild-type. The percentage of persis- persistence, transcriptomic analysis of persister cells have shown ters in a given culture can be as high as 1% of stationary-phase cells that persistence is associated with the down-regulation of biosyn- for several microorganisms (Kim and Wood,2010). Establishment thetic genes and the up-regulation of several TA modules (RelBE, of a persistent subpopulation does not require previous antibiotic MazEF, DinJYafQ, YgiU; Lewis, 2010), Some of these TA systems exposure. are known to affect translation (Christensen et al., 2001; Peder- The presence of persister cells in infections increase the chance sen et al., 2002), which explains their effect in dormancy and of survival of the bacteria in the presence of antibiotics, an issue consequently on antibiotic resistance. The SOS response, which particularly relevant in the case of chronic infections. Because of up-regulates DNA repair functions, also induces several TA genes this, the analysis of the genes and mechanisms responsible for the in Escherichia coli, whose promoters contain a Lex box: symER, development of a persister phenotype is of relevance to imple- hokE, yafN/yafO, and tisAB/istR (Dorr et al., 2010). This means ment novel therapeutic approaches based on the eradication of that TA systems, and hence the formation of persister cells, can antibiotic resistant persistent cells. be activated by factors that trigger the SOS system. It is known The first gene described to be involved in the development of that some antibiotics, such as quinolones, induce the emergence persistence was hipA (Moyed and Bertrand, 1983)inEscherichia of persister cells (Dorr et al., 2010). Since these antibiotics pro- coli. HipA is a toxin that belongs to the hipBA toxin/antitoxin duce DNA damage and activate the SOS system, it is likely possible system (TA systems; Correia et al., 2006), which overexpression that the effect of quinolones on persistence is SOS-dependent. The inhibits cell growth and induces antibiotic resistance by persister role of other stresses on persistence has been studied. This is the formation. Other TA systems as MqsRA (Kim and Wood, 2010) case of oxidative stress that is important during infection due to or TisAB (Dorr et al., 2010; Lewis, 2010) have been also associated the immune response. However, this stress only induces persis- to the formation of persister cells, indicating that a misbalance in tence against fluoroquinolones but not for other antibiotics such the production of toxin and antitoxin may be in the basis of this as kanamycin. Surprisingly, this phenotype is due to the overex- phenotype. pression of the MDR efflux pump AcrAB–TolC (Wu et al., 2012). Toxin/antitoxin systems are highly distributed among differ- This indicates that transient expression of classic antibiotic resis- ent bacterial species. Usually, these systems are formed by two tance mechanisms forming part of the intrinsic resistome may be components, a toxin that inhibits cell growth and an antitoxin of relevance for the establishment of persistence. that impedes the activity of the toxin. At the moment, three One important issue for avoiding persistence would be finding general TA systems have been described. The antitoxins of TA elements which mutation impede the development of persistent types I and III are small RNAs, whereas the toxins of the type cells. However, the analysis of a transposon mutants library of II TA systems are inhibited by protein antitoxins (Gerdes et al., Escherichia coli (Hu and Coates, 2005; Hansen et al., 2008) and of 1997, 2005; Blower et al., 2011). Recent work has shown that type the comprehensive Kei collection (Baba et al., 2006), did not allow II TA systems are highly relevant for developing persistence in identifying any gene which mutation fully impede the phenotype Escherichia coli and that the simultaneous deletion of 10 TA loci of persistence. These data suggest a great degree of redundancy from the chromosome of Escherichia coli reduced the fraction in the elements and pathways involved in the development of of persistent cells by at least 100-fold (Maisonneuve et al., 2011; persistence. Even though TA systems could be good candidates Gerdes and Maisonneuve, 2012). to decrease the fraction of persister cells in a given population, In addition to TA systems, other factors modulate the devel- their high number, with 671 TA systems already identified in 126 opment of persistence. Among them, elements involved in the prokaryotic genomes (Pandey and Gerdes, 2005) makes difficult regular bacterial metabolism have shown to be relevant. This is developing drugs directed to reduce persistence by targeting these the case of phoU, which encodes a negative global regulator that systems. suppresses the cellular metabolic activity, altering expression of Another approach for eliminating persisters can be by shifting several elements with relevance for bacterial physiology, ranking their metabolic state. A recent report shows that this is feasi- from flagella-encoding genes to genes encoding energy production ble by specific metabolic stimuli that allow the recovery of the enzymes (Li and Zhang, 2007). proton motive force of persistent cells, enabling their killing by In depth analysis on the mechanisms of persistence has shown aminoglycosides (Allison et al., 2011). that there are two types of persistent cells (Balaban et al., 2004), which further supports the idea that there exist different routes BIOFILM toward the development of persistence. Type I persisters consti- Biofilm is a structured population of bacteria embedded in a tute a pre-existing population of resting cells that are generated matrix, which is composed by polysaccharides, proteins, and extra- at stationary phase. These cells when are inoculated into fresh cellular DNA. It has been shown than cells growing in biofilms are medium from stationary phase switch back to growing cells with less susceptible to antibiotics than those growing planktonically

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(Mah and O’Toole, 2001; Amini et al., 2011). This phenotypic characteristic of swarmer cells consists on their reduced suscep- resistance is relevant for the treatment of infections on intubated tibility to different antibiotics (Kim et al., 2003; Overhage et al., or catheterized patients, as well as in infections of prosthesis 2008b; Lai et al., 2009). and some chronic infections as those of cystic fibrosis patients Transcriptomic analyses of P. aeruginosa have shown that that involve the colonization of surfaces (Costerton et al., 1999) several genes change their expression during swarm cell differ- in which biofilm formation is frequent. The antibiotic resistance entiation. Some of these genes encode porins and efflux pumps, associated with biofilms depends on several causes, some due to such as MexGHI-OpmD. A differential expression of these genes the structure of the extracellular matrix, some other to the phys- in swarmer cells might be involved in their phenotype of reduced iological state of biofilm-growing bacteria; which is different to susceptibility to antibiotics (Overhage et al., 2008a). Nevertheless, that of planktonic cells. Even inside the biofilm, bacteria show the reasons for this transient phenotype of antibiotic resistance are different metabolic states, because there is a gradient of nutri- still far to be fully understood, since there are other elements that ents and oxygen between the surface of the biofilm and its deeper might be involved in the phenotype. For instance, several proteases region. (Lon, AsrA, PfpI, ClpS, and ClpP) that affect swarming motility are The extracellular matrix may change the activity of the antibi- also relevant for the formation of biofilms, and therefore for phe- otics by two different reasons; by diminishing the diffusion of the notypic antibiotic resistance (Marr et al., 2007; Kindrachuk et al., antibiotic or by sequestering it through its binding to the matrix. 2011; Fernandez and Hancock, 2012). This is not a general trend, since in several occasions, slow dif- Quorum-sensing seems to play a key role in swarming differen- fusion of the antibiotic is not the most important element in the tiation. It has been shown that PvdQ, an acylase that hydrolyzes the phenotypic resistance displayed by biofilms (Walters et al., 2003; QS signal 3-oxo-C12-HSL [N-(3-oxo-dodecanoyl)-l-homoserine Stewart et al., 2009; Singh et al., 2010). Another aspect in which lactone] is involved in swarming. The analysis of cells either over- the extracellular matrix may participate in the phenotype of resis- expressing or lacking PvdQ showed that PvdQ reduced P. aerug- tance of biofilms is by triggering specific mechanisms of resistance. inosa outer membrane permeability, thereby elevating antibiotic DNA, a macromolecule capable of chelate cations, is one of the resistance under swarming conditions, upon which this protein is components of the extracellular matrix. Since a reduced concen- up-regulated (Wang et al., 2013). A similar effect of reduced per- trations of divalent cations trigger expression of the regulator of meability has been shown for Salmonella enterica swarmer cells. resistance to cationic antimicrobials PhoP–PhoQ (Mulcahy et al., In particular the expression of the porin OmpA, which is used for 2008), the extracellular matrix trigger itself resistance to these the entrance of nutrients and some antibiotics is low in swarming drugs. cells (Sugawara and Nikaido, 1992; Kim and Surette, 2004), sug- When analyzing the role of the metabolic state of bacteria on gesting that changes in the permeability of the cellular envelopes, the phenotypic resistance of biofilms, it has been shown that the in response to nutrients’ availability, might be in the basis of the degree of resistance depends on the region of the biofilm and on antibiotic resistance phenotype displayed by swarmer cells. the antibiotic involved. Different regions of the biofilms contain There are some common factors that might induce the differ- subpopulations in different metabolic stages that mainly depend ent situations of phenotypic resistance above described. Changes on the oxygen and nutrients availability (Huang et al., 1995; Stern- in the environment as the lack of nutrients or low oxygen levels, berg et al., 1999). It has been described than oxygen-rich regions of which reduce the growth rates, occur in stationary phase and this P. aeruginosa biofilms are highly susceptible to quinolones whilst induces persisters, which are much more abundant in this growth cells in these regions are phenotypically resistant to cationic pep- phase. These nutritional cues also take place in some regions of tides, and the opposite occurs at regions of the biofilms with low bacterial biofilms, mainly at their deepest zone, and are relevant oxygen tension (Rani et al., 2007). Altogether, these results indi- for their reduced susceptibility to antibiotics. QS signaling is a cate that the phenotypic resistance of bacterial biofilms depend on relevant system in the basis of biofilm formation, which is also several factors that operate simultaneously. involved in triggering the bacterial physiological reprogramming that occurs in swarmer cells. Expression of TA systems occurs SWARMING in response to external signaling, and is involved in the genera- Swarming is a specific type of movement of bacterial popula- tion of persister cells, but also are differentially expressed during tions. It is characterized by the formation of hyper-flagellated cells biofilm formation, directly or via QS signaling. Cellular damage in nutrient-rich environments. It is supposed that this type of produced by toxic compounds, antibiotics included, may induce motility allows the colonization of such environments. Conse- the SOS response, which triggers expression of reparation sys- quently, swarming is just the most visible phenotype of a complex tems and usually also reduces metabolic activity, a situation that physiological adaptation process that is dependent on cell–cell induces the formation of persister cells and is also foreseen in signaling [quorum-sensing (QS)] and on nutrients availability biofilms. (Fraser and Hughes, 1999). The capability of swarm has been It thus seems that bacterial populations have developed a bat- described in different microorganisms, including Escherichia coli, tery of mechanisms to respond to stress and these mechanisms are Serratia marcescens, Burkholderia thailandensis, Bacillus subtilis, also useful to transiently resist the activity of antibiotics. Salmonella enterica serovar Typhimurium, and P. aeruginosa (Kim et al., 2003; Overhage et al., 2008a; Lai et al., 2009), and it might INHIBITORS OF RESISTANCE DETERMINANTS be of relevance for the colonization of surfaces during infection, One of the areas under development in the search for novel drugs as for instance in the lungs of cystic fibrosis patients. A relevant to fight infections is the study of inhibitors of resistance (Baquero

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et al., 2011; Martinez et al., 2011b; Martinez, 2012). The use of by the diazabicyclooctanes (DBOs). There are two compounds of these drugs in combination with those antibiotics against which this family under clinical trials: MK-7655 and NXL104 (avibac- the mechanisms of resistance operate, increases bacterial suscepti- tam) (Coleman, 2011). MK-7655 is a potent inhibitor of class bility to the antimicrobials. The first inhibitors of resistance were A and C β-lactamases, that can be used in combination with developed against mechanisms acquired through HGT, in partic- imipenem to kill AmpC and KPC (Klebsiella pneumoniae car- ular plasmid-encoded β-lactamases (Bush, 1988). However, the bapenemase) producers (Hirsch et al., 2012). Avibactam is a definition of intrinsic resistome offers the possibility of develop- promising BLI that inhibits class A and C β-lactamases, includ- ing inhibitors that increase susceptibility to all isolates of a given ing BlaC from Mycobacterium tuberculosis (Xu et al., 2012). In species (Garcia-Leon et al., 2012). For instance, the inhibition of combination with ceftazidime, avibactam protects β-lactams from MDR efflux pumps, which makes Gram-negative bacteria suscep- hydrolysis in β-lactamase-producing Enterobacteriaceae and P. tible to macrolides, will allow the use of these compounds for aeruginosa (Dubreuil et al., 2012; Levasseur et al., 2012). More treating infections by Gram-negative microorganisms. recently it has been proposed that a triple combination avibactam, Although clavulanic acid, the first inhibitor of β-lactamases ceftazidime, and metronidazole will be useful for treating compli- was described more than 40 years ago (Reading and Cole, 1977), cated intra-abdominal infections, which may present a mixed pop- few other inhibitors, all of them inhibiting the same type of β- ulation of Enterobacteriaceae and anaerobes (Dubreuil et al., 2012; lactamases, are already in the market. Main efforts have focused on Lucasti et al., 2013). the inhibitors of other β-lactamases (Drawz and Bonomo, 2010) Finding common inhibitors for all MBLs is a difficult task, and MDR efflux pumps (Lomovskaya and Watkins, 2001). In the due to the diversity of class B β-lactamases (Drawz and Bonomo, case of aminoglycoside-inactivating enzymes, the development of 2010). However, different structural families of inhibitors are inhibitors is problematic due to the large number and variability under development, including tetrahydropyrimidine-2-thione of families of these resistance elements. and pyrrole derivatives (Hussein et al., 2012), 3-mercapto-1,2,4- Based on the Ambler’s classification (Ambler et al., 1991), the β- triazoles and N-acylated thiosemicarbazides (Faridoon et al., lactamases are divided into four classes: class A, C, and D being ser- 2012), N-heterocyclic dicarboxylic acids and pyridylmercaptoth- ine β-lactamases, and class B being metallo-β-lactamases (MBLs). iadiazoles (Feng et al., 2012), 2-substituted 4,5-dihydrothiazole-4- The commercially available β-lactamase inhibitors (BLIs), clavu- carboxylic acids (Chen et al., 2012), or mercaptoacetate (Wachino lanic acid, sulbactam and tazobactam, are only effective against et al., 2012). class A β-lactamases. However, their combinations with β-lactam Although most inhibitors of β-lactamases target a specific fam- antibiotics are still effective, despite the emergence of resistance, ily of these enzymes, efforts have been made to develop drugs either because of gene-dosage effect (Martinez et al., 1987, 1989; capable to inactivate a large range of β-lactamases. Among them Reguera et al., 1991), either because of mutations that make β- single molecules as mercaptomethylpenicillinates (Buynak et al., lactamase resilient to inhibition (Blazquez et al.,1993; Canica et al., 2004) or reverse hydroxamates and oximes (Ganta et al., 2009) 1998; Salverda et al., 2010; Frase et al., 2011; Li et al., 2012). are under development as well as combinations of compounds The discovery that carbapenems may inhibit some β-lactamases as BAL30376, which is a mixture of BAL19764, a siderophore opened the possibility of finding antibiotics with dual activities, monobactam active to class B MBLs, BAL29880, a bridged antimicrobial and inhibitor of resistance (Buynak, 2006). How- monobactam active to class C β-lactamases, and clavulanate ever, this can be a difficult task, since antibiotics as cefoxitin may (Livermore et al., 2010; Page et al., 2011). either inhibit or induce expression of β-lactamases depending on In addition to traditional methods in searching enzyme’s their concentration (Tsuey-Ching et al., 2012). Because of this, inhibitors, information derived from other studies may help in mathematical models are being implemented to establish reliable developing β-lactamases inhibitors. In this regard, the finding dosage regimes (Bhagunde et al., 2012) and novel derivatives are of a small protein, produced by the same producer of clavulanic being developed to surpass these problems. Among them, N-acyl acid, Streptomyces clavuligerus (Reading and Cole,1977; Yuanet al., β-sultams obtained, by sulfonylation of β-lactams and addition 2011) and capable to inhibit class A β-lactamases opens the pos- of an acyl group, inhibit Enterobacter cloacae class C β-lactamase sibility of developing peptides or haptamers capable to inhibit (Page et al., 2003). Penicillin and cephalosporin sulfone deriva- β-lactamases. Similarly, the finding that the active site structures tives are also part of the pipeline of BLIs. LN-1-255 is a penicillin and the catalytic mechanisms of N-terminal nucleophile hydro- sulfone that is active against OXA-type β-lactamases and is being lase (a component of the bacterial proteasome) and β-lactamases used as model for further improvements in this type of inhibitors are similar, allows discovering cross-inhibition of both enzymes (Pattanaik et al., 2009; Drawz et al., 2010). by compounds as O-aryloxycarbonyl hydroxamates (Pelto and In addition to β-lactams that inhibit β-lactamases, there are Pratt, 2008) or 1,3,4-oxathiazol-2-ones (Adediran et al., 2012). also non-β-lactams able to inhibit these enzymes. One of the This opens the possibility of testing already known inhibitors of families receiving more attention is the one formed by boronic the bacterial proteasome as inhibitors of β-lactamases. acids (Kiener and Waley, 1978), which are potential inhibitors Beta-lactamases inhibitors will be very useful, but only for treat- of all types of serine β-lactamases (Tan et al., 2010), and seem ing infections by those organisms presenting a β-lactamase. A to be effective even against penicillin-binding proteins (PBPs) wider spectrum of activity might have the efflux pumps inhibitors resistant to β-lactams (Zervosen et al., 2012). Nevertheless clin- (EPIs). MDR efflux pumps are present in all bacterial species ical use of these compounds is compromised because of the contributing to intrinsic and acquired (when overexpressed) toxicity of boron. Another class of non-β-lactam BLIs is formed resistance to all family of drugs. Since any single efflux pump

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can extrude a wide range of antibiotics belonging to different MexAB-OprM of P. aeruginosa (Yoshida et al., 2007), 1-(1- structural families, its inhibition will simultaneously increase naphthylmethyl)-piperazine reversed multidrug resistance in A. the bacterial susceptibility to several antibiotics (Vila and Mar- baumannii but not in P. aeruginosa (Pannek et al., 2006), or 13- tinez, 2008). In vitro work has shown that inhibition of efflux cyclopentylthio-5-OH tetracycline (13-CPTC), a semisynthetic pumps makes bacteria more susceptible to antibiotics and also tetracycline analogue that binds TetB pump of Escherichia coli reduces the probability of emergence of antibiotic resistant (Nelson and Levy, 1999). mutants (Lomovskaya et al., 2001). In addition, since some efflux The finding that plant-produced compounds are substrates and pumps contribute to the virulence of bacterial pathogens, their inducers of efflux pumps (Matilla et al., 2007) suggests that these inhibition will also impair their capability for producing infec- compounds may also inhibit MDR determinants. Indeed, it has tions (Hirakata et al., 2009), including the formation of biofilms been shown that plant extracts contain a variety of EPIs (Tegos (Baugh et al., 2012). et al., 2002; Musumeci et al., 2003; Lewis and Ausubel, 2006; Stavri Theoretically, there are different alternatives for inhibiting et al.,2007), which may be useful for increasing the susceptibility to MDR determinants (Fernandez and Hancock, 2012). One that antibiotics of different bacterial species (Groblacher et al., 2012a,b; could serve for inhibiting all efflux pumps would be the inhibi- Holler et al., 2012a; Roy et al., 2012; Zhou et al., 2012). tion of energy sources required for pumps activity. Unfortunately In addition of inhibitors targeting specifically classical resis- activity of MDR efflux pumps is coupled either to the membrane tance elements, the study of the intrinsic resistome opens potential, either to ATP, both of which are key element for any the possibility of looking for inhibitors of targets that con- eukaryotic of prokaryotic cell. This means that most of these tribute to resistance despite they are not classical resistance potential inhibitors will be too toxic to be used. There are, how- determinants. This is the case of the P. aeruginosa cyanide- ever, some elements that are specific for bacteria and could be used insensitive terminal oxidase. Mutants defective in this gene as potential targets. This is the case of TonB, a protein involved are hypersusceptible to antibiotics (Tavankar et al., 2003). Con- in the activity of P. aeruginosa MDR efflux pumps by coupling sequently, inhibition of this component of the respiratory the energized state of the membrane to the operation of bacterial chain will increase the overall susceptibility of P. aeruginosa transporters (Zhao et al., 1998). to antibiotics. Same situation happens with global regulators Other ways of inhibiting the activity of efflux pumps include the as the P. aeruginosa Crc post-transcriptional repressor (Morales interference with the pump assembly or the blockage of its activ- et al., 2004; Moreno et al., 2009). Mutants defective in this ity, for instance using antibodies as has been described for SmrA gene are hypersusceptible to different antibiotics (Linares et al., from Stenotrophomonas maltophilia (Al-Hamad et al., 2011), the 2010); hence its inhibition will increase susceptibility to these development of effectors precluding the release of the MDR repres- antibiotics. sor from its operator DNA, and the competition with antibiotics An interesting approach in the search of inhibitors of resis- transported by the efflux pump. tance is the screening of non-antibiotic compounds, which had One of the first EPIs with potential therapeutic use is the syn- been already tested for other diseases, and may be used as helper thetic dipeptide amide phenylalanine-arginyl-β-naphthylamide, compounds to improve efficacy of antibiotics (Martins et al., which inhibits several Gram-negative efflux pumps (Lomovskaya 2008). The benefit of using these compounds is that their use et al., 2001; Mahamoud et al., 2006), although not all of them has been already approved, so as novel long and costly toxi- (Sanchez et al., 2003). This molecule is a competitive inhibitor that cological trials are not needed. Among them some anesthetics, binds to the same site used by the pump to bind the antibiotic. antihistaminic, and psychotherapeutic compounds have demon- Some efforts have been made in the optimization of diamide- strated to improve the activity of antibiotics (Kristiansen and containing EPIs (Watkins et al., 2003). However, it has been Amaral, 1997). Within this group of compounds some of them shown that the moieties that are responsible for their unfavor- change the permeability of the bacterial membrane (Martins able toxicological properties, are also essential for their activity, a et al., 2008), as the anti-inflammatory drug diclofenac (Dutta situation that impedes their therapeutic use (Lomovskaya and et al., 2007) or the anti-psychotic chlorpromazine (Blair and Pid- Bostian, 2006). dock, 2009), whereas for others, the mechanism of action is not Pyridopyrimidines and arylpiperazines have also been assayed known. as EPIs of MDR pumps and major efforts have been performed A more recent approach for improving the activity of the for their optimization (Nakayama et al., 2004a,b; Yoshida et al., antibiotics is by enhancing the cellular responses associated to 2006a,b, 2007). Differing to the previously described dipeptide the antibiotics induced cell death pathway. It has been proposed amides, that only impede the action of the antibiotics they com- that bactericidal antibiotics induce a cell pathway that involves the pete, pyridopyrimidines increase the susceptibility to all substrates generation of oxygen reactive species (Kohanski et al., 2007, 2010). of the efflux pumps, indicating a different mechanism of action Understanding this pathway may reveal targets for adjuvants that (Lomovskaya and Bostian, 2006). improve the efficacy of the antibiotics (Farha and Brown, 2013). Although some efforts on the development of EPIs against By using Escherichia coli whole-genome metabolic models and efflux pumps for Gram-positive organisms, as Staphylococcus further experimental validation of predicted targets, it has been aureus NorA, have been made (Markham et al., 1999; Holler shown that inactivation of some elements increase susceptibility to et al., 2012b; Sabatini et al., 2012), most EPIs so far described oxidants and antibiotics (Brynildsen et al., 2013), which opens the inhibit efflux pumps from Gram-negative organisms with some possibility of searching a new family of drugs capable to increase degree of specificity. Among them, pyridopyrimidines inhibit the activity of antibiotics.

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ACKNOWLEDGMENTS HEALTH-F3-2010-241476 (PAR) from the European Union. Work in our laboratory is supported by grants BIO2011- Guillermo Garcia-Leon is the recipient of a FPI fellowship, Ale- 25255 from the Spanish Ministry of Science and Innova- jandra Bernardini is the recipient of a “La Caixa” fellowship tion, PROMPT from CAM, REIPI from the Instituto de and Fernando Corona is the recipient of a JAE predoctoral Salud Carlos III, and HEALTH-F3-2011-282004 (EVOTAR) and fellowship.

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the beta-lactamase from Mycobac- Yoshida, K., Nakayama, K., Oht- (2012). Genome-wide identification commercial or financial relationships terium tuberculosis. Biochemistry 51, suka, M., Kuru, N., Yokomizo, Y., of ampicillin resistance determi- that could be construed as a potential 4551–4557. Sakamoto, A., et al. (2007). MexAB- nants in Enterococcus faecium. PLoS conflict of interest. Yim, G., Wang, H. H., and Davies, OprM specific efflux pump inhibitors Genet. 8:e1002804. doi: 10.1371/ J. (2006). The truth about antibi- in Pseudomonas aeruginosa. Part 7: journal.pgen.1002804 Received: 15 February 2013; accepted: 11 otics. Int. J. Med. Microbiol. 296, highly soluble and in vivo active qua- Zhao, Q., Li, X. Z., Mistry, A., Sriku- April 2013; published online: 30 April 163–170. ternary ammonium analogue D13- mar, R., Zhang, L., Lomovskaya, 2013. Yim, G., Wang, H. H., and 9001, a potential preclinical candi- O., et al. (1998). Influence of the Citation: Olivares J, Bernardini A, Davies, J. (2007). Antibiotics date. Bioorg. Med. Chem. 15, 7087– TonB energy-coupling protein on Garcia-Leon G, Corona F, Sanchez MB as signalling molecules. Philos. 7097. efflux-mediated multidrug resistance and Martinez JL (2013) The intrin- Trans. R. Soc. B Biol. Sci. 362, Yuan, J., Chow, D. C., Huang, W., in Pseudomonas aeruginosa. Antimi- sic resistome of bacterial pathogens. 1195–1200. and Palzkill, T. (2011). Identifica- crob. Agents Chemother. 42, 2225– Front. Microbiol. 4:103. doi: 10.3389/ Yoshida, K., Nakayama, K., Kuru, N., tion of a beta-lactamase inhibitory 2231. fmicb.2013.00103 Kobayashi, S., Ohtsuka, M., Take- protein variant that is a potent Zhou, X., Jia, F., Liu, X., and This article was submitted to Fron- mura, M., et al. (2006a). MexAB- inhibitor of Staphylococcus PC1 beta- Wang, Y. (2012). Total alka- tiers in Antimicrobials, Resistance and OprM specific efflux pump inhibitors lactamase. J. Mol. Biol. 406, loids of Sophorea alopecuroides- Chemotherapy, a specialty of Frontiers in in Pseudomonas aeruginosa. Part 5: 730–744. induced down-regulation of AcrAB- Microbiology. carbon-substituted analogues at the Zervosen, A., Bouillez, A., Herman, A., ToLC efflux pump reverses suscep- Copyright © 2013 Olivares, Bernardini, C-2 position. Bioorg. Med. Chem. 14, Amoroso, A., Joris, B., Sauvage, E., tibility to Ciprofloxacin in clinical Garcia-Leon, Corona, Sanchez and Mar- 1993–2004. et al. (2012). Synthesis and evalua- multidrug resistant Escherichia coli tinez. This is an open-access article dis- Yoshida, K., Nakayama, K., Yokomizo, tion of boronic acids as inhibitors of isolates. Phytother. Res. 26, 1637– tributed under the terms of the Creative Y., Ohtsuka, M., Takemura, M., penicillin binding proteins of classes 1643. Commons Attribution License, which Hoshino, K., et al. (2006b). MexAB- A, B and C. Bioorg. Med. Chem. 20, permits use, distribution and reproduc- OprM specific efflux pump inhibitors 3915–3924. tion in other forums, provided the origi- in Pseudomonas aeruginosa. Part 6: Zhang, X., Paganelli, F. L., Bier- Conflict of Interest Statement: The nal authors and source are credited and exploration of aromatic substituents. schenk, D., Kuipers, A., Bon- authors declare that the research was subject to any copyright notices concern- Bioorg. Med. Chem. 14, 8506–8518. ten, M. J., Willems, R. J., et al. conducted in the absence of any ing any third-party graphics etc.

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