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REVIEW Stress responses as determinants of antimicrobial resistance in Pseudomonas aeruginosa: multidrug efflux and more1 Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Laurentian University on 12/07/14 For personal use only.

Keith Poole

Abstract: Pseudomonas aeruginosa is a notoriously antimicrobial-resistant organism that is increasingly refractory to antimicrobial chemotherapy. While the usual array of acquired resistance mechanisms contribute to resistance development in this organism a multitude of endogenous genes also play a role. These include a variety of multidrug efflux loci that contribute to both intrinsic and acquired antimicrobial resistance. Despite their roles in resistance, however, it is clear that these efflux systems function in more than just antimicrobial efflux. Indeed, recent data indicate that they are recruited in response to environmental stress and, therefore, function as components of the organism’s stress responses. In fact, a number of endogenous resistance-promoting genes are linked to environmental stress, functioning as part of known stress responses or recruited in response to a variety of environmental stress stimuli. Stress responses are, thus, important determinants of antimicrobial resistance in P. aeruginosa. As such, they represent possible therapeutic targets in countering antimicrobial resistance in this organism. Key words: Pseudomonas aeruginosa, antimicrobial resistance, efflux, stress responses. Résumé : Pseudomonas aeruginosa est un organisme antibiorésistant notoire qui est de plus en plus récalcitrant a` l’antibiothérapie. Une panoplie habituelle de mécanismes de résistance acquis contribuent au développement de la résistance chez cet organisme, sans compter la multitude de gènes endogènes y jouant également un rôle. Du nombre, mentionnons divers locus d’efflux multimédicament contribuant a` l’antibiorésistance innée et acquise. Outre leurs rôles dans la résistance, il est manifeste que ces systèmes d’efflux sont voués a` une fonction autre que celle de l’antibiorésistance. En effet, des données récentes indiquent qu’ils seraient recrutés en réponse a` des stress environnementaux et feraient partie de la réponse au stress chez l’organisme. En fait, bon nombre de gènes endogènes favorisant la résistance sont liés aux stress environnementaux, a` titre d’éléments reconnus des réponses aux stress ou encore recrutés en réponse a` divers stimulus environnementaux de nature stressante. Les réponses aux stress sont donc d’importants déterminants de l’antibiorésistance chez P. aeruginosa. À cet égard, elles représentent des cibles thérapeutiques potentielles aptes a` neutraliser l’antibiorésistance chez cet organisme. [Traduit par la Rédaction] Mots-clés : Pseudomonas aeruginosa, antibiorésistance, efflux, réponses au stress.

Pseudomonas aeruginosa — a very resistant organism Pseudomonas aeruginosa is a common nosocomial pathogen (Azzopardi et al. 2014; Bereket et al. 2012; Berezin and Solorzano 2014; Bursle et al. 2014; Chen and Hsueh 2012; Chung et al. 2011; Hidron et al. 2008; Hirsch et al. 2012; Karakoc et al. 2013; Ploypetch et al. 2013; Ranjan et al. 2014; Rattanaumpawan et al. 2013; Rello et al. 2014; Resende et al. 2013; Sandiumenge and Rello 2012; Zhanel et al. 2008, 2010) that causes infections with a high mortality rate (Chung et al. 2011; Kerr and Snelling 2009; Lambert et al. 2011; Mahar et al. 2010; Mutlu and Wunderink 2006; Ranjan et al. 2014; Rattanaumpawan et al. 2013). This latter effect is, in part, attributable to (i) the organism’s intrinsically high resistance to many antimicrobials (Giamarellos-Bourboulis et al. 2006) and (ii) the development of increased, particularly multidrug, resistance in health care settings (Bodro et al. 2013, 2014; Chaisathaphol and Chayakulkeeree 2014; Chen et al. 2013; Chittawatanarat et al. 2014; Folgori et al. 2014; Medell et al. 2012; Pena et al. 2013; Pourakbari et al. 2012; Xiao et al. 2012), both of which complicate antipseudomonal chemotherapy (Chaisathaphol and Chayakulkeeree 2014; Chittawatanarat et al. 2014; Chung et al. 2011; Folgori et al. 2014; Hirsch et al. 2012; Hirsch and Tam 2010; Kallen et al. 2010; Keen et al. 2010; Kerr and Snelling

2009; Morata et al. 2012; Resende et al. 2013; Shorr 2009; Tumbarello et al. 2011). Not surprisingly, P. aeruginosa has been identified by the Antimicrobial Availability Task Force & Infectious Diseases Society of America as a problematic organism with limited treatment options owing to increased incidence and increasing antimicrobial resistance (Bodro et al. 2013, 2014; Boucher et al. 2009; Pendleton et al. 2013; Talbot et al. 2006). Antimicrobial resistance in bacteria typically derives from the acquisition of antibiotic-degrading (e.g., ␤-lactamases) or -modifying (e.g., aminoglycoside-modifying enzymes) enzymes, the alteration of antibiotic targets by mutation or chemical modification, or reduced accumulation owing to membrane impermeability or active efflux (Wright 2003), all of which have been described in P. aeruginosa (Poole 2011).

Multidrug efflux — not just for resistance Efflux-mediated resistance can be drug-specific, mediated by transporter proteins encoded by genes acquired by horizontal transfer, or multidrug, with a single, typically chromosomally encoded transporter accommodating a range of structurally diverse antimicrobials (Poole 2005b). Bacterial multidrug efflux systems are grouped into 5 families that include the Major Facilitator family, the Small Multidrug Resistance family, the ATP-Binding Cassette family,

Received 2 October 2014. Accepted 9 October 2014. K. Poole. Department of Biomedical and Molecular Sciences, Botterell Hall, Queen’s University, Kingston, ON K7L 3N6, Canada. E-mail for correspondence: [email protected]. 1This article is based on a presentation by Dr. Keith Poole at the 64th Annual Meeting of the Canadian Society of Microbiologists in Montréal, Quebec, on 28 July 2014. Dr. Poole was the 2014 recipient of the CSM Murray Award for Career Achievement. Can. J. Microbiol. 60: 783–791 (2014) dx.doi.org/10.1139/cjm-2014-0666

Published at www.nrcresearchpress.com/cjm on 9 October 2014.

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the Multidrug and Toxic Compound Export family, and the Resistance Nodulation Division (RND) family (Li and Nikaido 2009). While examples of all of these have been reported in P. aeruginosa (Poole 2013), by far the most significant contributors to resistance to clinically relevant agents and in clinical isolates are in the RND family (Poole 2001, 2004a, 2004b, 2005b, 2007; Poole and Srikumar 2001). Twelve RND family efflux systems have been described in P. aeruginosa (Poole 2013), although only 4, MexAB–OprM (Hamzehpour et al. 1995; Poole et al. 1993), MexCD–OprJ (Poole et al. 1996a), MexEF-OprN (Köhler et al. 1997), and MexXY–OprM (Aires et al. 1999; Mine et al. 1999; Westbrock-Wadman et al. 1999), contribute significantly to intrinsic or acquired resistance (Poole 2005a, 2011, 2013). Expression of these efflux systems is highly regulated (Cao et al. 2004; Evans et al. 2001; Köhler et al. 1999; Matsuo et al. 2004; Morita et al. 2006a; Ochs et al. 1999; Poole et al. 1996b; Purssell et al. 2014; Purssell and Poole 2013), though typically not by antimicrobials, an indication that drug efflux is not their intended function (Poole 2008). Indeed, evidence increasingly points to these functioning as components of adaptive stress responses (Poole 2012b).

Stress and resistance Environmental conditions can and do impact P. aeruginosa susceptibility to antimicrobials (Johnson et al. 2012; Macfarlane et al. 1999; Narten et al. 2012). Environmental stress in particular, which effects a myriad of adaptive and protective responses in this organism, alters gene expression patterns (Chang et al. 2005a; Firoved et al. 2004; Wood and Ohman 2009) and cell physiology (Macdonald and Kuehn 2013; Sabra et al. 2002; Wood et al. 2007) in ways that can profoundly influence antimicrobial susceptibility. Indeed, stress is increasingly identified as a defining signal for recruitment, not only of multidrug efflux systems but other resistance determinants in P. aeruginosa, as well as other bacteria, with these determinants functioning as components of stress responses (Poole 2012a, 2012b). Thus, stress responses are themselves resistance determinants in P. aeruginosa, with a potential to make significant contributions to resistance development in this organism. This can occur as a result of the stress-dependent recruitment of resistance determinants (e.g., antimicrobial efflux) (Fetar et al. 2011; Fraud et al. 2008; Fraud and Poole 2011), changes to antimicrobial targets (Moskowitz et al. 2004), amelioration of the adverse consequences of antimicrobial action (Khakimova et al. 2013; Krahn et al. 2012; Nguyen et al. 2011), alterations to the membrane barrier functions (Krahn et al. 2012), or promotion of resistant growth modes (biofilms) (de la Fuente-Nunez et al. 2013, 2014).

Oxidative stress and antimicrobial resistance Organisms such as P. aeruginosa that grow aerobically are routinely exposed to oxidative stress in the form of reactive oxygen species (ROS) (e.g., peroxide, superoxide) that are the unavoidable byproducts of aerobic respiration. ROS damage a variety of cellular macromolecules and, thus, elicit adaptive oxidative stress responses in bacteria intended to permit survival in the presence of this stressor (Imlay 2013). Interestingly, expression in P. aeruginosa of 2 multidrug efflux systems that have been linked to antimicrobial resistance in clinical strains (Poole 2013), mexAB–oprM (Chen et al. 2008) and mexXY–oprM (Fraud and Poole 2011), is positively impacted by agents of oxidative stress, with these efflux systems possibly playing some role in ameliorating the effects of this stress. Stress-responsive antioxidant mechanisms also contribute to antimicrobial resistance. Since antimicrobials are known to generate ROS that are key to the often-lethal effects of these agents (Kolodkin-Gal et al. 2008; Kohanski et al. 2007), such antioxidant responses are protective and antimicrobial resistance-promoting. Efflux In the case of mexAB–oprM, exposure of the organism to the ROS peroxide or cumene hydroperoxide was associated with a modest

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increase mexAB–oprM expression (Chen et al. 2008), apparently owing to oxidation of the MexR repressor (Evans et al. 2001) of this multidrug efflux operon, which caused the protein to dissociate from its target mexAB–oprM promoter (Chen et al. 2008). Expression of mexAB–oprM is also induced by chlorinated phenols like pentachlorophenol (Ghosh et al. 2011; Muller et al. 2007; Starr et al. 2012), an uncoupler of oxidative phosphorylation that may also promote oxidative stress that impacts the redox-sensitive MexR repressor. Pentachlorophenol has, for example, been shown to dramatically increase O2 flux in P. aeruginosa, generating an oxidative stress that could impact MexR activity (McLamore et al. 2010). Still, there was no indication that oxidative stress enhanced MexAB– OprM-mediated antimicrobial resistance in P. aeruginosa (Chen et al. 2008), and earlier transcriptomic studies of P. aeruginosa genes responsive to oxidative stress did not identify mexAB–oprM as being significantly (>2-fold) ROS-inducible (Chang et al. 2005a, 2005b). Oxidative stress induction of the mexXY genes of the P. aeruginosa MexXY–OprM multidrug efflux system is mediated by the oxidative-stress-inducible (Chang et al. 2005a, 2005b) PA5471 (armZ) gene product (Fraud and Poole 2011) that functions as the antirepressor of MexZ (Hay et al. 2013), the repressor of mexXY expression (Matsuo et al. 2004). This efflux system somewhat uniquely accommodates aminoglycosides (AGs) (Lau et al. 2014a) and is commonly associated with AG resistance in clinical isolates, particularly cystic fibrosis (CF) lung isolates where it is the primary determinant of AG resistance (Poole 2005a; Sobel et al. 2003). This latter is an intriguing observation given that the CF lung is known to be rich in ROS (Ciofu et al. 2005), which may, thus, be promoting the development of PA5471- and MexXYmediated AG resistance in CF lung isolates. In agreement with this, in vitro exposure of P. aeruginosa to peroxide enhanced the frequency with which MexXY-dependent AG-resistant mutants could be recovered (Fraud and Poole 2011), in this way demonstrating a positive association between oxidative stress and antimicrobial resistance. Intriguingly, the mexXY operon is also induced by antimicrobials that target the ribosome (e.g., tetracycline, chloramphenicol, erythromycin, AGs) (Morita et al. 2006b, 2009), as a result of antimicrobial disruption of the ribosome (Jeannot et al. 2005), and by mutational disruption of the ribosome (Lau et al. 2012; Morita et al. 2006b); these means of mexXY induction are similarly mediated by the PA5471 (armZ) gene product (Lau et al. 2012; Morita et al. 2006b), which is also induced by these agents (Morita et al. 2006b). In reconciling mexXY induction by both oxidative stress and ribosome disruption, one possible explanation is that oxidative stress also disrupts ribosome function or in some way perturbs translation. In this vein, ROS have been shown to reduce translational fidelity in E. coli by interfering with the editing activity of an aminoacyl-tRNA synthetase (Ling and Soll 2010). Alternatively, since ribosome disruption via mutation or antibiotic exposure leads to the production of aberrant polypeptides that are prone to oxidative modification in E. coli (Dukan et al. 2000), it may be that oxidatively modified or damaged proteins are the common trigger for PA5471 and mexXY induction, with MexXY–OprM possibly playing some role in ridding the cells of these damaged, aberrant polypeptides. Antioxidant mechanisms Despite some controversy surrounding ROS and oxidative stress being central to bacterial killing by bactericidal antimicrobials (Keren et al. 2013; Kohanski et al. 2007; Liu and Imlay 2013), it would appear that ROS do contribute to antibiotic lethality (Dwyer et al. 2014), and thus, antioxidant or oxidative stress protective responses in bacteria should promote antimicrobial resistance. In this vein, Nguyen and co-workers have shown that the antibiotic tolerance of nutrient-limited biofilm cells of P. aeruginosa is attributed, in part, to antioxidant defenses that include catalases and their turnover of antibiotic-generated ROS (Khakimova et al. 2013; Nguyen et al. 2011). These results suggest that starved, nongrowing cells may Published by NRC Research Press

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be at greater risk from oxidative stress or oxidative killing, so they take steps to protect themselves from the oxidative killing that is promoted by bactericidal antimicrobials. Similarly, production of H2S by P. aeruginosa has been linked to antibiotic resistance and appears to work by bolstering the organism’s antioxidant capacity, both by stimulating superoxide dismutase and catalase production and (or) activity and by reducing the Fenton-reaction-mediated generation of ROS (Shatalin et al. 2011). Still, regulation of H2S production and (or) activity or expression of H2S synthetic proteins or genes was not assessed in this instance, so it is not clear whether this antimicrobial resistance mechanism will be recruited in response to oxidative stress or antibiotic-generated ROS and, thus, represents another example of a stress-inducible mechanism of antimicrobial resistance.

Nitrosative stress and antimicrobial resistance Denitrifying organisms and pathogens such as P. aeruginosa are exposed to nitrosative stress in the form of nitric oxide, a reactive nitrogen species (RNS) that is produced during denitrification and by immune cells. RNS damage a variety of cellular constituents in bacteria and elicit an adaptive response that serves to detoxify or evade the effects of RNS (Bowman et al. 2011). The mexEF–oprN multidrug efflux locus is induced by nitrosative stress (e.g., in the presence of the nitrosating agent S-nitrosoglutathione or the nitric oxide (NO)-generating agent diethylamine triamine NONOate) (Fetar et al. 2011). This induction is dependent upon the MexT transcriptional activator (Köhler et al. 1999; Ochs et al. 1999). Moreover, several of the MexT targets identified in an array study of the MexT regulon (Tian et al. 2009) were also shown to be induced in response to nitrosative stress (Fetar et al. 2011), suggesting that MexT controls expression of a regulon with some function in a nitrosative stress response. Interestingly, chloramphenicol but not the related compound, florfenicol, which lacks a nitro group, induces mexEF–oprN expression, again dependent upon MexT (Fetar et al. 2011). This highlights the importance of the nitro moiety of chloramphenicol for this induction, an interesting observation given that some common products of nitrosative stress in bacteria are nitrated amino acids (i.e., chloramphenicol may resemble a nitrated nitrosative stress product that is an intended signal for MexT and substrate for MexEF–OprN).

Envelope stress and antimicrobial resistance The literature is ripe with descriptions of stress responses that facilitate survival and adaptation to environmental conditions that result in bacterial membrane (outer or cytoplasmic) or cell wall damage (Poole 2012a). Given that antibiotics must penetrate and cross these structures to access their cellular targets and often target them as part of their static or cidal activities (Bush 2012), the protective envelope or cell wall changes manifested by envelope stress responses can and do compromise antibiotic activity and, thus, promote resistance (Poole 2012a). In P. aeruginosa, a number of envelope stress-responsive systems are linked to antimicrobial resistance, including the mexCD–oprJ multidrug efflux locus (Fraud et al. 2008), the AmgRS 2-component system (TCS) that protects the cytoplasmic membrane from damage promoted by AGs (Krahn et al. 2012; Lee et al. 2009; Lau et al. 2014b), and the ParRS (Fernandez et al. 2010) and CprRS (Fernandez et al. 2012) TCSs, that protect the outer membrane (OM) from cationic antimicrobial peptides (CAPs) such as polymyxin B and colistin. MexCD–OprJ Originally shown to be induced by membrane-damaging biocides (chlorhexidine and benzalkonium chloride) and dyes (ethidium bromide and rhodamine) (Morita et al. 2003), mexCD–oprJ was more recently shown to be inducible by a variety of membrane-damaging agents that included detergents, organic solvents, biocides, and CAPs (Fraud et al. 2008). Membrane-damaging agent induction of this ef-

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flux system was mediated by the algU-encoded envelope stress response sigma factor (Fraud et al. 2008), a functionally equivalent homologue of E. coli RpoE (Yu et al. 1995) that was first described as a regulator of alginate biosynthesis in P. aeruginosa (Hershberger et al. 1995; Martin et al. 1993), an indication that envelope stress is the signal for MexCD–OprJ recruitment. AlgU is also required for the hyperexpression of this efflux system seen in strains carrying inactivating mutations in the nfxB gene encoding the repressor of mexCD– oprJ expression (Fraud et al. 2008), an indication that efflux gene expression requires both the active sigma factor and alleviation of NfxB-mediated repression. What the actual inducing signal is for the latter is unknown but is likely to be related to some feature of envelope stress. While the role of MexCD–OprJ in the AlgU-regulated envelope stress response is unclear, the exometabalome of a mexCD– oprJ-hyperexpressing nfxB mutant reveals elevated levels of longchain fatty acids, which have been proposed as possible MexCD–OprJ substrates (Stickland et al. 2010). Perhaps this efflux system plays a role in fatty acid export as part of a system for exchanging these components of membrane lipids as the cell responds to envelope stress and restructures its membranes accordingly. Recently, a second mexCD–oprJ repressor, EsrC, was described (Purssell et al. 2014). Inducible by envelope stress, dependent on AlgU, and also negatively regulated by NfxB, EsrC appears to moderate mexCD–oprJ expression under inducing conditions (Purssell et al. 2014). This may reflect the fact that mexCD–oprJ hyperexpression seems to be detrimental to the cell — pump-deficient “revertants” of mexCD–oprJ-hyperexpressing nfxB null mutants are readily selected during passage of these mutants in vitro (Purssell and Poole 2013). AmgRS The amgRS locus in P. aeruginosa encodes a homologue of the E. coli OmpR–EnvZ TCS and was first identified in a screen of transposon-insertion mutants susceptible to AGs (Krahn et al. 2012; Lee et al. 2009). However, unlike OmpR–EnvZ, AmgRS regulates a number of membrane transporter and protease genes, reminiscent of the E. coli CpxRA envelope stress response (Raivio 2014), and does so in response to AG exposure (Lee et al. 2009). It has been suggested that AmgRS responds to envelope stress mediated by aberrant polypeptides that are proposed to accumulate upon AG treatment (Kohanski et al. 2008), with the AmgRS-controlled stress response functioning to protect cells from aberrant peptide-mediated membrane damage (Lee et al. 2009), which has been shown to occur (Davis et al. 1986). In support of this, an amgR null mutant showed enhanced AG-promoted membrane damage and increased AG susceptibility (Krahn et al. 2012) while mutational activation of this TCS promoted AG resistance (Lau et al. 2013) and reduced AG-promoted membrane damage (Lau et al. 2014b). ParRS and CprRS Membrane-disrupting CAPs, including polymyxin B and proteins of innate immunity, also trigger stress responses whose outcome is a strengthening of membranes and, in the case of Gramnegative organisms, a modification of the lipopolysaccharide (LPS) to prevent CAP binding (Gunn 2001), LPS being the initial site of CAP interaction with cells (Gutsmann et al. 2005). In P. aeruginosa, exposure of the cell to subinhibitory levels of CAPs, such as polymyxin B, colistin, and CAPs of innate immunity, promoted resistance to these agents by activating expression of the arnBCADTEF-ugd (a.k.a. pmrHFIJKLM-ugd and PA3552-59; hereafter arn (Moskowitz et al. 2004)) locus (Fernandez et al. 2010), a homologue of the well-studied pmr locus that is associated with CAP resistance in Salmonella (Gunn 2008; Richards et al. 2010). This locus is responsible for decorating LPS with 4-amino arabinose (Moskowitz et al. 2004), a cationic molecule whose presence interferes with CAP binding to LPS (Shafer et al. 1984). CAP-promoted expression of the arn locus is dependent on the TCS ParRS (Fernandez et al. 2010). Interestingly, ParRS also mediates CAP induction of the mexXY multidrug efflux operon that is linked to AG resistance in P. aeruginosa Published by NRC Research Press

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(Muller et al. 2011), thus enhancing AG resistance as well (Fernandez et al. 2010). More recently, CprRS, a second TCS mediating CAPinducible arn expression and, thus, CAP resistance, has been described (Fernandez et al. 2012). Responding to different antimicrobial peptides or offering a differential response to the same peptides relative to ParRS, CprRS, like ParRS, is required for polymyxin B and colistin induction of the arn locus and, thus, adaptive resistance to these polycationic antimicrobials.

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Nutrient stress antimicrobial resistance Generally speaking, antibiotics preferentially kill rapidly replicating bacteria (Eng et al. 1991), and it has been suggested that reduced growth and metabolic activity associated with nonoptimal (e.g., nutrient-limited) growth environments might explain resistance in these instances. Certainly, at least some of the resistance attributable to the biofilm mode of growth in P. aeruginosa arises from established biofilm cells being largely oxygen-starved and occurring in an anaerobic stationary phase or nongrowing state that renders them resistant to antimicrobials; by comparison, young biofilm cells are more aerobic, are growing, and thus are antimicrobial-sensitive (Borriello et al. 2004). Similarly, screening of active and dormant cells within a P. aeruginosa biofilm reveals that the dormant cells are more tolerant to the AG tobramycin (Kim et al. 2009). A contributing factor to the antibiotic tolerance of anoxic P. aeruginosa biofilm cells appears to be their reduced metabolic activity — addition of arginine (a fermentable nutrient) or nitrate or nitrite (to stimulate anaerobic respiration) enhanced antimicrobial killing of mature anaerobic (but not aerobic) biofilm cells (Borriello et al. 2006). Nonetheless, nutrient limitation can also impact antimicrobial resistance as a result of its activation of stress responses that promote resistance by recruiting antioxidant and biofilm resistance mechanisms (e.g., the stringent response) or by modifying the cell surface so as to prevent antimicrobial binding and entry into the cell (e.g., the Mg2+ limitation-responsive TCSs PhoPQ and PmrAB). Stringent response Typically activated by nutrient deficiency, most notably amino acid deprivation, the stringent response (SR) is characterized by reduced expression of genes associated with growth and increased expression of survival genes that economize the use of scarce nutrients (Chatterji and Ojha 2001; Sharma and Chatterji 2010). Associated with increased production of the alarmones guanosine 5=-(tri)diphosphate, 3=-diphosphate ((p)ppGpp) (Dalebroux and Swanson 2012), the SR has a myriad of effects on bacterial cell physiology and perhaps, not surprisingly, impacts antimicrobial susceptibility (Wu et al. 2010). In P. aeruginosa, ppGpp and the SR has been linked to tolerance to several antimicrobials in nutrient-limited planktonic and biofilm cells of this organism (Nguyen et al. 2011). Nguyen and co-workers show that nutrient-limited mutant cells that are deficient in ppGpp production are markedly less antimicrobial tolerant than their wild-type counterparts, with ppGpp-deficient biofilm cells showing reduced tolerance to several classes of antimicrobials, including an AG (gentamicin), a ␤-lactam (meropenem), a CAP (colistin), and a fluoroquinolone (ofloxacin). The lack of ppGpp was correlated with an increase in ROS production and oxidative stress, an indication that the tolerance of wild-type ppGpp+ cells was linked to SR-promoted recruitment of antioxidant defenses, which protect cells from antimicrobial-generated ROS that ultimately contribute to antimicrobial lethality. In agreement with this, the antimicrobialsusceptible ppGpp– cells showed reduced levels of catalase and superoxide dismutase activity relative to their antimicrobial-resistant ppGpp+ counterparts (Nguyen et al. 2011). More recently, the SR regulation of catalases and their involvement in the antimicrobial resistance of nutrient-limited biofilm P. aeruginosa was confirmed (Khakimova et al. 2013). These results suggest that starved, nongrowing cells may be at greater risk from oxidative stress or oxidative killing, so they take steps to avoid it and in so doing

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promote protection from the oxidative killing that is promoted by bactericidal antimicrobials. Mg2+ limitation and LPS remodelling Divalent cations such as Mg2+ play a central stabilizing role in the barrier function of the OM of Gram-negative bacteria via an interaction with the LPS constituent of this membrane (Nikaido 2003). As such, Mg2+ deficiency triggers an adaptive response that involves remodelling of the LPS to maintain OM integrity and stability in the face of this potentially LPS-perturbing nutrient limitation (Kato and Groisman 2008; McPhee et al. 2006). In P. aeruginosa, as well as other organisms, this remodelling includes substitution of the lipid A component of LPS with the CAP and polymyxin resistance-promoting 4AA species (Moskowitz et al. 2004; Richards et al. 2010). As in Salmonella, where it is best studied (Gunn 2008; Richards et al. 2010), the arn locus and 4AA modification of LPS is inducible by low Mg2+ in P. aeruginosa (Ernst et al. 1999; McPhee et al. 2003), consistent with earlier reports of Mg2+ deficiency promoting resistance to polymyxin B (Macfarlane et al. 1999) and innate immunity CAPs (Macfarlane et al. 2000) in this organism. Mg2+ regulation of polymyxin and CAP resistance, in part via influences on arn expression (McPhee et al. 2003), is mediated by the products of 2 TCSs, PhoPQ (Macfarlane et al. 1999, 2000) and PmrAB (McPhee et al. 2003; Moskowitz et al. 2004), both of which are themselves inducible under conditions of Mg2+ deficiency (Macfarlane et al. 1999; McPhee et al. 2003). Significantly, the PhoPQ- and PmrAB-regulated LPS modification is an important determinant of polymyxin resistance in clinical strains. Indeed, there are several reports of phoQ (Barrow and Kwon 2009; Miller et al. 2011), pmrB (Abraham and Kwon 2009; Barrow and Kwon 2009; Lee et al. 2012; Moskowitz et al. 2012), and phoP (Lee et al. 2012) mutations responsible for polymyxin B (Abraham and Kwon 2009; Barrow and Kwon 2009; Lee et al. 2012; Moskowitz et al. 2012) and colistin (Lee et al. 2012; Miller et al. 2011; Moskowitz et al. 2012) resistance in clinical isolates that is dependent on LPS modification by the arn locus (Barrow and Kwon 2009; Miller et al. 2011; Moskowitz et al. 2012). Low Mg2+ stimulation of resistance to polycationic antimicrobials, including polymyxin and AGs in P. aeruginosa, resulting from synthesis and binding of polyamines (i.e., spermidine) to the cell surface, has also been reported (Johnson et al. 2012). Synthesized by the products of the low Mg2+-inducible PA4773-4774 (speDE) genes (Johnson et al. 2012), spermidine binding to LPS presumably interferes with the binding and, thus, uptake of polycationic antimicrobials. Nutrient limitation and biofilms Pseudomonas aeruginosa commonly exists in the infected host in biofilms (Mulcahy et al. 2014), structures that amongst other benefits provide resistance to antimicrobials (Hoiby et al. 2010). Environmental factors that promote bacterial biofilm formation will, thus, contribute positively to antimicrobial resistance. A variety of stresses have been linked to biofilm formation in P. aeruginosa, with biofilm formation itself possibly being a stress response in this organism (de la Fuente-Nunez et al. 2013). Nutrient limitation (i.e., nutrient stress) in the form of Mg2+ limitation, for example, has a positive impact on biofilm formation in P. aeruginosa (Mulcahy and Lewenza 2011). Similarly, the SR and ppGpp have been linked to biofilm formation and maintenance in P. aeruginosa—ppGpp deficiency in a mutant strain was shown to hamper biofilm formation, while loss of ppGpp synthesis in a 2-day-old biofilm promoted dispersal (de la Fuente-Nunez et al. 2014). As well, a peptide that targeted and promoted the degradation of ppGpp blocked biofilm formation and promoted dispersal of preformed biofilms (de la Fuente-Nunez et al. 2014), highlighting both the importance of ppGpp and the SR to biofilm formation and the utility of targeting this stress response in countering biofilm-mediated antimicrobial resistance. Published by NRC Research Press

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Heat shock and the general stress response and antimicrobial resistance The heat shock response, best studied in E. coli (Guisbert et al. 2008) but also described in P. aeruginosa (Allan et al. 1988), is a transient response to temperature increase and the adverse consequences this can have on protein folding, with heat shock components typically playing a role in facilitating protein refolding or turning over misfolded proteins. Interestingly, AGs such as tobramycin have been shown to induce expression of heat shock genes in P. aeruginosa (Kindrachuk et al. 2011). This is mediated by the alternate Lon protease AsrA, whose expression is induced in response to tobramycin or heat shock, dependent upon the RpoH heat shock sigma factor (Kindrachuk et al. 2011). Intriguingly, asrA overexpression in an engineered strain had a modest positive impact on AG resistance (Kindrachuk et al. 2011), raising the possibility that the heat shock response might protect, to some extent, against AG challenge. One explanation for the link between AGs and heat shock is that components of the heat shock response charged with eliminating misfolded proteins might target the aberrant mistranslated polypeptides that are produced by AGdisrupted ribosomes and that insert into and disrupt bacterial membranes (Davis et al. 1986). AG-mediated membrane damage has been reported (Davis et al. 1986) and is likely a key step in the lethal activity of these agents (Kohanski et al. 2008; Lee et al. 2009); therefore, elimination of aberrant polypeptides that may be responsible would certainly reduce their toxicity to cells. RpoS, a general stress response sigma factor that responds to heat shock, hyperosmolarity, and prolonged peroxide exposure in P. aeruginosa (Suh et al. 1999), has also been linked to antibiotic resistance in this organism. Its overexpression was shown to partially restore ofloxacin tolerance in an ofloxacin-sensitive LasIR– strain of P. aeruginosa defective in acylhomoserine lactone production and quorum-sensing-dependent gene expression (Kayama et al. 2009). RpoS has also been linked to carbapenem tolerance in P. aeruginosa — an rpoS mutant strain showed enhanced susceptibility to carbapenems in stationary phase (Murakami et al. 2005). Similarly, a heat-shock-promoted increase in carbapenem tolerance in this organism was also shown to be dependent on rpoS (Murakami et al. 2005). Still, the RpoS targets responsible for quinolone and carbapenem resistance in P. aeruginosa are unknown. Concluding remarks While a link between stress and known antimicrobial resistance determinants has in many cases been established, the contribution of the resistance genes to the stress response itself is often not known. For example, while several of the RND family multidrug exporters of P. aeruginosa are stress-inducible, their roles in the corresponding stress responses and the identities of the inducing effector and efflux substrate molecules remain uncertain. Knowledge of the environmental circumstances and (or) effector molecules responsible for recruiting multidrug efflux systems is key to our understanding of the biology of these transporters and vital to an appreciation of those environments (e.g., in hospital or at a site of infection) where their induction may compromise antimicrobial chemotherapy. In many cases, stressor induction of these efflux systems does not enhance resistance (Fetar et al. 2011; Fraud et al. 2008; Fraud and Poole 2011); their documented contributions to resistance is limited to circumstances of mutational overexpression (Poole 2011). Nonetheless, since these resistance determinants are components of stress responses, the relevant stresses will provide a selective pressure for efflux-expressing antimicrobial-resistant mutants even in the absence of antimicrobials. Indeed, in vitro exposure of P. aeruginosa to ROS selects for MexXY-expressing pan-AG-resistant mutants (Fraud and Poole 2011). This may explain the preponderance of MexXY-mediated AG resistance in CF lung isolates of P. aeruginosa, which are chronically exposed to ROS in the inflamed CF lung (Poole 2005a). The possibility

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that environmental stress (in this case oxidative) at the site of infection can drive resistance development independent of antimicrobial exposure is worrying, limiting as it does our ability to manage resistance (e.g., by avoiding use of the antibiotic for a period of time, which would typically remove the selective pressure for resistance development and maintenance) and, thus, to maintain long-term drug efficacy. Given the link between stress and resistance and the potential for stress responses to contribute to antimicrobial resistance development, stress response pathways may be suitable targets for therapeutic intervention. In support of this, inactivation of the AmgRS regulators of an envelope stress response in P. aeruginosa increases susceptibility to AGs (Krahn et al. 2012), loss of the AlgU envelope stress response sigma factor reverses the multidrug resistance of a mutant P. aeruginosa hyperexpressing MexCD–OprJ (Fraud et al. 2008), and inactivation of the SR in P. aeruginosa enhances susceptibility to several antimicrobials (Nguyen et al. 2011) and blocks biofilm formation (de la Fuente-Nunez et al. 2014). Still, a better understanding of the link between stress and antimicrobial resistance, including the identification of stress-induced effectors that recruit resistance determinants and promote resistance, and the gene(s) involved, is needed to fully appreciate the importance of stress responses as resistance determinants, their value as therapeutic targets, and how best to target them.

Acknowledgements Work in the author’s laboratory on multidrug efflux systems and stress response determinants of antimicrobial resistance has long been funded by The Canadian Institutes of Health Research and Cystic Fibrosis Canada, whose support is gratefully acknowledged.

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