Mechanisms of -lactam Resistance Among Pseudomonas aeruginosa

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1 Send rders of Reprints at Current Pharmaceutical Design, 2013, 19, Mechanisms of -lactam Resistance Among Pseudomonas aeruginosa Daniel J. Wolter 1 and Philip D. Lister 2, * 1 Department of Pediatrics, University of Washington, Seattle, WA; 2 Department of Medical Microbiology and Immunology, Creighton University School of Medicine, 2500 California Plaza, maha, NE 68178, USA Abstract: Treatment of serious P. aeruginosa infections becomes more challenging with each passing year. As this pathogen acquires more transferrable resistance mechanisms and continues to rapidly adapt and emerge resistant during the course of antimicrobial therapy, we face the growing threat of pan-resistance. This review has focused on those mechanisms that directly impact the future of -lactam antibiotics, including the production of -lactamases, porin-mediated resistance, and/or the overexpression of RND efflux pumps. With the pipeline of new anti-pseudomonal agents diminishing, it is essential that novel therapeutic strategies be explored. These include targeting biofilm formation and maintenance, virulence factors, and resistance mechanisms. Furthermore, we must continue to search for effective antibacterial combinations to not only prevent further emergence of resistance but also treat resistant strains already in the environment. Keywords: Pseudomonas aeruginosa, -lactam, resistance. INTRDUCTIN Antibacterial resistance is an increasing threat to the treatment of gram-positive and gram-negative pathogens. Among the gramnegatives, P. aeruginosa has historically been one of our most difficult therapeutic challenges. This pathogen is not only armed with an impressive arsenal of chromosomally-encoded resistance mechanisms, but has the propensity to acquire a wide range of resistance genes from its environment. The focus of this review will be on the diversity of resistance mechanisms that threaten the clinical use of -lactams for P. aeruginosa infections, including -lactamases, porin-mediated resistance, and efflux-mediated resistance. For some of these mechanisms, P. aeruginosa has the ability to alter production and/or activity and emerge resistant during the course of treating a patient. Furthermore, with the cooperative effects of multiple mechanisms, the threat of pan- -lactam resistant P. aeruginosa is a growing concern. CLINICAL SIGNIFICANCE AND THERAPEUTIC CHAL- LENGE F PSEUDMNAS AERUGINSA P. aeruginosa is a ubiquitous microbial pathogen that can be isolated from a wide variety of environmental sources including plants, animals, and humans. As a nosocomial threat, P. aeruginosa has been isolated from respiratory therapy equipment, antiseptics, sinks, medicines, and hydrotherapy pools [1]. Community reservoirs include swimming pools, humidifiers, hot tubs, and contact lens solution [2-4]. Although P. aeruginosa is not normally a part of the normal flora, a significant proportion of hospitalized patients can become colonized [1], especially if protective barriers of skin or mucous membranes have been breached or if the patient is immunocompromised [3, 5] Patient populations at particular risk are those on mechanical ventilators, patients with catheters, surgical patients, patients with severe burn wounds, and seriously ill patients in the intensive care unit [6-12]. In addition, patients for whom the normal protective flora has been disrupted by antimicrobial therapy are also at increased risk for colonization [6, 13, 14]. Although P. aeruginosa can cause community-acquired infections, the majority of serious infections are nosocomial. According to the CDC National Nosocomial Infections Surveillance System (NNISS), P. aeruginosa ranks fifth among nosocomial pathogens for overall frequency of isolation in U.S. hospitals [15, 16]. *Address correspondence to this author at the Department of Medical Microbiology and Immunology, Creighton University School of Medicine, USA; Tel: ; plister@cnm.edu For the subsets of nosocomial pneumonia, health-care associated pneumonia, and ventilator associated pneumonia, P. aeruginosa is the second most common pathogen identified [17, 18]. In addition, P. aeruginosa is a leading cause of infections among patients in the intensive care unit [11, 12]. Selection of appropriate antibacterial therapy is essential to optimizing the treatment of serious infections with P. aeruginosa [19, 20]. The initial challenge with P. aeruginosa centers on its intrinsic antibiotic resistance that is not observed in most other gram-negative bacilli. This inherent resistance is due to low permeability of its outer membrane [21] and intrinsic basal expression of chromosomal resistance mechanisms [22]. Complicating selection of appropriate therapy is the propensity for P. aeruginosa to develop resistance to multiple classes of antibacterial agents. Multidrug resistant strains are isolated with alarming frequency, and these strains develop a multi-drug resistant phenotype through the acquisition of resistance genes on mobile genetic elements and/or through mutational events influencing the expression and/or activity of chromosomal resistance mechanisms [22]. An even greater concern is the propensity for P. aeruginosa to emerge resistant during the course of therapy, leading to clinical failure and potentially doubling the length of hospital stay and the cost of patient care [23]. This review will focus specifically on mechanisms of -lactam resistance among P. aeruginosa. Figure 1 summarizes the general mechanisms by which P. aeruginosa expresses resistance to this antibacterial class. -lactam antibiotics enter the periplasmic space of P. aeruginosa by direct penetration across the outer membrane or through porins located in the outer membrane (Fig. 1A). nce in the periplasmic space, -lactams exert their antibacterial effect by binding to and inactivating the penicillin-binding proteins involved in cell wall biosynthesis. Although mutational changes impacting the affinity of penicillin-binding proteins for -lactams may play a role in resistance for some strains, the primary mechanisms of lactam resistance are 1) production of an inactivating enzyme ( lactamase), 2) porin-mediated resistance, and 3) efflux-pump mediated resistance (Fig. 1B). -LACTAMASES F Pseudomonas aeruginosa The production of drug-inactivating enzymes is the most common mechanism of -lactam resistance among gram-negative bacteria. Molecular and genetic analyses of clinical isolates have shown that P. aeruginosa can produce a diversity of -lactamases, some of which are encoded by chromosomal genes and others on mobile genetic elements. Although the -lactamases produced by P /13 $ Bentham Science Publishers

2 210 Current Pharmaceutical Design, 2013, Vol. 19, No. 2 Wolter and Lister A. M PG PS PBP PBP PBP PBP CM B. X M PG PBP PBP PBP PS CM Fig. (1). Mechanisms of -lactam Resistance Among P. aeruginosa. Panel A: Interactions of -lactams with wild-type susceptible P. aeruginosa. lactam molecules penetrate into the periplasmic space either by passing directly through the outer membrane or entering through specific porins, e.g. prd for carbapenems ( ). nce they enter the periplasmic space, -lactams can interact with their target penicillin binding proteins (PBP) located on the outside of the cytoplasmic membrane. Panel B: Mechanisms of resistance to -lactams. The primary mechanisms of -lactam resistance include the production of lactamases ( ), decrease or loss of prd porin in the outer membrane, and overproduction of RND efflux pumps ( ) aeruginosa can vary with respect to their spectra and efficiency of hydrolysis, these characteristics alone do not define the resistance profiles associated with different enzymes. Instead, -lactamaseassociated resistance in P. aeruginosa is also dependent upon the efficiency of drug penetration, ability of this pathogen to minimize drug accumulation in the periplasmic space (porin-mediated resistance and/or extrusion by efflux pumps), and the cooperative effects of different -lactamases within the same cell. Therefore, it is not surprising that the resistance profile associated with a -lactamase in one strain of P. aeruginosa may differ from the resistance profile associated with that same -lactamase in a different strain. With this in mind, the following sections will summarize the different families of -lactamases that have been detected in clinical isolates of P. aeruginosa and the general resistance threat they provide when produced by this pathogen. The -lactamases have been categorized based on the recently updated classification of Bush and Jacoby [24], and are summarized in Table 1. Group 1 Chromosomal AmpC Cephalosporinase. AmpC is a serine-based -lactamase belonging to molecular class C and is characterized by its more efficient hydrolysis of cephalosporins than penicillin and its relative resistance to inactivation by clavulanate and tazobactam [24]. The AmpC of P. aeruginosa is naturally an inducible enzyme. In the absence of an inducing -lactam, wild-type strains of P. aeruginosa produce low basal levels of AmpC and may be susceptible to the anti-pseudomonal penicillins, penicillin-inhibitor combinations, cephalosporins and carbapenems if no other resistance mechanisms are operative [25]. However, the increased production of AmpC in P. aeruginosa can cause resistance to virtually all -lactams, except to the carbapenems [22, 26]. Although data have suggested that AmpC plays a role in the intrinsic level of susceptibility of P. aeruginosa to carbapenems [27-30], overproduction of AmpC does not significantly decrease P. aeruginosa susceptibility to carbapenems [27, 30-33]. Resistance to the carbapenems usually requires the cooperation of additional resistance mechanisms, e.g. efflux pump overproduction, outer membrane porin decrease, and/or co-production of additional carbapenem-hydrolyzing enzymes [22]. ne pathway to overproduction of AmpC is through the reversible induction of ampc expression during exposure with certain -lactams (cephamycins and carbapenems) and the -lactamase

3 Antibacterial Resistant Pseudomonas aeruginosa Current Pharmaceutical Design, 2013, Vol. 19, No Table 1. -lactamases of P. aeruginosa Functional Group Molecular Class Potential Resistance Impact Representative Enzymes Group 1 C Penicillins Penicillin-Inhibitor Combinations Cephalosporins Cephamycins Monobactams Group 1e C Penicillins Penicillin-Inhibitor Combinations Cephalosporins Cephamycins Monobactams Carbapenems Group 2b A Penicillins Early cephalosporins Group 2be A Penicillins Early cephalosporins Extended-spectrum cephalosporins Monobactams Chromosomal AmpC Cephalosporinase Extended-Spectrum AmpC Cephalosporinase TEM-1, TEM-2, SHV-1 TEM-4, TEM-21, TEM-24, TEM-42, TEM-116 SHV-2, SHV-2ª, SHV-5, SHV-12 CTX-M-1, CTX-M-2, CTX-M-43 PER-1, PER-2 VEB-1, VEB-1a, VEB-1b, VEB-2 GES-1, GES-8, GES-9 BEL-1, BEL-2 Group 2c A Penicillins Ticarcillin-clavulanate Group 2d D Penicillins Penicillin-Inhibitor Combinations PSE-1, PSE-3, PSE-4, PSE-5 CARB-3, CARB-4 XA-1, XA-2, XA-3, XA-4, XA-5, XA- 6, XA-10, XA-13, XA-20, XA-46, XA- 50, XA-56, LCR-1 Group 2de D Penicillins Penicillin-Inhibitor Combinations Cephalosporins Monobactams Group 2df D Penicillins Penicillin-Inhibitor Combinations Narrow-spectrum Cephalosporins Carbapenems XA-11, XA-14 to XA-19, XA-28, XA-31, XA-32, XA-35, XA-45, XA-74, XA-147, XA-161 XA-40, XA-50-type

4 212 Current Pharmaceutical Design, 2013, Vol. 19, No. 2 Wolter and Lister (Table 1) Contd... Functional Group Molecular Class Potential Resistance Impact Representative Enzymes Group 2f A Penicillins KPC Penicillin-Inhibitor Combinations Cephalosporins Carbapenems Group 3 B Penicillins Penicillin-inhibitor combinations Cephalosporins Cephamycins Carbapenems GES-2, GES-5 IMP-1, IMP-2, IMP-4, IMP-6, IMP-7, IMP-9, IMP-10, IMP-11, IMP-13, IMP-14, IMP-15, IMP- 16, IMP-18, IMP-22, VIM-1 to VIM-11, VIM-13, VIM-15 to VIM-18, SPM-1 GIM-1 inhibitor clavulanate [34-38]. Although several components of the ampc regulatory system have been identified and characterized, the precise pathway of induction remains unclear [22]. Induction of ampc expression provides P. aeruginosa with resistance to cefoxitin, but alone does not provide resistance to the carbapenems. The clinical significance of clavunate s induction of ampc is complex. Due to induction by clavulanate, Enterobacteriaceae that produce an inducible ampc are usually more susceptible to ticarcillin than ticarcillin-clavulanate [39]. By contrast, MICs of ticarcillinclavulanate against P. aeruginosa remain similar to those of ticarcillin alone despite the induction of ampc [36]. The reason for this discrepancy appears to relate to the concentration of clavulanate used in CLSI-recommended susceptibility assays (2 μg/ml), a concentration which is too low to induce sufficient levels of AmpC that would impact the potency of ticarcillin in the combination [36]. However, pharmacodynamic studies have demonstrated that the level of induction achieved with pharmacokinetically-relevant concentrations of clavulanate is sufficient to negatively impact the antibacterial activity of the combination [36]. A more serious therapeutic threat is when the regulation of ampc is lost through derepression, defined as constitutive highlevel ampc expression. As a result, P. aeruginosa becomes resistant to virtually all -lactams. The pathway to derepression of ampc usually involves genetic mutations that alter proteins responsible for regulating ampc expression [22], and these mutational events can lead to the emergence of resistance during therapy. Emergence of resistance due to ampc derepression has been reported in up to 56% of patients treated with anti-pseudomonal penicillins, penicillin-inhibitor combinations, extended-spectrum cephalosporins, and aztreonam [40-48], and most commonly occurs during treatment of infections outside the urinary tract and in patients with cystic fibrosis and neutropenia. Group 1e Extended-Spectrum AmpC Cephalosporinases. verexpression of ampc alone does not always impact susceptibility of P. aeruginosa to the carbapenems. However, recently described mutational variants of AmpC (extended-spectrum AmpC) have been characterized that can directly influence carbapenem susceptibility. These AmpC variants were first described in Enterobacteriaceae [49-53], and more recently among P. aeruginosa [54]. Although these enzymes can exhibit increased hydrolytic activity against cephalosporins and imipenem, overproduction of extendedspectrum AmpCs seems to be a requirement for carbapenem resistance [54]. Characterization of 32 carbapenem-intermediate and resistant P. aeruginosa isolates from France demonstrated that 21 (65%) overexpressed an extended-spectrum AmpC cephalosporinase [55]. Group 2b -lactamases. Group 2b -lactamases are serinebased enzymes from molecular class A and include the common plasmid-encoded enzymes TEM-1, TEM-2, and SHV-1 [24]. These -lactamases were first found in members of the Enterobacteriaceae [56, 57], and later identified in strains of P. aeruginosa [58]. Group 2b -lactamases most readily hydrolyze the penicillins and the early cephalosporin antibiotics. Group 2be Extended-Spectrum -lactamases. Group 2be includes a wide variety of serine-based extended-spectrum lactamases (ESBLs) from molecular class A. The first ESBLs characterized were variants of the Group 2b SHV-1, TEM-1, and TEM- 2 -lactamases and were discovered in members of the Enterobacteriaceae [59-61]. These ESBLs are able to hydrolyze the penicillins and early cephalosporins like the Group 2b enzymes, but also demonstrate an extension of their hydrolytic capabilities to include extended-spectrum oxyimino-cephalosporins (ceftazidime and cefotaxime), and the monobactam aztreonam [62, 63]. ESBLs have also been identified in P. aeruginosa. The first TEM-derived ESBL reported in P. aeruginosa was TEM-42 from a clinical isolate in France [64]. Since that first report, only four other TEM-derived ESBLs have been described, including TEM-4, TEM- 21, TEM-24, and TEM-116 [65-68]. In addition, four SHV-derived ESBLs (SHV-2, SHV-2a, SHV-5, and SHV-12) have been identified in clinical isolates of P. aeruginosa [69-72]. The CTX-M-family of -lactamases represents a group of enzymes that are not closely related to either the TEM- or SHVderived ESBLs, but are classified as ESBLs based upon their hydrolytic profiles [73]. These enzymes share homology with the chromosomal AmpC of Kluyvera ascorbata [74]. To date, three CTX- M-family ESBLs have been identified in P. aeruginosa, and include CTX-M-1, CTX-M-2, and CTX-M-43 [65, 75, 76]. Additional Group 2be ESBLs that show limited homology with TEM- or SHV-derived ESBLs include the PER-type, VEB-type, GES-type, BEL-type, and PME-1 -lactamases. PER-1 was first described in 1993 in a strain of P. aeruginosa from France [77]. A closely related enzyme, PER-2, has also been identified in P. aeruginosa [75]. The first VEB-type ESBL found in P. aeruginosa was VEB-1, also from a clinical isolate in France [78]. Subsequently, three other VEB-like ESBLs (VEB-1a, VEB-1b, and VEB-2) have been identified in P. aeruginosa and share > 99% amino acid sequence homology with VEB-1 [79, 80]. The GES-1 ESBL was

5 Antibacterial Resistant Pseudomonas aeruginosa Current Pharmaceutical Design, 2013, Vol. 19, No discovered in a strain of K. pneumoniae from French Guiana [81], and then subsequently in P. aeruginosa [82]. Extended-spectrum lactamases GES-8, GES-9, and GES-13 have also been identified in isolates of P. aeruginosa [83-85]. The extended-spectrum lactamase BEL-1 was discovered in 2005 in a clinical isolate of P. aeruginosa from Belgium [86], and more recently, BEL-2 was found in another P. aeruginosa isolate from Belgium [87]. The most recent ESBL discovered, PME-1 (Pseudomonas aeruginosa ESBL 1) was described in a clinical isolate of P. aeruginosa from Pennsylvania and shares 50% amino acid identity with the L2 lactamase of Stenotrophomonas maltophilia [88]. Group 2c Penicillinases: The Group 2c enzymes encompass a relatively small group of serine-based penicillinases from molecular class A [24]. Group 2c enzymes identified in P. aeruginosa include PSE-1, PSE-3, PSE-4, PSE-5, CARB-3, and CARB-4. Although these enzymes are inhibited by clavulanate, they hydrolyze ticarcillin with such efficiency that they can provide P. aeruginosa with resistance to ticarcillin-clavulanate [89-91]. In contrast, strains of P. aeruginosa that produce PSE-type enzymes are more susceptible to piperacillin-tazobactam, most likely due to the decreased hydrolysis of piperacillin allowing tazobactam to be a more effective inhibitor [90]. Group 2d XA-type Penicillinases: The Group 2d -lactamases are molecular class D enzymes class A enzymes with respect to the active site serine, but they are only weakly inhibited by clavulanate and tazobactam [24]. The characteristics and clinical importance of these enzymes have been extensively reviewed by Poirel et al. [92]. Although these enzymes were first distinguished by the increased hydrolytic activity against cloxacillin and oxacillin, thus the designation XA-type -lactamases, this phenotype does not define all enzymes in the family [92]. The XA-family of enzymes represents one of the largest and most diverse groups of -lactamases, ranking second behind the TEM-family of enzymes [24]. Although different XA-type enzymes have been isolated from a variety of gram-negative bacterial species [92], many of the XA-type lactamase variants can be found in isolates of Acinetobacter baumannii and P. aeruginosa [92]. Table 1 includes representative Group 2d XA-type -lactamases detected in P. aeruginosa. Due to their limited hydrolytic profiles and lack of inhibition by tazobactam and clavulanate, the Group 2d XA-type -lactamases primarily provide P. aeruginosa resistance to the anti-pseudomonal penicillins and inhibitor-penicillin combinations [92]. Group 2de XA-type Extended-Spectrum -lactamases: The first published extended-spectrum XA-type -lactamase was XA-11 [93]. This enzyme is a variant of the narrow-spectrum XA-10 (formerly PSE-2) and was first identified in 1991 in a P. aeruginosa isolate from Turkey [93]. ver the past two decades, several other extended-spectrum XA-family enzymes have been identified, primarily among clinical isolates of P. aeruginosa [92, 94] (Table 1). Although the hydrolytic profiles can vary substantially, the XA-type ESBLs are characterized by their increased hydrolysis of the oxyimino-cephalosporins and aztreonam and can provide resistance to these drugs [92]. Group 2df XA-type Carbapenemases: The carbapenemhydrolyzing XA-type -lactamases have been identified most frequently among isolates of A. baumannii, and most are encoded by chromosomal genes [95]. To date, only a few XA-type carbapenemases have been reported in P. aeruginosa (Table 1) [96, 97]. The XA-40 carbapenemase described in P. aeruginosa was shown to be identical to the enzyme found in A. baumannii [97]. Although the bla XA-40 gene is found on similar plasmids in both P. aeruginosa and A. baumannii [97], it was originally described as a chromosomal gene in A. baumannii [98]. P. aeruginosa also encodes for a chromosomal XA-50 enzyme, of which sequence variants have been described and characterized as carbapenemases [96]. Although the XA-type carbapenemases have a high affinity for carbapenems, they do not efficiently hydrolyze these substrates and their clinical significance is difficult to evaluate [95, 99]. Group 2f Carbapenemases: The Group 2f carbapenemases are serine-based -lactamases belonging to molecular class A [24, 100]. In addition to the carbapenems, these -lactamases are capable of hydrolyzing the penicillins, cephalosporins, and aztreonam, but they are inhibited by tazobactam and clavulanic acid [24, 99]. Although some Group 2f enzymes are encoded by chromosomal genes (e.g. SME), the class A carbapenemases of clinical importance to P. aeruginosa are carried on plasmids. The first family includes the GES -lactamases, of which GES- 1 was discovered as an ESBL in an isolate of K. pneumonia [81]. Two variants, GES-2 and GES-5, have been identified among P. aeruginosa clinical isolates and nosocomial outbreaks from different geographic regions [ ]. Although they were first considered ESBLs based on their hydrolytic profiles, these enzymes demonstrate an extension of their hydrolytic activity to include imipenem [99, 100, 104]. The second family includes the Klebsiella pneumoniae carbapenemases (KPCs), which were discovered in and most commonly found among clinical isolates of K. pneumonia and other members of the Enterobacteriaceae [99, 100]. Currently, 12 different variants of KPCs (KPC-2 through KPC-13) have been identified. The first KPC-producing P. aeruginosa were reported among clinical isolates in Colombia, and these isolates produced KPC-2 [106]. Subsequently, KPC-2 has been identified among isolates of P. aeruginosa from Puerto Rico [107], Trinidad and Tobago [108], China [109], and the United States [110], and KPC-5 was characterized in a clinical isolate of P. aeruginosa from Puerto Rico [111]. Strains of P. aeruginosa that produce KPCs can exhibit resistance to all lactam antibiotics. Group 3 Metallo- -lactamases: The Group 3 metallo- lactamases are molecular class B enzymes that are characterized by their requirement for a zinc ion in the active site [24, 112]. As a result of their broad-spectrum hydrolytic capacity and lack of inhibition by tazobactam or clavulanate, metallo- -lactamases can provide resistance to the penicillins, inhibitor-penicillin combinations, and carbapenems [24, 112]. However, metallo- -lactamases do not efficiently hydrolyze monobactams [24, 112]. The history and clinical significance of metallo- -lactamases have been the focus of two recent reviews [99, 112]. Similar to the class A carbapenemases described above, metallo- -lactamases can be encoded on both the chromosome and mobile genetic elements. The first transferable metallo- -lactamase, IMP-1, was identified in 1988 in a clinical isolate P. aeruginosa from Japan [113]. Since this first report, dozens of transferrable metallo- -lactamases have been described globally among isolates of P. aeruginosa, and they belong to four distinct families, namely IMP, VIM, GIM, and SPM [112]. PRIN-MEDIATED RESISTANCE The outer membrane of P. aeruginosa is an effective antibacterial barrier that is only 8% as permeable as the outer membrane of Escherichia coli [114]. To aid in the passage of nutrients and other desired molecules into the cell, P. aeruginosa utilizes a collection of porin channels. Based on the completed sequence of the P. aeruginosa genome, 64 known or putative porins have been identified [114]. The following discussion will primarily focus on the wellcharacterized association between the prd porin and carbapenem susceptibility of P. aeruginosa, but also touch upon the potential role of prf in altering -lactam susceptibility. prd and Susceptibility of P. aeruginosa to Carbapenems The prd porin of P. aeruginosa is a substrate-specific porin that facilitates the diffusion of basic amino acids, small peptides, and carbapenems into the cell [115, 116]. Studies suggest that prd

6 214 Current Pharmaceutical Design, 2013, Vol. 19, No. 2 Wolter and Lister is the preferred portal of entry for carbapenems across the outer membrane, as decreases or loss of prd significantly decrease the susceptibility of P. aeruginosa to available carbapenems [30, ]. However, recent studies have described strains of P. aeruginosa that exhibit a discordance between prd absence and susceptibility to carbapenems [33, 120], suggesting other pathways of carbapenem penetration. The complex pathways used by P. aeruginosa to regulate levels of prd in the outer membrane has been recently reviewed [22], and include mechanisms that impact oprd expression and mechanisms that influence the production of a functional prd channel. Regardless of the pathway, the impact of prd-mediated resistance on the carbapenems can be analyzed by comparing carbapenem susceptibilities according to clinically-significant breakpoints (established by the CLSI) with prd levels. Although some investigators have concluded from the analysis of non-controlled clinical isolates that meropenem is unique in having alternative pathways of penetration [121], data from a more recent study of controlled isogenic wild-type and prd-deficient mutant pairs demonstrated that loss of prd impacted susceptibility to meropenem to a greater degree than imipenem or doripenem [122]. When considering the clinical impact of prd-mediated resistance, loss of prd alone is usually sufficient to push MICs of imipenem above the resistance breakpoint. This is not surprising since the MIC 50 for imipenem against P. aeruginosa is already at 1 μg/ml [123, 124]. By comparison, wild-type P. aeruginosa are generally 4-fold more susceptible to meropenem and doripenem than they are to imipenem, and the impact of prd-mediated resistance on potency to these carbapenems does not always push MICs above the susceptible breakpoint. Additional resistance mechanisms, e.g. efflux pump overproduction and/or -lactamase production, may be required to provide clinical resistance to meropenem and doripenem. For example, studies using laboratory-derived mutants have shown that a combination of prd loss with either MexAB-prM overexpression or AmpC overproduction resulted in significantly higher MICs to meropenem and doripenem, respectively, compared to porin loss alone [125]. Potential Decrease of -lactam Susceptibility Through prf Loss The porin, prf, is a major constituent of the P. aeruginosa outer membrane and participates in the maintenance of cell shape [126, 127], supports growth in low osmolarity conditions [128], potentially serves as a general or nonspecific porin for the passage of various compounds [128, 129], and has a proposed role in adherence to epithelial cells [130]. Yoon et al. also demonstrated a role for oprf in biofilm formation, anaerobic growth, and nitrate uptake [131]. Similar to the effect of prd loss on carbapenem susceptibilities, loss of prf has been suggested in a few studies to decrease the susceptibility of P. aeruginosa to certain -lactams. In the first study, Piddock et al. reported that a multi-drug resistant strain (including resistance to several -lactams such as cefotaxime, ceftazidime, and carbenicillin) of P. aeruginosa that lacked prf was isolated from a patient following therapy [132]. Restoration of prf production conincided with reversion of -lactam susceptibilities to pre-therapy levels [132]. Secondly, a laboratory-derived prf deficient strain of P. aeruginosa had modest increases in MICs to several -lactams including carbenicillin and cefotaxime [133]. Loss of prf may have decreased -lactam susceptibility in these studies by causing either: 1) diminished -lactam uptake directly through the prf channel or 2) altered permeability as the indirect result of defects in membrane architecture since prf serves in anchoring the outer membrane to the peptidoglycan layer [128, 134]. Regardless, prf may be another mechanism that can influence -lactam susceptibilities. Additional studies are needed to address this possibility. EFFLUX-MEDIATED RESISTANCE -lactams that freely diffuse into the P. aeruginosa cell and escape inactivation by resident -lactamases may encounter another formidable obstacle in route to their target, extrusion by efflux pumps. The P. aeruginosa PA1 genome possesses efflux encoding genes belonging to five different superfamilies [135], but thus far, only efflux pumps belonging to the resistance-nodulationdivision (RND) family have been shown to participate in -lactam resistance. The RND family represents the highest number of predicted pumps within the P. aeruginosa chromosome, 12 total including two divalent metal cation transporters, which far exceeds the RND pumps present on the E. coli chromosome (4 total) [135]. f these, 3 RND efflux pumps are capable of exporting -lactams, namely MexAB-prM, MexCD-prJ, and MexXY. The P. aeruginosa RND pumps are assembled with three separate components, a periplasmic membrane fusion protein (MFP), an outer membrane factor (MF), and a cytoplasmic membrane (RND) transporter, that form a channel spanning across both the inner and outer membranes (Fig. 2) [22]. Although the RND pump creates a passage way from the extracellular milieu to the cytoplasm from which substrates may be removed, the architecture of the pump also permits the recognition and export of compounds from the periplasmic space, the site of -lactam activity. Crystallographic examination of a RND component in E. coli, AcrB (homologous to MexB in P. aeruginosa) [136, 137], and H + MFP RND MP mfp rnd Transcription omp M PG Fig. (2). Structure and function of RND efflux pumps in P. aeruginosa. RND pumps typically exist in a tripartite system consisting of an RND cytoplasmic membrane transporter (RND), a membrane fusion protein (MFP), and outer membrane factor (MF). The genes which encode the pump components are organized into operons, and following translation of the polycistronic message, the pump components assemble into a channel that spans across the entire membrane. -lactams enter into the RND transporter through openings known as vestibules and are exported through the channel to the extracellular milieu using proton-motive force. Figure revised and reprinted from Lister et al. [22] with permission from the American Society of Microbiology. PS CM

7 Antibacterial Resistant Pseudomonas aeruginosa Current Pharmaceutical Design, 2013, Vol. 19, No protein modeling of the MexB component in P. aeruginosa [138] have shown them to exist as a homotrimeric complex with transmembrane helices lodged into the cytoplasmic membrane (transmembrane domain) and a periplasmic domain that forms a central cavity. A route from the periplasm to the central cavity exists through openings, referred to as vestibules, between monomers along the surface of the cytoplasmic membrane [139]. -lactams in the periplasm may enter into the RND transporter through these vestibules. Initial chimeric and mutational studies of E. coli and P. aeruginosa pumps linked substrate specificity to the cytoplasmic RND transporter and suggest the location of substrate recognition occurs within the central cavity [140, 141]. However, more recent studies have identified a binding pocket within the periplasmic domain that is connected to the vestibule through an additional channel, and this pocket serves as the substrate recognition site [142, 143]. nce inside the binding pocket, RND transporters utilize proton-motive force to translocate substrates (e.g. -lactams) through the MF to the extracellular environment. Addition of compounds that interfere with the proton gradient, such as the proton uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), prevents the efflux of substrates from within the cell [144]. The efflux encoding genes within P. aeruginosa are organized into operons on the bacterial chromosome (Fig. 2) [22]. mexaboprm, mexcd-oprj, and mexxy each possess a gene that codes for the MFP (mexa, mexc, and mexx) and RND transporter (mexb, mexd, and mexy). The mexab-oprm and mexcd-oprj operons also contain a gene for the MF (oprm and oprj) whose product completes the formation of the tripartite efflux systems. However, an MF encoding gene is absent from the mexxy operon. Therefore, MexXY must associate with other outer membrane protein components to create a functional pump. prm was shown to serve as the MF for MexXY [ ] and several other candidate MFs have been proposed including pmb, pmg, pmh, and possibly pmi [146, 148]. The regulatory genes mexr, nfxb, and mexz reside directly upstream of mexab-oprm, mexcd-oprj, and mexxy, respectively, and are transcribed divergently from the operon [22]. The products of these regulatory genes bind to operator sites within the intergenic regions to control expression of the efflux operons (discussed below). MexAB-prM MexAB-prM, the first discovered P. aeruginosa RND pump, contributes to both the intrinsic and adaptive resistance of P. aeruginosa to the -lactam class of antibiotics. MexAB-prM is able to export the widest range of -lactams among the RND pumps such as carbenicillin and ticarcillin (carboxypenicillins), piperacillin (ureidopenicillin), aztreonam (monobactam), ceftazidime and cefotaxime (3rd generation cephalosporins), faropenem (penem), and the carbapenems meropenem and panipenem (excluding imipenem and biapenem). A recent study showed that overproduction of MexAB-prM in a defined laboratory mutant led to slightly elevated MICs for doripenem compared to the parental P. aeruginosa strain suggesting the recognition and export of this drug at a low level [125]. -lactam inhibitors (e.g. clavulanate and cloxacillin) are also substrates for MexAB-prM [149]. Intrinsic resistance to these -lactams is attributed to the constitutive expression of mexaboprm in wild-type cells [150]. Deletion of any one component (mexa, mexb, or oprm) through genetic knockout renders P. aeruginosa hypersusceptible to the -lactams listed above [151, 152]. Expression of mexab-oprm is growth-phase dependent in wild-type cells and is regulated by the N-Butyryl-L-homoserine lactone (C4- HSL) quorum sensing autoinducer [153, 154]. Although expressed at sufficient levels to influence -lactam susceptibility, transcription of the mexab-oprm operon in wild-type P. aeruginosa is not fully derepressed. Expression of mexab-oprm is initiated from two separate promoters, a distal mexa promoter that overlaps with the mexr promoter and a proximal mexa promoter [155]. However, each promoter is occupied by regulatory proteins that repress the transcription of the efflux operon. MexR, a regulatory protein of the MarR family [156], binds to cis-acting sites that encompass the distal mexa promoter, and a trans-acting repressor of the TetR family, NalD, binds to sequences encompasssing the mexa proximal promoter [157]. Loss of MexR-mediated regulation of the distal mexa promoter causes mexab-oprm hyperexpression in mutants termed nalb-type and nalc-type. In nalb-type strains, mutations within mexr can either truncate the production of full length protein, potentially diminish protein stability, or compromise MexR functionality by preventing protein dimerization or DNA binding [158, 159]. nalctype mutants retain a wild-type mexr, but mutations in another gene, nalc (formerly PA3721), indirectly impacts the ability of MexR to bind to the distal mexa promoter. NalC, a member of the TetR/AcrR family, suppresses the expression of the PA3720- PA3719 (renamed ArmR), and inactivation of NalC results in ArmR overexpression [160, 161]. ArmR is a 53-amino acid antirepressor that binds inside a hydrophobic cavity within the MexR dimer and allosterically inhibits MexR from docking with the distal mexa promoter [162]. nalc-type mutants overexpress mexab-oprm at lower levels in comparison to nalb-type mutants, and as a result, do not have as great of an impact on -lactam susceptibility than nalb-type mutants [163]. Hyperexpression of mexab-oprm from the proximal mexa promoter occurs in nald-type mutants as a result of mutations within nald. These mutations presumably interfere with the ability of NalD from binding to the operator sequence associated with the mexa-proximal promoter [157]. MexCD-prJ In comparison to MexAB-prM, the MexCD-prJ efflux pump has a more restricted substrate profile for the -lactams. MexCD-prJ is capable of exporting the fourth generation cephalosporins (i.e. cefepime, cefpirome, and cefozopran) [164, 165]. Studies have suggested that additional -lactams, such as the carbapenems meropenem and doripenem, may also be exported by MexCD-prJ [166, 167]. However, substrate specificities in these studies were determined using isogenic mutants containing deletions of other efflux systems. The possibility that these deletions may have triggered changes in other mechanisms that could impact drug susceptibility can not be ruled out. Nonetheless, overproduction of MexCD-prJ does not decrease the susceptibility of P. aeruginosa mutants to these additional -lactams [164], but rather, only impacts the fourth generation cephalosporins. MexCD-prJ also differs from MexAB-prM in that the pump is not detectable in wild-type cells [165, 168]. As such, MexCD-prJ does not contribute to intrinisic -lactam resistance [169, 170]. Expression of the mexcd-oprj operon is tightly controlled by the regulatory factor NfxB, a protein that displays similarity to the LacI-GalR family [165]. NfxB binds to a cis-acting site within the nfxb-mexc intergenic region [171] and represses expression of the efflux operon [165, 171]. Mutations within nfxb (e.g. base substitutions, deletions, IS element disruption) have been suggested to alleviate repression of mexcd-oprj leading to its overexpression in mutants termed nfxb-type [165, ]. verproduction of MexCD-prJ has been shown to occur at two different levels in mutants defined as type A and type B. Type A mutants produce lower amounts of MexCD-prJ than type B mutants, and as a result, have a smaller decrease in susceptibility to antibiotics exported by this pump in comparison to type B mutants [164]. Regardless, overproduction of MexCD-prJ in both types of mutants is dependent on alterations within NfxB [164]. MexXY The third RND efflux system responsible for -lactam resistance in P. aeruginosa is the MexXY pump. As mentioned above, an MF encoding gene is absent from the mexxy operon, but in-

8 216 Current Pharmaceutical Design, 2013, Vol. 19, No. 2 Wolter and Lister stead, MexXY associates with prm [ ] and possibly other candidate MFs [146, 148] to form a tripartite pump. MexXY has a similar substrate profile to the MexCD-prJ pump with its ability to export fourth generation cephalosporins [166, 174] but differs from MexCD-prJ in that MexXY accommodates the removal of the "fifth generation" cephalosporin ceftobiprole [175, 176]. mexxy expression in P. aeruginosa is inducible when cells are exposed to ribosomal inhibitors (e.g. tetracycline, erythromycin, and gentamicin) [177], but expression does not increase when challenged with sub-inhibitory concentrations of cefepime [178]. Thus, MexXY most likely does not contribute to intrinsic -lactam resistance in wild-type cells. Expression of mexxy is negatively regulated by MexZ whose gene is located directly upstream of the efflux operon. MexZ, a member of the TetR regulatory family, binds to an inverted repeat sequence within the mexz-mexx intergenic region and blocks access of the RNA polymerase to the putative mexxy promoter [179]. verexpression of mexxy has been associated with mutations in mexz or the mexz-mexx intergenic region in mutants termed agrztype [180]. However, P. aeruginosa strains hyperexpressing mexxy but lacking mutations within these sites, called agrw-type mutants, have also been described [180]. mexxy overexpression in agrwtype mutants has recently been linked to a two-component regulatory system, ParRS [181]. Contribution of Efflux to -lactam Resistance in Clinical Isolates Numerous studies have detected the overexpression of an RND efflux pump in -lactam resistant P. aeruginosa clinical isolates suggesting their participation in the resistant phenotype. MexAB- prm hyperexpressing clinical isolates have been observed for each of the nal (B, C, and D)-type mutants and have often been identified in studies examining the mechanism(s) associated with carbapenem resistance in P. aeruginosa [ ]. mexxy overexpressing agrz- and agrw-type mutants from the clinical setting have also been encountered frequently [ ]. MexCD-prJ overproducing clinical isolates have been described in the literature [ ] but are not as common as the other pump mutants, possibly suggesting a disadvantage for P. aeruginosa that utilize this resistance mechanism. Indeed, studies have shown that nfxb-type mutants have diminished fitness and virulence traits [ ]. verexpression of the RND efflux pumps typically reduces the susceptibility (2-8-fold decrease in MIC) of P. aeruginosa to lactams that are substrates for a particular pump, but clinical resistance (according to CLSI breakpoints) often requires the involvement of an additional resistance mechanism. For example, nalbtype mutants have decreased susceptibility to meropenem, but full resistance occurs with a concomittant loss of prd [117]. The combination of prd loss and MexAB-prM overepxression has been detected in several meropenem resistant clinical isolates [ ]. Additional combinations of -lactam resistance mechanisms have been reported including the simultaneous overexpression of two efflux pumps. Several groups have detected the cohyperexpression of MexAB-prM and MexXY in resistant P. aeruginosa [180, 201, 202]. Efflux pump overexpression has been discovered in isolates with concurrent chromosomal AmpC derepression [ ] or when harboring an acquired -lactamase (e.g. XA, GES) [174, 207]. Isolates have even been encountered containing at least three different -lactam resistance mechanisms (prd loss, pump hyperexpression, and -lactamase production) [107, 208, 209]. The repeated identification of efflux overexpressing P. aeruginosa clinical isolates, either alone or in combination with other mechanisms, highlights their importance in -lactam resistance. Pseudomonas aeruginosa -LACTAM RESISTME The mechanisms discussed so far in this review are typically considered as the predominant contributors to -lactam resistance in P. aeruginosa and have been analyzed quite extensively as a result of their impact on drug susceptibility. However, recent studies using more global screening methods (e.g. analysis of transposon mutant libraries and proteomics) have indicated that additional genetic determinants may participate in decreasing -lactam susceptibility [ ]. ne such library, the Harvard PA14 nonredundant library, represents a collection of mutants in which nonessential genes have been inactivated by a single transposon insertion [213]. This library has been used by two independent research groups to identify genes that directly cause -lactam resistance when inactivated or indirectly by increasing the mutational frequency [210, 211]. Susceptibility testing of mutants in the PA14 library to various -lactams revealed novel genes responsible for altering -lactam susceptibility. Many of these genes participate in the synthesis of the bacterial cell wall or lipopolysaccharide (LPS) including: galu (PA1 ortholog PA2023), a UDP-glucose pyrophosphorylase responsible for synthesis of a complete LPS core, and wbpm (PA1 ortholog PA3141), a UDP-N-acetylglucosamine 4,6-dehydratase essential for the biosynthesis of B-band LPS. Inactivation of genes within a large 17 RF gene cluster (PA4996 PA5012) that are involved in the assembly of the core oligosaccharide component of LPS were also identified by both research groups as causing decreased -lactam susceptibility [210, 211]. Alterations in cell wall and LPS architecture are believed to decrease the penetration of lactams into the periplasmic space. Various modifications in LPS composition were previously shown to either decrease [214, 215] or increase [216] -lactam susceptibility in P. aeruginosa supporting the role of LPS in antibiotic uptake. Besides genes involved with cell wall and LPS synthesis, analysis of the PA14 library has identified additional genes with diverse functions that alter -lactam susceptibility when deleted. Inactivation of genes that participate in chemotaxis and biofilm formation (wspe, a histidine kinase-response regulator and wspr, a diguanylate cyclase), amino acid metabolism (arob, a 3- dehydroquinate synthetase), carbon compound catabolism and energy metabolism (PA5192, a phosphoenolpyruvate carboxykinase), and transcriptional regulation (PA0479, regulator of the LysR family) all decreased susceptibility of P. aeruginosa to several lactams [210, 211]. It is unknown at this time how the loss of these genes influence -lactam susceptibility. However, these results illustrate the vast array of mechanisms capable of altering susceptibility of P. aeruginosa to -lactams. CNFLICT F INTEREST The authors confirm that this article content has no conflicts of interest. ACKNWLEDGEMENT Declared none. REFERENCES [1] Pollack M. Pseudomonas aeruginosa. In: Mandell GL, Bennett JE, Dolin R, ed.^eds., Principles and Practice of Infectious Diseases. Churchill Livingstone: New York 1995; pp [2] Pitt TL. Pseudomonas, Burkholderia, and Related Genera. In: Duerden BI, ed.^eds., Microbiology and Microbial Infections. xford University Press Inc: New York City 1998; pp [3] Pollack M. Pseudomonas aeruginosa. In: Mandell GL, Dolan R, Bennett JE, ed.^eds., Principles and Practices of Infectious Diseases. 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