Activity of a New Cephalosporin,CXA-101 (FR264205), against β-Lactam-Resistant Pseudomonas aeruginosa Mutants Selected In Vitro and after Antipseudomonal Treatment of Intensive Care Unit Patients

CXA-101, previously designated {"type":"entrez-nucleotide","attrs":{"text":"FR264205","term_id":"258272820","term_text":"FR264205"}}FR264205, is a new antipseudomonal cephalosporin. We evaluated the activity of CXA-101 against a highly challenging collection of β-lactam-resistant Pseudomonas aeruginosa mutants selected in vitro and after antipseudomonal treatment of intensive care unit (ICU) patients. The in vitro mutants investigated included strains with multiple combinations of mutations leading to several degrees of AmpC overexpression (ampD, ampDh2, ampDh3, and dacB [PBP4]) and porin loss (oprD). CXA-101 remained active against even the AmpD-PBP4 double mutant (MIC = 2 μg/ml), which shows extremely high levels of AmpC expression. Indeed, this mutant showed high-level resistance to all tested β-lactams, except carbapenems, including piperacillin-tazobactam (PTZ), aztreonam (ATM), ceftazidime (CAZ), and cefepime (FEP), a cephalosporin considered to be relatively stable against hydrolysis by AmpC. Moreover, CXA-101 was the only β-lactam tested (including the carbapenems imipenem [IMP] and meropenem [MER]) that remained fully active against the OprD-AmpD and OprD-PBP4 double mutants (MIC = 0.5 μg/ml). Additionally, we tested a collection of 50 sequential isolates that were susceptible or resistant to penicillicins, cephalosporins, carbapenems, or fluoroquinolones that emerged during treatment of ICU patients. All of the mutants resistant to CAZ, FEP, PTZ, IMP, MER, or ciprofloxacin showed relatively low CXA-101 MICs (range, 0.12 to 4 μg/ml; mean, 1 to 2 μg/ml). CXA-101 MICs of pan-β-lactam-resistant strains ranged from 1 to 4 μg/ml (mean, 2.5 μg/ml). As described for the in vitro mutants, CXA-101 retained activity against the natural AmpD-PBP4 double mutants, even when these exhibited additional overexpression of the MexAB-OprM efflux pump. Therefore, clinical trials are needed to evaluate the usefulness of CXA-101 for the treatment of P. aeruginosa nosocomial infections, particularly those caused by multidrug-resistant isolates that emerge during antipseudomonal treatments.The growing threat of Pseudomonas aeruginosa antimicrobial resistance results from, on the one hand, the extraordinary capacity of this microorganism for developing resistance to almost every available antibiotic by the selection of mutations in chromosomal genes and, on the other hand, the increasing prevalence of transferable resistance determinants, particularly those encoding class B carbapenemases (or metallo-β-lactamases [MBLs]) or extended-spectrum β-lactamases (ESBLs), frequently cotransferred with genes encoding aminoglycoside-modifying enzymes (16, 19).Particularly noteworthy among the mutation-mediated resistance mechanisms are those leading to the repression or inactivation of the porin OprD, conferring resistance to carbapenems (5, 7, 24, 26), or those leading to the hyperproduction of the chromosomal cephalosporinase AmpC, such as AmpD or PBP4 inactivation (11, 20), causing resistance to penicillins and cephalosporins. In addition, mutations leading to the upregulation of one of several efflux pumps encoded in the P. aeruginosa genome may confer resistance or reduced susceptibility to multiple agents, including almost all β-lactams, fluoroquinolones, and aminoglycosides (3, 18, 25). Furthermore, the accumulation of these chromosomal mutations can lead to the emergence of multidrug-resistant (MDR) (or even pan-antibiotic-resistant) strains which eventually may be responsible for outbreaks in the hospital setting (4). Indeed, sequential development of resistance to almost all available antipseudomonal agents is a not uncommon outcome of the treatment of severe P. aeruginosa infections, frequently occurring in intensive care unit (ICU) patients or patients with hematological disease (2, 10).Unfortunately, over the last 2 decades there has been very limited progress in developing novel antipseudomonal agents which can overcome MDR in P. aeruginosa (19). CXA-101 (previously designated {"type":"entrez-nucleotide","attrs":{"text":"FR264205","term_id":"258272820","term_text":"FR264205"}}FR264205), a new cephalosporin with promising characteristics for the treatment of P. aeruginosa infections, appears stable against the most common resistance mechanisms driven by mutation in this species (27, 28).The objective of this study was to investigate the activity of CXA-101 against β-lactam-resistant P. aeruginosa mutants selected in vitro and after antipseudomonal treatment of ICU patients. The in vitro mutants investigated included a highly challenging collection of strains with multiple combinations of mutations leading to various levels of AmpC overexpression (ampD, ampDh2, ampDh3, and dacB [PBP4]) and porin loss (oprD). Additionally, a well-characterized collection of 50 sequential isolates susceptible or resistant to penicillicins, cephalosporins, carbapenems, or fluoroquinolones (first-line antipseudomonal agents) that emerged during treatment of ICU patients was tested.     

Activity of a New Antipseudomonal Cephalosporin,CXA-101 (FR264205), against Carbapenem-Resistant and Multidrug-Resistant Pseudomonas aeruginosa Clinical Strains

The activity of the new cephalosporin CXA-101 (CXA), previously designated {"type":"entrez-nucleotide","attrs":{"text":"FR264205","term_id":"258272820","term_text":"FR264205"}}FR264205, was evaluated against a collection of 236 carbapenem-resistant P. aeruginosa isolates, including 165 different clonal types, from a Spanish multicenter (127-hospital) study. The MICs of CXA were compared to the susceptibility results for antipseudomonal penicillins, cephalosporins, carbapenems, aminoglycosides, and fluoroquinolones. The MIC of CXA in combination with tazobactam (4 and 8 μg/ml) was determined for strains with high CXA MICs. The presence of acquired β-lactamases was investigated by isoelectric focusing and PCR amplification followed by sequencing. Additional β-lactamase genes were identified by cloning and sequencing. The CXA MIC₅₀/MIC₉₀ for the complete collection of carbapenem-resistant P. aeruginosa isolates was 1/4 μg/ml, with 95.3% of the isolates showing an MIC of ≤8 μg/ml. Cross-resistance with any of the antibiotics tested was not observed; the MIC₅₀/MIC₉₀ of CXA-101 was still 1/4 when multidrug-resistant (MDR) strains (42% of all tested isolates) or AmpC-hyperproducing clones (53%) were analyzed. Almost all (10/11) of the strains showing a CXA MIC of >8 μg/ml produced a horizontally acquired β-lactamase, including the metallo-β-lactamase (MBL) VIM-2 (one strain), the extended-spectrum β-lactamase (ESBL) PER-1 (one strain), several extended-spectrum OXA enzymes (OXA-101 [one strain], OXA-17 [two strains], and a newly described OXA-2 derivative [W159R] designated OXA-144 [four strains]), and a new BEL variant (BEL-3) ESBL (one strain), as identified by cloning and sequencing. Synergy with tazobactam in these 11 strains was limited, although 8 μg/ml reduced the mean CXA MIC by 2-fold. CXA is highly active against carbapenem-resistant P. aeruginosa isolates, including MDR strains. Resistance was restricted to still-uncommon strains producing an acquired MBL or ESBL.The increasing prevalence of nosocomial infections caused by multidrug-resistant (MDR) Pseudomonas aeruginosa strains severely compromises the selection of appropriate antibacterial treatments and is therefore associated with significant morbidity and mortality (13, 20). The growing threat of antimicrobial resistance in P. aeruginosa results from the extraordinary capacity of this microorganism to develop resistance to almost any available antibiotic by the selection of mutations in chromosomal genes and from the increasing prevalence of transferable resistance determinants, particularly those encoding class B carbapenemases (or metallo-β-lactamases [MBLs]) or extended-spectrum β-lactamases (ESBLs) (14, 18). VIM and IMP MBLs and OXA and PER ESBLs have disseminated among P. aeruginosa strains from diverse geographic areas (6, 11, 16, 22, 29, 32, 39). These resistance genes are frequently carried in integrons, together with aminoglycoside resistance determinants that are often located in transferable elements such as plasmids or transposons (12, 24, 25, 26, 31). Noteworthy among the mutation-mediated resistance mechanisms are those leading to the repression or inactivation of the porin OprD, conferring resistance to carbapenems (5, 8, 23, 30, 41), or those leading to the hyperproduction of the chromosomal cephalosporinase AmpC, such as AmpD or PBP4 inactivation (10, 19), determining resistance to penicillins and cephalosporins. In addition, mutations leading to the upregulation of one of several efflux pumps encoded in the P. aeruginosa genome may confer resistance or reduced susceptibility to multiple agents, including almost all β-lactams, fluoroquinolones, and aminoglycosides (2, 17, 28). The accumulation of these chromosomal mutations can lead to the emergence of MDR (or even pan-antibiotic-resistant) strains which eventually may be responsible for outbreaks in the hospital setting (4, 9).Unfortunately, in the last 2 decades, little progress has occurred in developing novel antipseudomonal agents that can overcome MDR in P. aeruginosa. CXA-101, previously designated {"type":"entrez-nucleotide","attrs":{"text":"FR264205","term_id":"258272820","term_text":"FR264205"}}FR264205, is a new promising cephalosporin intended for the treatment of P. aeruginosa infections that appears to be stable against the most common resistance mechanisms driven by mutation in this species (35, 36). The objective of this study was to evaluate the activity of CXA-101 against a collection of well characterized carbapenem-resistant and MDR P. aeruginosa isolates obtained in a large multicenter study in Spain (8, 33). Additionally, this study also aimed to investigate the possible mechanisms of resistance to CXA-101.(This study was presented in part at the 49th Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, CA, September 2009.)     

Changes to Its Peptidoglycan-Remodeling Enzyme Repertoire Modulate β-Lactam Resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is a leading cause of hospital-acquired infections and is resistant to many antibiotics. Among its primary mechanisms of resistance is expression of a chromosomally encoded AmpC β-lactamase that inactivates β-lactams. The mechanisms leading to AmpC expression in P. aeruginosa remain incompletely understood but are intricately linked to cell wall metabolism. To better understand the roles of peptidoglycan-active enzymes in AmpC expression—and consequent β-lactam resistance—a phenotypic screen of P. aeruginosa mutants lacking such enzymes was performed. Mutants lacking one of four lytic transglycosylases (LTs) or the nonessential penicillin-binding protein PBP4 (dacB) had altered β-lactam resistance. mltF and slt mutants with reduced β-lactam resistance were designated WIMPs (wall-impaired mutant phenotypes), while highly resistant dacB, sltB1, and mltB mutants were designated HARMs (high-level AmpC resistant mutants). Double mutants lacking dacB and sltB1 had extreme piperacillin resistance (>256 μg/ml) compared to either of the single knockouts (64 μg/ml for a dacB mutant and 12 μg/ml for an sltB1 mutant). Inactivation of ampC reverted these mutants to wild-type susceptibility, confirming that AmpC expression underlies resistance. dacB mutants had constitutively elevated AmpC expression, but the LT mutants had wild-type levels of AmpC in the absence of antibiotic exposure. These data suggest that there are at least two different pathways leading to AmpC expression in P. aeruginosa and that their simultaneous activation leads to extreme β-lactam resistance.     

Affinity of the New Cephalosporin CXA-101 to Penicillin-Binding Proteins of Pseudomonas aeruginosa

CXA-101, previously designated {"type":"entrez-nucleotide","attrs":{"text":"FR264205","term_id":"258272820","term_text":"FR264205"}}FR264205, is a new antipseudomonal cephalosporin. The objective of this study was to determine the penicillin-binding protein (PBP) inhibition profile of CXA-101 compared to that of ceftazidime (PBP3 inhibitor) and imipenem (PBP2 inhibitor). Killing kinetics, the induction of AmpC expression, and associated changes on cell morphology were also investigated. The MICs for CXA-101, ceftazidime, and imipenem were 0.5, 1, and 1 μg/ml, respectively. Killing curves revealed that CXA-101 shows a concentration-independent bactericidal activity, with concentrations of 1× the MIC (0.5 μg/ml) producing a >3-log reduction in bacterial load after 8 h of incubation. Live-dead staining showed that concentrations of CXA-101 as low as 0.5× the MIC stopped bacterial septation and induced an intense filamentation, which is consistent with the documented high affinity of PBP3. CXA-101 was found to be a potent PBP3 inhibitor and showed affinities ≥2-fold higher than those of ceftazidime for all of the essential PBPs (1b, 1c, 2, and 3). Compared to imipenem, in addition to the obvious inverse PBP2/PBP3 affinities, CXA-101 showed a significantly higher affinity for PBP1b but a lower affinity for PBP1c. Furthermore, CXA-101, like ceftazidime and in contrast to imipenem, was found to be a very weak inducer of AmpC expression, consistent with the low PBP4 affinity documented.Pseudomonas aeruginosa is intrinsically resistant to several antibiotics due to the low permeability of its outer membrane, the constitutive expression of several efflux pumps, and the production of antibiotic-inactivating enzymes (8, 15-17). Intrinsic resistance to β-lactam antibiotics occurs via the induction of chromosomally encoded AmpC β-lactamase (8-10, 16).Antipseudomonal penicillins (piperacillin) and cephalosporins (cefepime or ceftazidime) are active against P. aeruginosa because they are very weak inducers of this chromosomal β-lactamase, although these compounds are certainly hydrolyzed by AmpC (15). Furthermore, mutants hyperproducing AmpC are frequently selected during treatment with antipseudomonal β-lactams, leading to the failure of antimicrobial therapy with these antibiotics (3, 7, 12).The activity of β-lactams against P. aeruginosa (and most other Gram-negative pathogens) will mainly depend in the overall balance of three key attributes: (i) concentration reached in the periplasmic space, dependent on the balance between diffusion through the outer membrane and extrusion by efflux pumps; (ii) resistance to the chromosomally encoded AmpC, dependent on whether the β-lactam is stable to hydrolysis and/or whether it does or does not prevent the induction of the β-lactamase; and (iii) most importantly, the affinity against the main targets of β-lactam antibiotics, the essential penicillin-binding proteins (PBPs), which are PBP1b, PBP1c, PBP2, and PBP3 in the case of P. aeruginosa (25). The PBP inhibition profile is therefore crucial for establishing the qualitative and quantitative dimensions of β-lactam bactericidal activity.Although high-molecular-weight PBPs (1b, 1c, 2, and 3) carry essential functions, the low-molecular-weight penicillin-binding proteins function as dd-carboxypeptidases and/or dd-endopeptidases and are not essential for cell viability (4, 21, 25, 27). Antipseudomonal cephalosporins such as ceftazidime or cefotaxime bind preferentially to PBP3 (1, 4, 23), leading to filament formation, while carbapenems bind to PBP2 (5, 30), ultimately leading to the conversion of rod-shaped cells into spherical cells.Recent works, however, show that it might not be wise to inhibit all PBPs, since at least one of them, PBP4, is shown to be a trap target for β-lactams that is connected to the AmpC induction pathway (19).CXA-101, previously designated {"type":"entrez-nucleotide","attrs":{"text":"FR264205","term_id":"258272820","term_text":"FR264205"}}FR264205, is a new cephalosporin currently under clinical development that shows promising characteristics for the treatment of P. aeruginosa infections. Several recent surveys have revealed a potent in vitro activity of CXA-101 against P. aeruginosa, including cystic fibrosis and multidrug-resistant strains (2, 13, 18, 20, 28, 31). In addition, in vitro studies have shown that CXA-101 appears to be stable against the most common resistance mechanisms driven by mutation in this species, particularly noteworthy the overexpression of the chromosomal cephalosporinase AmpC driven by ampD and/or dacB (PBP4) mutations (18, 20, 28, 29). Recent studies have shown that, consistent with this observation, rates of P. aeruginosa mutation to CXA-101 resistance are extremely low (<5 × 10⁻¹¹), sharply contrasting with the high mutation rates documented for other antipseudomonal cephalosporins or carbapenems (24). Only certain transferable β-lactamases, such as extended-spectrum β-lactamases or class B carbapenemases, which are still infrequent in P. aeruginosa, appear to compromise CXA-101 activity (6, 13, 18). Thus far, no study has been performed to evaluate the binding affinity of CXA-101 to its target PBPs in P. aeruginosa or other pathogens. The objective of the present study was to determine the PBP inhibition profile of CXA-101 compared to those of ceftazidime (a PBP3 inhibitor) and imipenem (a PBP2 inhibitor). Killing kinetics, associated changes on cell morphology, and AmpC-inducing properties were also investigated.     

Mutations in β-Lactamase AmpC Increase Resistance of Pseudomonas aeruginosa Isolates to Antipseudomonal Cephalosporins

Mutation-dependent overproduction of intrinsic β-lactamase AmpC is considered the main cause of resistance of clinical strains of Pseudomonas aeruginosa to antipseudomonal penicillins and cephalosporins. Analysis of 31 AmpC-overproducing clinical isolates exhibiting a greater resistance to ceftazidime than to piperacillin-tazobactam revealed the presence of 17 mutations in the β-lactamase, combined with various polymorphic amino acid substitutions. When overexpressed in AmpC-deficient P. aeruginosa 4098, the genes coding for 20/23 of these AmpC variants were found to confer a higher (2-fold to >64-fold) resistance to ceftazidime and ceftolozane-tazobactam than did the gene from reference strain PAO1. The mutations had variable effects on the MICs of ticarcillin, piperacillin-tazobactam, aztreonam, and cefepime. Depending on their location in the AmpC structure and their impact on β-lactam MICs, they could be assigned to 4 distinct groups. Most of the mutations affecting the omega loop, the R2 domain, and the C-terminal end of the protein were shared with extended-spectrum AmpCs (ESACs) from other Gram-negative species. Interestingly, two new mutations (F121L and P154L) were predicted to enlarge the substrate binding pocket by disrupting the stacking between residues F121 and P154. We also found that the reported ESACs emerged locally in a variety of clones, some of which are epidemic and did not require hypermutability. Taken together, our results show that P. aeruginosa is able to adapt to efficacious β-lactams, including the newer cephalosporin ceftolozane, through a variety of mutations affecting its intrinsic β-lactamase, AmpC. Data suggest that the rates of ESAC-producing mutants are ≥1.5% in the clinical setting.     

Differential Selection of Single-Step AmpC or Efflux Mutants of Pseudomonas aeruginosa by Using Cefepime,Ceftazidime, or Ceftobiprole

Single-step Pseudomonas aeruginosa mutants, selected with ceftobiprole, ceftazidime, or cefepime, were generated at frequencies of 10⁻⁶ to <10⁻⁹ at two and four times the MIC. The chromosomal AmpC β-lactamase activity was increased in all ceftazidime-selected mutants. Mutants selected with cefepime either increased AmpC activity or upregulated expression of the mexXY efflux genes. Mutants selected with ceftobiprole did not overexpress AmpC; 90% of these produced elevated levels of mexXY RNA, indicating that increased efflux, not AmpC derepression, is the predominant response to ceftobiprole during first-step mutations in P. aeruginosa.Pseudomonas aeruginosa is a pathogen known for both intrinsic resistance and acquired resistance to antibiotics (15). A common cause of resistance to extended-spectrum cephalosporins in P. aeruginosa is overexpression of the chromosomal AmpC β-lactamase (5). In addition, downregulation of porins and increased expression of efflux pumps have roles in resistance to β-lactams and other types of antibiotics (2, 8, 27). Recent studies with clinical isolates indicated that some extended-spectrum cephalosporins, such as ceftobiprole and cefepime, but not ceftazidime, are substrates of the MexXY-OprM efflux pump (1). For these P. aeruginosa isolates, reduced susceptibility to cefepime and/or ceftobiprole was associated with overexpression of the MexXY efflux system (1, 9, 14). These substrate profiles from clinical isolates correlate with genetic experiments that showed that cefepime, and not ceftazidime, was extruded by MexXY (17).Ceftobiprole, an anti-methicillin-resistant Staphylococcus aureus (MRSA) cephalosporin with broad-spectrum activity, has successfully completed clinical trials for the treatment of complicated skin and skin structure infections and pneumonia (22-24). The activity of ceftobiprole against P. aeruginosa (MIC₅₀, 2 μg/ml) is similar to those of cefepime (MIC₅₀, 4 μg/ml) and ceftazidime (MIC₅₀, 2 μg/ml) (7). Surveillance studies have shown that MICs of cefepime and ceftobiprole are lower than those of ceftazidime for Enterobacteriaceae with AmpC hyperproduction (26); however, there are limited data for P. aeruginosa with overexpressed AmpC. The goal of this study was to analyze the resistance mechanisms present in P. aeruginosa mutants selected by exposure to ceftobiprole, ceftazidime, or cefepime and to evaluate the MIC profiles for mutants that overexpressed either AmpC β-lactamase or the MexXY efflux system.(Part of this study was previously presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, October 2008.)     

Potent in vitro activity of tomopenem (CS-023) against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa

Tomopenem (formerly CS-023) is a novel 1β-methylcarbapenem with broad-spectrum coverage of gram-positive and gram-negative pathogens. Its antibacterial activity against European clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa was compared with those of imipenem and meropenem. The MICs of tomopenem against MRSA and P. aeruginosa at which 90% of the isolates tested were inhibited were 8 and 4 μg/ml, respectively, and were equal to or more than fourfold lower than those of imipenem and meropenem. The antibacterial activity of tomopenem against MRSA was correlated with a higher affinity for the penicillin-binding protein (PBP) 2a. Its activity against laboratory mutants of P. aeruginosa with (i) overproduction of chromosomally coded AmpC β-lactamase; (ii) overproduction of the multidrug efflux pumps MexAB-OprM, MexCD-OprJ, and MexEF-OprN; (iii) deficiency in OprD; and (iv) various combinations of AmpC overproduction, MexAB-OprM overproduction, and OprD deficiency were tested. The increases in the MIC of tomopenem against each single mutant compared with that against its parent strain were within a fourfold range. Tomopenem exhibited antibacterial activity against all mutants, with an observed MIC range of 0.5 to 8 μg/ml. These results suggest that the antibacterial activity of tomopenem against the clinical isolates of MRSA and P. aeruginosa should be ascribed to its high affinity for PBP 2a and its activity against the mutants of P. aeruginosa, respectively.     

Unexpected Challenges in Treating Multidrug-Resistant Gram-Negative Bacteria: Resistance to Ceftazidime-Avibactam in Archived Isolates of Pseudomonas aeruginosa

Pseudomonas aeruginosa is a notoriously difficult-to-treat pathogen that is a common cause of severe nosocomial infections. Investigating a collection of β-lactam-resistant P. aeruginosa clinical isolates from a decade ago, we uncovered resistance to ceftazidime-avibactam, a novel β-lactam/β-lactamase inhibitor combination. The isolates were systematically analyzed through a variety of genetic, biochemical, genomic, and microbiological methods to understand how resistance manifests to a unique drug combination that is not yet clinically released. We discovered that avibactam was able to inactivate different AmpC β-lactamase enzymes and that bla_PDC regulatory elements and penicillin-binding protein differences did not contribute in a major way to resistance. By using carefully selected combinations of antimicrobial agents, we deduced that the greatest barrier to ceftazidime-avibactam is membrane permeability and drug efflux. To overcome the constellation of resistance determinants, we show that a combination of antimicrobial agents (ceftazidime/avibactam/fosfomycin) targeting multiple cell wall synthetic pathways can restore susceptibility. In P. aeruginosa, efflux, as a general mechanism of resistance, may pose the greatest challenge to future antibiotic development. Our unexpected findings create concern that even the development of antimicrobial agents targeted for the treatment of multidrug-resistant bacteria may encounter clinically important resistance. Antibiotic therapy in the future must consider these factors.     

Antipseudomonal Agents Exhibit Differential Pharmacodynamic Interactions with Human Polymorphonuclear Leukocytes against Established Biofilms of Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common pathogen infecting the lower respiratory tract of cystic fibrosis (CF) patients, where it forms tracheobronchial biofilms. Pseudomonas biofilms are refractory to antibacterials and to phagocytic cells with innate immunity, leading to refractory infection. Little is known about the interaction between antipseudomonal agents and phagocytic cells in eradication of P. aeruginosa biofilms. Herein, we investigated the capacity of three antipseudomonal agents, amikacin (AMK), ceftazidime (CAZ), and ciprofloxacin (CIP), to interact with human polymorphonuclear leukocytes (PMNs) against biofilms and planktonic cells of P. aeruginosa isolates recovered from sputa of CF patients. Three of the isolates were resistant and three were susceptible to each of these antibiotics. The concentrations studied (2, 8, and 32 mg/liter) were subinhibitory for biofilms of resistant isolates, whereas for biofilms of susceptible isolates, they ranged between sub-MIC and 2 × MIC values. The activity of each antibiotic alone or in combination with human PMNs against 48-h mature biofilms or planktonic cells was determined by XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay. All combinations of AMK with PMNs resulted in synergistic or additive effects against planktonic cells and biofilms of P. aeruginosa isolates compared to each component alone. More than 75% of CAZ combinations exhibited additive interactions against biofilms of P. aeruginosa isolates, whereas CIP had mostly antagonistic interaction or no interaction with PMNs against biofilms of P. aeruginosa. Our findings demonstrate a greater positive interaction between AMK with PMNs than that observed for CAZ and especially CIP against isolates of P. aeruginosa from the respiratory tract of CF patients.     

Effect of sodium mercaptoacetic acid on different antimicrobial disks in the sodium mercaptoacetic acid double disk synergy test for detection of IMP-1 metallo-β-lactamase-producing Pseudomonas aeruginosa isolates in Japan

We determined the optimal antimicrobial in the sodium mercaptoacetic acid double disk synergy test (SMA-DDST) for the detection of IMP-1-producing Pseudomonas aeruginosa isolates in Japan and evaluated the performance of the test.Fifty-four P. aeruginosa clinical isolates were tested, including 39 IMP-1 producers and 15 non-metallo-β-lactamase (MBL)-producing carbapenem- and ceftazidime (CAZ)-resistant isolates. The SMA-DDST was performed with CAZ, cefepime (CFPM), imipenem (IPM), meropenem (MEPM), doripenem (DRPM), or biapenem (BIPM)-containing disks. The sensitivity of the SMA-DDST with CAZ, CFPM, IPM, MEPM, DRPM, and BIPM was 39/39 (100%), 36/39 (92%), 18/39 (46%), 8/39 (21%), 19/39 (49%), and 36/39 (92%), respectively. The specificity was 15/15 (100%) for all SMA-DDSTs. This suggests that the isolates may have a resistance mechanism other than MBL production for IPM, MEPM, or DRPM. Since the CAZ resistance mechanism in P. aeruginosa is the same as that of CFPM, but differs from that of carbapenems, we conclude that combining CAZ with BIPM SMA-DDSTs can prevent any failure in the detection of IMP-1-producing P. aeruginosa.     

Extended-Spectrum Cephalosporinases in Pseudomonas aeruginosa

The characterization of AmpC-type β-lactamases was performed in a collection of 32 clinical Pseudomonas aeruginosa isolates with intermediate susceptibility or resistance to imipenem and ceftazidime. Twenty-one out of those 32 isolates overexpressed AmpC β-lactamase, and the MICs of ceftazidime and imipenem were reduced after cloxacillin addition. Cloning and sequencing identified 10 AmpC β-lactamase variants. Reduced susceptibility to imipenem, ceftazidime, and cefepime was observed only with recombinant P. aeruginosa strains expressing an AmpC β-lactamase that had an alanine residue at position 105. The catalytic efficiencies (k_cat/K_m) of the AmpC variants possessing this residue were increased against oxyiminocephalosporins and imipenem. In addition, we show here that those AmpC variants constitute a favorable background for the in vitro selection of imipenem-resistant strains. This report identified a novel resistance mechanism that may contribute to imipenem resistance in P. aeruginosa.Most AmpC-type β-lactamases naturally produced by gram-negative bacteria hydrolyze amino- and ureido-penicillins, cephamycins (cefoxitin or cefotetan), and, at low levels, oxyiminocephalosporins such as ceftazidime, cefotaxime, or ceftriaxone, and monobactams such as aztreonam (4). Zwitteronic cephalosporins such as cefepime and cefpirome and carbapenems such as imipenem and meropenem usually are excluded from the spectrum of activity of AmpC β-lactamases (10).However, cephalosporinases with broadened substrate activity have been reported in several enterobacterial isolates, including Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, and Escherichia coli (16, 23). These extended-spectrum AmpC (ESAC) β-lactamases confer reduced susceptibility to all cephalosporins, including cefepime and cefpirome (16, 23). Those enzymes differ from wild-type cephalosporinases by amino acid substitutions or insertions in four regions in the vicinity of the active site: the Ω loop, the H-10 helix, the H-2 helix, and the C-terminal end of the protein (1, 2, 7, 8, 11, 16, 18-21, 24, 25, 28, 29).In P. aeruginosa, the overexpression of the naturally occurring AmpC is associated with a decreased susceptibility or resistance to expanded-spectrum cephalosporins such as ceftazidime. Resistance to those cephalosporins also may be related to clavulanic acid-inhibited extended-spectrum β-lactamases (ESBLs) (31). Resistance to cefepime and susceptibility to ceftazidime have been related to the overexpression of an efflux pump (MexXY-OprM) or to the production of OXA-30-like β-lactamases (12). Resistance to imipenem is associated mostly with structural changes or the loss of the OprD outer membrane protein and rarely to metallo-β-lactamases or specific Ambler class A β-lactamases such as GES derivatives and KPC-2 (30, 31).Recently, several ESACs have been described from E. coli contributing to reduced susceptibility to imipenem (17). The aim of this study was to characterize the AmpC β-lactamases expressed by P. aeruginosa clinical isolates that were of intermediate susceptibility or resistant to imipenem and to search for the putative involvement of those AmpC proteins in that resistance pattern.     

Bactericidal Activity,Absence of Serum Effect,and Time-Kill Kinetics of Ceftazidime-Avibactam against β-Lactamase-Producing Enterobacteriaceae and Pseudomonas aeruginosa

Avibactam, a non-β-lactam β-lactamase inhibitor with activity against extended-spectrum β-lactamases (ESBLs), KPC, AmpC, and some OXA enzymes, extends the antibacterial activity of ceftazidime against most ceftazidime-resistant organisms producing these enzymes. In this study, the bactericidal activity of ceftazidime-avibactam against 18 Pseudomonas aeruginosa isolates and 15 Enterobacteriaceae isolates, including wild-type isolates and ESBL, KPC, and/or AmpC producers, was evaluated. Ceftazidime-avibactam MICs (0.016 to 32 μg/ml) were lower than those for ceftazidime alone (0.06 to ≥256 μg/ml) against all isolates except for 2 P. aeruginosa isolates (1 bla_VIM-positive isolate and 1 bla_OXA-23-positive isolate). The minimum bactericidal concentration/MIC ratios of ceftazidime-avibactam were ≤4 for all isolates, indicating bactericidal activity. Human serum and human serum albumin had a minimal effect on ceftazidime-avibactam MICs. Ceftazidime-avibactam time-kill kinetics were evaluated at low MIC multiples and showed time-dependent reductions in the number of CFU/ml from 0 to 6 h for all strains tested. A ≥3-log₁₀ decrease in the number of CFU/ml was observed at 6 h for all Enterobacteriaceae, and a 2-log₁₀ reduction in the number of CFU/ml was observed at 6 h for 3 of the 6 P. aeruginosa isolates. Regrowth was noted at 24 h for some of the isolates tested in time-kill assays. These data demonstrate the potent bactericidal activity of ceftazidime-avibactam and support the continued clinical development of ceftazidime-avibactam as a new treatment option for infections caused by Enterobacteriaceae and P. aeruginosa, including isolates resistant to ceftazidime by mechanisms dependent on avibactam-sensitive β-lactamases.     

Involvement of the MexXY-OprM efflux system in emergence of cefepime resistance in clinical strains of Pseudomonas aeruginosa

Cefepime (FEP) and ceftazidime (CAZ) are potent β-lactam antibiotics with similar MICs (1 to 2 μg/ml) for wild-type strains of Pseudomonas aeruginosa. However, recent epidemiological studies have highlighted the occurrence of isolates more resistant to FEP than to CAZ (FEP^r/CAZ^s profile). We thus investigated the mechanisms conferring such a phenotype in 38 clonally unrelated strains collected in two French teaching hospitals. Most of the bacteria (n = 32; 84%) appeared to stably overexpress the mexY gene, which codes for the RND transporter of the multidrug efflux system MexXY-OprM. MexXY up-regulation was the sole FEP resistance mechanism identified (n = 12) or was associated with increased levels of pump MexAB-OprM (n = 5) or MexJK (n = 2), synthesis of secondary β-lactamase PSE-1 (n = 10), derepression of cephalosporinase AmpC (n = 1), coexpression of both OXA-35 and MexJK (n = 1), or production of both PSE-1 and MexAB-OprM (n = 1). Down-regulation of the mexXY operon in seven selected strains by the plasmid-borne repressor gene mexZ decreased FEP resistance from two- to eightfold, thereby demonstrating the significant contribution of MexXY-OprM to the FEP^r/CAZ^s phenotype. The six isolates of this series that exhibited wild-type levels of the mexY gene were found to produce β-lactamase PSE-1 (n = 1), OXA-35 (n = 4), or both PSE-1 and OXA-35 (n = 1). Altogether, these data provide evidence that MexXY-OprM plays a major role in the development of FEP resistance among clinical strains of P. aeruginosa.     

Nationwide surveillance of antimicrobial susceptibility patterns of pathogens isolated from surgical site infections (SSI) in Japan

To investigate the trends of antimicrobial resistance in pathogens isolated from surgical site infections (SSI), a Japanese surveillance committee conducted the first nationwide survey. Seven main organisms were collected from SSI at 27 medical centers in 2010 and were shipped to a central laboratory for antimicrobial susceptibility testing. A total of 702 isolates from 586 patients with SSI were included. Staphylococcus aureus (20.4 %) and Enterococcus faecalis (19.5 %) were the most common isolates, followed by Pseudomonas aeruginosa (15.4 %) and Bacteroides fragilis group (15.4 %). Methicillin-resistant S. aureus among S. aureus was 72.0 %. Vancomycin MIC 2 μg/ml strains accounted for 9.7 %. In Escherichia coli, 11 of 95 strains produced extended-spectrum β-lactamase (Klebsiella pneumoniae, 0/53 strains). Of E. coli strains, 8.4 % were resistant to ceftazidime (CAZ) and 26.3 % to ciprofloxacin (CPFX). No P. aeruginosa strains produced metallo-β-lactamase. In P. aeruginosa, the resistance rates were 7.4 % to tazobactam/piperacillin (TAZ/PIPC), 10.2 % to imipenem (IPM), 2.8 % to meropenem, cefepime, and CPFX, and 0 % to gentamicin. In the B. fragilis group, the rates were 28.6 % to clindamycin, 5.7 % to cefmetazole, 2.9 % to TAZ/PIPC and IPM, and 0 % to metronidazole (Bacteroides thetaiotaomicron; 59.1, 36.4, 0, 0, 0 %). MIC₉₀ of P. aeruginosa isolated 15 days or later after surgery rose in TAZ/PIPC, CAZ, IPM, and CPFX. In patients with American Society of Anesthesiologists (ASA) score ≥3, the resistance rates of P. aeruginosa to TAZ/PIPC and CAZ were higher than in patients with ASA ≤2. The data obtained in this study revealed the trend of the spread of resistance among common species that cause SSI. Timing of isolation from surgery and the patient’s physical status affected the selection of resistant organisms.     

Interplay between Chromosomal β-Lactamase and the MexAB-OprM Efflux System in Intrinsic Resistance to β-Lactams in Pseudomonas aeruginosa

We investigated the role of chromosomal β-lactamase and the MexAB-OprM efflux system in intrinsic resistance to β-lactams in Pseudomonas aeruginosa. Determination of the susceptibilities of a series of isogenic mutants with impaired production of the β-lactamase and the efflux system to 16 β-lactams including penicillins, cephems, oxacephems, carbapenems, and a monobactam demonstrated that the intrinsic resistance of P. aeruginosa to most of the β-lactams is due to the interplay of both factors.     

In Vivo Emergence of Multidrug-Resistant Mutants of Pseudomonas aeruginosa Overexpressing the Active Efflux System MexA-MexB-OprM

During a 6-month period, 21 pairs of Pseudomonas aeruginosa isolates susceptible (pretherapy) and resistant (posttherapy) to antipseudomonal β-lactam antibiotics were isolated from hospitalized patients. In vivo emergence of β-lactam resistance was associated with the overexpression of AmpC β-lactamase in 10 patients. In the other 11 patients, the posttherapy isolates produced only low, basal levels of β-lactamase and had increased levels of resistance to a variety of non-β-lactam antibiotics (e.g., quinolones, tetracyclines, and trimethoprim) compared with the levels of β-lactamase production and resistance of their pretherapy counterparts. These data suggested the involvement of the MexA-MexB-OprM active efflux system in the multidrug resistance phenotype of the posttherapy strains. Immunoblotting of the outer membrane proteins of these 11 bacterial pairs with a specific polyclonal antibody raised against OprM demonstrated the overexpression of OprM in all the posttherapy isolates. To determine whether mutations in mexR, the regulator gene of the mexA-mexB-oprM efflux operon, could account for the overproduction of the efflux system, sequencing experiments were carried out with the 11 bacterial pairs. Eight posttherapy isolates were found to contain insertions or deletions that led to frameshifts in the coding sequences of mexR. Two resistant strains had point mutations in mexR that yielded single amino acid changes in the protein MexR, while another strain did not show any mutation in mexR or in the promoter region upstream of mexR. Introduction of a plasmid-encoded wild-type mexR gene into five posttherapy isolates partially restored the susceptibility of the bacteria to selected antibiotics. These results indicate that in the course of antimicrobial therapy multidrug-resistant active efflux mutants overexpressing the MexA-MexB-OprM system may emerge as a result of mutations in the mexR gene.     

Antimicrobial Activity of Ceftazidime-Avibactam against Gram-Negative Organisms Collected from U.S. Medical Centers in 2012

The activities of the novel β-lactam–β-lactamase inhibitor combination ceftazidime-avibactam and comparator agents were evaluated against a contemporary collection of clinically significant Gram-negative bacilli. Avibactam is a novel non-β-lactam β-lactamase inhibitor that inhibits Ambler class A, C, and some D enzymes. A total of 10,928 Gram-negative bacilli—8,640 Enterobacteriaceae, 1,967 Pseudomonas aeruginosa, and 321 Acinetobacter sp. isolates—were collected from 73 U.S. hospitals and tested for susceptibility by reference broth microdilution methods in a central monitoring laboratory (JMI Laboratories, North Liberty, IA, USA). Ceftazidime was combined with avibactam at a fixed concentration of 4 μg/ml. Overall, 99.8% of Enterobacteriaceae strains were inhibited at a ceftazidime-avibactam MIC of ≤4 μg/ml. Ceftazidime-avibactam was active against extended-spectrum β-lactamase (ESBL)-phenotype Escherichia coli and Klebsiella pneumoniae, meropenem-nonsusceptible (MIC ≥ 2 μg/ml) K. pneumoniae, and ceftazidime-nonsusceptible Enterobacter cloacae. Among ESBL-phenotype K. pneumoniae strains, 61.1% were meropenem susceptible and 99.3% were inhibited at a ceftazidime-avibactam MIC of ≤4 μg/ml. Among P. aeruginosa strains, 96.9% were inhibited at a ceftazidime-avibactam MIC of ≤8 μg/ml, and susceptibility rates for meropenem, ceftazidime, and piperacillin-tazobactam were 82.0, 83.2, and 78.3%, respectively. Ceftazidime-avibactam was the most active compound tested against meropenem-nonsusceptible P. aeruginosa (MIC₅₀/MIC₉₀, 4/16 μg/ml; 87.3% inhibited at ≤8 μg/ml). Acinetobacter spp. (ceftazidime-avibactam MIC₅₀/MIC₉₀, 16/>32 μg/ml) showed high rates of resistance to most tested agents. In summary, ceftazidime-avibactam demonstrated potent activity against a large collection of contemporary Gram-negative bacilli isolated from patients in U.S. hospitals in 2012, including organisms that are resistant to most currently available agents, such as K. pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae and meropenem-nonsusceptible P. aeruginosa.     

Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance

Penicillin-binding protein 5 (PBP5) is one of the most abundant PBPs in Pseudomonas aeruginosa. Although its main function is that of a cell wall dd-carboxypeptidase, it possesses sufficient β-lactamase activity to contribute to the ability of P. aeruginosa to resist the antibiotic activity of the β-lactams. The study of these dual activities is important for understanding the mechanisms of antibiotic resistance by P. aeruginosa, an important human pathogen, and to the understanding of the evolution of β-lactamase activity from the PBP enzymes. We purified a soluble version of P. aeruginosa PBP5 (designated Pa sPBP5) by deletion of its C-terminal membrane anchor. Under in vitro conditions, Pa sPBP5 demonstrates both dd-carboxypeptidase and expanded-spectrum β-lactamase activities. Its crystal structure at a 2.05-Å resolution shows features closely resembling those of the class A β-lactamases, including a shortened loop spanning residues 74 to 78 near the active site and with respect to the conformations adopted by two active-site residues, Ser101 and Lys203. These features are absent in the related PBP5 of Escherichia coli. A comparison of the two Pa sPBP5 monomers in the asymmetric unit, together with molecular dynamics simulations, revealed an active-site flexibility that may explain its carbapenemase activity, a function that is absent in the E. coli PBP5 enzyme. Our functional and structural characterizations underscore the versatility of this PBP5 in contributing to the β-lactam resistance of P. aeruginosa while highlighting how broader β-lactamase activity may be encoded in the structural folds shared by the PBP and serine β-lactamase classes.     

In Vitro Susceptibility to Ceftazidime-Avibactam of Carbapenem-Nonsusceptible Enterobacteriaceae Isolates Collected during the INFORM Global Surveillance Study (2012 to 2014)

The activity of ceftazidime-avibactam was assessed against 961 isolates of meropenem-nonsusceptible Enterobacteriaceae. Most meropenem-nonsusceptible metallo-β-lactamase (MBL)-negative isolates (97.7%) were susceptible to ceftazidime-avibactam. Isolates that carried KPC or OXA-48-like β-lactamases, both alone and in combination with extended-spectrum β-lactamases (ESBLs) and/or AmpC β-lactamases, were 98.7% and 98.5% susceptible to ceftazidime-avibactam, respectively. Meropenem-nonsusceptible, carbapenemase-negative isolates demonstrated 94.7% susceptibility to ceftazidime-avibactam. Ceftazidime-avibactam activity was compromised only in isolates for which carbapenem resistance was mediated through metallo-β-lactamases.