[Show abstract][Hide abstract] ABSTRACT: Background:
Most available information on carbapenemase-producing Enterobacteriaceae (CPE) is usually associated with specific types of infection or patient or with descriptions of outbreaks. The aim of this study was to comprehensively analyse the clinical epidemiology, clinical features and outcomes of colonisation and infections due to CPE in Spain.
A multicentre prospective cohort study was carried out in 34 Spanish hospitals from February to May 2013. All new patients testing positive for CPE in clinical samples were included. Logistic regression was used to identify predictors of mortality.
Overall, 245 cases were included. The most frequent organism was Klebsiella pneumoniae (74%) and the carbapenemases belonged to the OXA-48 (74%), metallo-β-lactamase (MBL) (24%) and KPC (2%) groups. Acquisition was nosocomial in 145 cases (60%) and health care-associated (HCA) in 91 (37%); 42% of the latter were nursing home residents, in whom OXA-48-producing K. pneumoniae ST405 predominated. MBLs and OXA-48 predominated in ICU and medical patients, respectively. Overall, 67% of patients had infections. The most frequent infections identified in this study were urinary tract (43%) and skin structure (21%) infections, and 10% of infections were bacteraemic. Crude mortality was 20%. Inappropriate antibiotic therapy was independently associated with an increased risk of death (OR=3.30; 95% CI: 1.34-8.11).
We found some differences in the epidemiology of CPE depending on the type of carbapenemase produced. Although a low proportion of CPE infections were bacteraemic, active antibiotic therapy was a protective factor for reducing mortality.
The Journal of infection 11/2015; DOI:10.1016/j.jinf.2015.10.008 · 4.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives:
The main objective of this study was to investigate the relationship among the in vivo acquisition of antimicrobial resistance in Pseudomonas aeruginosa clinical isolates, the underlying molecular mechanisms and previous exposure to antipseudomonal agents.
PFGE was used to study the molecular relatedness of the strains. The MICs of ceftazidime, cefepime, piperacillin/tazobactam, imipenem, meropenem, ciprofloxacin and amikacin were determined. Outer membrane protein profiles were assessed to study OprD expression. RT-PCR was performed to analyse ampC, mexB, mexD, mexF and mexY expression. The presence of mutations was analysed through DNA sequencing.
We collected 17 clonally related paired isolates [including first positive samples (A) and those with MICs increased ≥4-fold (B)]. Most B isolates with increased MICs of imipenem, meropenem and ceftazidime became resistant to these drugs. The most prevalent resistance mechanisms detected were OprD loss (65%), mexB overexpression (53%), ampC derepression (29%), quinolone target gene mutations (24%) and increased mexY expression (24%). Five (29%) B isolates developed multidrug resistance. Meropenem was the most frequently (71%) received treatment, explaining the high prevalence of oprD mutations and likely mexB overexpression. Previous exposure to ceftazidime showed a higher impact on selection of increased MICs than previous exposure to piperacillin/tazobactam.
Stepwise acquisition of resistance has a critical impact on the resistance phenotypes of P. aeruginosa, leading to a complex scenario for finding effective antimicrobial regimens. In the clinical setting, meropenem seems to be the most frequent driver of multidrug resistance development, while piperacillin/tazobactam, in contrast to ceftazidime, seems to be the β-lactam least associated with the selection of resistance mechanisms.
Journal of Antimicrobial Chemotherapy 08/2015; DOI:10.1093/jac/dkv228 · 5.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
The type III secretion system (TTSS) is a major virulence determinant of Pseudomonas aeruginosa. The objective of this study was to determine whether the TTSS genotype is a useful prognostic marker of P. aeruginosa bacteremia mortality. We also studied the potential association between TTSS genotypes and multidrug-resistant (MDR) profiles, and how this interaction impacts the outcome of bloodstream infections.
We performed a post hoc analysis of a published prospective multicenter cohort of P. aeruginosa bloodstream infections. The impact in mortality of TTSS genotypes (exoS, exoT, exoU, and exoY genes) and resistance profiles was investigated. Cox regression analysis was used to control for confounding variables.
Among 590 patients, the 30-day mortality rate was 30% (175 patients), and 53% of them died in the first 5 days (early mortality). The unadjusted probabilities of survival until 5 days was 31.4% (95% confidence interval [CI], 17.4%-49.4%) for the patients with exoU-positive isolates and 53.2% (95% CI, 44.6%-61.5%) for exoU-negative isolates (log rank P = .005). After adjustment for confounders, exoU genotype (adjusted hazard ratio [aHR], 1.90 [95% CI, 1.15-3.14]; P = .01) showed association with early mortality. In contrast, late (30-day) mortality was not influenced by TTSS genotype but was independently associated with MDR profiles (aHR,1.40 [95% CI, 1.01-1.94]; P = .04). Moreover, the exoU genotype (21% of all isolates) was significantly less frequent (13%) among MDR strains (particularly among extensively drug-resistant isolates, 5%), but was positively linked to moderately resistant (1-2 antipseudomonals) phenotypes (34%).
Our results indicate that the exoU genotype, which is associated with specific susceptibility profiles, is a relevant independent marker of early mortality in P. aeruginosa bacteremia.
[Show abstract][Hide abstract] ABSTRACT: During a Spanish surveillance study, two natural variants of DHA β-lactamases, DHA-6 and DHA-7, were found, with the replacements
Ala226Thr and Phe322Ser, respectively, with respect to DHA-1. The DHA-6 and DHA-7 enzymes were isolated from Escherichia coli and Enterobacter cloacae clinical isolates, respectively. The aim of this study was to genetically, microbiologically, and biochemically characterize
the DHA-6 and DHA-7 β-lactamases. The blaDHA-6 and blaDHA-7 genes were located in the I1 and HI2 incompatibility group plasmids of 87.3 and 310.4 kb, respectively. The genetic contexts
of blaDHA-6 and blaDHA-7 were similar to that already described for the blaDHA-1 gene and included the qnrB4 and aadA genes. The MICs for cephalothin, aztreonam, cefotaxime, and ceftazidime were 8- to 32-fold lower for DHA-6 than for DHA-1
or DHA-7 expressed in the same isogenic E. coli TG1 strain. Interestingly, the MIC for cefoxitin was higher in the DHA-6-expressing transformant than in DHA-1 or DHA-7.
Biochemical studies with pure β-lactamases revealed slightly lower catalytic efficiencies of DHA-6 against cephalothin, ceftazidime,
and cefotaxime than those of DHA-1 and DHA-7. To understand this behavior, stability experiments were carried out and showed
that the DHA-6 protein displayed significantly higher stability than the DHA-1 and DHA-7 enzymes. The proximity of Thr226
to the N terminus in the tertiary protein structure in DHA-6 may promote this stabilization and, consequently, may induce
a slight reduction in the dynamic of this enzyme that primarily affects the hydrolysis of some of the bulkiest antibiotics.
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas aeruginosa is a ubiquitous versatile environmental microorganism with a remarkable ability to grow under diverse environmental conditions. Moreover, P. aeruginosa is responsible for life threatening infections in immunocompromised and cystic fibrosis patients, where the extraordinary capacity of this pathogen to develop antimicrobial resistance dramatically limits our therapeutic arsenal. Its large genome carries an outstanding number of genes belonging to regulatory systems, including multiple two component sensor-regulator systems modulating the response to the different environmental stimuli. Here we show that one of such systems, designated CreBC (carbon-source responsive) or BlrAB (beta -lactam resistance), might be of particular relevance. We first identified the stimuli triggering the activation of the CreBC system, specifically responding to PBP4 inhibition by certain β-lactam antibiotics. Second, through the analysis of a large comprehensive collection of mutants we demonstrate an intricate interconnection between the CreBC system, the peptidoglycan recycling pathway and the expression of the concerning chromosomal β-lactamase AmpC. Third, we show that the CreBC system, and particularly its effector inner membrane protein CreD, plays a major role in bacterial fitness and biofilm development, especially in the presence of subinhibitory concentrations of β-lactams. Finally, global transcriptomics reveals broad regulatory functions of CreBC in basic physiological aspects, particularly anaerobic respiration, both in the presence and absence of antibiotics. Therefore, the CreBC system is envisaged as a potentially interesting target for improving the efficacy of β-lactams against P. aeruginosa infections.
[Show abstract][Hide abstract] ABSTRACT: We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, mutS-) Pseudomonas aeruginosa. The strains were incubated for 24h with 0.5-64x MIC concentrations of each antibiotic in triplicate experiments. Tubes from the highest antibiotic concentration showing growth were re-inoculated in fresh medium containing concentrations up to 64x MIC for 7 consecutive days. Susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic and strain. Ceftolozane-tazobactam resistant mutants were further characterized by whole genome analysis through RNA-seq. High-level resistance development was fastest for ceftazidime followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed crossresistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower and high-level resistance was only observed for the mutator strain. Ceftolozane-tazobactam moderately resistant (MICs 4-8 μg/ml) PAO1 derivatives showed only 2-4 mutations, that determined global pleiotropic effects associated with a severe fitness cost. High-level resistant (MICs 32-128 μg/ml) PAOMS derivatives showed 45-53 mutations. Major changes in global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1-4 mutations in conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, when compared with wild-type ampC. Therefore, development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC.
[Show abstract][Hide abstract] ABSTRACT: A limited number of Pseudomonas aeruginosa genotypes (mainly ST-111, ST-175, and ST-235), known as high-risk clones, are responsible for epidemics of nosocomial infections by multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains worldwide. We explored the potential biological parameters that may explain the success of these clones. A total of 20 isolates from each of 4 resistance groups (XDR, MDR, ModR [resistant to 1 or 2 classes], and MultiS [susceptible to all antipseudomonals]), recovered from a multicenter study of P. aeruginosa bloodstream infections performed in 10 Spanish hospitals, were analyzed. A further set of 20 XDR isolates belonging to epidemic high-risk clones (ST-175 [n = 6], ST-111 [n = 7], and ST-235 [n = 7]) recovered from different geographical locations was also studied. When unknown, genotypes were documented through multilocus sequence typing. The biological parameters evaluated included twitching, swimming, and swarming motility, biofilm formation, production of pyoverdine and pyocyanin, spontaneous mutant frequencies, and the in vitro competition index (CI) obtained with a flow cytometry assay. All 20 (100%) XDR, 8 (40%) MDR, and 1 (5%) ModR bloodstream isolate from the multicenter study belonged to high-risk clones. No significant differences were observed between clonally diverse ModR and MultiS isolates for any of the parameters. In contrast, MDR/XDR high-risk clones showed significantly increased biofilm formation and mutant frequencies but significantly reduced motility (twitching, swimming, and swarming), production of pyoverdine and pyocyanin, and fitness. The defined biological markers of high-risk clones, which resemble those resulting from adaptation to chronic infections, could be useful for the design of specific treatment and infection control strategies.
[Show abstract][Hide abstract] ABSTRACT: A novel class C β-lactamase (FOX-8) was isolated from a clinical strain of Escherichia coli. The FOX-8 enzyme possessed a unique substitution (Phe313Leu) when compared to FOX-3. Isogenic E. coli strains carrying FOX-8 showed an 8 fold reduction in resistance to ceftazidime relative to FOX-3. In kinetic analysis FOX-8 displayed a 33 fold reduction in kcat/Km for ceftazidime compared to FOX-3. In the FOX family of β-lactamases, the Phe313 residue located in the R2 loop affects ceftazidime hydrolysis and alters the phenotype of E.coli strains carrying this variant.
[Show abstract][Hide abstract] ABSTRACT: A panel of 29 multidrug-resistant (MDR) Pseudomonas aeruginosa isolates recovered from seven hospitals as part of a country-wide surveillance of antimicrobial resistance in Bulgarian hospitals was studied. Molecular typing through multiple-locus variable number tandem-repeat analysis (MLVA6) yielded 23 different profiles. Phenotypic and genotypic tests for the detection of acquired carbapenemases yielded negative results in all cases. In contrast, 76% of the isolates produced other acquired β-lactamases, including extended-spectrum β-lactamases (ESBLs). Namely, 6 of the isolates (21%) produced a VEB-1 ESBL; 14 (48%) produced an OXA-10-type enzyme (7 OXA-10 and 7 OXA-10 ESBL variants, including 2 OXA-17 [A218G], 2 OXA-74 [C197T, A218G], and 3 OXA-142 [A218G, G470A]); 8 (28%) an OXA-2-type enzyme (all OXA-2); and 1 (3%) a PSE-1 carbenicillinase. Further analysis through multilocus sequence typing (MLST) revealed that the six VEB-1-producing strains, recovered from four hospitals, belonged to ST111 or ST244 international high-risk clones. Additionally, nearly all of the isolates (97%) lacked OprD production, explaining carbapenem resistance. Overexpression of AmpC was documented in 5 (17%) of the isolates, including most of the MDR isolates not producing any acquired β-lactamase. Particularly noteworthy was the very high prevalence of MexXY-OprM overexpression, documented in 72% of the isolates, whereas the prevalence of MexAB-OprM overexpression was lower (21%). In summary, while the production of metallo-β-lactamases is uncommon among P. aeruginosa isolates from Bulgarian hospitals, MDR profiles frequently result from the production of ESBLs combined with the lack of production of the carbapenem porin OprD and the overexpression of the MexXY-OprM efflux pump.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this investigation was to determine the prevalence of plasmid-mediated AmpC (pAmpC) and carbapenemases in Enterobacteriaceae collected from 35 hospitals in Spain and to establish their epidemiological relationships. We conducted a prospective multi-centre study on pAmpC- or carbapenemase-producing Enterobacteriaceae isolates from clinical samples collected from February to July 2009. The strains suspected to carry pAmpC were resistant or showed intermediate susceptibility to co-amoxiclav and second- or third-generation cephalosporins. Strains suspected to carry a carbapenemase were selected because they showed a minimum inhibitory concentration (MIC) to imipenem >1 mg/L. Polymerase chain reaction (PCR) and a sequencing strategy were used to characterise the enzymes. The clonal relationships between isolates was analysed by pulsed field gel electrophoresis (PFGE). Among 100,132 Enterobacteriaceae isolates collected, 1,654 were compatible with the production of pAmpC or carbapenemases. We found a prevalence of 0.64 % of pAmpC (n = 635) and 0.04 % of carbapenemases (n = 43). The most prevalent pAmpC enzymes were CMY-type (78.3 %), DHA-type (19.5 %), ACC-type (1.6 %) and FOX-type (0.6 %). The CMY-type was the most frequent in Escherichia coli and Proteus mirabilis species, whereas the DHA-type was mainly found in Klebsiella spp. The enzymes involved in carbapenem resistance were VIM-1, IMP-22 and the new IMP-28. Nine new bla genes were described: bla (CMY-54), bla (CMY-55), bla (CMY-56), bla (CMY-57), bla (CMY-96), bla (DHA-6), bla (DHA-7), bla (FOX-8) and bla (IMP-28). The prevalence of pAmpC or carbapenemases found is not negligible. The CMY-types were the predominant pAmpC, whereas the VIM or IMP enzymes were the predominant carbapenemases. Furthermore, we observed a great genetic diversity among pAmpC-producing strains and a close clonal relationship between carbapenemase-producing strains.
European Journal of Clinical Microbiology 09/2012; 32(2). DOI:10.1007/s10096-012-1737-0 · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We investigated the mechanisms leading to Pseudomonas aeruginosa pan-β-lactam resistance (PBLR) development during the treatment of nosocomial infections, with a particular focus on the modification of penicillin-binding protein (PBP) profiles and imipenem, ceftazidime, and ceftolozane (former CXA-101) PBP binding affinities. For this purpose, six clonally related pairs of sequential susceptible-PBLR isolates were studied. The presence of oprD, ampD, and dacB mutations was explored by PCR followed by sequencing and the expression of ampC and efflux pump genes by real-time reverse transcription-PCR. The fluorescent penicillin Bocillin FL was used to determine PBP profiles in membrane preparations from all pairs, and 50% inhibitory concentrations (IC(50)s) of ceftolozane, ceftazidime, and imipenem were analyzed in 3 of them. Although a certain increase was noted (0 to 5 2-fold dilutions), the MICs of ceftolozane were ≤4 μg/ml in all PBLR isolates. All 6 PBLR isolates lacked OprD and overexpressed ampC and one or several efflux pumps, particularly mexB and/or mexY. Additionally, 5 of them showed modified PBP profiles, including a modified pattern (n = 1) or diminished expression (n = 1) of PBP1a and a lack of PBP4 expression (n = 4), which correlated with AmpC overexpression driven by dacB mutation. Analysis of the essential PBP IC(50)s revealed significant variation of PBP1a/b binding affinities, both within each susceptible-PBLR pair and across the different pairs. Moreover, despite the absence of significant differences in gene expression or sequence, a clear tendency toward increased PBP2 (imipenem) and PBP3 (ceftazidime, ceftolozane, imipenem) IC(50)s was noted in PBLR isolates. Thus, our results suggest that in addition to AmpC, efflux pumps, and OprD, the modification of PBP patterns appears to play a role in the in vivo emergence of PBLR strains, which still conserve certain susceptibility to the new antipseudomonal cephalosporin ceftolozane.
[Show abstract][Hide abstract] ABSTRACT: An isolate of Klebsiella oxytoca carrying a novel IMP metallo-β-lactamase was discovered in Madrid, Spain. The bla(IMP-28) gene is part of a chromosomally located class I integron. The IMP-28 k(cat)/K(m) values for ampicillin, ceftazidime, and cefepime and, to a lesser extent, imipenem and meropenem, are clearly lower than those of IMP-1. The His306Gln mutation may induce important modifications of the L3 loop and thus of substrate accessibility and hydrolysis and be the main reason for this behavior.
[Show abstract][Hide abstract] ABSTRACT: We investigated the presence of OprD mutations in 60 strains of metallo-ß-lactamase-negative Pseudomonas aeruginosa intermediately susceptible (IS [n = 12]; MIC = 8 μg/ml) or susceptible (S [n = 48]; MICs ≤ 1 to 4 μg/ml) to imipenem and/or meropenem that were isolated from patients with bacteremia in order to evaluate
their impact on carbapenem susceptibility profiles. The presence of mutations in oprD was detected by sequencing analysis. OprD expression was assessed by both outer membrane protein (OMP) analysis and real-time
PCR (RT-PCR). Fourteen (23%) isolates had an OprD identical to that of PAO1, and OprD modifications were detected in 46 isolates
(77%). Isolates were classified as OprD “full-length types” (T1 [n = 40, including both wild-type OprD and variants showing several polymorphisms]) and OprD “deficient types” (T2 [n = 3 for OprD frameshift mutations] and T3 [n = 17 for premature stop codons in oprD]). RT-PCR showed that 5 OprD type T1 isolates presented reduced transcription of oprD (0.1- to 0.4-fold compared to PAO1), while oprD levels increased more than 2-fold over that seen with PAO1 in 4 OprD type T1 isolates. A total of 50% of the isolates belonging
to OprD “deficient types” were susceptible to both carbapenems, and 40% were susceptible to meropenem and intermediately susceptible
to imipenem. Only one isolate (5%) within this group was intermediately susceptible to both carbapenems, and one (5%) was
susceptible to imipenem and intermediately susceptible to meropenem. We concluded that OprD inactivating mutations in clinical
isolates of P. aeruginosa are not restricted only to carbapenem-resistant isolates but are also found in isolates with imipenem or meropenem MICs of
only 0.06 to 4 μg/ml.
[Show abstract][Hide abstract] ABSTRACT: A multidrug resistance (MDR) conjugative plasmid of ca. 50 kb (designated pERGB) was detected in a linezolid and methicillin-resistant
Staphylococcus aureus strain with sequence type 125 (ST125-MRSA-IVc). This strain was detected in two patients with chronic obstructive pulmonary
disease, previously treated with multiple antimicrobials, including linezolid. pERGB was transferable by conjugation and carried
the resistance genes cfr (oxazolidinones, phenicols, lincosamides, pleuromutilins, and streptogramin A), ant(4′)-Ia (tobramycin), tet(L) (tetracycline), and dfrK (trimethoprim). A novel genetic structure, linking all of these resistance genes for the first time, was elucidated through
sequencing of a 15,259-bp fragment from pERGB. Active surveillance to prevent the dissemination of such highly concerning
MDR transferable elements is needed.
[Show abstract][Hide abstract] ABSTRACT: The prevalence and impact of the overexpression of AmpC and efflux pumps were evaluated with a collection of 190 Pseudomonas aeruginosa isolates recovered from bloodstream infections in a 2008 multicenter study (10 hospitals) in Spain. The MICs of a panel of
13 antipseudomonal agents were determined by microdilution, and the expressions of ampC, mexB, mexY, mexD, and mexF were determined by real-time reverse transcription (RT)-PCR. Up to 39% of the isolates overexpressed at least one of the
mechanisms. ampC overexpression (24.2%) was the most prevalent mechanism, followed by mexY (13.2%), mexB (12.6%), mexF (4.2%), and mexD (2.2%). The overexpression of mexB plus mexY, documented for 5.3% of the isolates, was the only combination showing a significantly (P = 0.02) higher prevalence than expected from the frequencies of the individual mechanisms (1.6%). Additionally, all imipenem-resistant
isolates studied (25 representative isolates) showed inactivating mutations in oprD. Most of the isolates nonsusceptible to piperacillin-tazobactam (96%) and ceftazidime (84%) overexpressed ampC, while mexB (25%) and mexY (29%) overexpressions gained relevance among cefepime-nonsusceptible isolates. Nevertheless, the prevalence of mexY overexpression was highest among tobramycin-nonsusceptible isolates (37%), and that of mexB was highest among meropenem-nonsusceptible isolates (33%). Regarding ciprofloxacin-resistant isolates, besides the expected
increased prevalence of efflux pump overexpression, a highly significant link to ampC overexpression was documented for the first time: up to 52% of ciprofloxacin-nonsusceptible isolates overexpressed ampC, sharply contrasting with the 24% documented for the complete collection (P < 0.001). In summary, mutation-driven resistance was frequent in P. aeruginosa isolates from bloodstream infections, whereas metallo-β-lactamases, detected in 2 isolates (1%) producing VIM-2, although
with increasing prevalences, were still uncommon.
[Show abstract][Hide abstract] ABSTRACT: Constitutive AmpC hyperproduction is the most frequent mechanism of resistance to the weak AmpC inducers antipseudomonal penicillins and cephalosporins. Previously, we demonstrated that inhibition of the β-N-acetylglucosaminidase NagZ prevents and reverts this mechanism of resistance, which is caused by ampD and/or dacB (PBP4) mutations in Pseudomonas aeruginosa. In this work, we compared NagZ with a second candidate target, the AmpG permease for GlcNAc-1,6-anhydromuropeptides, for their ability to block AmpC expression pathways. Inactivation of nagZ or ampG fully restored the susceptibility and basal ampC expression of ampD or dacB laboratory mutants and impaired the emergence of one-step ceftazidime-resistant mutants in population analysis experiments. Nevertheless, only ampG inactivation fully blocked ampC induction, reducing the MICs of the potent AmpC inducer imipenem from 2 to 0.38 μg/ml. Moreover, through population analysis and characterization of laboratory mutants, we showed that ampG inactivation minimized the impact on resistance of the carbapenem porin OprD, reducing the MIC of imipenem for a PAO1 OprD mutant from >32 to 0.5 μg/ml. AmpG and NagZ targets were additionally evaluated in three clinical isolates that are pan-β-lactam resistant due to AmpC hyperproduction, OprD inactivation, and overexpression of several efflux pumps. A marked increase in susceptibility to ceftazidime and piperacillin-tazobactam was observed in both cases, while only ampG inactivation fully restored wild-type imipenem susceptibility. Susceptibility to meropenem, cefepime, and aztreonam was also enhanced, although to a lower extent due to the high impact of efflux pumps on the activity of these antibiotics. Thus, our results suggest that development of small-molecule inhibitors of AmpG could provide an excellent strategy to overcome the relevant mechanisms of resistance (OprD inactivation plus AmpC induction) to imipenem, the only currently available β-lactam not significantly affected by P. aeruginosa major efflux pumps.