No abstract

This study aimed to characterize the role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, ␤-lactam resistance, and ampC regulation. For this purpose, we constructed all single and multiple mutants of dacB, dacC, pbpG, and ampC from the wild-type P. aeruginosa PAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC), ampC expression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of ␤-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, only dacC mutation led to significantly increased pentapeptide levels, showing that PBP5 is the major DD-carboxypeptidase in P. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role as DD-carboxypeptidase only if PBP5 is absent, and their DD-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase in ampC expression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the ␤-lactam susceptibility profiles of the LMM PBP mutants correlated well with the ampC expression data. However, the inactivation of ampC in these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic ␤-lactam resistance. In summary, in addition to assessing the effect of P. aeruginosa LMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on ␤-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, ␤-lactam trapping, and DD-carboxypeptidase/␤-lactamase activity. Pseudomonas aeruginosa is a frequent cause of nosocomial infections, especially affecting patients in intensive care units (ICUs) with mechanical ventilation-associated pneumonia or burn wound infections, both of which are associated with a high mortality rate (1). This pathogen is also the major cause of chronic respiratory infections in patients with cystic fibrosis and other underlying chronic respiratory diseases (2). One of the most striking features of P. aeruginosa is its extraordinary capacity for developing resistance to almost any available antibiotic by the selection of mutations in chromosomal genes (3). Among the mutationmediated ␤-lactam resistance mechanisms, particularly noteworthy are those leading to the constitutive overexpression of the inducible chromosomal cephalosporinase AmpC, which confers resistance to penicillins, cephalosporins, and monobactams (4). Additionally, mutations that lead to the repression or inactivation of the porin OprD, acting synergistically with inducible or cons...

In addition to providing new insights into β-lactam resistance mechanisms and evolution, our findings should be helpful for guiding future strategies to combat P. aeruginosa infections.

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Objectives We analysed the dynamics and mechanisms of resistance development to imipenem alone or combined with relebactam in P. aeruginosa WT (PAO1) and mutator (PAOMS; ΔmutS) strains. Methods PAO1 or PAOMS strains were incubated for 24 h in Mueller–Hinton Broth with 0.125–64 mg/L of imipenem ± relebactam 4 mg/L. Tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64 mg/L of imipenem ± relebactam for 7 days. Two colonies per strain, replicate experiment and antibiotic from early (Day 1) and late (Day 7) cultures were characterized by determining the susceptibility profiles, WGS and determination of the expression of ampC and efflux-pump-coding genes. Virulence was studied in a Caenorhabditis elegans infection model. Results Relebactam reduced imipenem resistance development for both strains, although resistance emerged much faster for PAOMS. WGS indicated that imipenem resistance was associated with mutations in the porin OprD and regulators of ampC, while the mutations in imipenem/relebactam-resistant mutants were located in oprD and regulators of MexAB-OprM. High-level imipenem/relebactam resistance was only documented in the PAOMS strain and was associated with an additional specific (T680A) mutation located in the catalytic pocket of ponA (PBP1a) and with reduced virulence in the C. elegans model. Conclusions Imipenem/relebactam could be a useful alternative for the treatment of MDR P. aeruginosa infections, potentially reducing resistance development during treatment. Moreover, this work deciphers the potential resistance mechanisms that may emerge upon the introduction of this novel combination into clinical practice.

Imipenem and imipenem-relebactam MICs were determined for 1,445 Pseudomonas aeruginosa clinical isolates and a large panel of isogenic mutants showing the most relevant mutation-driven β-lactam resistance mechanisms. Imipenem-relebactam showed the highest susceptibility rate (97.3%), followed by colistin and ceftolozane-tazobactam (both 94.6%). Imipenem-relebactam MICs remained ≤2 μg/ml in all 16 isogenic PAO1 mutants and in 8 pairs of extensively drug-resistant clinical strains that had developed resistance to ceftolozane-tazobactam and ceftazidime-avibactam due to mutations in OXA-10 or AmpC.

The increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) is frequently linked to widespread international strains designated high-risk clones. In this work, we attempted to decipher the interplay between resistance profiles, high-risk clones, and virulence, testing a large ( = 140) collection of well-characterized isolates from different sources (bloodstream infections, nosocomial outbreaks, cystic fibrosis, and the environment) in a infection model. Consistent with previous data, we documented a clear inverse correlation between antimicrobial resistance and virulence in the model. Indeed, the lowest virulence was linked to XDR profiles, which were typically linked to defined high-risk clones. However, virulence varied broadly depending on the involved high-risk clone; it was high for sequence type 111 (ST111) and ST235 but very low for ST175. The highest virulence of ST235 could be attributed to its type III secretion system (TTSS) genotype, which was found to be linked with higher virulence in our model. Other markers, such as motility or pigment production, were not essential for virulence in the model but seemed to be related with the higher values of the statistical normalized data. In contrast to ST235, the ST175 high-risk clone, which is widespread in Spain and France, seems to be associated with a particularly low virulence in the model. Moreover, the previously described G154R AmpR mutation, prevalent in ST175, was found to contribute to the reduced virulence, although it was not the only factor involved. Altogether, our results provide a major step forward for understanding the interplay between resistance profiles, high-risk clones, and virulence.

Background Searching for new strategies to defeat Pseudomonas aeruginosa is of paramount importance. Previous works in vitro showed that peptidoglycan recycling blockade disables AmpC-dependent resistance and enhances susceptibility against cell-wall–targeting immunity. Our objective was to validate these findings in murine models. This study shows for the first time in different murine models of infection that blocking the peptidoglycan recycling in Pseudomonas aeruginosa causes an important virulence impairment and disables AmpC-mediated resistance, being hence validated as a promising therapeutic target. Methods Wildtype PAO1, recycling-defective AmpG and NagZ mutants, an AmpC hyperproducer dacB mutant, and their combinations were used to cause systemic/respiratory infections in mice. Their survival, bacterial burden, inflammation level, and effectiveness of ceftazidime or subtherapeutic colistin to treat the infections were assessed. Results Inactivation of AmpG or NagZ significantly attenuated the virulence in terms of mice mortality, bacterial load, and inflammation. When inactivating these genes in the dacB-defective background, the β-lactam resistance phenotype was abolished, disabling the emergence of ceftazidime-resistant mutants, and restoring ceftazidime for treatment. Subtherapeutic colistin was shown to efficiently clear the infection caused by the recycling-defective strains, likely due to the combined effect with the mice cell-wall– targeting immunity. Conclusions This study brings us one step closer to new therapies intended to disable P. aeruginosa AmpC-mediated resistance and dampen its virulence, and strongly support the interest in developing efficient AmpG and/or NagZ inhibitors.