Carbapenems

The ftsI locus of gram-negative bacteria encodes penicillin-binding protein 3 (PBP3), a common target of β-lactam antibiotics. Recent genomic surveillance of carbapenem-resistant Pseudomonas aeruginosa revealed that 17% of strains possessed nonsynonymous substitution in ftsI. However, the contribution of ftsI nonsynonymous substitution alone to the antimicrobial susceptibility of P. aeruginosa has not been formally evaluated. Therefore, we assessed the effect of ftsI nonsynonymous substitutions alone on β-lactam susceptibility in recombinant P. aeruginosa strains. The nonsynonymous substitutions C265G (Leu89Val), C1379T (Met460Thr), T1412G (Val471Gly), C1510T (Arg504Cys), C1579A (Pro527Thr), C1579T (Pro527Ser), C1599A (Phe533Leu), and G1609C (Val537Leu) were introduced into ftsI in an antimicrobial-susceptible strain via homologous recombination. Then, their β-lactam susceptibilities were investigated using the broth microdilution method and growth parameter analysis for the growth curves. The introduction of the N-terminal Leu89Val mutation did not alter β-lactam susceptibility. By contrast, the introduction of penicillin-binding (PB) domain mutations (Met460Thr, Val471Gly, Arg504Cys, Pro527Thr, Pro527Ser, Phe533Leu, or Val537Leu) reduced the susceptibility to meropenem and doripenem by two- to eightfold in MIC. Arg504Cys, the most common mutation encountered clinically, reduced susceptibility to cefepime by fourfold. Growth parameter analysis revealed that, conversely, these PB domain mutations generally increase the susceptibility to cefiderocol and piperacillin. The cocrystal structures of PBP3 with β-lactams suggest that the positions of PB domain mutations can affect the mobility of previously proposed key residues, namely, Tyr409, Arg489, Tyr503, Tyr532, Phe533, and the KSGT motif, via intramolecular interactions. These data demonstrated that seven specific variants in ftsI are genetic signals of bacterial adaptation to carbapenems.IMPORTANCEPrevious studies have reported the occurrence of ftsI mutations under various genetic backgrounds in Pseudomonas aeruginosa clinical isolates. However, the nature of ftsI mutations by themselves has not been thoroughly investigated under a common genetic background using recombinant strains. This study presents experimental data on the effect of ftsI point mutations alone on bacterial susceptibility to a wide range of antipseudomonal β-lactams. This study found that seven specific penicillin-binding domain mutations in FtsI/PBP3 reduced the efficacy of carbapenems but increased the efficacies of piperacillin and cefiderocol. This key finding will facilitate the development of therapeutic options for carbapenem-resistant P. aeruginosa-associated infections. Furthermore, this study contributes to the development of a reliable database collating resistance-associated point mutations, which would aid the interpretation of genomic data on P. aeruginosa clinical isolates.

β-Lactam/β-lactamase inhibitor combinations and carbapenems are the first-line treatments for multidrug-resistant Pseudomonas aeruginosa (P. aeruginosa) infections. However, carbapenem resistance is increasing globally at an alarming rate, which is especially concerning given the pivotal role of these agents. This study comprehensively evaluated the global distribution of carbapenem resistance in clinical P. aeruginosa isolates. The keywords including 'Pseudomonas', P. aeruginosa', 'P. aeruginosa', 'resistance', 'susceptibility', 'carbapenem antibiotics', 'carbapenems', 'imipenem', 'meropenem', 'ertapenem', 'doripenem', as well as 'prevalence' and 'incidence' were searched in electronic databases as the appropriate keywords. After screening, 160 studies were excluded, with 87 eligible studies from diverse geographic regions retained for final analysis. A comprehensive meta-analysis was then conducted on the data collected. The mean resistance rates (95% CI) were 33.3% (imipenem), 23.3% (meropenem), 60.9% (ertapenem), and 36.7% (doripenem). The time trend analysis showed that the resistance to meropenem has increased from the year 1997 to 2023. Meta-analysis showed substantial heterogeneity (I2 = 92%, p P. aeruginosa isolates. The increasing prevalence of carbapenem-resistant P. aeruginosa is a major global health threat requiring urgent action through new antimicrobials and improved antibiotic stewardship to protect these last-line drugs.

Pseudomonas aeruginosa can acquire carbapenem resistance through various mechanisms, including genomic mutations leading to the overexpression of efflux pumps, intrinsic AmpC-β-lactamase, and/or reduced permeability, and/or through the acquisition of plasmid-mediated carbapenemases and/or extended-spectrum-β-lactamases (ESBLs). Unfortunately, carbapenemase/ESBL-producing-carbapenem-resistant-P. aeruginosa (CP/ESBL-CRPA) cannot be differentiated from non-CP/ESBL-CRPA based solely on susceptibility testing results of conventional β-lactam (BL)-antibiotics. Knowing that these two groups display different activity profiles toward novel BL/β-lactamase-inhibitor (BLI) combinations, we developed and verified a cost-effective and easy-to-use diagnostic algorithm for screening and differentiation of carbapenemase and ESBL production in CRPA. We determined disc diffusion inhibition zones and gradient strip minimal inhibitory concentration values of 136 whole-genome-sequenced CRPA (70 metallo-β-lactamase-[MBL-], 1 GES-5-, 1 KPC-2-, 12 ESBL-, and 53 AmpC-hyper-producing isolates). We used the following BL-BLI combinations: ceftolozane-tazobactam (C-T), ceftazidime-avibactam, imipenem-relebactam (I-R), meropenem-vaborbactam, cefepime-enmetazobactam (C-E), and aztreonam-avibactam. We also included a lateral flow immunoassay (Carba-5, NG-Biotech) for confirmation of MBL production and double disc synergy testing (DDST) to improve ESBL detection. C-T was the most effective screening antibiotic for distinguishing MBL and ESBL producers from AmpC-hyperproducing CRPA, achieving a sensitivity of 100% for both MBL and ESBL producers. I-R reliably confirmed MBL production in C-T positive screened CRPAs, with a sensitivity of 92.8% and specificity of 100%. Incorporating Carba-5 into the phenotypic algorithm improved sensitivity for confirming MBL production to 100%. For the remaining C-T positive but I-R negative isolates, C-E showed 75% sensitivity and 78.6% specificity in detecting ESBL production. The DDST further confirmed ESBL production in six out of nine ESBL producers (66.6%). In conclusion, we established a simple and cost-effective diagnostic algorithm, enabling screening and confirmation of carbapenemase and ESBL production in CRPA.IMPORTANCECarbapenem-resistant Pseudomonas aeruginosa (CRPA) is a major global health threat, and rapid identification of its resistance mechanisms is crucial for effective treatment and infection control. Differentiating between carbapenemase-producing (CP), extended-spectrum β-lactamase-producing (ESBL), and AmpC-hyperproducing CRPA is challenging, as conventional susceptibility testing cannot reliably distinguish these resistance mechanisms. Our study presents a simple, cost-effective, and easy-to-implement phenotypic diagnostic algorithm that enables accurate screening and confirmation of CP and ESBL production in CRPA. This method is particularly valuable for laboratories lacking access to molecular diagnostics, as it provides a practical alternative for routine testing. By facilitating the early detection of resistant P. aeruginosa strains, this approach has the potential to improve patient outcomes, optimize antimicrobial therapy, and enhance global surveillance efforts against multidrug-resistant pathogens.

Metallo-β-lactamases (MBLs) hydrolyze the beta-lactam ring in beta-lactam antibiotics, rendering them ineffective. Infections caused by MBL-harboring bacteria result in higher mortality, more costs and prolonged hospital stays due to the limited treatment options. Recently, antimicrobial susceptibility testing using RPMI-1640 has been found to have more accuracy in predicting in vivo efficacy due to its zinc deficiency. We sought to expand the previous studies of accessing in vivo efficacy using zinc-limited media versus conventional media to more antimicrobial agents and MBL-producing strains. The susceptibility of isolates was determined by performing minimum inhibitory concentration (MIC) assays using traditional cation-adjusted Mueller Hinton broth (CAMHB) or zinc-limited media. In vivo outcomes were evaluated using the Galleria mellonella infection model and neutropenic mouse thigh infection model. The MICs of MBL-harboring strains decreased in zinc-limited media compared to in nutrient-rich media, suggesting susceptibility of a subset of resistant strains when tested in zinc-limited media. Notably, we observed statistically significant MICs decreasing against imipenem, which demonstrated the best efficacy among the six tested antibiotics. Additionally, the outcomes of in vivo tests in both the G. mellonella model and the mouse model were better predicted with in vitro MIC assays performing in zinc-limited media. The use of zinc-limited media may lead to increased accuracy of the prediction of in vivo efficacy of beta-lactams against MBL-harboring bacteria.