In vitro and in vivo activity of ceftazidime/avibactam and aztreonam alone or in combination against mcr-9, serine- and metallo-ß-lactamases-co-producing carbapenem-resistant Enterobacter cloacae complex.

PURPOSE: Enterobacteriaceae carrying mcr-9, in particularly those also co-containing metallo-ß-lactamase (MBL) and TEM type ß-lactamase, present potential transmission risks and lack adequate clinical response methods, thereby posing a major threat to global public health. The aim of this study was to assess the antimicrobial efficacy of a combined ceftazidime/avibactam (CZA) and aztreonam (ATM) regimen against carbapenem-resistant Enterobacter cloacae complex (CRECC) co-producing mcr-9, MBL and TEM. METHODS: The in vitro antibacterial activity of CZA plus ATM was evaluated using a time-kill curve assay. Furthermore, the in vivo interaction between CZA plus ATM was confirmed using a Galleria mellonella (G. mellonella) infection model. RESULTS: All eight clinical strains of CRECC, co-carrying mcr-9, MBL and TEM, exhibited high resistance to CZA and ATM. In vitro time-kill curve analysis demonstrated that the combination therapy of CZA + ATM exerted significant bactericidal activity against mcr-9, MBL and TEM-co-producing Enterobacter cloacae complex (ECC) isolates with a 100% synergy rate observed in our study. Furthermore, in vivo survival assay using Galleria mellonella larvae infected with CRECC strains co-harboring mcr-9, MBL and TEM revealed that the CZA + ATM combination significantly improved the survival rate compared to the drug-treatment alone and untreated control groups. CONCLUSION: To our knowledge, this study represents the first report on the in vitro and in vivo antibacterial activity of CZA plus ATM against CRECC isolates co-harboring mcr-9, MBL and TEM. Our findings suggest that the combination regimen of CZA + ATM provides a valuable reference for clinicians to address the increasingly complex antibiotic resistance situation observed in clinical microorganisms.

Activities of aztreonam in combination with several novel ß-lactam-ß-lactamase inhibitor combinations against carbapenem-resistant Klebsiella pneumoniae strains coproducing KPC and NDM.

Isolates coproducing serine/metallo-carbapenems are a serious emerging public health threat, given their rapid dissemination and the limited number of treatment options. The purposes of this study were to evaluate the in vitro antibacterial activity of novel ß-lactam-ß-lactamase inhibitor combinations (BLBLIs) against carbapenem-resistant Klebsiella pneumoniae (CRKP) coproducing metallo-ß-lactamase and serine-ß-lactamase, and to explore their effects in combination with aztreonam, meropenem, or polymyxin in order to identify the best therapeutic options. Four CRKP isolates coproducing K. pneumoniae carbapenemase (KPC) and New Delhi metallo-ß-lactamase (NDM) were selected, and a microdilution broth method was used to determine their susceptibility to antibiotics. Time-kill assay was used to detect the bactericidal effects of the combinations of antibiotics. The minimum inhibitory concentration (MIC) values for imipenem and meropenem in three isolates did not decrease after the addition of relebactam or varbobactam, but the addition of avibactam to aztreonam reduced the MIC by more than 64-fold. Time-kill assay demonstrated that imipenem-cilastatin/relebactam (ICR) alone exerted a bacteriostatic effect against three isolates (average reduction: 1.88 log10 CFU/mL) and ICR combined with aztreonam exerted an additive effect. Aztreonam combined with meropenem/varbobactam (MEV) or ceftazidime/avibactam (CZA) showed synergistic effects, while the effect of aztreonam combined with CZA was inferior to that of MEV. Compared with the same concentration of aztreonam plus CZA combination, aztreonam/avibactam had a better bactericidal effect (24 h bacterial count reduction >3 log10CFU/mL). These data indicate that the combination of ATM with several new BLBLIs exerts powerful bactericidal activity, which suggests that these double ß-lactam combinations might provide potential alternative treatments for infections caused by pathogens coproducing-serine/metallo-carbapenems.

Increased Expression and Amplification of blaKPC-2 Contributes to Resistance to Ceftazidime/Avibactam in a Sequence Type 11 Carbapenem-Resistant Klebsiella pneumoniae Strain.

Ceftazidime/avibactam (CAZ/AVI) is regarded as an effective alternative antibiotic for the clinical treatment of Klebsiella pneumoniae carbapenemase (KPC)-producing isolates. As resistance has been reported in some strains, it is critical to understand the key mechanisms contributing to the acquired resistance to CAZ/AVI. From January 2018 to April 2020, 127 KPC-producing carbapenem-resistant Klebsiella pneumoniae strains (CRKPs) were isolated at a university hospital in Chongqing, China, and 25 strains showed reduced susceptibility to CAZ/AVI. All reduced-susceptibility CRKPs were deficient in Ompk35 and Ompk36 porins, and 24 strains had a premature termination at amino acid position 63 in Ompk35 and 134 to 135 glycine and aspartic acid (GD) insertion in OmpK36, while the blaKPC-2 expression level showed no significant difference compared to that of strain BAA-1705. Four reduced-susceptibility strains evolved resistance under selective pressure of CAZ/AVI with the blaKPC-2 expression level increased, and two of these strains had mutations in the Ω-loop. The study found a strain of CRKP55 with changes in the resistance phenotype during conjugation, evolving from reduced sensitivity to high-level resistance to CAZ/AVI. Through plasmid sequencing and reverse transcription-quantitative PCR, it was speculated that insertion sequence (IS)26-mediated blaKPC-2 gene amplification caused the MIC value change in the conjugant JKP55. Our findings illustrated the potential of CAZ/AVI resistance under antibiotic stress and demonstrated that IS26 may mediate blaKPC-2 replication transposition, leading to high-level resistance during horizontal gene transfer. Investigation of CAZ/AVI resistance mechanisms may offer a unique opportunity to study the horizontal evolutionary trajectories of K. pneumoniae high-risk clones. IMPORTANCE Klebsiella pneumoniae carbapenemase (KPC) production is the most common mechanism of K. pneumoniae resistance to carbapenems in China. Currently, CAZ/AVI is considered a potential alternative therapeutic option for infections caused by these isolates. However, there have been increasing reports of resistant or reduced-sensitivity strains since the approval of this agent. In this study, resistance to CAZ/AVI was induced under drug-selective pressure and was caused by blaKPC-2 overexpression and/or substitutions in the Ω-loop of KPC. Additionally, it was demonstrated that a conjugative plasmid carrying blaKPC-2 could transfer horizontally between species, and perhaps, IS26-derived tandem amplification of blaKPC-2 during this period led to high-level resistance to CAZ/AVI. Our research suggests that IS26-mediated resistance evolution may have important implications in guiding clinical antibiotic use.