Dataset Information

Ertapenem Supplemented Selective Media as a New Strategy to Distinguish β-Lactam-Resistant Enterobacterales: Application to Clinical and Wastewater Samples.

ABSTRACT: The increase in carbapenem-resistant Enterobacterales (CRE) is mostly driven by the spread of carbapenemase-producing (CP) strains. In New Caledonia, the majority of carbapenemases found are IMP-type carbapenemases that are difficult to detect on routine selective media. In this study, a culture-based method with ertapenem selection is proposed to distinguish non-CRE, non-CP-CRE, and CP-CRE from samples with very high bacterial loads. Firstly, assays were carried out with phenotypically well-characterized β-lactam-resistant Enterobacterales isolates. Then, this approach was applied to clinical and environmental samples. Presumptive CP-CRE isolates were finally identified, and the presence of a carbapenemase was assessed. In a collection of 27 phenotypically well-characterized β-lactam-resistant Enterobacterales, an ertapenem concentration of 0.5 µg·mL^-1 allowed distinguishing CRE from non-CRE. A concentration of 4 µg·mL^-1 allowed distinguishing CP-CRE from non-CP-CRE after nine hours of incubation. These methods allowed isolating 18 CP-CRE from hospital effluents, including the first detection of a KPC in New Caledonia. All these elements show that this cost-effective strategy to distinguish β-lactam-resistant Enterobacterales provides fast and reliable results. This could be applied in the Pacific islands or other resource-limited settings, where limited data are available.

SUBMITTER: Bourles A

PROVIDER: S-EPMC9952050 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Ertapenem Supplemented Selective Media as a New Strategy to Distinguish β-Lactam-Resistant Enterobacterales: Application to Clinical and Wastewater Samples.

Bourles Alexandre A Kainiu Malia M Ukeiwe Damaris D Brunet Nina N Despaux Camille C Biron Antoine A Gourinat Ann-Claire AC Goarant Cyrille C Colot Julien J

Antibiotics (Basel, Switzerland) 20230215 2

The increase in carbapenem-resistant Enterobacterales (CRE) is mostly driven by the spread of carbapenemase-producing (CP) strains. In New Caledonia, the majority of carbapenemases found are IMP-type carbapenemases that are difficult to detect on routine selective media. In this study, a culture-based method with ertapenem selection is proposed to distinguish non-CRE, non-CP-CRE, and CP-CRE from samples with very high bacterial loads. Firstly, assays were carried out with phenotypically well-cha ...[more]

PMID: 36830303

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Project description:As safe agents of last resort, carbapenems are reserved for the treatment of infections caused by multidrug-resistant organisms. The impact of β-lactam antibiotics, cefotaxime, and meropenem on the frequency and diversity of carbapenemase-producing organisms recovered from environmental samples has not been fully established. Therefore, this methodological study aimed at determining β-lactam drugs used in selective enrichment and their impact on the recovery of carbapenemase-producing Enterobacterales (CPE) from untreated wastewater. We used a longitudinal study design where 1L wastewater samples were collected weekly from wastewater treatment plant (WWTP) influent and quarterly from contributing sanitary sewers in Columbus, Ohio USA with 52 total samples collected. Aliquots of 500 mL were passed through membrane filters of decreasing pore sizes to enable all the water to pass through and capture bacteria. For each sample, the resulting filters were placed into two modified MacConkey (MAC) broths, one supplemented with 0.5 μg/mL of meropenem and 70 μg/mL of ZnSO4 and the other supplemented with 2 μg/mL cefotaxime. The inoculated broth was then incubated at 37° C overnight, after which they were streaked onto two types of correspondingly-modified MAC agar plates supplemented with 0.5 μg/mL and 1.0 μg/mL of meropenem and 70 μg/mL of ZnSO4 and incubated at 37°C overnight. The isolates were identified based on morphological and biochemical characteristics. Then, up to four distinct colonies of each isolate's pure culture per sample were tested for carbapenemase production using the Carba-NP test. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) MALDI-TOF MS was used to identify carbapenemase-producing organisms. In total 391 Carba-NP positive isolates were recovered from the 52 wastewater samples: 305 (78%) isolates had blaKPC, 73 (19%) carried blaNDM, and 14 (4%) harbored both blaKPC and blaNDM resistance genes. CPE genes of both blaKPC and blaNDM were recovered in both types of modified MAC broths, with 84 (21%) having a blaKPC gene, 22 (6%) carrying blaNDM and 9 (2%) harbored both a blaKPC and blaNDM of isolates recovered from MAC medium incorporated with 0.5ug/mL meropenem and 70ug/mL ZnSO4. The most prevalent isolates were Klebsiella pneumoniae, Escherichia coli, and Citrobacter spp.

Project description:Carbapenem-resistant Enterobacterales (CRE), specifically those resistant to only ertapenem among carbapenems (ETP-mono-resistant), are increasingly reported, while the optimal therapy options remain uncertain. To investigate the prevalence and characteristics of ETP-mono-resistant CRE, CRE strains were systematically collected from 102 hospitals across China between 2018 and 2021. A 1:1 randomized matching study was conducted with ETP-mono-resistant strains to meropenem- and/or imipenem-resistant (MEM/IPM-resistant) strains. Antimicrobial susceptibility testing, whole-genome sequencing, carbapenem-hydrolysing activity and the expression of carbapenemase genes were determined. In total, 18.8% of CRE strains were ETP-mono-resistant, with relatively low ertapenem MIC values. ETP-mono-resistant strains exhibited enhanced susceptibility to β-lactams, β-lactam/β-lactamase inhibitor combinations, levofloxacin, fosfomycin, amikacin and polymyxin than MEM/IPM-resistant strains (P < 0.05). Phylogenetic analysis revealed high genetic diversity among ETP-mono-resistant strains. Extended-spectrum β-lactamases (ESBLs) and/or AmpC, as well as porin mutations, were identified as potential major mechanisms mediating ETP-mono-resistance, while the presence of carbapenemases was found to be the key factor distinguishing the carbapenem-resistant phenotypes between the two groups (P < 0.001). Compared with the MEM/IPM-resistant group, limited carbapenemase-producing CRE (CP-CRE) strains in the ETP-mono-resistant group showed a significantly lower prevalence of ESBLs and porin mutations, along with reduced expression of carbapenemase. Remarkably, spot assays combined with modified carbapenem inactivation method indicated that ETP-mono-resistant CP-CRE isolates grew at meropenem concentrations eightfold above their corresponding MIC values, accompanied by rapidly enhanced carbapenem-hydrolysing ability. These findings illustrate that ETP-mono-resistant CRE strains are relatively prevalent and that caution should be exercised when using meropenem alone for treatment. The detection of carbapenemase should be prioritized.

Project description:ObjectivesThis study assessed in vitro activities of cefepime/taniborbactam and comparator antimicrobial agents against ertapenem-non-susceptible Enterobacterales (ENSE) clinical isolates collected from the CANWARD study 2007-19, and associations between MIC and various mechanisms of β-lactam resistance identified using WGS.MethodsA total of 179 ENSE (MIC ≥ 1 mg/L) isolates underwent susceptibility testing using reference CLSI broth microdilution. WGS was performed using the Illumina NextSeq platform. Carbapenemases, ESBLs and other β-lactamases were identified using ResFinder 4.0. Alterations in ompC/F and ftsI (PBP3) were identified by comparing extracted sequences to the appropriate NCBI reference gene. Porin alterations were analysed with Provean v1.1.3. Specific alterations of interest in PBP3 included a YRIN or YRIK insertion after P333.ResultsCefepime/taniborbactam was highly active (MIC50/MIC90, 0.5/2 mg/L; 177/179 isolates inhibited at ≤ 8 mg/L) against ENSE with various antimicrobial resistance phenotypes. Thirteen (7.3%) of the 179 ENSE isolates demonstrated cefepime/taniborbactam MIC values ≥ 4 mg/L and possessed combinations of β-lactam resistance mechanisms, including a carbapenemase and/or ESBL and/or other β-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3. Of the two Escherichia coli isolates that demonstrated a cefepime/taniborbactam MIC of 32 mg/L, one possessed NDM-5, OXA-181 and TEM-1B, an OmpC alteration and P333_Y334insYRIN in PBP3, while the second contained CTX-M-71, a truncated OmpF and a large alteration in OmpC (F182_R195delinsMTTNGRDDVFE).ConclusionsCefepime/taniborbactam was highly active against ENSE with various antimicrobial resistance phenotypes/genotypes. ENSE isolates with cefepime/taniborbactam MIC values ≥ 4 mg/L possessed combinations of β-lactam resistance mechanisms, including β-lactamase genes, as well as alterations in OmpC and/or OmpF and/or PBP3.

Dataset Information

Ertapenem Supplemented Selective Media as a New Strategy to Distinguish β-Lactam-Resistant Enterobacterales: Application to Clinical and Wastewater Samples.

Publications

Ertapenem Supplemented Selective Media as a New Strategy to Distinguish β-Lactam-Resistant Enterobacterales: Application to Clinical and Wastewater Samples.

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