Characterization of carbapenem-resistant gram-negative bacterial isolates from Nigeria by whole genome sequencing.

This study characterized the mechanisms of carbapenem resistance in gram-negative bacteria isolated from patients in Yola, Nigeria. Whole genome sequencing (WGS) was performed on 66 isolates previously identified phenotypically as carbapenem-non-susceptible. The patterns of beta-lactamase resistance genes identified were primarily species-specific. However, blaNDM-7 and blaCMY-4 were detected in all Escherichia coli and most Providencia rettgeri isolates; blaNDM-7 was also detected in 1 Enterobacter cloacae. The E. coli and E. cloacae isolates also shared blaOXA-1, while blaOXA-10 was found in all P. rettgeri, one Pseudomonas aeruginosa and 1 E. coli. Except for Stenotrophomonas maltophilia isolates, which only contained blaL1, most species carried multiple beta-lactamase genes, including those encoding extended-spectrum beta-lactamases, AmpC and OXA in addition to a carbapenemase gene. Carbapenemase genes were either class B or class D beta-lactamases. No carbapenemase gene was detected by WGS in 13.6% of isolates.

Does the presence of multiple ß-lactamases in Gram-negative bacilli impact the results of antimicrobial susceptibility tests and extended-spectrum ß-lactamase and carbapenemase confirmation methods?

OBJECTIVES: Many multidrug-resistant Gram-negative bacilli (MDR-GNB) harbour multiple ß-lactamases. The aim of this study was to assess the impact of multiple ß-lactamase carriage on the accuracy of susceptibility tests and extended-spectrum ß-lactamase (ESBL) and carbapenemase confirmation methods. METHODS: A total of 50 MDR-GNB, of which 29 carried multiple ß-lactamases, underwent broth microdilution (BMD) and disk diffusion (DD) testing as well as confirmation tests for ESBLs and carbapenemases. Whole-genome sequencing (WGS) was used for ß-lactamase gene identification. RESULTS: Categorical agreement of BMD and DD testing results ranged from 86.5 to 97.7% for 10 ß-lactam agents. BMD and DD algorithms for ESBL detection were highly variable; 6 of 8 positive strains carried an ESBL plus a carbapenemase or an AmpC enzyme, which may confound antimicrobial selection. The sensitivity and specificity of the modified carbapenem inactivation method (mCIM) were both 100%, whilst mCIM and EDTA-modified carbapenem inactivation method (eCIM) when used together to differentiate serine from metallo-ß-lactamase carriage were both 96%. Xpert® Carba-R results (in vitro diagnostic test) were consistent with WGS results. Predicting phenotypic carbapenem resistance from WGS data overall showed 100% specificity but only 66.7% sensitivity for Enterobacterales isolates that were non-susceptible to imipenem and meropenem. CONCLUSIONS: Multiple ß-lactamases in MDR-GNB does not impact DD results, the utility of mCIM/eCIM tests, or Xpert Carba-R results. However, ESBL algorithms produced inconsistent results and predicting carbapenem resistance from WGS data was problematic in such strains.

Characterisation of carbapenem-resistant Gram-negative organisms from clinical specimens in Yola, Nigeria.

OBJECTIVES: This study aimed to identify carbapenem-resistant Gram-negative bacteria from clinical specimens of patients in Yola, Nigeria. METHODS: Routine clinical specimens were screened for the presence of carbapenem-resistant Gram-negative bacteria using chromogenic agar plates. Susceptibility of all presumptive isolates to carbapenems was tested by MIC and disk diffusion methods. Real-time PCR was used to test for the presence of carbapenemase genes. RESULTS: Screening of 1741 clinical specimens yielded 119 (6.8%) presumptive carbapenem-resistant Gram-negative bacteria. Antimicrobial susceptibility testing confirmed carbapenem resistance in 105 of these isolates. New Delhi metallo-ß-lactamase (blaNDM) gene was detected in 26 isolates and Verona integron-encoded metallo-ß-lactamase (blaVIM) gene was detected in four. The mechanism of resistance could not be identified in approximately two thirds of the carbapenem-resistant isolates. CONCLUSION: While blaNDM and blaVIM accounted for 28.6% of the resistance seen, further molecular-based studies are needed to characterise the other mechanisms of carbapenem resistance in these isolates.