Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia

Stewart, A. G.; Price, E. P.; Schabacker, K.; Birikmen, M.; Burnard, D. M.; Harris, P. N.; Choong, K.; Subedi, S.; Sarovich, D. S.

doi:10.1101/2020.02.20.958728

bioRxiv · 10.1101/2020.02.20.958728

Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia

Stewart, A. G.·Price, E. P.·Schabacker, K.·Birikmen, M.·Burnard, D. M.·Harris, P. N.·Choong, K.·Subedi, S.·Sarovich, D. S.

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Abstract

SynopsisThird-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 264 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using rapid ([~]40 min) real-time PCR assays targeting the most common extended spectrum {beta}-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). AmpC CMY {beta}-lactamase prevalence was also examined. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance were identified in 97.0% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, most (98.3%) isolates harbored at least one {beta}-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. A subset of isolates (n=98) were subjected to whole-genome sequencing (WGS) to identify the presence of cryptic resistance determinants, and to verify genotyping accuracy. WGS of {beta}-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

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Stewart, A. G., Price, E. P., Schabacker, K., Birikmen, M., Burnard, D. M., Harris, P. N., Choong, K., Subedi, S., Sarovich, D. S.. 2020-02-23. Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia. https://doi.org/10.1101/2020.02.20.958728

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