Molecular Epidemiology of Third-Generation-Cephalosporin-Resistant Enterobacteriaceae in Southeast Queensland, Australia

Abstract

Third-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 β-lactamases (ESBLs; blaCTX-M-1 and blaCTX-M-9 groups, plus blaTEM, blaSHV, and an internal 16S ribosomal DNA control). AmpC CMY β-lactamase (blaCMY) 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 β-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 β-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.

Rapid fluoroquinolone resistance detection inPseudomonas aeruginosausing mismatch amplification mutation assay-based real-time PCR

“…One ulcer and one ear infection isolate, both from Brisbane, were obtained from the 1,000 International P. aeruginosa Consortium collection (18). Ethics approvals were obtained as previously described (16,17,19).…”

“…Isolates were DNA-extracted using the DNeasy kit (Qiagen, Chadstone Centre, VIC, Australia), followed by Illumina paired-end whole-genome sequencing (WGS) (19). Quality-filtered reads (21) were assembled with MGAP v1.1 (https://github.com/dsarov/MGAP---Microbial-Genome-Assembler-Pipeline) or SPAdes (22).…”

Express yourself: Quantitative real-time PCR assays for rapid chromosomal antimicrobial resistance detection in Pseudomonas aeruginosa

“…Instead, nucleic acid-based detection platforms such as real-time PCR remain vital -and substantially cheaper, simpler, and more accessible in routine clinical microbiology services than NGS -for the rapid identification of pathogens and their AMR determinants (12). In addition to identifying AMR-conferring single-nucleotide polymorphisms [SNPs] (13,14), insertions/deletions (15), copy-number variants (16), gene gain (17,18), or gene loss (19), targeting altered RNA expression of key AMR loci (20)(21)(22)(23) provides a rapid way to identify the phenotypic consequences of the myriad genotypic variants that underpin AMR. For example, a quantitative real-time PCR (qPCR) targeting upregulation of three key AMR resistance-nodulationdivision (RND) efflux pumps in the melioidosis pathogen, Burkholderia pseudomallei, enables the simultaneous identification of meropenem (MEM) resistance, and decreased susceptibility towards doxycycline and co-trimoxazole, in a single reaction (24).…”

“…[SNPs] (13, 14), insertions/deletions (15), copy-number variants (16), gene gain (17, 18), or gene loss (19), targeting altered RNA expression of key AMR loci (20–23) provides a rapid way to identify the phenotypic consequences of the myriad genotypic variants that underpin AMR. For example, a quantitative real-time PCR (qPCR) targeting upregulation of three key AMR resistance-nodulation-division (RND) efflux pumps in the melioidosis pathogen, Burkholderia pseudomallei , enables the simultaneous identification of meropenem (MEM) resistance, and decreased susceptibility towards doxycycline and co-trimoxazole, in a single reaction (24).…”

Diminished Susceptibility to Cefoperazone/Sulbactam and Piperacillin/Tazobactam in Enterobacteriaceae Due to Narrow-Spectrum β-Lactamases as Well as Omp Mutation

“…extended-spectrum β-lactamases (ESBLs) (Stewart et al 2021), are the leading cause of CSL and TZP-resistant Enterobacteriaceae isolates. Many of these enzymes also hydrolyze third-generation cephalosporins and most CSL and TZP-nonsusceptible Enterobacteriaceae are also resistant to ceftriaxone (CRO).…”