27Third-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major 28 threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates 29 from 258 patients presenting at a regional Australian hospital over a 14-month period.
30Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates 31 were examined using a rapid (~40 min) pentaplex real-time PCR assay targeting the most 32 common extended spectrum β-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, 33 SHV, and an internal 16S ribosomal DNA control). Additionally, AmpC CMY β-lactamase 34 prevalence was examined using a singleplex PCR. A subset of isolates, including all 3GC-R 35 isolates obtained from the intensive care unit, were subjected to whole-genome sequencing 36 (WGS) to assess transmission dynamics, the presence of unidentified resistance 37 determinants, and genotyping accuracy. Escherichia coli (80.2%) and Klebsiella pneumoniae 38 (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter 39 cloacae infrequently identified. Ceftriaxone and cefoxitin resistance was identified in 97% 40 and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings 41 in Enterobacteriaceae, the majority (98.3%) of isolates harbored at least one β-lactamase 42 gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 43 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY genes. WGS of β-lactamase negative or 44 carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including45 blaNDM and blaIMP, and confirmed all PCR-positive genotypes. No evidence of transmission 46 among intensive care unit patients was identified. We demonstrate that our PCR assays 47 enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has 48 important infection control and therapeutic implications.49 52 determinants within and among bacterial populations [1]. Both infection and colonization 53 with these globally-disseminated organisms are associated with poor clinical outcomes and 54 death [2]. 3GC-R Enterobacteriaceae prevalence rates vary considerably among hospitals 55 and countries, with particularly high rates in India, Asia, and the Middle East [3]. Australia 56 has historically observed a relatively low frequency of 3GC-R Enterobacteriaceae. However, 57 this pattern may be changing, with an extended spectrum β-lactamase (ESBL) phenotype 58 detected in 13.3% of E. coli and 9.8% of K. pneumoniae in blood culture isolates in 2018 [4], 59 up from 2013 rates of 7.6% and 6.3%, respectively. Moreover, ceftriaxone resistance was 60 seen in 13.4% of E. coli and 9.4% of K. pneumoniae, with 86.3% and 82.6% containing CTX-M-61 type ESBL genes, respectively [4]. 62 63 AMR in 3GC-R Enterobacteriaceae is generally conferred by the presence of an ESBL or the 64 over-expression of a chromosomal or plasmid-borne AmpC β-lactamase; however, the 65 prevalence of these determinants var...