ChloS-HRM, a novel assay to identify chloramphenicol-susceptible Escherichia coli and Klebsiella pneumoniae in Malawi

Abstract

Objectives Chloramphenicol is a broad-spectrum antimicrobial widely available in sub-Saharan Africa. With susceptibility re-emerging among Enterobacteriaceae in Blantyre, Malawi, we designed and evaluated a new high-resolution melt (HRM) RT-PCR assay, ChloS-HRM, to identify chloramphenicol-susceptible infections in a hospital setting. Methods Seventy-two previously whole-genome sequenced isolates of Escherichia coli and Klebsiella pneumoniae from the Queen Elizabeth Central Hospital, Malawi, were subjected to determination of chloramphenicol MICs. Primers were designed to detect 18 chloramphenicol resistance genes that produce seven distinct peaks correlating with different gene groups ( catA1, catA2, catA3, catB2, catB group 3, cmlA and floR ) following HRM analysis. ChloS-HRM results were compared with MIC and WGS results. Results ChloS-HRM correctly identified 15 of 17 phenotypically susceptible isolates and 54 of 55 resistant isolates, giving an accuracy of 88% in identifying susceptibility and 98% in identifying resistance. WGS identified 16 of 17 susceptible and 54 of 55 resistant isolates, giving an accuracy of 94% in identifying susceptibility and 98% in identifying resistance. The single false-susceptible result had no detectable gene by ChloS-HRM or WGS. Compared with WGS, ChloS-HRM had 100% sensitivity and specificity for catA ( catA1–3 ), cmlA and floR , and 96% specificity for catB ; sensitivity could not be estimated due to the lack of catB in the clinical sample collection. The overall agreement between MIC and HRM was 96% and between MIC and WGS it was 97%. Conclusions ChloS-HRM could support antimicrobial stewardship in enabling de-escalation from third-generation cephalosporins by identifying chloramphenicol-susceptible infections. This would be valuable in areas with chloramphenicol-susceptible MDR and XDR Enterobacteriaceae.

Piperacillin/tazobactam resistant, cephalosporin susceptibleEscherichia colibloodstream infections are driven by multiple acquisition of resistance across diverse sequence types

“…Recent work on methicillin resistant Staphylococcus aureus has described frequent collateral sensitivity to narrow spectrum penicillin/inhibitor combinations, highlighting that targeted de-escalation rather than escalation can be possible when treating organisms highly resistant to first line drugs (55). Molecular diagnostics and whole genome sequencing can be used to rapidly detect AMR genes to predict AMR phenotype (56, 57). However, it is essential to match the phenotypic resistance to the genotypic mechanisms.…”

“…Whilst molecular diagnostics can be used to rapidly detect AMR genes 58,64 Our study has some limitations, including that we only sequenced TZP resistant/3GC susceptible isolates at our site, and utilised a large and UK wide collection of isolates for comparison. These comparison isolates were collected between 2001 and 2012, and so are not contemporary with the study isolates collected between 2014 and 2017 but serve as the best available large-scale comparator of the same demographics.…”

Molecular Surveillance and Dissemination of Klebsiella pneumoniae on Frequently Encountered Surfaces in South African Public Hospitals

“…There are two different types of CAT enzymes, namely, CAT-A and CAT-B, encoded by different genes. Generally, the genes catA1, catA2, catA3, catB2 and catB3 are commonly detected in species belonging to the Enterobacteriaceae family and have been noted in Klebsiella species (Roberts & Schwarz, 2016;Williams et al, 2019).…”

A novel air-dried multiplex high-resolution melt assay for the detection of extended-spectrum β-lactamase and carbapenemase genes

“…PCR-based detection of ESBL and carbapenemase genes provides a faster detection of AMR than phenotypic methods, which might in turn generate more timely information for treatment decisions [ 13 , 14 ]. Whist molecular methods for the detection and characterisation of AMR genes are becoming increasingly established, with good agreement with phenotypic methods and producing faster results [15] , [16] , [17] , their use in clinical settings is hampered by the high degree of multiplexing needed due to the many genes involved in an antimicrobial-resistant phenotype. Additionally, PCR requires a cold chain to maintain the integrity of reagents as well as equipment and trained staff, which are often unavailable in low- and middle-income countries (LMICs).…”