Molecular epidemiology and mechanisms of colistin and carbapenem resistance in Enterobacteriaceae from clinical isolates, the environment and porcine samples in Pretoria, South Africa

Introduction: Carbapenems and colistin are the last-line antibiotics for treating Gram-negative bacterial infections. However, increasing reports of resistance to these antibiotics is being reported in clinical settings, the environment and in animals. In this paper, we describe the molecular epidemiology and resistance mechanisms of colistin and carbapenem resistance in clinical, veterinary, and environmental Enterobacterales isolates in Pretoria, South Africa. Method: One hundred VITEK®-2-confirmed colistin and carbapenem-resistant clinical isolates were collected from the departmental isolate bank at the National Health Laboratory Service. A total of 88 porcine (stool) and 11 environmental (effluents) samples were collected in November 2018 and again in March 2019 from a farm in Pretoria. Both the porcine and environmental samples were screened using Eosin methylene blue agar with colistin and ertapenem disks. All isolates were identified and a minimum inhibitory concentration of colistin and carbapenems was determined using the MicroScan® WalkAway system. Isolates resistant to colistin were confirmed by the broth microdilution method. Isolates phenotypically resistant to colistin and carbapenems were selected for whole genome sequencing to determine the resistome and phylogenetic trees were drawn to determine the relatedness of isolates. Results: A total of 275 Gram-negative isolates were identified from the clinical (100), environmental (57) and veterinary (118) samples using the MicroScan® WalkAway system. The MicroScan® WalkAway system’s minimum inhibitory concentration results for clinical isolates revealed 88% and 93% resistance to colistin and carbapenems, respectively. BMD was found to be more reliable in all isolates, and it recorded higher MICs (increased resistance) than the MicroScan® WalkAway system. Overall, colistin susceptibility was higher among animal isolates compared to the clinical and environmental samples. Genomic analysis identified several resistance genes associated with resistance among the isolates and the CTX-M family were the dominant resistance genes. Phylogenomic analysis demonstrated closer evolutionary relationship between EB008 (environment), SW10B (animals), and C080 and C084 (both humans) strains as well as with strains from the United States of America, Canada, China, Russia and Durban (South Africa). Conclusion: The study established multiple resistance genes from different antibiotics to mediate resistance in Enterobacterales isolates from humans, animals and the environment. The presence of carbapenemases in animals is alarming and poses a public health concern. Strains EB008 (environment), SW10B (animals) and C080 and C084 (both human) were phylogenetically related with strains from the United States of America, China and Durban (South Africa) more commonly. Therefore, One Health approach studies are significant to ascertain colistin and carbapenem transmission from human to animals/the environment and vice versa to combat increasing resistance in Enterobacterales.Dissertation (MSc)--University of Pretoria, 2020.National Research Foundation (NRF)National Health Laboratory Service research grantMedical MicrobiologyMScUnrestricte