Molecular epidemiology of antibiotic-resistant ESKAPEE pathogens in surface water in proximity to informal settlements: a tale of two cities.

Abstract

Drug-resistant Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp and Escherichia coli (ESKAPEE) are increasingly identified in wastewater and surface water of rivers and streams, presenting a transmission risk to humans, animals, and plants. Using whole genome sequencing and bioinformatics analysis, we investigated the resistome, mobilome, and phylogenetic relationships of antibiotic-resistant ESKAPEE bacteria in surface water from two cities. Water samples (500 mL) from streams near informal settlements in Durban and Pietermaritzburg were filtered through a 0.45 μm filter membrane. The ESKAPEE were identified on selective media, purified and tested for antibiotic susceptibility using the VITEK® 2 platform. DNA was extracted from isolates for whole genome sequencing, followed by bioinformatics analysis using the open-source CARD, CGE, RAST, BV-BRC and PubMLST tools. Eleven E. faecium, 12 E. coli, four K. pneumoniae and one Enterobacter isolate were molecularly identified. Cephalosporin-resistant E. coli was found in Durban with the AcrAB-TolC efflux pump that conferred resistance to multiple antibiotic classes. The ARGs identified in E. coli were blaTEM- 1, qnrB19 and qnrS1, sul1, sul3, dfrA12, tet(A), cmlA1, aadA1 and aadA2. ARGs aac(6)- Ii, ant(6)-Ia and aph(3”)-III, tet(M) and tet(L), msr(C) and erm(B) and dfrG were detected in E. faecium. The Durban K. pneumoniae isolates were MDR harbouring blaSHV-75, blaSHV-110, blaSHV-81, blaCTXM-14, blaCTX-M-15, blaTEM-1B, and blaOXA-1. E. kobei only harboured blaACT and tet(A) genes that showed phenotypic resistance against piperacillin- tazobactam. ARGs and MGEs in E. faecium were mostly carried on chromosomes. Plasmid-carried ARGs were associated with IS1, IS1B, IS6, IS256 and ISKpn19, and the Tn3 transposons in E. coli. Of all identified ESBL genes in K. pneumonaie, only blaTEM, blaCTX-M-14 and blaCTX-M-15 were co-carried on plasmids and associated with ISKpn25, ISNCY, IS3, IS1, IS5075, IScep1, and Tn3. Phylogenetic analysis revealed close relationships with other South African human, animal and environmental isolates. The identified ARGs and their associations with MGEs present potential transmission routes of these resistance genes within and across bacterial species in aquatic environments, making these surface waters a potential reservoir for antibiotic resistance transmission.