Characterisation of mobile colistin resistance genes (mcr-3 and mcr-5) in river and storm water in regions of the Western Cape of South Africa

Abstract

Background Colistin is regarded as a last-resort antimicrobial against multi-drug resistant Gram-negative bacteria (GNB), therefore the dissemination of colistin resistance in the environment is of great concern. Horizontal transfer of mobile colistin resistance (mcr) genes to potential pathogens poses a serious problem. This study aimed to describe the presence of colistin resistant GNB and mcr genes in river and storm water in regions of the Western Cape. Methods Water samples were collected from three rivers during May 2019 and January 2020 and two storm water samples were collected in November 2019. Colistin resistant GNB were cultured on MacConkey agar containing colistin and identified by MALDI-TOF. Colistin resistance was confirmed using broth microdilution (BMD). mcr-1-5 genes were detected by PCR performed directly on the water samples and on the colistin resistant isolates. mcr functionality was assessed by BMD after cloning the mcr genes into pET-48b(+) and expression in SHuffle T7 E. coli. Results mcr-5.1 and various mcr-3 gene variants were detected in the Plankenburg-, Eerste- and Berg rivers and in storm water from Muizenberg, and only mcr-5.1 was detected in storm water from Fish Hoek. Colistin resistant GNB were isolated from all of the water sources. Aeromonas spp. were the most common colistin resistant organisms detected in the water sources; 25% (6/24) of colistin resistant Aeromonas spp. isolated from the Berg river contained novel mcr-3 variants; mcr-3.33 (n = 1), mcr-3.34 (n = 1) mcr-3.35 (n = 1) mcr-3.36 (n = 2) and mcr-3.37 (n = 1), which were confirmed to confer colistin resistance. Conclusions The mcr-5.1 and mcr-3 colistin resistance gene variants were present in widely dispersed water sources in regions of the Western Cape. The mcr genes were only detected in water sampled downstream of and alongside communities, suggesting that their presence is driven by human influence/contamination. This is the first documentation of mcr-3 and mcr-5 gene variants in any setting in South Africa. Spill-over of these genes to communities could result in horizontal gene transfer to pathogenic bacteria, exacerbating the challenge of controlling multidrug resistant GNB infections.

Escherichia coliB-strains are intrinsically resistant to colistin and not suitable for characterization and identification ofmcrgenes

“…We found that four E.coli B-strains, BL21, BL21D, T7, and Shuffle are intrinsically resistant to colistin with MICs of 8-16 μg/mL. This observation was surprising, as previous studies have used B-strains like BL21D and Shuffle to report MIC values for mcr variants and indicated that the strains were susceptible to colistin (26,30,31). However, our findings are consistent with studies by Trent et al (38) and Xu et al (37), who reported that B-strains BLR(DE3) (obtained from Novagen) and BL21(DE3) (obtained from both Novagen and Stratagene), used in their respective studies, were resistant to colistin.…”

“…To determine whether a given gene confers colistin resistance and to assess the level of colistin resistance conferred by a given variant or sub-variant, authors oftentimes express mcr genes in heterologous laboratory E. coli strains using either the native plasmid (18, 28, 29) or cloning the gene into a plasmid downstream of an inducible promoter like the T7 RNA polymerase promoter with the lac operator (e.g., pET plasmids) (30, 31). This method is necessary when discovering new variants from whole genome sequence (WGS) data or when the isolate is not available or culturable.…”

Molecular Detection of Integrons, Colistin and β-lactamase Resistant Genes in Salmonella enterica Serovars Enteritidis and Typhimurium Isolated from Chickens and Rats Inhabiting Poultry Farms

“…In general, the current study detected high prevalence of ESBL encoding genes in Salmonella isolates. The significance of detecting β-lactamase resistance genes raises public health concerns by limiting the therapeutic choices for treating salmonellosis in animals and humans [47], and COL raises major health concerns, as it is used as a treatment of last resort [18,19].…”

“…Antibiotic resistance against colistin (COL), is becoming more common and a point of concern because it is a last-resort antibiotic used against difficult-to-treat pathogens such as Acinetobacter baumanni, Klebsiella pneumoniae, and Pseudomonas aeruginosa [15][16][17][18][19][20]. Although COL use in humans is uncommon in Africa, it is widely used in livestock [16,19].…”

More than mcr: Canonical Plasmid- and Transposon-Encoded Mobilized Colistin Resistance (mcr) Genes Represent a Subset of Phosphoethanolamine Transferases

“…Colistin minimum inhibitory concentration (MIC) data were collected from the literature as follows (Supplementary Table S8) (Di Pilato et al, 2016; Liu et al, 2016; Xavier et al, 2016; AbuOun et al, 2017; Borowiak et al, 2017; Carattoli et al, 2017; Ling et al, 2017; Liu et al, 2017; Lu et al, 2017; Poirel et al, 2017; Tijet et al, 2017; Yang et al, 2017; Yin et al, 2017; Zhao et al, 2017; Alba et al, 2018; Carattoli et al, 2018; Chavda et al, 2018; Dortet et al, 2018; Duggett et al, 2018; Eichhorn et al, 2018; Fernandes et al, 2018; Garcia-Graells et al, 2018; Hammerl et al, 2018; Kieffer et al, 2018; Li et al, 2018; Liu et al, 2018; Poirel et al, 2018; Rebelo et al, 2018; Shen et al, 2018; Teo et al, 2018; Wang et al, 2018b; Wang et al, 2018c; Wise et al, 2018; Xiang et al, 2018; Xu et al, 2018b; Xu et al, 2018c; Yang et al, 2018; Abdul Momin et al, 2019; Bitar et al, 2019; Chavda et al, 2019; Cui et al, 2019; Deshpande et al, 2019; Hadjadj et al, 2019; Li et al, 2019; Long et al, 2019; Wang et al, 2019; Yang et al, 2019; Yuan et al, 2019; Zhang et al, 2019; Zheng et al, 2019; Bitar et al, 2020; Cha et al, 2020; Fan et al, 2020; Garcia-Menino et al, 2020; Hatrongjit et al, 2020; Lei et al, 2020; Martins-Sorenson et al, 2020; Neumann et al, 2020; Ngbede et al, 2020; Wang et al, 2020; Anyanwu et al, 2021; Hu et al, 2021; Khanawapee et al, 2021; Leangapichart et al, 2021; Snyman et al, 2021; Stosic et al, 2021; Uddin et al, 2021; Yu et al, 2021). A search of NCBI’s PubMed database (…”