Molecular quantification and characterisation of aminoglycoside resistant bacteria and genes from aquatic environments

Masters in Environmental Science, North-West University, Potchefstroom CampusAntibiotic resistance bacteria and antibiotic resistance genes (ARGs) could be disseminated and selected for in the environment, particularly aquatic ecosystems. This is due to the interplay between humans, animals and this ecosystem. The aim of this study was to investigate the levels and characteristics of aminoglycoside resistant bacteria and associated ARGs, isolated from surface water and sediment of the Crocodile and Marico Rivers. Levels of kanamycin resistant bacteria were determined by plating samples on nutrient agar, supplemented with kanamycin. Isolates were purified and resistance profiles to three other aminoglycosides were determined. Multi-aminoglycoside resistant isolates were identified using Gram staining and 16S rRNA gene sequencing. These were screened for nptII and related ARGs (intI 1, ampC and msrA/B efflux pump). Kanamycin resistant bacteria levels ranged from a few (10 CFU/ml) to very high (2.0 x 106 CFU/ml) in both river systems. No nptII genes were detected using this method. However, the efflux pump gene (msrA/B) were detected among some of the isolates. Additionally, the microbial populations at various sites were screened for these selected ARGs using culture-dependent and cultureindependent methods. The culture-dependent method involved enrichment either supplemented with or without kanamycin. Plasmid, as well as genomic DNA, was extracted. Environmental DNA was also extracted directly from filtered water samples (eDNA). This DNA (enriched plasmid, as well as genomic DNA and eDNA) was analysed by end-point PCR, real-time PCR (qPCR), as well as droplet digital PCR (ddPCR). Results indicated that nptII could be quantified in plasmid and genomic DNA of the samples (both with and without kanamycin). Levels determined by qPCR ranged from undetectable to 1.58 x 104 copies per nanogram of input DNA. ddPCR yielded copy numbers ranging from undetectable to 3.70 x 10-5 copies per nanogram of input DNA. In the case of ampC quantification in plasmid DNA, qPCR results indicated levels ranging from undetectable to 4.90 x 109 copies per nanogram of input DNA, whereas ddPCR ranged from undetectable to 6.55 x 10-3 copies per nanogram of input DNA. Quantification of nptII using the eDNA, qPCR results indicated levels ranging from undetectable to 1.23 x 105 copies per nanogram of input DNA. No samples were quantifiable using ddPRC. Relevant ARGs (msrA/B efflux pump, β-lactam ampC and integrase class one (intI), were detected using the culture-dependent, as well as cultureindependent approaches. This is significant, since the class 1 integrase gene is the most ubiquitous among multidrug resistant bacteria. Bacteria containing this gene are able to harbour multiple resistant gene cassettes and could serve as a proxy for anthropogenic pollution. Overall, the results from this study indicated that the culture-based enrichment method provided the best resolution of resistance gene diversity in the two Rivers; however, the culture-independent method indicated ubiquity of the intI 1 gene, demonstrating the potential transferability of ARGs. This study emphasizes the importance of examining antibiotic resistance in the environment.Master