Antimicrobial resistant bacteria and genes in selected surface water bodies of the North West Province

Abstract

It has recently been established that apart from the over or misuse of antibiotics, metal pollution in the natural environment may also contribute to antibiotic resistance even when antibiotics are absent. The Wonderfonteinspruit (WFS) is highly impacted by a century of gold mining activities taking place in South Africa. Therefore, this system was identified as a possible driver for metal and antibiotic resistance. The system is also impacted on by agricultural activities and urbanization. The overall aim of this study was to investigate antimicrobial (metals and antibiotics) resistant heterotrophic plate count (HPC) bacterial levels in the mining impacted WFS and receiving Mooi River (MR) system and to identify and characterize multiple antibiotic resistant (MAR) bacteria based on their resistance levels and detection antibiotic resistance genes (ARGs) they may host. Surface water sampling took place at six sites in close vicinity to the confluence of the WFS and MR on three sampling occasions in 2015. R2A agar and R2A agar supplemented with antimicrobials (ampicillin, copper (Cu), iron (Fe), lead (Pb) and zinc (Zn)) individually were used to isolate HPC bacteria. Various physico-chemical properties were measured using standard methods and brought into context with antimicrobial HPC levels. Morphologically distinct antimicrobial resistant isolates were purified and screened for antibiotic susceptibility to seven antibiotics (ampicillin, amoxicillin, tetracycline, erythromycin, streptomycin, trimethoprim and chloramphenicol) from six antibiotic classes by a disc diffusion method. Selected MAR isolates were identified by 16S rRNA amplification, sequencing and comparison to the BLASTn database. The MIC ranges for the identified isolates towards the original antimicrobial they were isolated from and other (amoxicillin, tetracycline, erythromycin and streptomycin) antibiotics were determined by agar dilution. Finally, the presence of five ARGs (blaTEM, ampC, tetA, tetL, tetK) were screened for by PCR amplification of the gene and sequencing verification. Physico-chemical parameters generally exceeded the recommended water quality objectives for the catchment. From the statistical analysis of physico-chemical and HPC results it was evident that most of the HPC results related to the mining impacted site WFS 1. Co-resistance was observed as 82% of the isolates isolated from metal containing media were resistant to at least one antibiotic and over 30% of all the antimicrobial resistant isolates were MAR at all of the sites. A large proportion of isolates were resistant to all 7 antibiotics tested. Phyla detected among the 72 MAR isolates were Proteobacteria, Firmicutes, Bacteriodetes and Actinobacteria in descending order. Pseudomonas and Acinetobacter genera from the Gammaproteobacteria class were most frequently identified among the isolates. High minimum inhibitory concentration (MIC) levels for metals and antibiotics were detected amongst all the genera. In general, it was observed that the bacterial community was most susceptible to Cu and most resistant to Pb. Resistance to the β-lactam antibiotics were most prevalent and most of the identified MAR isolates had high levels of resistance (MIC >100 mg/L) to the antibiotics of this class. blaTEM was most prevalent among the ARGs and found in 78% of the MAR bacteria. The ampC and tetA genes were detected in four isolates each, whereas tetL and tetK were not detected among the MAR isolates of the current study. The study could successfully conclude that metal and antibiotic resistance co-occurred in isolates from all of the sites. β-lactamase resistance was widespread as was found in previous studies. However, antimicrobial resistance was more prevalent in the mining impacted WFS sites compared to the MR and it is therefore concluded that there is a form of co-selection taking place for metal and antibiotic resistance.