Characteristics of antibiotic resistant bacteria in raw and drinking water from water production facilities

MSc (Microbiology), North-West University, Potchefstroom CampusWater is a limited resource, and the quality of raw water has an impact on drinking water production. Regular testing is done to ensure that the quality is compliant with regulations. Heterotrophic bacteria are widely used as indicators of drinking water quality. According to SANS 241 (2015), the amount of heterotrophic plate count (HPC) bacteria in drinking water should not exceed more than 1000 colony forming units (CFU)/ml. There is currently a growing concern about the presence of HPC bacteria in drinking water. Many of these might be potentially pathogenic microorganisms, and they are associated with secondary infections in immuno-compromised individuals. The South African population has a higher potential infection rate of HPC bacteria compared to other countries due to the escalating percentage of patients living with HIV. These potential pathogens have been reported in drinking water systems. Therefore, this current study aimed to determine antibiotic resistant bacteria and potentially pathogenic HPC bacteria in the drinking water. Samples were collected from two water production facilities, one in the North West (NW-D) and the other in Gauteng (GH-T) Province. Physico-chemical parameters measured were free chlorine, TDS, pH, nitrate, nitrite, COD, phosphates and temperature. HPC bacteria were isolated and purified with a culture-based method, identified by Gram stain and 16S rRNA sequencing, and tested for α- or β-haemolysis on blood agar. Further tests were done for the production of extracellular enzymes such as DNase, hyaluronidase, lipase, proteinase, chondroitinase and lecithinase. Total coliforms and faecal coliforms were enumerated on MLG Agar, using standard procedures. The levels of these were only used for water quality purposes. Antibiotic susceptibility of the HPC bacteria and whether antibiotic resistant genes (ARGs) could be associated with antibiotic resistance phenotypes were determined. Environmental DNA (eDNA) was also isolated to determine if ARGs could be detected directly in selected samples. Turbidity and nitrite levels were alarmingly high, and in some cases, both exceeded the SANS 241 (2015) of drinking water. The other physico-chemical parameters were mainly within the recommended levels. All microbiological parameters were detected at levels below the standard in drinking water, however the higher levels of heterotrophic bacteria after treatment should not be ignored. Fifty one percent (51%) of the isolated HPC bacteria displayed α- or β-haemolysis. These haemolytic isolates revealed the production of various enzymes: proteinase (74% from GH-T), DNase (58% from NW-D) for the 2017 sampling run and chondroitinase (58% from NW-D) for 2016. Both DNase and proteinase enzymes were produced by most of the haemolysin producing isolates. Hyaluronidase and lecithinase were the least detected enzymes. Among all the isolates, resistance to individual antibiotics were observed in the following order of decrease: ampicillin (89%), trimethroprim (77%), cephalothin (62%) and penicillin G (53%) in water after treatment and drinking water. There was high a percentage susceptibility, and low intermediate resistance percentage of HPC isolates to neomycin, erythromycin, and streptomycin. Penicillin and ciprofloxacin were detected at low levels by liquid chromatography-mass spectrometry analysis. Isolates were resistant to more than two antibiotics classes. The most prevalent MAR phenotype was AMP-TM-KF-PEN-G-VAN-OT. Genes encoding for macrolide-lincosamide-streptogramin (MLS) (ermF and ermB), were detected. However, ermB was more prevalent in the MAR isolates (50%), while ermF gene was found in only one isolate (3%). The tetM and Intl 1 genes could not be detected in any of the isolates. β-lactam (blaTEM and ampC) genes were also detected in samples from the water treatment system. Moreover, ermB and intl 1 was found in raw water when eDNA was tested. The identified species included Bacillus cereus, Bacillus thuringiensis, Bacillus pumilus, Bacillus licheniformis, Micrococcus luteus, Shinella sp., Chryseobacterium sp., Paenibacillus chitinolyticus, Bacillus anthracis, Bacillus wiedmannii, Bacillus Toyonensis and Bacillus pumilus. The predominant genus was Bacillus, for all the sampling sites in both production facilities. These species are linked to various infections, including wound infections and skin and soft-tissue infections. This study confirmed the presence of potentially pathogenic HPC bacteria in the raw and treated water from two water production facilities. The obtained results showed that ARGs were not completely removed during the drinking water production process, and they can also thus be present in the distribution system. This is a cause for concern, particularly for the immune-compromised individuals. The results show a need for education and awareness programmes in the communities about contaminants entering the water source, and ways to treat water at house level. This will help prevent water-borne diseases but particularly the spread of antibiotic resistance through water systems.Master