Antibiotic resistant bacteria and -genes in raw water, and the implications for drinking water production

Abstract

The introduction of antibiotic treatment in the 1940s was a revolutionary breakthrough in medicine. However, the wide and inappropriate use of antibiotics ever since have created the perfect conditions for increasing the selective pressures on bacteria to develop resistance against antibiotics. Antibiotic resistance truly threatens the success of modern medicine and finding such antibiotic resistant bacteria in drinking water distribution systems (DWDS) in South Africa has recently been demonstrated. The aim of this study was thus to determine the general drinking water quality and antibiotic resistance profiles of isolated heterotrophic bacteria in two DWDS in South Africa. Samples were collected in 2016 and 2017 and included raw water, treated water and sampling points in the distribution system. Selected physico-chemical properties that must adhere to the South African National Standards (SANS 241) for drinking were measured for each sample. Heterotrophic plate count (HPC) bacteria were enumerated on R2A Agar using standard methods. These were isolated by a successive streak plate method to purify the isolates. The isolates were identified by sequencing the 16S rRNA gene. The Kirby Bauer disc diffusion method was used to determine antibiotic susceptibility of the isolates. Conventional PCRs were run in order to detect antibiotic resistance genes in the isolates such as ampC, TEM1, ermB, ermF, tetM and int1. The presence of antibiotics within water samples were determined. The potential pathogenicity of isolates was determined by testing for the production of haemolysins, proteinase, lecithinase, lipase and DNase. Environmental DNA (eDNA) was isolated from raw water, treated water and distribution water then conventional PCR was used to detect presence of the same six antibiotic resistance genes. Six Bacillus isolates isolated from the raw water, treated water and drinking water were used for whole genome sequencing. The physico-chemical properties mostly complied to the SANS 241 (2015) except for the turbidity levels that in some cases reached 3.30 mg/L and 3.85 mg/L at the both drinking water treatment plants (DWTP) respectively. Also the nitrites showed high levels that reached levels of 5.00 mg/L and 10.50 mg/L for both treatment plants respectively. Identification of HPC isolates showed that Bacillus spp. represented 35% and 31% of HPC bacteria in the raw water at both DWTP respectively. In the drinking water Bacillus spp. represented 73% and 39% of HPC bacteria at both DWTP respectively. High resistance patterns were observed for ampicillin, cephalothin, penicillin and trimethoprim. The average number of isolates showing resistance against these four antibiotics in the raw water, treated water and distribution water ranged from 47% - 100%. A clear trend was observed that the average number of isolates resistant to antibiotics increased from the raw water into the drinking water (treated water and distribution water). The average multiple antibiotic resistance (MAR) index showed values higher than 0.2 at both DWTP in all the samples indicating antibiotic pollution. Only one isolate amplified the ampC gene whereas the rest of the antibiotic resistant genes were not detected. For eDNA, only the int1 gene was detected in the raw water and distribution water at one of the DWTP. For both DWTP more than 76% of isolates tested positive for haemolysins, proteinase and lecithinase whereas between 19% and 50% of the isolates tested positive for lipase and DNase. Nine isolates produced all the enzymes. The high levels of turbidity and nitrites might be an indication of biofilm formation in the distribution system or ineffective filtering of water. A clear trend can be observed where selective pressure takes place from the raw water to the drinking water regarding heterotrophic bacteria and antibiotic resistance. Together with that we prove that heterotrophic bacteria can produce enzymes that can make them potentially pathogenic. Antibiotic resistance is a phenomenon that presents itself even within drinking water of which we are dependent on a daily basis. Together with its pathogenicity potential it can pose health risks to the immunocompromised, elderly as well as young children