Characteristics of Enterobacteriaceae in surface water from the North West Province

Abstract

Faecal pollution of surface water systems poses a significant risk to the environment and public health. According to the latest Green Drop report in South Africa, the North West province has been identified as a high-risk area. Contributing factors include rapid urbanisation, inadequate sanitation infrastructure, informal settlements, ageing wastewater treatment plants with minimal maintenance, and the overloading of treatment capacities. Faecal pollution is evidenced by the presence of indicator microorganisms such as coliforms, which highlight the prevalence of Escherichia coli (including its pathogenic serovar E. coli O157), alongside other Enterobacteriaceae genera, including Hafnia, Klebsiella, Enterobacter, Citrobacter, and Serratia species. Many of these non-E. coli Enterobacteriaceae have gained clinical significance due to their ability to harbour antibiotic-resistant genes (ARGs) and produce extended-spectrum beta-lactamases (ESBLs). These pathogens have the potential to cause diseases such as urinary tract infections, diarrhoea, neonatal meningitis, sepsis, and respiratory infections, posing a severe threat to public health and placing a burden on healthcare systems. Therefore, this study aimed to investigate the characteristics of Enterobacteriaceae isolated from selected surface water. The study investigated the water quality and microbiological hazards within three key surface water systems in the North West province: the Mooi River, the Schoonspruit River, and the Setumo Dam. Physico-chemical and microbial parameters were measured and compared against the water quality targets recommended by the Department of Water and Sanitation (1996). Alarming findings included highly alkaline pH levels exceeding 9 in recreational sites, posing eye, skin, and mucosal irritation risks, and exceptionally high total dissolved solids (2 197 mg/L) at Site S1 of the Setumo Dam, likely linked to untreated wastewater discharge. Furthermore, total coliform and E. coli counts consistently exceeded acceptable thresholds for wastewater effluent and recreational use, with the lowest E. coli levels recorded at the Schoonspruit River's dolomitic eye (58 MPN/100 ml), indicative of widespread faecal contamination. A total of 3 532 E. coli isolates were recovered from the water systems, including 818 putative E. coli O157 colonies. However, Pearson's correlation analysis (r = 0.61, p = 0.27) revealed no significant relationship between the overall E. coli counts and the presence of E. coli O157. Thirty-four haemolytic Enterobacteriaceae isolates, selected for their ability to lyse erythrocytes, were further characterised. Twenty-eight isolates were confirmed to be E. coli on EMB agar and exhibited distinct extracellular enzymatic activities, including haemolysin, β-galactosidase, and DNase production. Notably, isolates from the Setumo Dam also exhibited catalase, lipase, and serine protease activity, suggesting adaptations to nutrient availability in this environment. Biochemical profiling identified citrate utilisation and urea hydrolysis as common metabolic traits, while the Enteropluri® test provided genus and species-level identifications. Enterobacter was the dominant genus among the identified isolates, while nearly half of the isolates remained unidentified. Antibiotic susceptibility testing using the Kirby-Bauer method against 14 antibiotics from 8 classes revealed prevalent resistance to β-lactam antibiotics (amoxicillin, cefazolin, and ampicillin) and sulphamethoxazole, likely linked to their widespread use in human and veterinary medicine. Furthermore, ESBL production was confirmed in 21 isolates, predominantly from the Mooi River (18 isolates) and the Setumo Dam (3 isolates), with key resistance genes (blaTEM, blaCTX, Intl1, AmpC, sul1, and sul2) detected via PCR and gel electrophoresis. Importantly, screening for carbapenemase genes (blaOXA, blaNDM, and blaKPC) revealed their presence in selected isolates, raising concerns about future treatment challenges for ESBL-producing pathogens. Sanger sequencing of the 16S rRNA gene identified the Citrobacter spp. as the most abundant genus (44%), followed by Rahnella spp. (17%), Enterobacter spp. (12%), and others. Leveraging on technological advancements such as whole genome sequencing, four genomes were identified from the Citrobacter genus: C. braakii (S21 and S23), C. murliniae (S24), and C. portucalensis (S25). These four genomes demonstrated ANI values exceeding 98%, confirming a close genomic relationship to genomes in the NCBI database. Interestingly, genomic strain S25 was identified as Citrobacter portucalensis by pubMLST and it shared 90% of its genetic make-up with Citrobacter freundii, highlighting its close evolutionary association. All four genomes were characterised as ESBL-producing Citrobacter species, harbouring the blaCMY gene, which is a key determinant of β-lactam antibiotic resistance. This study also highlighted the virulence genes associated with adhesion, biofilm formation, siderophore production, environmental stress response, and iron acquisition. Genes encoding cell surface localisation components of the capsular polysaccharide system (CPS), such as vexABCDE, and capsule polysaccharide biosynthesis genes (tviBCDE) were uniquely identified in the C. braakii genomes, providing distinguishing molecular markers for this species. In contrast, the C. murliniae strain (S24) exhibited a distinct virulence profile, characterised by the presence of the iroABCDEN gene cluster, which encodes the heme receptor protein shuA, integral to the iron-salmochelin siderophore system. In addition to the antibiotic resistance observed, these virulence genes and patterns confirm the pathogenetic characteristics of these four genomes. This was supported by results from the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, which aligned with observations from phenotypic analysis. This findings highlight the prevalence and diversity of antibiotic-resistant Enterobacteriaceae in surface water systems, highlighting their contribution to water quality degradation and public health risks. The findings demonstrate the urgent need for continuous water quality monitoring through comprehensive epidemiological efforts, targeted wastewater management interventions, and robust antibiotic resistance mitigation strategies to safeguard human, animal, and environmental health within the One Health framework. Moreover, the presence of antibiotic-resistant Enterobacteriaceae in these water systems poses a significant risk of contamination to crops irrigated directly with this water. This could have far-reaching implications, including reduced food quality, compromised food safety, and potential threats to food security, particularly in vulnerable communities.