*4.2. Identification of Virulence Genes among* E. coli *Isolates*

Pathogenic *E. coli* strains are a major cause of infections worldwide, the most common of which are diarrhoeal diseases. All the 100 *E. coli* isolates from the tanks tested positive for one or more VGs. The most detected pathotype was the NMEC (*ibeA*; 28%) which is responsible for neonatal meningitis and endothelial cell invasion [35]. The *ibeA* gene is also reportedly found in avian pathogenic *E. coli* (APEC) and causes avian colibacillosis, which is the most significant infectious bacterial disease of poultry worldwide [35]. The detection of the *ibeA*-positive strains in this study possibly indicates that the observed pathotype may be due to the presence of birds around the HRW systems. Although

the present study did not investigate whether the *ibeA* gene detected was of human or avian origin, the presence of *ibeA*-positive isolates in the HRW systems is still of health concern given that there could be a possibility of zoonotic infections arising from the consumption of untreated rainwater containing these strains. Genes pertaining to other pathotypes of public health concern were also detected in the present study. For example, the *flicH7* (22%) and *Stx2* (14%) genes of EHEC were also detected in the isolates. Members of the EHEC group have been involved in many diarrhoeal disease outbreaks around the world, and they are known to cause hemorrhagic colitis and hemolytic uremic syndrome in humans [36].

The EHEC pathotype showed high prevalence across all the sampling sites except for the sites located in Grahamstown west. Both T1 and T6 which yielded a high percentage in VGs detection were situated at Rhodes University. Prevalence of the virulence gene *ipaH* (26%) (pathotype EIEC) was also noticeable in 4 tanks; 3 of the tanks were situated on campus and 1 in Kenton-on-sea. A previous study conducted in Cape Town, South Africa, reported that EPEC and EHEC (3% each) were detected in lower numbers, whereas EIEC was not identified in any of the rainwater tanks tested in their study [12]. The results differ from the findings of the current study where EIEC (26%) was the second most detected pathotype. This shows that the location of the tank could affect the pathotypes detected. Due to the detection of *E. coli* pathotypes in the current study, there is a great need to create awareness on household treatment technologies among users of HRW. Available treatment options which have proven to be successful in the treatment of HRW such as boiling, closed-couple solar pasteurizer, and solar disinfection (SODIS) can be used to decontaminate HRW [37–39]. In this study, all the rainwater tanks did not have any treatment option fitted, such as first-flush diverters and filters, except for T5 which had a chlorinator. However, due to limited maintenance of the rainwater-harvesting systems, the chlorinator in T5 was clogged in the middle of the sampling season and the *E. coli* counts increased going forward. The interruption of the treatment option observed in this study is also a clear indication of lack of proper maintenance of the HRW systems.
