**4. Discussion**

#### *4.1. Concentration of* E. coli *in Harvested Rainwater*

Faecal coliform bacteria such as *E. coli* have been widely used as indicator organisms to assess the possibility of pathogen presence in water [29]. Therefore, the presence of *E. coli* in roof-harvested rainwater in the Eastern Cape, South Africa, was monitored. All the 11 tanks monitored in this study were contaminated with varying concentrations of *E. coli* (0.85 ± 0.26–3.02 ± 0.21 MPN/100 mL)*.* Other scholars have previously reported on the high detection of *E. coli* from roof-harvested rainwater (2 to 986 CFU/100 mL; 1 to 99 MPN/100 mL and 0 to 41 CFU/100 mL) [1,30,31]. None of the tanks monitored in this study met the guidelines for drinking-water quality, as the *E. coli* amounts exceeded the South African drinking-water quality guidelines of 0 CFU/100 mL. The considerable amounts of *E. coli* in the harvested rainwater samples indicate possible faecal contamination.

The variations in the number of *E. coli* contamination in different HRW systems could be attributed to the fact that some of the HRW systems (Rhodes University) had a constant presence of birds which could have landed and dropped faecal matter on the roof, thereby contaminating tank water. Bird faecal droppings may negatively impact roof-harvested rainwater quality due to the presence of zoonotic pathogens [32]. A study conducted in South Africa investigated antibiotic resistance in *E. coli* isolates from roof-harvested rainwater tanks and urban pigeon faeces as the likely source of contamination and concluded that urban pigeons, the most likely source of HRW contamination, are also reservoirs of multiple antibiotic-resistant bacteria [33]. The findings of the South African study on bird faeces and antibiotic-resistant *E. coli* have a similar conclusion to our study where bird faecal matter was suspected to contribute to the contamination of HRW. In cases where the sources of faecal pollution in rainwater tanks are suspected to be from birds, the application of bird faecal markers may have the potential to confirm the sources of faecal contamination in a rainwater tank [32]. In another study to identify the likely sources of potential clinically significant *E. coli* in rainwater tanks, a source-tracking approach was used where a biochemical-fingerprinting method for typing of *E. coli* strains revealed that of the 43 strains from rainwater tank samples, 14 (from 7 tanks) and 9 (from 6 tanks) had identical biochemical phenotypes to those found in bird and possum faecal samples, respectively [34]. Furthermore, five strains from 4 rainwater tanks were identical to those isolated from both bird and possum faecal samples [34].

The rainwater tanks in the current study are used for various purposes such drinking and toilet flushing (for tanks situated at Rhodes University). Tanks situated at Grahamstown west were mainly used for gardening and sometimes drinking, depending on the availability of the municipal supply, while Kenton-on-sea tanks were used for indoor potable uses such dish-washing and laundry. In order to reduce or limit the risk of pathogenic and antimicrobial resistant *E. coli*, constant cleaning and maintenance of the catchment area may significantly improve the quality of the HRW, as the catchment area is suspected to contribute largely to the deterioration of the HRW in the Eastern Cape due to birds landing on the roof. Installation of first flush diverters may also help to improve the quality of the HRW. A study conducted in South Africa on the quality of HRW reported that 100% of the samples tested for *E. coli* exceeded the recommended standard of 0 CFU/100 mL [12]. Their results were similar to the ones observed in this study where all of the samples showed high levels of *E. coli*. In the Eastern Cape, where harvested rainwater is used for various household purposes including drinking, the presence of *E. coli* in the rainwater tanks is a major health concern as the presence of *E. coli* could imply the presence of other bacterial pathogens which may be detrimental to the health of rainwater users. The findings of the current study are of significant health concern as antibiotic-resistant pathogenic *E. coli* isolates may cause diseases if the users of the HRW consume the water without treatment. Furthermore, resistance of the isolated pathogenic *E. coli* to commonly used antibiotics in South Africa may lead to antibiotic treatment failure with serious public health implications for the population and the country.
