*4.3. Detection of Antibiotic-Resistant* E. coli *in Harvested Rainwater*

Results of the antibiotic-resistance profiling of the isolates from harvested rainwater analysed in the current study revealed that most of the *E. coli* isolates were resistant to the commonly prescribed antibiotics in South Africa. In areas such as the Eastern Cape where most of the population rely on harvested rainwater, exposure to antibiotic-resistant bacteria can further increase the health risk, particularly to children, the elderly and immune-compromised individuals. Antibiotic resistance is on the increase worldwide as most microorganisms now exhibit resistance to a large number of known antibiotics. The *E. coli* isolates from harvested rainwater in this study revealed resistance to Cephalothin (76%), Tetracyclines (51%), Colistin sulphate (47%), Ampicillin (50%) and Streptomycin (40%). The antibiotics most used in South Africa are the penicillins (Cephalothin) and fluoroquinolones, (Ciprofloxacin and glycopeptides) [40]. Tetracyclines and trimethoprim are also extensively used in the treatment of bacterial infections in both human and animals [41].

Cephalothin belongs to the β-Lactam class of antibiotics which are characterised by a β-lactam ring in their molecular structure [42]. Resistance to beta-lactam antibiotics has been highly documented as bacterial strains that produce extended-spectrum beta-lactamases have become more common [43]. Extended-spectrum beta-lactamase (ESBL)-producing *E. coli* are highly resistant to an array of antibiotics and infections by these strains are difficult to treat [43]. Furthermore, genes for ESBLs are most often encoded on plasmids, which can readily be transferred between bacteria [44]. Given that most of the isolates carrying virulence genes, especially the *ibeA* gene, were also resistant to Cephalothin, this could suggest that most of the isolated *E. coli* strains may carry the ESBL genes with the possibility of transfer to related organisms within the rainwater tanks. However, it is necessary to conduct further studies to ascertain such ARGs' transfer within harvested-rainwater systems. Results of such studies would highlight the need for implementation of appropriate treatment options and

better policies for the safe use of harvested rainwater, especially where such water is the main source of water for personal and household uses, thus protecting the lives of users of harvested rainwater.

In the current study, the tested *E. coli* isolates showed resistance to one or more antibiotics with the highest *E. coli* resistance recorded against Cephalothin, Ampicillin and Tetracyclines. Also, there was evidence of MAR *E. coli* in almost all the HRW systems with some isolates showing simultaneous resistance to a panel of up to nine antibiotics. These results indicate that in the case of infections occurring due to the consumption of contaminated harvested rainwater, treatment may fail because of the persistent resistance of the *E. coli* isolates detected in the HRW systems. A similar study carried out in Pretoria and Johannesburg, South Africa, showed that the resistances most encountered were against Ampicillin, Gentamicin, Amikacin and Tetracyclines [34]. These results were not in agreement with our findings, where *E. coli* isolates were resistant to Cephalothin and 100% susceptible to Gentamicin, although the same method and concentration was used for Gentamicin in both studies. The difference in antibiotic resistance results from the two studies could be attributed to the fact that roof-harvested rainwater samples were collected from different locations (Gauteng and Eastern Cape). Our findings were, however, similar to the those of Chidamba and Korsten [34] in that the authors also reported a substantial prevalence of MAR. All the isolates tested in this study showed a MAR index greater than 0.2, suggesting that a greater proportion of the isolates were likely to be from a high-risk source such as faecal material. These results and the differences observed with other studies could inform those implementing antibiotic-resistance surveillance schemes that would address different geographical locations. Also, the presence of MAR *E. coli* in harvested rainwater could pose a severe health risk to the public in general, as antibiotic resistance decreases the efficiency of antibiotics used in the treatment of infections. These findings are of major concern, as more households are now reported to be using harvested rainwater for their daily water needs.
