Escherichia coli Harbouring Resistance Genes, Virulence Genes and Integron 1 Isolated from Athi River in Kenya

Rivers can act as reservoirs of highly resistant strains and facilitate the dissemination of resistance, virulence and integron 1 genes. A cross-sectional study was carried out where 318 water samples were collected (53 from each site) and from the samples, 318 E. coli isolates were analysed for resistance genes, virulence genes and integron 1 using Polymerase Chain Reaction. 22% of the isolates had blaTEM, 33% had blaCTX-M and 28% had blaCMY. Prevalence of typical Enteropathogenic E. coli strains (carrying both eae and bfp genes) was 5% while the prevalence of atypical Enteropathogenic E. coli (carying only eae) was 1.8%. The prevalence of Enteroaggregative E. coli carrying the aggr genes was 11%. The prevalence of Enterotoxigenic E. coli encoding only lt toxin was 16 (5%) and while those carrying only st toxin was 6.9%. The prevalence of Enteroinvasive E. coli strains encoding as IpaH was 5% while that of strains, adherent invasive E. coli, carrying adherent invasive gene inv was 8.7%. 36% isolates were positive for class 1 integrons which were mostly isolated near the sewage effluent from waste treatment plant. Anthropogenic activities and close proximity to sewage treatment plant were found to play a key role in pollution of water body and accumulation of resistance and virulence genes. These results suggest that waste treatment plant may act as reservoir of resistance, virulence and integron 1 genes and is a potential risk to human and animal health in the region.


Introduction
Water bodies and aquatic systems have great potential as sources of infectious bacteria to people who use the water for recreational activities, fishing, drinking, bathing and irrigation of crops, especially those eaten raw [1]. E. coli is an important indicator organism for fecal pollution in environmental waters and has also been useful in monitoring antimicrobial resistance patterns in gram-negative bacteria and pollution of water bodies with resistance and virulence genes, especially those suspected to be of human origin requires evaluation [2] [3]. Some E. coli strains have acquired virulence genes that allow them to cause various infections such as diarrhea and hemolytic-uremic syndrome (HUS). If such pathotypes find their way into the water systems, their potential for spread could be multiplied. Clinical isolates can find their way into water systems through fecal matter from humans and animals. This is particularly the case if the sewerage systems are not properly designed or where water treatment is poor.
The presence of antimicrobial resistance genes on mobile genetic elements leads to their dissemination and possible development of multi-resistance phenotype. The dissemination of resistance is associated with genetic mobile elements, such as plasmids, that may also carry virulence determinants. A combination of resistance genes and virulence factors enable a host to replicate and disseminate these genes to other hosts with ease. As a result of using antimicrobials, bacteria can evolve resistance that can be passed to commensal and other pathogens sharing the same ecosystem, e.g. the human gut. It is assumed that virulent MDR strains are more difficult to control than other strains and this impacts on patient's chemotherapeutic success. Presence of such bacteria in healthcare, animal and environmental setting is a major public health concern because such bacteria are highly virulent and untreatable using antimicrobials [4].
The ability of E. coli to carry plasmid-borne integron 1 suggests that the encoded antimicrobial resistance genes can easily be transferred among bacteria and even between pathogenic commensal strains and environment E. coli trains.
It is of interest to note that if such highly resistant strains enter aquatic systems; their chance of spread is highly increased.
Rivers and aquatic systems are important environments for exchange of resistance determinants among enteric and environmental isolates due to activities along a water body such as drainage of sewage containing heavy metal that results in natural selection of resistant strains, humans extensive use of antibiotics in agriculture and health that promote displacement of susceptible strains with resistant ones which find their way to the aquatic environments as well as use of detergents which can select for MDR strains in sections of rivers where domestic activities such as washing clothes and household items with detergents take place [5].
Athi River in Machakos county is a heavily polluted water system mainly as a result of contamination from sewerage originating from Westlands and Kasarani  [6]. However, little is known about the genetic basis of resistance from this river. The aim of this cross-sectional study was therefore to determine the molecular basis of resistance to selected antimicrobials and carriage of virulence genes and integron 1 gene among the isolates.

Materials and Methods
The study was carried out along the banks of River Athi within the Athi River Township in Machakos County as shown in Figure 1.

Sampling
The sampling points were selected based on prevailing human activities such as washing, drinking points for livestock, points where residents fetch water for domestic use, and points heavily contaminated with industrial effluent. Sampling was also done along sections of the river passing through virgin lands that have no obvious evidence of recent interference by human activity, agriculture or settlement as shown in Figure 1.

Isolation of E. coli
For isolation of E. coli, 25

PCR Amplification
PCR amplification was carried out using quagen PCR kit. The thermo-cycler PCR conditions were primer specific.

Statistical Analysis
Statistical analysis was conducted using the SPSS Version 20.0 software.

Coliforn Forming Units (CFUs) across the Sites
Results for CFUs from this study had been published work previously [12] but the importance of the data plays a role in accessing the contamination of the river and therefore the data will be shown in order to get clear picture. Sewage effluent area had the highest mean E. coli counts of 9.5 × 10 3 while near virgin land had the lowest mean of 9.5 × 10 2 as shown in Figure 2.

Prevalence of Extended Spectrum Beta Lactamases (ESBLs)
There were a total of 7 out of 318 E. coli isolates that were ESBL-producer (2.2%). Figure 3 shows an isolate that was an ESBL producer. ESBLs hydrolyse third generation cephalosporins and azreonam but are inhibited by clavulanic acid.

Resistance Genes and Integron Class 1 across the Sites
E. coli isolates that had resistance genes bla TEM , bla CTX-M and bla CMY were 265 (83%) and those that had no resistance genes were 53 (17%). Bla CTX-M had the highest prevalence of 106 (33%) while bla TEM had lowest prevalence of 70 (22%). Resistance genes bla TEM , bla CTX-M and bla CMY were highest in sewage effluent and  near road and farming while bla CMY gene was relatively higher near sewage effluent 32 (12%) and near road and farming 27 (10.2%). bla TEM had lower prevalence compared to the other two across sites as shown in Figure 4. Of the total isolates analysed, 114 (36%) had integron 1 while 204 (64.0%) did not have integron 1. Integron 1 gene was highest in near sewage effluent 11(30.6%) and lowest in site where water is used for domestic purposes 3(8.3%) as shown in Figure  5. Table 2 summarizes the mean CFUs, ESBL, resistance genes and inte-gron1that were found across the sites. Gel images for resistance genes bla CMY , bla TEM and bla CTX-M are shown in Figure 6 and Figure 7. Gel image of integron 1 gene is shown in Figure 8.

Distribution of Pathotypes
Isolates that harbored virulence genes were 140 (44%) while those that did not have any virulence genes were 178 (56%). EAEC pathotype had the highest prevalence of 35 (25.0%) while atypical EPEC had the lowest prevalence of 6 (4.3%). EAEC pathotype was highest near road and farming site and were not isolated in site used for domestic purposes. AIEC pathotype with inv gene were most in virgin land and not present in industrial zone as shown in Figure 9. Gel images for virulence genes are shown in Figure 10 and Figure 11.

Discussion
The resistance genes that were isolated from this study were bla TEM (22%), bla CTX-M (33%) and bla CMY (28%). Evidence has shown that antibiotic resistant bacteria and antibiotic resistance genes (ARGs) are ubiquitous in natural environments, including sites considered pristine. This finding is similar to this study because AGR genes were found in virgin land [13]. A study done in China        Near road and farming 1.9 × 10 3 53 0 (0) 22 (7) 30 ( [18].
Pathogenic E. coli has also been isolated in other studies from river water [19]. Presence of pathogenic E. coli in water creates a potential risk for infections in humans and animals especially if the water is used for irrigation, drinking and for recreational purposes [20] [21] [22]. Increase in presence of multi-drug resistant pathogenic E. coli in water that was seen in San Pedro River in Mexico [23] as well as in India where surface, municipal and ground water were collected [24].
It is likely that multiple exposure pathways are involved in transmitting pathogenic E. coli to humans and river water play a significant role as it is highly contaminated. While the presence of virulence genes (VGs) in E. coli isolates alone is insufficient to determine pathogenicity, the presence of diarrheagenic E. coli pathotypes in high frequency could lead to increased health risks if untreated rain water were to be used for nonpotable purposes and recreational activities. The prevalence of integron 1 was 36% in this study. The int 1 gene was found mostly near sewage treatment plant as well as after the treatment plant. This could be attributed by the effluent of the treatment plant becoming a reservoir of int 1 gene. This phenomenon was described by a previous study done in Poland [21] where the number of integron 1 genes were higher downstream the discharge of WWTPS effluent [21]. Integron 1 in E. coli from aquatic environment has also been reported in previous study done in Kenya using water from different sources (47.4%, 18/38) [25] which was higher compared to this study.

Conclusions
E. coli isolates harboured resistance genes, virulence genes and integron class 1 genes. Resistance genes, virulence genes and integron class 1 were more near the sewage effluent and therefore there is a need to decontaminate the area. From this study therefore, it can be concluded that anthropogenic activities along wa- ter bodies can play a role in the contamination of water and spread of antimicrobial resistance genes and virulence genes. Because river water flows from upstream to downstream, activities done upstream have potential to affect people living many kilometres downstream of the river who use the water for drinking, farming and other recreational purposes The sewage treatment plant played a key role in contamination of the river and isolation of resistance and virulence genes. Better treatment and quality control of waste water should be emphasized in this region and more modern technologies employed in the sewage treatment plant which can help to remove contaminants together with virulence and resistance genes from waste water. It should be noted that however, chemical disinfection methods may cause an undesirable selection of antimicrobial resistance by themselves as seen in previous studies.