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.
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 [
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 [
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 [
Athi River in Machakos county is a heavily polluted water system mainly as a result of contamination from sewerage originating from Westlands and Kasarani areas in Nairobi [
The study was carried out along the banks of River Athi within the Athi River Township in Machakos County as shown in
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
Water samples were collected from each site in varying dates between September 2014 and January 2015. Sampling of each site was done only once. The samples were transported to Kenya Medical Research Institute Centre for Microbiology laboratory in an insulated cool box (4˚C - 8˚C) and processed within 24 hrs.
For isolation of E. coli, 25 ml of sample water was inoculated into 225 mL of buffered peptone water (BPW) (Oxoid, UK) and incubated at 37˚C for 24 hrs. A loop-full of broth (10 µl) was then streaked on MacConkey’s agar (Oxoid, UK)
and incubated at 37˚C for 24 hrs. Suspect colonies (medium sized pink non-mucoid colonies) were then picked from the plates by use of sterile wire loop and identified as E. coli using standard morphological and biochemical tests for Enterobacteriaceae.
Pure colonies of each isolate selected for further analysis were suspended in 300 µl of DNA extraction buffer and boiled for 15 minutes at 95˚C using a heating block. After lysis, centrifugation was done at 14,000 rmp for 5 minutes at 4˚C. The DNA containing supernatant was stored in −20˚C and later used as the source of DNA template for further PCR amplification experiments [
PCR amplification was carried out using quagen PCR kit. The thermo-cycler PCR conditions were primer specific.
Oligonucleotide name | Oligonucleotide 5’-3’ | Target gene | PCR annealing temperature | Amplification product (bp) | Accession number/reference |
---|---|---|---|---|---|
Resistance genes | |||||
blaTEM | ATGAGTATTCAACAT TTC CG-F CCAATGCTTAATCAG TGA-R | blaTEM | 53.5 | 840 | EF125012 |
cmy | ATGATGAAAAAATCGTTATGC-F TTGCAGCTTTTCAAGAATGCG-R | BlaCMYs | 55 | 1200 | U77414 |
Ctx-m | ATGTGCAGYACCAGTAARGTK-F ATGGCRAARTARGTSACCAGA-R | BlaCTX-M | 60 | 593 | Y10278 |
eae | CTGAACGGCGATTACGCGAA-F CGAGAGACGATACGATCCAG-R | eae | 54 | 917 | [ |
bfp | AATGGTGCTTGCGCTTGCTGC-F GCCGCTTTATCCAACCTGGTA-R | bfp | 54 | 326 | [ |
Virulence genes | |||||
Aggr | GTATACACAAAGAAGGAAGC-F ACAGAATCGTCAGCATCAGC-R | Aggrks a1, Aggrks a2 | 54 | 254 | [ |
lt | GCACACGGAGCTCCTCAGTC-F TCCTTCATCCTTTCAATGGCTTT-R | lt | 54 | 218 | [ |
st | GCTAAACCAGTAGASTCTTCAAAA-F CCCGGTACARGCAGGATTACAACA-R | st | 54 | 147 | [ |
Ipa H | CTCGGCACGTTTTAATAGTCTGG-F GTGGAGAGCTGAAGTTTCTCTGC-R | Ipa H | 54 | 933 | [ |
Inv | ATATCTCTATTTCCAATCGCGT-F GATGGCGAGAAATTATATCCCG-R | Inv | 54 | 382 | [ |
Int 1 | TCGGTCAAGGTT-F AACTTTCAGCACATG-R | Int 1 | 50 | 923 | U12338 |
Electrophoresis was carried out in 1.5% agarose .The gel was observed under UV light and image captured using a digital camera. Statistical analysis was conducted using the SPSS Version 20.0 software.
Statistical analysis was conducted using the SPSS Version 20.0 software.
Results for CFUs from this study had been published work previously [
There were a total of 7 out of 318 E. coli isolates that were ESBL-producer (2.2%).
E. coli isolates that had resistance genes blaTEM, blaCTX-M and blaCMY were 265 (83%) and those that had no resistance genes were 53 (17%). BlaCTX-M had the highest prevalence of 106 (33%) while blaTEM had lowest prevalence of 70 (22%). Resistance genes blaTEM, blaCTX-M and blaCMY were highest in sewage effluent and
near road and farming while blaCMY gene was relatively higher near sewage effluent 32 (12%) and near road and farming 27 (10.2%). blaTEM had lower prevalence compared to the other two across sites as shown in
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
The resistance genes that were isolated from this study were blaTEM (22%), blaCTX-M (33%) and blaCMY (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 [
site | Mean cfus | No. of isolates analysed | ESBL n (%) | blaTEM n (%) | blaCTX-M n (%) | blaCMY n (%) | Integron 1n (%) |
---|---|---|---|---|---|---|---|
Domestic use | 5.2 × 103 | 53 | 0 (0) | 3 (1) | 6 (2) | 13 (5) | 3 (8.3) |
settlements | 1.6 × 103 | 53 | 2 (0.6) | 0 (0) | 16 (6) | 19 (7) | 4 (11.1) |
Virgin land | 9.5 × 102 | 53 | 1 (0.3) | 6 (2) | 10 (3) | 19 (7) | 6 (16.7) |
Sewerage effluent | 9.5 × 103 | 53 | 2 (0.6) | 10 (3) | 19 (7) | 32 (12) | 11 (30.6) |
Near road and farming | 1.9 × 103 | 53 | 0 (0) | 22 (7) | 30 (11) | 27 (12) | 7 (19.4) |
Industrial zone | 7.6 × 103 | 53 | 2 (0.6) | 16 (5) | 17 (10) | 0 (0) | 5 (13.9) |
CFUs were highest in area near sewage effluent which also harboured the highest number of integron1 gene. Areas near road and farming had the highest number blaTEM and BlaCMY genes. ESBLs were found in equal numbers in areas close to human settlements, near sewage effluent and area near industrial zone.
supported the theory of bacterial resistance being strongly influenced by discharge of waste water [
Both blaTEM and blaSHV genes have been reported mostly from clinical samples and from environmental samples like farm animals and estuarine waters. Interestingly, majority of the isolates in this study were positive for blaCTX-M while none were positive for blaSHV. The abundance of blaCTX-M in aquatic environments has been reported in other studies done in Switzerland and Malaysia [
Entero aggregative E. coli (EAEC) had the highest prevalence (11%) compared to other pathotypes screened in this study. Although no study in Kenya has analyzed prevalence of diarrheagenic E. coli in river water, our findings concur with others done in Kenya using clinical isolates. A recent study in Kenya demonstrated EAEC (8.9%), as the most frequent followed by ETEC (1.2%) and EIEC (0.6%) [
Pathogenic E. coli has also been isolated in other studies from river water [
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 [
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 water 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.
I would like to thank centre of microbiology in Kenya medical research institute personnel for offering me lab space and equipments. The work was supported by National Council for Science and Technology (NACOSTI).
PW came up with the concept for the work and designed the study. She did the lab work and drafted the manuscript. JK participated in the study design and helped in drafting the manuscript. VM corrected the proposal and helped in its coordination. All authors read and approved final manuscript.
PW is a master’s student in Kenya studying medical microbiology, Prof. VM is the dean of biological sciences in Jomo Kenyatta University of agriculture and technology who has a vast experience in proposal development and microbial techniques and Dr. JK is a researcher in Centre of Microbiology Research Centre in Kenya Medical Research Institute. He helped in actualizing the study and fine tuning the manuscript
The authors declare there is no conflict of interest regarding the publishing of this manuscript.
Approved.
Wambugu, P., Kiiru, J. and Matiru, V. (2018) Escherichia coli Harbouring Resistance Genes, Virulence Genes and Integron 1 Isolated from Athi River in Kenya. Advances in Microbiology, 8, 846-858. https://doi.org/10.4236/aim.2018.811056