Seasonal Prevalence, Molecular Characterization, Antimicrobial Resistance and Factors Associated With Thermophilic Campylobacter in Cattle, Chicken and Water in Kajiado County, Kenya

Abstract

Members of the genus Campylobacter are frequently associated with abortion, gastroenteritis and/or diarrhoea in livestock. The bacteria also present a substantial public health problem resulting in major financial constraints to the health care system and economic impacts due to lost productivity. Despite the relevance of this bacterium, there is limited epidemiological information and genotypic relatedness on their occurrence in livestock in Kenya. The objectives of this study were to: (1) determine seasonal prevalence of thermophilic Campylobacter species among cattle, chicken and water in Kajiado County; (2) identify risk factors associated with their occurrence in cattle herds raised on integrated small-scale farms; (3) assess antimicrobial usage in cattle and chicken production systems, susceptibility profiles and resistance genes in Campylobacter spp. isolated from water, cattle and chicken faecal samples; and (4) determine virulence factors and genetic relatedness of Campylobacter isolates from the water, cattle and chicken faecal samples in Kajiado County, Kenya. A total of 457 samples comprising 265 cattle rectal swabs, 142 chicken cloacal swabs, and 50 cattle trough water samples were aseptically collected from 55 randomly selected smallholder farms practicing mixed farming. A pre-tested semi-structured questionnaire was used to collect data on respective farm characteristics and management practices (as potential risk factors for campylobacter colonization in cattle), antimicrobial use, disease history and animal health-seeking behavior among farm owners. Individual samples were subjected to standard techniques for isolation, biochemical tests, followed by singleplex-polymerase chain reaction (sPCR) assays for identification and confirmation of Campylobacter genus and species. Out of the 162 isolates that were recovered and confirmed by 16S rRNA-PCR assay, 103 speciated isolates [Campylobacter coli (n =29) and C. jejuni (n = 74)] were assayed for phenotypic antimicrobial

susceptibility profiling using Kirby–Bauer disk-diffusion method against: ampicillin (AX), tetracycline (TE), gentamicin (GEN), erythromycin (E), ciprofloxacin (CIP) and nalidixic acid (NA). Additionally, detection of antimicrobial resistance genes (tetracycline resistance ribosomal protection [tet(O)], beta-lactamase (blaOXA-61), aminoglycoside phosphotransferase (aph-3-1), gyrase subunit A (gyrA), and multi-drug efflux pump (cmeB) encoding resistance to multiple antibiotics) and virulence genes [encoding for motility (flaA), adhesion (cadF), invasion (ciaB) and cytotoxin production (cdtA)] among the 103 isolates were detected by PCR and amplicon sequencing. To evaluate the genetic relatedness, 36 isolates including 20 isolates based on 16S ribosomal RNA (16S rRNA) primer specific for genus Campylobacter, 8 isolates based on lipoprotein (ceuE) primer for C. coli and 8 isolates based on hippurate hydrolase (hipO) primer for C. jejuni were sanger-sequenced at Inqaba laboratories (South Africa). Phylogenetic analysis was done on sequences of cattle, chicken and water isolates using molecular evolutionary genetic analysis version 11 (MEGA 11). Overall, thermophilic Campylobacter prevalence was 35.4% [95% confidence interval (95% CI) = 31.0–39.8]; with C. jejuni dominating at 55.6% (95% CI=47.9–63.3%) over C. coli in all sample types. The highest thermophilic Campylobacter prevalence was observed in chicken at 44.4% (95% CI=36.2–52.6%), followed by cattle at 30.9% (95% CI=25.3–36.5%). Thermophilic Campylobacter species were isolated in both seasons; with higher prevalence [39.8% (95% CI=33.6–45.9)] recorded during cold-rainy season. There was significant (P<0.05) association between season and thermophilic Campylobacter occurrence. Farm stocking dairy breeds [adjusted odds ratio (AOR) = 12.7, 95%CI: 3.2-60] were significantly associated with Campylobacter carriage in cattle. There was a significant interaction between farms that kept companion animals and those that did not co-graze cattle with other ruminants; modifying the

odds of acquisition of thermophilic Campylobacter in cattle by 10 times (AOR: 10, 95%CI=1.2–95.9). Tetracyclines, aminoglycosides and β-lactam-based antimicrobials were the most commonly used antimicrobials; with 54.5% of the farms generally reporting using antimicrobials in chicken production systems than in cattle. Overall, antimicrobial resistance among Campylobacter isolates ranged between 100% for ampicillin to 11.7% for gentamicin. Multi-drug resistance (MDR) was observed in 99 of 103 (96.1%) isolates; with the AX-TE-E-CIP being the most common MDR pattern at 29.1%. All the C. coli isolates irrespective of source and all the chicken isolates irrespective of Campylobacter species displayed multi-drug resistance. The tet(O), gyrA and cmeB genes were detected at 93.2%, 61.2% and 54.4% in all the Campylobacter isolates, respectively. Of the virulence genes; ciaB and flaA were the most detected genes in C. jejuni (89.7% and 62.2%, respectively), and in C. coli (81.1% and 62.1%, respectively) isolates irrespective of source. Phylogenetic analysis showed that Campylobacter sequences of the isolates from cattle, chicken and water sources were highly related (i.e. similar lineage), suggesting existence of a complex web transmission. In conclusion, the study provides insight into Campylobacter spp. in livestock and environmental reservoirs, possible transmission dynamics and the relevance for therapeutic regimens in Kajiado County. Seasonality and/or animal husbandry practices play a role in the epidemiology of the organisms in livestock and environment; therefore, understanding pathogen-specific seasonal patterns and associated risk factors is important for improving existing disease prevention and control strategies. Moreover, the existence of major virulence genes associated with the pathogenicity of Campylobacter spp., demonstrates that they can potentially infect humans. Consequently, this calls for implementation of one-health approach to reduce the impact of this foodborne zoonotic bacterium for the wellbeing of human and animal health.