Epidemiology, Molecular Characterization, Virulence Gene, and Antimicrobial Resistance Profiles of Thermophilic Campylobacter Species in Puppies in the Nairobi Metropolitan Region, Kenya

Abstract

Dogs are the most popular companion animals in many parts of the world and their ownership has psychological and physical benefits. However, despite the benefits, their close contact with humans poses a major public health concern due to transmission of zoonotic agents such as Campylobacter species that are important gastrointestinal pathogens which cause diarrhea in puppies. Campylobacter species are amongst the prioritized human and animal pathogens with regards to antimicrobial resistance. Although puppies are considered an important reservoir of Campylobacter pathogens, the current status of Campylobacter species infecting puppies in Kenya remains unclear. The objectives of this study were to determine the prevalence, risk factors, virulence genes, antimicrobial resistance profiles, and molecular epidemiology of thermophilic Campylobacter species in puppies in the Nairobi Metropolitan Region, Kenya. A cross-sectional study was conducted in the Nairobi Metropolitan Region, Kenya from January 2021 to August 2021 where a total of 260 puppies were randomly selected from breeding kennels, shelters and those presented to the University of Nairobi Veterinary Teaching and Referral Hospital for treatment, vaccinations, routine check-up, and boarding. Rectal swabs were collected from the puppies and a pretested questionnaire was used to collect data on potential risk factors for Campylobacter infection. Conventional culture and biochemical tests were done to identify Campylobacter species isolates. Genomic DNA was extracted and tested for presence of the Campylobacter DNA through amplification of the 16S rRNA gene and species specific genes using Polymerase chain reaction (PCR). Genetic identities of the Campylobacter species were confirmed through BLASTn analysis and phylogenetic reconstruction. Polymerase chain reaction (PCR) was done to assess for the presence of potential virulence genes associated with

motility, adherence, invasion, and cytotoxicity. Resistant phenotypes and genotypes were determined by disc diffusion and molecular methods respectively. In this study, 150 presumptive Campylobacter species isolates were identified by conventional culture and biochemical tests yielding a prevalence of 57.7% (150/260). Polymerase chain reaction (PCR) detected 64 Campylobacter species isolates (24.6%, 64/260) by targeting the 16S rRNA gene specific for these microorganisms. Campylobacter coli was the predominant species at 13% (33/260) followed by C. jejuni at 4% (10/260). The frequency of the occurrence of selected virulence genes, i.e. flaA, cadF, ciaB, iam, pldA, as well as genes responsible for the formation of the cytolethal distending toxin (CDT), i.e. cdtA, cdtB, and cdtC was determined. flaA, ciaB, and cdtB putative genes were detected in 25, 46.9, and 43.8% of strains respectively. Resistant phenotypes were detected in Campylobacter species with 100% resistance to ampicillin, 96.9% to tetracyclines, 96.9% to erythromycin, and 82.8% to trimethoprim-sulphamethoxazole. The corresponding resistance genes detected in the phenotypic-resistant Campylobacter species isolates included 23S rRNA gene (67.7%) for erythromycin, gyrA gene (31.3%) for ciprofloxacin, tet(C)/tet(O) genes (21%, 69.4%) for tetracycline, blaOXA-61 gene (32.8%) for ampicillin, and aph-3-1 gene (11.1%) for gentamicin. This is the first study in Kenya to report on the genotyping data, virulence, and antimicrobial resistance profiles of Campylobacter species in puppies in Kenya; it showed that pathogenic and multidrug-resistant Campylobacter jejuni and Campylobacter coli strains are present in puppies in the Nairobi Metropolitan Region, Kenya posing potential zoonotic risk. Great attention should therefore be paid in choosing the most appropriate antimicrobial therapy in small animal practice.