The virulence and antimicrobial resistance of Salmonella spp. isolates from rodents inhabiting chicken farms in Mafikeng, South Africa

Abstract

Rodents are known to carry a number of zoonotic pathogens of importance causing both human and animal diseases. Rodents inhabiting poultry houses have been shown to carry disease causative agents for salmonellosis, fowl pox, erysipelas and leptospirosis. The main aim of the current study was to determine the types and virulence of Salmonella spp. carried by rodents captured at poultry farms around Mafikeng, North West Province, South Africa. In order to achieve this, rodents were captured around poultry houses and identified. A total of 154 rodents were humanely captured from six selected poultry farms, processed and identified molecularly using the Cytochrome oxidase subunit 1 (COI) and the Cytochrome-b (Cyt-b) barcoding genes for species identification. Two rodent pest species namely; Rattus rattus known as the black rat and Rattus tanezumi (Asian Rat/Asian House Rat) were identified. Out of 154 rodents captured, the dominant population, 99 (64.3%), were identified as R. rattus and 55 (35.7%) were R. tanezumi. Of the two barcoding genes, Cyt-b gene was only able to identify 40 (25.97%) of the total samples while COI was more efficient and amplified all the samples. After identifying the types of rodents found in these farms, these rats were then checked for the presence of Salmonella. Fecal samples were collected from their caeca and analyzed for Salmonella using cultural methods and conventional PCR targeting the 16S rDNA gene. Sixty eight Salmonella spp. were detected, identified and confirmed by PCR. Overly, 38.2% were identified as S. typhimurium, 11.8% as S. newport, 17.6% as S. enteritidis, 10.3% as S. heidelberg, 8.8% as S. bongori, 5.9% as S. enteric serovar paratyphi B, 4.4% as S. tennessee and 2.9% as S. pullorum. Most of the Salmonella isolates were from R. rattus (63.3%) species and the rest were from R. tanezumi (36.8%). This, to our knowledge, is the first study to have isolated and determined Salmonella spp. in rats around poultry farms in South Africa, particularly from R. tanezumi. The Salmonella isolates were then checked for their virulence by detection of documented virulence genes. Isolates were screened for the presence of eleven (n=11) virulence genes that are known to confer pathogenicity to Salmonella, namely; invA, Sdf I, Spy, SpvC, hilA, spiC, misL, orfL, Ppb23, fliB and fliC. The virulence genes were detected by PCR using published primers. Out of the 68 invA positive Salmonella strains, 12 (18%), 25 (37%), 14 (21%), 34 (50%), 44 (65%), 32 (47%), 39 (57%) were positive for SdfI, spy, SpvC, hilA, misL, OrfL spiC genes, respectively. There were Salmonella serotypes which were carrying multi-virulence genes i.e. S. typhimurium, S. enteritidis, S. newport, S. heidelberg, S. bongori, and S. pullorum, with 7 (10.3%), 6 (8.8%), 2 (2.9%), 3 (4.4%), 2 (2.9%) and 3 (4.4%), respectively. The more the number of virulent genes detected in an isolate, the higher the risk of pathogenicity the isolate was likely to be, and so most of these strains were of high pathogenic potential. Finally, the isolates were assessed for antibiotic resistance by both phenotypic and genotypic methods. Most of the Salmonella isolates showed resistance to Rifampicin 68 (100%), Tetracycline 32 (47.1%), Ciprofloxacin 21 (30.9%), Sulphonamides 12 (17.6%), Cephalothin 12 (17.6%), Chloramphenicol 9 (13.2%), Streptomycin 8 (11.8%), Enrofloxacin 6 (8.8%), Ampicillin 3 (4.4%), Amoxicillin/clavulanic Acid 2 (2.9%) and Nalidixic acid 1 (1.5%). All Salmonella isolates were, however, susceptible to gentamicin. Several Salmonella serovars showed multiple drug resistance of up to four different antibiotics. Using molecular means, antibiotic resistance genes assessed included the following resistance genes; tet, cat, blaTEM, sul, qnrA and aadA. Each of the genes represents resistance to the antibiotics: Tetracycline, Chloramphenicol, β-lactams, Sulfonamide, Quinolones and Aminoglycoside, respectively. All these genes were detected from some of Salmonella isolates at varying levels. Seventy-seven percent (n=52) of the isolates were also confirmed as harbouring class 1 integrons, the presence of which indicates that these isolates were containing one/more genes that encode antibiotic resistance. In conclusion, this study has shown that two rodent types, namely, R. tanezumi and R. rattus are the common rodent species in poultry farms around Mafikeng. These rat types carry Salmonella spp. some of which are known for causing disease outbreaks in animals and humans. Their pathogenic potential is represented by the virulent genes that were detected. These Salmonellae spp. had varying levels of antibiotic responsiveness with some showing multiple drug resistance. These findings are very important in the control and treatment of Salmonella in poultry farms as well as its management at public health level. The findings of this study also highlight the significance of rodent control in order to control the occurrence of Salmonella in poultry farms.