Genotypic diversity and antimicrobial resistance in salmonella isolates from food animals and animal products

A THESIS SUBMITTED IN FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY OF SOKOINE UNIVERSITY OF AGRICULTURE. MOROGORO, TANZANIA.Salmonella infection is the second leading cause of foodborne illness in humans worldwide. Salmonella spp. as one of the foodborne pathogens are widely distributed in food animal production systems. Food animal products, therefore, are considered as the major reservoir for the pathogens. Salmonella spp. have the potential to contaminate food across the production chain. Most strains of commonly occurring serotypes, such as Typhimurium, have been shown to exhibit multi-drug resistance. Previous studies on antimicrobial resistance have shown the emergence of multi-drug resistant (MDR) Salmonella isolates in food of animal origin even when there was no history of using antimicrobials, as therapeutics or as growth promoters. This suggests the presence of other co-selective pressure risk factors, including the use of heavy metal micronutrients and different types of biocides that may play a role in persistence of MDR. The main objective of the thesis was to investigate the role of the potential co-selective agents, such as heavy metal micronutrients and biocides in the emergence and persistence of MDR Salmonella isolates in the swine production systems in the United States. In addition, the thesis study determined the prevalence of antimicrobial resistance and diversity of Salmonella isolates detected from food animal production in Tanzania as a baseline for future investigation of co-selective agents and risk factors. In this thesis, the feed and faecal levels of Cu2+ and Zn2+ were measured. The agar plate-dilution method was used to determine the minimum inhibitory concentrations (MIC) of CuSO4, ZnCl2, Quaternary ammonium compound (Biosentry® and Quaternary ammonium compound (QAC) with gulteraldehyde (Synergize®) against Salmonella isolates. Antimicrobial susceptibility was tested using the Kirby-Bauer disc diffusion method against 12 antimicrobial agents representing various antibiotic classes. Genetic markers that are known to confer tolerance against heavy metals and biocides such as pcoA, czcD, and qacEΔ1 that encode for Cu2+, Zn2+ and quaternary ammonium compounds (QAC) tolerance, respectively, were amplified. In the present study, the isolates of faecal origin were found more likely to be Cu2+ tolerant than those of feed origin (OR, 27.0; 95% CI, 2.8 to 250; P = 0.0042) or environmental origin (OR, 5.8; 95% CI, 3.1 to 11.1, P = 0.0002), suggesting the significance of gastrointestinal ecosystem and the role it may play as facilitating co-selective pressure. The PCR results on the level of czcD gene carriage by Salmonella isolates were concordant with the increase in the level of the Zn2+ MIC of Salmonella isolates (OR, 5.2; 95% CI, 2.4 to 11.5, P < 0.0001). Isolates with high zinc tolerance were more likely to carry the czcD, the zinc tolerance gene. The PCR results on the level of pcoA gene carriage by Salmonella isolates were also concordant with the increase in the level of the Cu2+ MIC of Salmonella isolates (OR, 5.83; 95% CI, 3.07 to 11.09, P < 0.0001), indicating that isolates with high copper tolerance were more likely to carry the pcoA, the copper tolerance gene. Salmonella isolates were detected from 13.9% and 6.7% of environmental drag swab samples at pre- and post-disinfection, and from 17.2% and 7.1% of the faecal samples from early and late finishing pigs, respectively. Barn-level prevalence of antimicrobial resistance among Salmonella isolates detected from floor swabs was 92.3%, whereas, the prevalence of antimicrobial resistance among Salmonella isolates recovered from the faeces was 98.02%. The odds of qac genes carriage for Salmonella isolates were much higher for MDR Salmonella isolates with high MICs of the biocide than non MDR isolates with low MICs. There was a strong association between the level of MDR Salmonella isolates and the level of disinfectant resistance genes detected from the MDR Salmonella isolates, indicating the involvement of qacEΔ1 as the one of the MDR efflux pumps commonly known to extrude a wide range of structurally dissimilar toxic compounds across the bacterial cell membranes. A cross-sectional design was carried out in a pastoral region of Tanzania with a large population of livestock and a principal meat and milk producer in the country. Salmonella isolates were recovered from 4.2% of the total of 1540 samples from apparently healthy animals, animal products, floor swabs and sewage. Salmonella isolates detected belonged to two subspecies, consisting of Salmonella enterica subsp. enterica (95.3%) and S. enterica subsp. salamae (4.7%). The predominant serotypes were Salmonella I 8, 20:i:- (32.8%), S. Hadar (10.9%), S. Colindale (6.3%), S. Anatum (6.3%) and S. Heidelberg (6.3%). About one-third of the Salmonella isolates were phenotypically resistant to at least one antimicrobial, of which 82.6% were multi-drug resistant (MDR). Two MDR Salmonella isolates were found to carry integrons (intI1) with gene cassettes aac(3)-Id-aadA7 and two pansusceptible Salmonella isolates were found to carry the non integron-borne resistance genes such as sul2, aadA1 and blaTEM-1B, blaTEM-1A. The pulsed field-gel electrophoresis (PFGE) DNA fingerprint patterns strongly indicated that the majority of Salmonella isolates were clonal. The occurrence of clonal Salmonella isolates in food animals and animal products from agro-pastoral communities indicates that animal products are important source of pathogens to the agro-pastoralist communities and public at large. This finding is of great public health concern since consumption of uncooked meat and unpasteurized milk is common in Tanzania. In addition, occurrence of antimicrobial resistance to different classes of antibiotics has led to emergence of MDR Salmonella isolates which are becoming a serious public health menace to the contemporary world. Therefore, the current study recommends a coordinated regular surveillances and monitoring on the use and sale of antimicrobials particularly in Tanzania. The laws and bylaws regarding the stocking, distribution and dispensing of the antimicrobials should be strengthened and enforced