Genotyping of Multi-drug Resistant Non-typhoidal Salmonella: Detection of Fluoroquinolone and Other Multi-drug Resistance Determinants
Munyalo, Janet A
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The genus Salmonella belongs to the family of Enterobacteriaceae. Within the genus Salmonella there are 2 species, Salmonella bongori and Samonella enterica. There are over 2500 different serotypes or serovars that have been identified to date belonging to the above mentioned genus. Non-typhoidal Salmonella (NTS) is an important pathogen that causes foodborne diseases in both humans and gastrointestinal illness in animals. In humans, multi-drug resistant (MDR) NTS strains present a major health concern particularly in health care areas with inadequate resources. There is doubt that these MDR strains of Salmonella may not be limited to humans and zoonotic spread may be stirring. Free-range pig production around homesteads is a regular way of farming in Kenya hence increases the danger of spread of infection to human beings. Therefore pigs’ serves as reservoirs of antimicrobial resistant Salmonella and slaughterhouse cross-contamination of pork becomes a food safety hazard. Tetracyclines, sulphonamides and trimethoprim, nitrofurans aminoglycosides, beta-lactams and the quinolones are the mostly used drugs in food-producing animals in the country. The in- feed use of co-selective agents (such as grave metal micronutrients copper and zinc) and antibiotics in swine production systems impose selective pressure and contribute to increased MDR strains. However, in Kenya and Eastern Africa there is inadequate data on the contribution of heavy metal micronutrients use in swine feed and their role on emergence of MDR Salmonella. There is also insufficient data on the antibiotic resistant genes in Salmonella isolated from swine fecal sources. This study aimed to characterize antimicrobial resistant genes in Salmonella isolated from swine faeces and the function of weighty metal micronutrients in the appearance of MDR Salmonella. A total of 171 Isolates were used in the study. Of these, 86 isolates originated from Kenya, 35 isolates from Ethiopia and 50 isolates from the United States of America (USA). Minimum Inhibitory Concentration (MIC) of copper-sulfate (CuSO4) and zinc-chloride (ZnCl2) was measured using agar dilution. Amplification of pcoA and czcD genes that encode for copper ions (Cu+2) and zinc ions (Zn+2) tolerance was done. Amplification and sequencing of quinolone resistance determining region of topoisomerases genes gyrA and plasmid mediated quinolone resistance gene qnrA were done. Amplification of class 1 integrons and Salmonella plasmid virulence (spvA) gene was done. Isolates from USA and Ethiopia had predetermined heavy metal tolerance (Cu+2 and Zn+2) MICs and were used as comparative test isolates. The 86 test isolates were all subjected to MIC determination for Cu2+ and Zn2+. 98% (N=84) of the isolates had an MIC of 4mM, 1.16% (N=1) 0.5 mM, and 1.16% (N=1) had MIC of 0.25 mM for Zncl2. 98% (N=84) had MIC of 8 mM, 1.16% (N=1) 1.0 mM and 1.16% (N=1) MIC of 2.0 mM for CuSO4. The zinc tolerance genes czcD and copper extrusion efflux gene pcoA were not detected in any of these isolates regardless of their MIC or MDR status. The findings demonstrated that there is no association between heavy metal resistance and antimicrobial resistance in NTS. Forty two(42) isolates out of the total 171 isolates selected with antibiotic resistance characteristic SulCip Cip and CipNa identified single and double amino-acid substitutions in gyrA at positions Ser83 (N = 29; →Phe, →Tyr, → Cysteine, → Gly, → Isol) and Asp87 (N = 33; →Asn, →Gly, →Ly, →Arg) and one (1) isolate carried qnrA gene by PCR amplification and sequencing. There was no strong relationship between NAL and CIP resistant isolates in amino acid substitutions and mutations in both positions Ser83 and Asp87. 31 out of 171 isolates were selected for integron detection with characteristic antibiotic resistance pattern SulTe by PCR amplification and 10 out of the 31 isolates were positive for Class 1 integrons of 1.2 kb and 1.5kb size. 38 isolates were selected for spvA gene detection with antibiotic resistance SulCip and 3 out of the 38 isolates were found to contain spvA gene. This study shows the potential significance of pigs as a source of sole and many antimicrobial-resistant Salmonella isolates and that the use of relatively low heavy metal ion concentrations in swine feed might not be sufficient to induce increased multidrug resistance nontyphoidal Salmonella serotypes in swine. In addition, the study provides important information for incorporation into public health policy to control and manage Salmonella swine production systems in controlling antibiotic resistance transfer from swine to humans.
university of nairobi