Determination of the Role of Salmonella enterica Plasmids in Antibiotic Susceptibility and Caenorhabditis elegans Pathogenicity

Poultry animals and poultry associated products are important risk sources for Salmonellosis. S.Kentucky and S.Infantis are among the serovars frequently isolated from retail chickens and were reported to be isolated in Turkey. In this study, the role of plasmids carried by S.Kentucky and S.Infantis isolates in antibiotic resistance profiles of the isolates and their pathogenicity on Caenorhabditis elegans nematode model system were investigated. The isolates used, 1 of Kentucky and 2 of Infantis serotypes, were selected among food-borne Salmonella isolated from chicken carcass in Edirne. All three isolates were previously shown to contain plasmids carrying multidrug resistance and were known to be pathogenic on C.elegans nematode model system. S.Kentucky A10 isolate was resistant to ampicillin, nalidixic acid, tetracycline, ciprofloxacin, trimethoprim and sulphonamide and carried one plasmid with 31.6 kb size. S.Infantis A15 isolate was resistant to ampicillin, streptomycine, nalidixic acid, tetracycline, neomycin, sulphonamide and kanamycin and carried a plasmid with 19.9 kb size while the other S.Infantis isolate (A16) was resistant to streptomycin, nalidixic acid, tetracycline, trimethoprim, neomycin, sulphonamide and kanamycin and carried 3 plasmids with 42.4, 1.5 and 1.2 kb sizes. Plasmid curing experiments were performed to investigate the role of plasmids in antibiotic resistance and pathogenicity in C.elegans. Ethidium bromide (EtBr) dye was used as the plasmid curing agent. Plasmids were isolated from cultures grown in LB broth with different concentrations of EtBr (50, 75, 100, 125 pg/ml) according to the Kado-Liu method and the most effective EtBr concentration was determined as 125 pg/ml. C.elegans pathogenicity assays were carried out using plasmid cured isolates. The time 50% of the nematode die (TD50) values of the nematode groups fed with plasmid cured isolates were compared with previously obtained TD50 values of the nematode groups fed with wild type Salmonella isolates. Student's t-test (p<0.05) was used to showthe level of significance between TD50 values of the two groups. TD50 values of the positive control group fed with S.Typhimurium ATCC 14028 and the negative control group fed with Escherichia coli OP50 were found as 4.0 +/- 0.4 and 8.0 +/- 0.02 days, respectively. The differences between TD50 values of nematode groups fed with wild type and plasmid cured isolates were statistically significant both for S.Kentucky (A10) (4.9 +/- 0.04-6.2 +/- 0.1) and S.Infantis (A16) (4.4 +/- 0.01-6.2 +/- 0.2) (p< 0.05) strains, but no significant difference was observed for the groups fed with wild type and plasmid cured S.Infantis (A15) (5.7 +/- 0.39-5.8 +/- 0.16) strain. Broth microdilution method was used to determine whether there was any change in minimal inhibitory concentrations (MIC) of the antibiotics for which the isolates were resistant before plasmid elimination. No significant difference was found between the MIC values of the resistant antibiotics among Salmonella isolates carrying plasmids and with cured plasmid. This study is important since the first in vivo results about the role of Kentucky and Infantis serovar plasmids on C.elegans nematode model system were presented.