Effect of tetracycline resistance genes on transfer and stability of ampicillin resistance genes in Salmonella enteritidis

The transfer of antibiotic resistance genes by plasmids has greatly contributed to the dissemination of multidrug resistant bacteria. The influence of individual resistance genes on the spread of multidrug resistance has not been evaluated. This study tested the hypothesis that tetracycline resistance genes promoted the transfer and stability of ampicillin resistance genes on plasmids. To eliminate the confounding effect of other antibiotic resistance genes, isolates with resistance to only tetracycline and ampicillin or ampicillin alone were used in the study. Three plasmid-bearing tetr-ampr S. enteritidis isolates, one with the tetA gene and two with the tetC gene, were mated to a nalidixic acid resistant S. enteritidis and their rates of transfer were compared to three isolates with resistance to ampicillin only. The three tetr-ampr isolates bore similar plasmids as shown by restriction enzyme analysis with AhdIHindIII or SmaI The rate of transfer of plasmid DNA of the tetA tetr-ampr parent was significantly higher that the amp resistant parents (p < 0.00 15), and the tetC (tetr-ampr) (p < 0.033) parents. PFGE was used to assess genetic events such as insertions, deletions, transpositions and acquisition of plasmid DNA. Alterations in PFGE patterns of XbaI digested total cellular DNA and plasmid DNA were compared between parents and transconjugants. Five of six transconjugants showed specific alterations in banding patterns of XbaI digested plasmids or total cellular DNA run by PFGE when compared to either parent. Southern blots confirmed the transfer of tetracycline resistance genes and ampicillin resistance genes on similar sized bands (∼50 kbp) in both plasmid and genomic DNA. This is evidence that specific bands in PFGE of total cellular DNA represent plasmid DNA. The nalr-tetr-amp r resistant transconjugant that harbored the tetA class resistance gene lost its resistance to tetracycline after ten days in media without antibiotics, yet retained its resistance to ampicillin. In contrast, transconjugants derived from the tetC parents retained tetracycline and ampicillin resistance after 15 days without antibiotics, as did the transconjugants from the amp r parents. Southern blots of plasmids of this transconjugant confirmed the loss of the tetA gene and the continued presence of the amp gene. Furthermore, PFGE of genomic DNA of this partially cured transconjugant was different from both parents and the original transconjugant, possessing a ∼100kb band and a lower molecular weight fragment that bound the amp probe. These results suggest that tetracycline resistance genes of the tetA class may be responsible for the increased prevalence of multidrug resistant S. enteritidis . However, this effect may be limited to environments that contain tetracycline, because in the absence of tetracycline the tetA gene was unstable. Also, the results show that tetracycline resistance genes did not affect the stability of ampicillin resistance genes in plasmids after transfer to the recipient strains. Finally, the results also demonstrate how PFGE of XbaI digests of total cellular DNA and plasmid DNA can be used together to identify elements in PFGE patterns of total cellular DNA that represent plasmid DNA. This approach will aid in the interpretation of differences in banding patterns in both epidemiologic investigations and in tracking the spread of antibiotic resistant bacteria.