Conjugal Transfer Of S.typhi Resistant Plasmid PR_ST98 To E.coli In Mouse

Objective: To compare the differences of conjugal transfer of Salmonellatyphi plasmid pR_ST98 to Escherichia coli in-vivo and in-vitro and inexpression of the anti-drug resistant gene.Methods: S. typhi only strictly infects human beings and has no animalmodel. So Salmonella typhimurium, a close relative to S. tyhpi with aclear genetic background, was used. Conjugant pR_ST98/RIA was createdby transferring pR_ST98 into an attenuated strain of S. typhimurium RIA.Then conjugal transfer of pR_ST98 to E. coli was studied by peroral (p.o.)infection of BALB/c mice.1. pR_ST98/E, coliK₁₂W₁₄₈₅, created by transferring pR_ST98 to E. coliK₁₂W₁₄₈₅(F^-)Rif ^rLac⁺, was used as donors, while S. typhimurium RIA asconjugation receipts, so as to form conjugant pR_ST98/RIA. Antimicrobialsusceptibility test and plasmid analysis were used to identify the existenceof conjugant.2. E. coli from naive mice were isolated by Salmonella and Shigellaculture. After biochemical identification, Antimicrobial susceptibility testand plasmid profile analysis were used to survey the antibiotic resistant ofthe isolated bacteria and the plasmid existence. Differences of drug resistant markers of E. coli in mice and pR_ST98 were compared to preparethe antibiotic selective SS culture.3. Twenty 5-week aged BALB/c mice were randomly assigned 5mice each to 4 groups. They were p.o. infected with 10⁸, 10⁷, 10⁶ or10⁵CFU/ml pR_ST98/RIA, respectively. From 2^nd day to 30^th, mice enteric E.coli were isolated each day by antibiotic selective SS culture plates, then5 red and lmm-diameter colonies were selected from each plate. Afterbiochemical identification, their drug resistant spectra and plasmid profilewere checked.Results: 1. Resistant plasmid pR_ST98 in S. typhi could be very easilytransferred from S. typhi to E. coliK₁₂W₁₄₈₅(F^-)Rif ^rLac⁺, and then thepR_ST98/E, coliK₁₂W₁₄₈₅ could also transferred its pR_ST98 to S. typhimuriumRIA. However, in different genera, conjugal transfer conditions weredifferent and the resistant markers coded by the same pR_ST98 in differentstrains varied. Conjugant pR_ST98/E, coliK₁₂W₁₄₈₅ didn't express Sm andGm resistant markers, and pR_ST98/RIA didn't express Cos except for Smand Gm.2. One hundred strains of E. coli were isolated from naive miceintestinal tract. All of them were in different drug resistant conditions.97% and 42% of them were Ap resistant and Sm resistant respectively.All of them were sensitive to Akn and Cp. We found that no isolatedbacterium has the same drug resistant spectra to pR_ST98, so Cm and Cp were selected to prepare antibiotic selective SS culture. We found that nobacterium has a same molecular weight plasmid to pR_ST98 by plasmidspectra analysis, too.3. From 2^nd day after p.o. infection, SS antibiotic selective cultureplates appeared red colony strains which were resistant to pR_ST98 codedantibiotics, and positive rates were 40% (8/20),65% (13/20) and 80%(16/20) on day 2^nd,3^rd and 4^th day. Three mice infected with 10⁸CFU/mlbacteria died on 8^th,11^th and 16^th day respectively. One infected with10⁷CFU/ml bacteria died on 13^th day. For 4 mice died, the positive ratechanged to 81% (13/16) on 30^th day. All of the isolated bacteria were E.coli identified by biochemical test. Antimicrobial susceptibility testshowed that the isolated bacteria's drug resistant spectra were changedand their new resistant markers are similar to pR_ST98. Plasmid analysisshowed that all conjugants formed in vivo have plasmid pR_ST98. And thenumber of conjugants was not found directly related to the p.o.pR_ST98/RIA quantity.Conelusion: 1. In-vitro, plasmid pR_ST98 could be transferred from S. typhito E. coliK₁₂W₁₄₈₅, and the conjugant pR_ST98/E, coliK₁₂W₁₄₈₅ could alsotransferred its pR_ST98 to S. typhimurium RIA. 2. The susceptibility testshowed that E. coli strains isolated from naive mice were resistant toantibiotics in different degrees. 3. pR_ST98/RIA was very easily transferredto E. coliK₁₂W₁₄₈₅ in mice, and the isolated conjugants were all E. coli identified by biochemical test. 4. In different genera, conjugal transferconditions of resistant plasmid pR_ST98 were different in or out of mice andthe resistant markers coded by the same pR_ST98 varied in different strains,which showed the diversity and complexity of drug resistant plasmidexpression.