| Salmonella is considered as the main food-borne pathogen. And Salmonella enterica serovar Typhimurium accounted for a high occurence rate in the infections induced by Salmonella. Fluoroquinolones(FQs) as the preferred drugs for the treatment of salmonellosis are extensively used in clinic settings. In the selection pressure of FQs, S. Typhimurium quickly produced resistance. The main resistance mechanisms associated are active multidrug resistant(MDR) efflux pump and target gene mutations within quinolone resistant determining regions(QRDRs).In this experiment, a S. typhimurium SH10 isolated from chicken, susceptible to quinolones and other common uesed drugs, was used as a parent strain to establish efflux pump gene knockout methods mediated by p KD46 plasmid and Phage P22. To investigate the influence of efflux pump Acr AB-Tol C and it's regulator genes on susceptibility of SH10, we sepreately knockouted these genes and determined these gene-deleted strains minimum inhibitory concentration(MIC) to 15 common used antibiotics by double agar dilution method. The result showed that deletion of the targeted genes did not change the susceptibilities to the tested drug. In addition,we measuered the migration of these mutants on semi-solid media. As the result displayed that the radius of migration shrink from 27 mm to 18.3-21.7mm(P < 0.05 or 0.01),when regulatory genes mar A, ram A and sox R inactivated. Conversely, the radius enlarged from 27 mm to 31-33.7mm(P < 0.05),as inactivating mar R, ram R and sox S,. These results proved that transcriptional regulators mar A, ram A and sox R played a positive role in the regulating migration ability of S. typhimurium SH10.To understand if there is the existing feedback regulation mechanism in S. typhimurium multi-drug resistant efflux pump Acr AB-Tol C, We examined SH10 acr AB-tol C expression level in the presence of efflux pump inhibitor PA?N, the other efflux pump genes expression levels when acr A or tol C interrupted, and expression of regulatory genes by real-time quantitative PCR.The results displayed that the expression level of acr A and acr B were 2.32-fold and 1.57-fold of parent strain, of mar A and ram A being original 1.40-fold and 2.94-fold in the presence of PNBN. When acr A inactivated, acr B and tol C expression levels elevated to 2.92-fold and 2.48-fold respectively, while mar A and ram A increased to 2.29 times and 2.89 times. When missing tol C, acr A and acr B expression levels improved to 2.5-fold while mar A and ram A increased to 3- fold separately. Therefor, when the function of S.typhimurium efflux pump Acr AB-Tol C impaired, the bacterial will activate expression of transcription activators ram A and mar A to promote the transcription of acr AB-tol C in feedback way.To explore the coordinated regulation between acr AB and other pumps the expression of acr F, acr D, mds B, mdt B, mac A, emr A, mdf A and mdt K in SH10 acr AB::kan mutan were determined by real-time quantitative PCR. the results showed that except mdt K, the rest seven efflux pump genes expression were upregulated to certain extent(1.59-2.26 times, P < 0.05).We further examined the eight efflux pump genes expression levels in three double-genes deleted strains ?acr ABmar A::kan, ?acr ABram A::kan and ?acr ABsox R::kan mediated by Phage P22. The results indicated that deletion of mar A, ram A and sox S in ?acr AB::kan could significantly reduce the expression levels of other efflux pump genes(0.26-0.85 fold, 0.18-0.88 fold, 0.27-1.26 fold, P < 0.05). Moreover, we also determined the expression levels of acr AB-tol C and regulatory genes mar A, ram A, sox S in efflux pump mutants ?acr EF::kan, ?acr D::kan, ?mdt ABC::kan, ?mds ABC::kan, ?mac AB::kan, ?emr AB::kan, ?mdf A::kan, and ?mdt K::kan. The data revealed that expression levels of acr AB-tol C, mar A and ram A improved significantly in these eight efflux deletion strains(P < 0.05), and some mutunts migration had aso been enhanced. The above results indicated that the function of Acr AB or the any of other efflux pumps could be coordinated and activated through transcriptional regulator genes mar A and ram A once any of them were interrupted.Different ciprofloxacin resistant level mutants derivated from S. typhimurium SH10 or S. typhimurium reference strain SL1344 were obtained under the ciprofloxacin pressure. These induced mutants were selected according to the MIC for ciprofloxacin, including susceptibility-reduced strains(MICcip were 0.125 ?g/m L and 0.25 ?g/m L respectively), low-level resistant strains(MICcip were 2 ?g/m L), and high level resistant strains(MICcip were 8 ?g/m L). Detection of target gene mutations within QRDRs among the selected mutants revealed that only single site mutation S83 F was detected. Subsequently, we examined the expression levels of acr AB-tol C and its main transcriptional activators. The results showed that the elevated resistance level to ciprofloxacin was parallel with the expression of acr ABtol C( P < 0.05), which activated by ram A. The mutants presenting high-level resistance to ciprofloxacin also possesed multidrug resistant phenotype. In addition, we also examined fitness costs in these induced mutants. As the results revealed, the growth abilities, invasion and migration abilities in these mutants were decreased accordingly with the increasing resistance in bacteria. Conclusion: When S. typhirumiun multidrug resistant pump acr AB function inactivated, on the one hand bacterial could promote expression of acr AB in a feedback way mediated by mar A and ram A, on the other hand, bacterial can also activate other relate efflux pumps expression, particularly other RND family pumps, to compensate for the loss of bacterial efflux function. However, inactivation of other efflux pumps will also influence the expression of acr AB through the global regulator genes mar A and ram A. The vitro resistance induced experiment comfirmed that multidrug acr AB-tol C playing a major role in the development of fluoroquinolones resistance in S. typhimurium SH10 and SL1344. |
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