| Salmonella enterica Serovar Typhimurium is a zoonotic pathogenic bacteria.The prevalence of multidrug resistant(MDR)S.enterica species in many areas of the world has become a great public health concern.Two-component signal transduction system CpxAR exists in Gram-negative bacteria widespreadly.It has been reported that CpxAR plays an important role in resistance of Escherichia coli to ?-lactams,and also contributes to the resistance of S.enterica Serovar Typhimurium to ceftriaxone.But the regulative mechanism of CpxAR on the MDR in S.enterica Serovar Typhimurium has not been elucidated.In order to explore the role of CpxAR in the MDR of S.enterica Serovar Typhimurium and further elucidate the regulative mechanism,experiments in the following aspects were carried out.(1)Firstly,cpxR gene deletion strain JS?cpxR was constructed by the method of one-step inactivation of chromosomal genes on basis of Red homologous recombination system.The cpxR complementary strain JS?cpxR/pcpxR was prepared through introducting the expression plasmid p BAD-CpxR into the mutant JS?cpxR.Meanwhile,phosphorylation sites mutations complementary strain(JS?cpxR/pcpxR*)was constructed to as a control.The minimal inhibitory concertrations(MICs)of 14 kinds of commonly used representative antibacterial agents Doxycycline(DOX),Oxyetramycin(OXY),Gentamicin(GEN),Amikacin(AMK),Apramycin(APR),Neomycin(NEO),Mequindox(MEQ),Olaquindox(OLA),Ciprofloxacin(CIP),Enrofloxacin(ENR),Florfenicol(FFC),Ceftriaxone(CRO),Ceftiofur(CEF),CEQ(Cefquinome),Colistin(COL)for JS and the above strains constructed in this study were determined with broth microdilution method.The results showed that strain JS?cpxR showed 2 to 4 folds decreases in the MICs of GEN,AMK,APR,NEO,CEF,CRO and CEQ,respectively,compared with that of the parent strain JS.With the increasing of L-arabinose concentration,the MICs of above 7 kinds of antibiotics for JS?cpxR/pcpxR were gradually restored to the level of parental strain JS,in a dose dependent manner,but that for JS?cpxR/pcpxR* MIC were not restored.It suggests that CpxR contributes to the resistance of S.enterica Serovar Typhimurium to GEN,AMK,APR,NEO,CRO,CEF,CEQ,and the phosphorylation of its receiver domain with an aspartate(D51)is necessary for its regulation function.(2)Secondly,the acr B gene deletion strain JS?acr B was constructed by using the same method as above.Then strain JS?cpxR::kan constructed in the previous study and JS?acr B were used as the donor and recipient respectively to constructed the double gene deletion strain JS?acr B?cpxR::kan with the help of P22 phage by using the phage transduction technology.Meanwhile,the cpxR complemental strain JS?acr B?cpxR::kan/pcpxR was prepared through introducting the expression plasmid p BAD-CpxR into the double gene mutant.The minimal inhibitory concertrations(MICs)of the above 14 kinds of commonly used representative antibacterial agents on JS and the above strains constructed in this study were determined with broth microdilution method.The results showed that strain JS?acr B showed 32,4,4,4,16,32,4,2,128 and 4-fold decreases in the MICs of DOX,GEN,AMK,ENR,FFC,CRO,CEF and CEQ respectively,compared with those of the parent strain JS.Strain JS?acr B?cpxR::kan showed 2,8,2,2 and 2-fold decreases in the MICs of DOX,GEN,AMK,APR,CEF compared with that of the mutant JS?acr B.While the complemental strain JS?acr B?cpxR::kan/pcpxR showed 4,16,2,4,4,2,4 and 4-fold increases in the MICs of DOX,GEN,AMK,APR,NEO,CRO,CEF and CEQ compared with those of JS?acr B?cpxR::kan.It suggests that cpxR influence the susceptibility of S.enterica Serovar Typhimurium to ?-lactam,aminoglycoside and DOX in the absence of acr B.In addition,significant increases(32-fold)in the susceptibility to colistin were abserved in strain JS?cpxR?acr B/pcpxR compared to strain JS?cpxR?acr B,it indicates that cpxR may enhance susceptibility to colistin in acr B deletion background.(3)The relative mRNA expression level of MDR related genes of strains JS,JS?cpxR,JS?cpxR/pcpxR were analyzed by Real Time PCR.The results showed that strain JS?cpxR showed no significance difference in the relative mRNA expression level of the tested genes compared to JS.Complementary strain JS?cpxR/pcpxR induced by 0.2% L-arabinose showed significant decreases in the mRNA expression levels of omp F,omp C,omp W,omp D,tol C,acr B,mar A,and sox S genes and significant increases in those of stm3031 and stm1530 genes compared to JS?cpxR,but the expression levels of Acr D,Mdt A,and Acr F in these three strains were all very low and almost undetectable.It suggested that CpxR overproduction up-regulated the expression levels of Omp F,Omp C,Omp D,Omp W,Acr B,Tol C,Mar A,and Sox S,down-regulated the expression levels of STM1530 and STM3031.After all strains were induced simultaneously by GEN to the 15 th passages at subinhibitory concentrations,the relative mRNA expression level of MDR related genes of them were also analyzed by Real Time PCR,and strain JS?cpxR/pcpxR induced by 0.2% L-arabinose showed significant increases in the mRNA expression levels of efflux pumps mdt A and acrD genes compared to JS?cpxR,but acr F were detected to be negative in all tested strains.It suggests that CpxR can upregulate the expression of acrD and mdt A,and had no effect on the expression of acr F under the induction of GEN at subinhibitory concentrations.(4)127 strains of Salmonella spp.supplied by Animal Husbandry Bureau of Henan province were collected from chicken farms in different regions(Zhengzhou,Xuchang,Kaifeng,Jiaozuo,Xinxiang)of Henan Province.27 strains of them were identified to be S.enterica Serovar Typhimurium by multiplex PCR method.9 strains were selected from the 27 strains S.enterica Serovar Typhimurium and named as SH1-9.Then strain JS?cpxR::kan and SH1-9 were used as the donor and recipient respectively to constructed the cpxR gene deletion strain SH1-9?cpxR with the help of P22 phage by using the phage transduction technology.The changes of MICs of the above antibiotics and the expression level of MDR related genes between SH1-9 and SH1-9?cpxR were analyzed.The results showed that,after cpxR was deleted,all isolates showed incr eased susceptibility to AMK,GEN,APR and CEF,6 isolates showed increased susceptibility to CRO,6 isolates showed increased susceptibility to CEQ,3 isolates showed increased susceptibility to DOX,while all isolates showed no changes of susceptibility to FFC and COL,as compared to their parental strain.The results indicate that CpxR also contributes to aminoglycosides and ?-lactams resistance in clinical isolates of S.enterica Serovar Typhimurium.In addition,after cpxR was deleted,6 isolates showed increased expression of Omp F,5 isolates showed increased expression of Omp C,3 isolates showed increased expression of Omp D,4 isolates showed increased expression of Omp W,only one isolates showed increased expression of STM1530,2 isolates showed increased expression of STM3031,5 isolates showed decreased expression of Acr D,6 isolates showed decreased expression of Mdt A,as compared to their parental strain.The results suggests that in S.enterica Serovar Typhimurium isolates,cpxR deletion can lead to increased expression of outer membrane protein Omp F,Omp C,Omp D,Omp W,decreased expression of efflux pump Mdt A and Acr D.(5)The growth curve of S.enterica serovar Typhimurium standard strain(JS),cpxR overexpression strain(JS/pcpxR)and phosphorylation site mutational cpxR overexpression strain(JS/pcpxR*)in two different energy levels of minimum medium were determined,and the influence of CpxR on the promoter activities of omp D and acrD gene at the subinhibitory concentrations of GEN were analyzed by using Lac Z fusion assay.The results showed that the logarithmic phase growth speed of strains JS,JS/pcpxR and JS/pcpxR* grown in M9-0.2% glycerol culture with subinhibitory concentrations of GEN were much lower than that of them grown in M9-0.2% glycerol culture,and the logarithmic phase growth speed of strains JS and JS/pcpxR* grown in M9-0.2% glucose culture with subinhibitory concentrations of GEN were also lower than that of them in M9-0.2% glucose culture,but the logarithmic phase growth speed of strain JS/pcpxR grown in M9-0.2% glucose culture is largely similar to that of it cultured in M9-0.2% glucose culture with subinhibitory concentrations of gentamicin.The results indicate that the overexpression of CpxR can improve the resistance of S.enterica serovar Typhimurium to GEN in the presence of glucose.Through the analysis of the target gene promoter sequences,some same or similar sequences with CpxR box were found in the promoter region of omp F?omp C?omp D and acrD genes.Then the influence of CpxR on the promoter activities of omp D and acrD gene at the subinhibitory concentrations of gentamicin were analyzed by using Lac Z fusion assay.The results showed that the ?-galactosidase activity of fusion reporter strain JS?cpxR/pcpxR/Pomp D was significantly lower than that of JS?cpxR/Pomp D regardless of the presence of GEN,while the ?-galactosidase activity of fusion reporter strain JS?cpxR/pcpxR/PacrD was significantly higher than that of JS?cpxR/PacrD after induced by subinhibitory concentrations of GEN,and approached that of JS/PacrD.It suggests that CpxR can decrease the activity of the promoter of omp D gene and increase activity of the promoter of acrD gene,and the increased transcription of efflux pump Acr D mediated by CpxR is an important regulative mechanism of CpxR on resistance of S.enterica Serovar Typhimurium to GEN.In conclusion,CpxR contributes to the formation of MDR including aminoglycosides,?-lactams and doxycycline resistance in S.enterica Serovar Typhimurium,the effect of CpxR on the expression levels of MDR-related genes(outer membrane protein genes ompF,ompC,ompD,ompW,stm1530 and stm3031,efflux pump genes acrD,mdt A and regulatory genes mar A and sox S)is closely associated with CpxR-mediated antibiotics resistance of S.enterica Serovar Typhimurium,and the upregulation effect of CpxR on the expression of Acr D is the main mechanism of resistance of S.enterica Serovar Typhimurium to aminoglycosides. |
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