| Fluoroquinolones are important drugs for the treatment of invasive Salmonella infection.The development of fluoroquinolone resistance in Salmonella is an endpoint results of the accumulation of multiple biochemical mechanisms.In order to study the role of multidrug efflux transporter AcrB in the development of fluoroquinolone resistance in Salmonella,the following experiments were performed.One hundred and eight Salmonella strains with different ciprofloxacin resistance levels were isolated during 2009-2014.All of the isolates were included in the survey to detect mutation in the acr B gene.The results showed that all isolates with high-level ciprofloxacin resistance contained AcrB mutations.Twelve(54.5%,12/22)strains carried the AcrB P319L mutation,and 10(45.5%,10/22)strains had M78I and P319L double mutationss.All strains carrying the acr B mutations are multidrug resistant as revealed by the drug susceptibility assay.The mechanisms of fluoroquinolone resistance and third-generation cephalosporins resistance in isolates with AcrB mutations were detected,including QRDR mutation,PMQR gene,role of efflux pump and ESBL genes.Plasmids carrying ESBL gene were also studied.The results showed that,all of the AcrB mutation positive strains contained gyr A double mutations and par C single mutation.The results of PMQR gene detection showed that,aac(6')-Ib-cr was the most predominant PMQR gene(77.3%),followed by oqx AB(50%).For the isolates containing AcrBM78I/P319L double mutations,the prevalence of oqx AB was higher(90%).In the presence of PA?N,the MIC of fluoroquinolones decreased by 2-64 folds,which indicated that the efflux pump played an important role in the development of high-level fluoroquinolone resistance in Salmonella.Resistance to the third-generation cephalosporins carrying the AcrBM78I/319L double mutant strain was mediated by P1-like bacteriophage carrying bla CTX-M-27.Plasmids that express AcrBWT,AcrBM78I,AcrBP319L and AcrBM78I/319L were constructed;The growth curves of those constructed strains with 0.1%or 1%bile salts and without bile salts were monitored by monitering the OD600.The growth curve in the medium with bile salts showed that AcrBP319L and AcrBM78I/P319L mutants were more tolerant to high concentrations of bile salts.Drug sensitivity results indicated that AcrBM78I and AcrBP319L mutations can increase the resistance to fluoroquinolones,tetracycline,minocycline,erythromycin,novobiocin,and bile salts.Circular dichroism was used to study the secondary structure of different AcrB mutants at 195-250nm and the stability of those AcrB proteins at 4-98°C;BN-PAGE were used to measure the trimer stabilities of those AcrB mutants.The results of circular dichroism spectroscopy indicated that the composition of the secondary structure of the Salmonella AcrB was not completely same with that of the AcrB protein of Escherichia coli.the AcrBM78I mutation had no effect on the CD spectrum of AcrB,and the effect of AcrBP319L on the CD spectrum of AcrB was not significant.The differences between AcrBM78I/P319L double mutant and AcrBWT is relatively more visible.Based on the ratio of?222nm/?208nm,we hypothesized that the AcrBP319L single mutation and the AcrBP319L/M78I double mutant protein may have changed the partial structures of AcrB protein;the percentages of coiled coils might be higher than the wild-type AcrB protein.The results of thermostability study showed that,although the amino acid similarity between wild-type AcrB of Escherichia coli and wild-type AcrB of Salmonella was 95%,the thermal stability of these two proteins was quite different,and their melting temperatures were 63.9°C and 60.2°C,respectively.The melting temperature of the AcrBP319L mutation was reduced by nearly 1°C,while the melting temperature was recoveried for the AcrBM78I/P319L double mutations protein.The results of BN-PAGE showed that the trimer stability of E.coli AcrB protein was much higher than that of AcrB protein in Salmonella.The AcrBM78I and P319L mutations did not change the trimer stabilitied compared with AcrBWT.The structure of Salmonella AcrB protein was obtained by homology modeling.The drug resistance mechanisms of AcrBM78I were studied by site-directed mutagenesis and fluorescent dye BODIPY-FL-maleimide labeling assay.The results of site-derected mutagenesis of AcrB site 78 combine with the results of MIC tests showed that the increased hydrophobicity of amino acid of site 78 improved the resistance of AcrB.Fluorescence labeling experiments showed that mutations in AcrBM78I enhanced binding of certain substrates to certain amino acid sites in the efflux pathway(eg,site Q89,E673 and F617),while weakened the binding to other amino acids(eg,S134 and N274).The structure of Acr AB mutants and their MIC,indicated that the reason why AcrBP319L had higher resistance level to LMMD and HMMD was that the more flexible side chain of Leucine made the functional rotation of AcrB more efficient,thus AcrB can efflux more substrates.In conclusion,the study showed that AcrB mutations M78I and P319L played important roles in high-level fluoroquinolone resistance in clinically isolated Salmonella.AcrBM78I and P319L mutations are prevalent in Salmonella with high-level fluoroquinolones resistance.More studies will be needed to investigate the mutations of AcrB in clinical bacterium. |
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