Investigation Of Selected Conditions And Mechanisms In The Development Of Resistance In Salmonella Enteritidis Under Sub-inhibitory Concentrations Of Enrofloxacin

The extensive use of antimicrobials in livestock production will lead to sub-inhibitory concentrations of antimicrobial agents in the livestock and poultry farms as well as around the farms.The problem of antimicrobial resistance caused by the use of veterinary antibacterial agents with sub-inhibitory concentrations has attracted widespread attention.However,the selected conditions and antimicrobial resistance mechanisms of Salmonella enteritidis to veterinary antimicrobials with sub-inhibitory concentrations has not been systematically studied.Salmonella is one of the most common food-borne pathogens,causing great economic loss to the global aquaculture industry and seriously threatening human health.Enrofloxacin,as a special animal medicine,has a good therapeutic effect on animal digestive tract diseases.So enrofloxacin is commonly used for the prevention and treatment of salmonellosis in veterinary clinic.This paper takes enrofloxacin as the representative veterinary antimicrobials,using Salmonella enteritidis as the tested bacteria strains,systematically to investigate the selected conditions and mechanisms in the development of resistance in Salmonella enteritidis under sub-inhibitory concentrations of enrofloxacin.The molecular mechanism of known fluoroquinolone resistance and transcriptometric sequencing were detected and analyzed for some drug-resistant bacteria induced by enrofloxacin of different concentrations,aiming to explore the selected conditions and molecular mechanism of Salmonella enteritidis resistance under sub-inhibitory concentrations of enrofloxacin in this study.1 Study on the selected Conditions in the development of resistance in Salmonella enteritidis under sub-inhibitory concentration of enrofloxacin To simulate sub-inhibitory concentrations of enrofloxacin in the farm and surrounding environment,the concentration of 1/2×MIC,1/4×MIC,1/8×MIC,1/16×MIC,1/32×MIC,1/64×MIC and 1/128×MIC of enrofloxacin was used to induce Salmonella enteritidis strain CICC21527 in vitro,and the resistance rate of the resistants was tested by the enrofloxacin-containing plates with the concentrations of 2~64×MIC of enrofloxacin every 100 generations.The effect of temperatures(12?,25?,37?,42?),p H(4.4,5.4,6.4,7.4),bile salt concentration(0.1%,0.3%,1.0%,2.0%),bacterial inoculation volume(102 CFU/m L,105 CFU/m L,108 CFU/m L)on the occurrence of resistance were conducted to simulate the temperatures(refrigerated temperature,room temperature,optimal temperature,livestock body temperature),livestock digestive tract p H(stomach p H,duodenum p H,caecum p H,ileum p H),livestock small intestine salt concentration at different time periods(before and after eating),the challenge doses(low,medium and high)of Salmonella enteritidis under 1/2×MIC enrofloxacin,respectively.The resistance rate of the resistants was also tested on the enrofloxacin-containing plates with the concentration of 2~32×MIC of enrofloxacin every 100 generations.The results showed that the resistance rate and resistance level of Salmonella enteritidis increased with the increase of enrofloxacin concentration and the extension of the induction generation within the range of sub-inhibitory concentrations of enrofloxacin(1/2×MIC~1/128×MIC).The results of the growth curve showed that the growth rate of Salmonella enteritidis was slower and the breeding period was prolonged with the increase of enrofloxacin concentration in the range of sub-inhibitory concentration of enrofloxacin.The growth of Salmonella enteritidis grew faster and faster as the temperature rises,and decreased after reaching the optimum temperature.The closer to the optimal temperature,the shorter the proliferation period.The growth of Salmonella enteritidis increased with the increase of p H and the reproduction period became shorter.The growth of Salmonella enteritidis was accelerated with the increase of bile salt concentration,and it will show a downward trend after reaching the optimum bile salt concentration and the reproduction period will be prolonged.The growth of Salmonella enteritidis was inhibited with the increase of inoculation and the breeding period was prolonged.The resistance rate and resistance level of Salmonella enteritidis increased with the increase of p H but the resistance rate and resistance level of Salmonella enteritidis showed a trend of increasing first and then decreasing with the temperature,bile salt,and the bacterial inoculum increased under the selective pressure of 1/2×MIC enrofloxacin.The occurrence of Salmonella enteritidis resistance was mainly affected by the enrofloxacin selection pressure,the time for the Salmonella enteritidis growth to reach the plateau period,and the induction time in the sub-inhibitory concentration range.That is,the greater the Salmonella enteritidis selection pressure,the longer the breeding period and the more the transmission algebra.The longer the number of passages and passages,the higher the resistance rate and resistance level.The influence of temperature,p H and bile salt on the resistance of Salmonella enteritidis under the selective pressure of sub-inhibitory concentration enrofloxacin mainly depends on the number of bacterial flora,thus affecting the number of resistants that can be selected.The higher the number of bacteria flora,the higher the resistance rate.The effect of inoculation amount on the occurrence of Salmonella enteritidis resistance was mainly influenced by the effect of inoculation amount at low dose and the length of logarithmic period at high dose.2 Detection of known resistance mechanisms of Salmonella enteritidis to Enrofloxacin Quinolone-resistant determining region(QRDR)of gyr A,gyr B,par C and par E gene of the resistant induced by sub-inhibitory concentration of enrofloxacin in vitro were analyzed by BLAST after PCR amplification.RT-PCR was used to detect the expression levels of the outer membrane porin(omp C,omp D,omp F)and efflux pumps(acr B,acr F,emr B,mdf A,mdt K)of each resistant strain of Salmonella enteritidis to determine whether the differential expression of each porin and efflux pump occurred in this study.The results indicated that only the bases of the gyr A gene were mutated,and none of the gyr B,par C and par E genes were mutated of the resistant of 2×MIC,4×MIC,8×MIC,16×MIC and 32×MIC strains induced by enrofloxacin(1/2×MIC,1/8×MIC,1/32×MIC,1/128×MIC).The mutation sites of gyr A gene are mainly Ser83 and Asp87.The low and medium-leve resistants(?8×MIC)are not all mutated,while all of the medium and high-level resistant(?16×MIC)are mutated.The expression of outer membrane proteins gene of omp C,omp D,omp F in low-level resistants(?4×MIC)was down-regulated,and the expression of outer membrane proteins gene of omp F in medium-and high-level resistants(?8×MIC)was down-regulated.The expression of efflux pump genes acr F and mdt K in low-level resistants(?4×MIC)was up-regulated,and the expression of acr B was up-regulated in 4×MIC resistants;the medium-high level resistants(?8×MIC)efflux pump gene acr B,emr B and mdf A were up-regulated.Therefore,we speculate that the main mechanism of resistance in low-level resistants was the lack of membrane pore protein to reduce drug intake,of which Omp F is the most important.With the increase of drug resistance,the efflux pump and the mutation of gyr A gene gradually became the main resistance mechanism.3 Analysis of transcriptome sequencing of Salmonella enteritidis resistant strain The transcriptome sequencing libraries of Salmonella enteritidis strain CICC21527 and the resistant of 32×MIC,16×MIC and 8×MIC strains were induced by enrofloxacin(1/2×MIC,1/8×MIC,1/128×MIC)induced by sub-inhibitory concentration of enrofloxacin in vitro was constructed in this study,and Illumina RNA-seq bidirectional sequencing was performed.The transcriptome sequencing data of Salmonella enteritidis strain CICC21527 and resistant strain induced by sub-inhibitory concentration of enrofloxacin in vitro was processed for gene expression annotation and differentially expressed genes(foldchange?2,P?0.05)screening,GO and KEGG pathway annotation analysis,protein interaction network analysis,SNP analysis,and CARD comparison analysis.The results showed that the expression of 2040 genes changed between Group C and B,1032 genes were up-regulated,and 1008 genes were down-regulated among them.There were 1497 gene expression changes between Group D and B,723 genes were up-regulated,and 774 genes were down-regulated among them.There were 1196 gene expression changes between Group E and B,644 genes were up-regulated,and 552 genes were down-regulated among them.There were 1785 gene expression changes between Group C and D,797 genes were up-regulated,and 988 genes were down-regulated among them.There were 1851 gene expression changes between Group C and E,852 genes were up-regulated,and 999 genes were down-regulated among them.There were 584 gene expression changes between Group D and E,296 genes were up-regulated,and 288 genes were down-regulated among them.The expression levels of gyr A and gyr B genes of Salmonella enteritidis resistants were up-regulated,and the higher the resistance level was,the higher the expression level was,showing a significant dose-effect relationship,while the expression levels of par C and par E were not significantly up-regulated in the transcriptome.The expression levels of acr A,acr B,acr E,and emr B in efflux pump genes were significantly up-regulated,and the expression levels of tol C in 32×MIC resistant strains were significantly up-regulated.The expression levels of other efflux pump genes were not significantly expressed.The expression of omp F in the outer membrane proteins gene was decreased,but the expression level of other genes were up-regulated.There were 573 differentially expressed genes among resistants.The protein protein interaction network results show that the differential proteins mainly concentrated in the ribosome,arginine and proline metabolism,metabolic pathways,purine metabolism,biosynthesis of secondary metabolites,biosynthesis of antibiotics,protein export,taurine and hypotaurine metabolism,one carbon pool by folate.SNP analysis showed that the sfm H,gyr A and lig A genes were mutated in all resistant strains.A total of 97 drug resistance-related genes were matched,including 79 antibiotic resistance genes,23 antibiotic action target genes,and 2 antibiotic biosynthesis genes with comparison of the CARD database.Among the SNP and indel loci matched with the CARD database,there were 3 resistance-related genes inculding bet I,ept A and gyr A,and 1 resistant indel loci of bet I.Therefore,the decrease of expression of omp F,the overexpression of Acr AB in the efflux pump,the mutation of gyr A and the up-regulation of gyr A and gyr B are the direct factors determining resistance.In summary,the occurrence of Salmonella drug resistance within the range of sub-inhibitory concentration is mainly affected by the selection pressure of enrofloxacin,the the length of breeding period and the induction generations.Among the low-level resistants,the lack and missing of outer membrane porins reduces drug intake as the main drug resistance mechanism,and Omp F is the most important mechanism of drug resistance in low-level resistants.With the increase of resistance level,up-regulated expression of efflux pump and gyr A gene mutation gradually become the main resistance mechanism.This study has certain reference significance for exploring the selected conditions and mechanisms in the development of resistance in Salmonella enteritidis under sub-inhibitory concentrations of Enrofloxacin.It provides a theoretical and practical basis for the mitigation and control of resistants induced by sub-inhibitory concentration antimicrobials.