| In recent years,the local chicken breeds are becoming more and more available in the market in China,and Guangxi is one of the provinces that has the most abundant local breeds resources and the highest yield.In the last few years,our research group has been monitoring the contamination of Salmonella in the whole production chain in the large-scale local chicken breeding companies in Guangxi.And it has been confirmed that there is widespread and serious Salmonella contamination/infection at different production stages,especially the Salmonella pullorum（SP）.So it is a crucial task for the effective prevention and control of Salmonella in the breeding company of the local chicken breeds.At present,the antimicrobial is still the main way used for the prevention and control of Salmonella infection in the breeding company.But with the increasing frequency of use and abuse,it is easy to induce antimicrobial resistance and antimicrobial multi-resistance（AMR）,which makes the increasingly difficult for the prevention and control the Salmonella infection.At the same time,the development of new antibiotics can’t keep up the speed of the bacterial resistance produced,and the resulting problems such as antibiotic residue,antimicrobial resistant genes（ARGs）transfer and other problems are also leaving safety risks to the health of human and animals.Therefore,it is very important to investigate the AMR mechanism of Salmonella.Quorum sensing（QS）system is a universal mechanism of information transmission between the bacterial cells,that is,the bacterial community behavior is regulated by the signaling molecules synthesized and secreted afterward.When the concentration of signal molecules reaches a certain threshold value,the expression of certain specific genes in the bacteria is initiated,including the regulation of efflux pump,biofilm（BF）and other non-specific antimicrobial resistant mechanisms to improve the bacterial antimicrobial resistance.The Lux S/AI-2 type is the most common QS system,regulating bacterial resistance and virulence in both Gram-negative and Gram-positive bacteria.However,the information on the mechanism of the Acr AB-Tol C efflux pump regulated by the Lux S/AI-2 type QS system in SP is limited.In this study,the recent dominating Salmonella is known as the SP,isolated from the whole production chain in many large-scale local chicken breeding companies in Guangxi,are studied to analyze the evolution of its drug resistance.And the possible AMR mechanism was also analyzed by the transcriptome sequencing.At the same time,the mechanism of the expression of the Acr AB-Tol C efflux pump genes and proteins are regulated by the key molecules Lux S,AI-2 and Lsr R of QS system of SP,is systematically studied.Also,drugs were screened and verified based on inhibiting QS signal expression.Finally,it will provide a theoretical basis for the research and development of new antibacterial drugs,and provide a novel route for the effective prevention and control of SP infection in the local chicken breed industry by exploring the AMR mechanism of SP isolates.Firstly,a total of 583 Salmonella isolates isolated from many large-scale local chicken breeding companies in Guangxi were tested for the resistance phenotypes and the ARGs,and the consistency between the presence of the ARGs and antimicrobial resistant phenotypes was analyzed.At the same time,the efflux pump genes,BF genes and BF formation ability of the strains were detected.And the differentially expressed genes of non-AMR strain and AMR strain of SP were compared,and the possible AMR mechanism was also analysed by the transcriptome sequencing.The results showed that 583 Salmonella isolates with different serotypes demonstrated varying degrees of resistance,and up to 98.11%（572/583）of the isolates showed AMR.The resistant phenotype profiles were evolved from"wide and less"to"narrow and more"as the years paased by and most of the isolates showed the resistant phenotype profile of AMP-AMC-TMP-SIZ-SXT-NAL,indicating that the resistance of isolates was evolved from widespread resistance to relatively concentrated resistance.Totally 216 isolates with different serotypes isolated from different times and different companies were selected and examined for the ARGs.The highest detection rate of the ARGs was par C（92.91%）,followed by gyr A（92.12%）,bla TEM-1（72.22%）,aph（3）-Ia（68.06%）,tet M（43.06%）,tet A（32.41%）and others.Of the antimicrobial resistant genes examined in this study,ten of them including tet B,tet G,sulⅢ,dfr12,aad A1,aad B,intⅠ2,intⅠ3,tnp U and blaCTX-M were not detected in2018.However,all of those resistant genes were detected in 2019 or 2020,although the positive rate was still relatively low.The coincidence rates between the antimicrobial resistant phenotypes and the corresponding ARGs detected in the Salmonella isolates against the three classes of antibiotics were higher than 70%.And the quinolone and fluoroquinolone had the highest coincidence rate,which was over100%in 2019,followed by the tetracyclin（77.11%）andβ-lactams（72.30%）.During the years 2018-2020,the coincidence rate ofβ-lactams was increased year after year,while the tetracyclin showed a trend of declining.However,the aminoglycosides and sulfonamides had lower coincidence rates of 66.27%and 29.30%,respectively.The results indicated that the resistance mechanism of the antimicrobials besides the quinolone and fluoroquinolone,might not been all regulated by the specific ARGs and that some other unknown resistance mechanisms of these antimicrobials may exsist.At the same time,the positive rate of effusion pump genes and BF genes in the SP isolates was higher than 90%,and these isolates had strong BF formation ability in vitro.Transcriptomic analysis showed that the differentially expressed genes of AMR strains and non-AMR strains of SP were mainly enriched in cell membrane and membrane components,biological processes,energy metabolism,signal transduction,membrane transport and other metabolic routes,and the expressions of efflux pump genes acr A（1.67 times up-reregulated）,acr B（2.24 times up-reregulated）,sox S（2.06times up-reregulated）and sox R（2.71 times down-reregulated）were changed significantly,while that of the BF genes were not.These results suggest that the AMR mechanism of these SP isolates may involve in the active efflux in the mechanism of non-specific antimicrobial resistance.In order to explore the non-specific antimicrobial resistance mediated by SP efflux pump and its regulation mechanism,the regulation effect of the efflux pump Acr AB-Tol C on its AMR based on QS system was studied.In the study,the lux S gene knockout mutant of SP was firstly constructed,and then the growth curve,antimicrobial resistance phenotype,and the efflux pump Acr AB-Tol C gene transcription level and protein expression level of the lux S-deleted strain were detected.Then the interaction between Lux S protein and the efflux pump regulatory proteins Sox S and Sox R was analyzed by the surface plasmon resonance（SPR）.The results showed that the SP lux S knockout mutant showed antimicrobial sensitivity differences,that is sensitive to the antibiotics AMP,AMC,TMP,SXT,SIZ and NAL,when compared to the resistant phenotypes of its original wild-type strain SP129.However,the growth rate of theΔlux S mutant did not change significantly compared to the wild-type strain.These results indicated that the lux S gene does not affect the growth of the strain but can regulate the resistance to the antimicrobials.Also,lux S gene knockout caused that the transcription levels of acr A,acr B and sox S genes and the expression level of Sox S protein were significantly decreased,while the transcription level of sox R gene and the expression level Sox R protein were significantly increased.This indicated that QS system can regulate the efflux pump regulation gene transcription and protein expression to affect the efflux pump gene expression by the Lux S protein.In addition,SPR experiment showed that there was no interaction between Lux S protein and the proteins Sox S and Sox R,indicating that Lux S protein can’t be directly acting on the efflux pump regulatory protein.The results of these experiment demonstrated that Lux S,a key molecule in the QS system,can effectively regulate the efflux pump to change the resistance of SP isolates,but it does not function by directly acting on the efflux pump regulatory proteins,and there may be other ways of regulation.The regulatory effect of the signaling molecule AI-2,a downstream molecule of the Lux S protein,and its target protein Lsr R in the QS system,on the SP efflux pump Acr AB-Tol C was investigated in the study.The signaling molecule AI-2 was synthesized in vitro,and the concentration and activity of AI-2 and its regulation effect on the growth of SP and the antimicrobial resistance were analyzed.Also,the m RNA transcription of acr A,acr B,tol C,sox S and sox R genes regulated by AI-2 was detected by q PCR and the effect of AI-2 on the expression of Sox S and Sox R proteins was detected by Weatern blotting（WB）.The interaction between AI-2 and the Sox S and Sox R proteins was further analyzed by the molecular docking technology.In addition,Lsr R protein was expressed in vitro and the interaction between Lsr R protein and sox S and sox R gene promoters was confirmed by the electrophoretic mobility shift assay（EMSA）.The results showed that the concentration of the signaling molecule AI-2 synthesized in vitro can reach 300μmol/L,and its biological activity was 3-fold higher than that of the positive control Vibrio harvey BB152.The growth rate of the isolates added the AI-2,was basically consistent with that of the un-added group.However,the the isolate 1817D23 and the isolate SP129 showed resistance to its originally sensitive antibiotics CTX,CAZ,GEN,AMK,KAN and TET.This indicated that AI-2 synthesized in vitro had certain activity and concentration,had not efect on the growth of the isolates,but it can regulate the antimicrobial resistance of the isolates.Also,after adding the AI-2,the m RNA transcription levels of the efflux pump genes acr A,acr B and its regulatory gene sox S were significantly increased,and the expression level of Sox S protein was also significantly increased,but the m RNA transcription of gene sox R and the protein sox R expression levels were not significantly changed.In addition,the molecular docking analysis showed that AI-2can be combined with proteins Sox S and Sox R by forming the hydrogen bonds between the amino acid residues of both molecules.This suggested that AI-2 can regulate the transcription of the efflux pump gene sox S and the expression of protein Sox S through the binding to protein Sox S,and promote the expression of the efflux pump genes to enhance the antimicrobial resistance.In addition,EMSA results showed that there was no interaction between protein Lsr R and the promoters of gene sox S and sox R,indicating that Lsr R,the target protein of AI-2,could not directly interact with sox S and sox R genes.These results indicated that the signaling molecule AI-2 can enhance the transcription level of sox S gene and the expression level of protein Sox S by binding with Sox S protein,thus promoting the expression of efflux pump gene to enhance the antimicrobial resistance of SP.In order to explore the effective control of the AMR of SP by inhibiting the expression of signal molecule AI-2 in QS system,the Lux S protein was selected as the target to screening the QS inhibitors（QSI）by the molecular docking technology.At the same time,the selected traditional Chinese medicine（TCM）monomer molecule was used for the antibacterial experiments and the antibacterial effects combined with antibiotics in vitro.And the effect of TCM monomer molecules on the m RNA transcription level of lux S gene was detected by the q PCR,and the expression level of Lux S protein was detected by the WB.The results showed that glycyrrhizic acid（GA）,combined with Lux S protein by forming the hydrogen bonds and the hydrophobic force through amino acid residues,was selected as the inhibitor of Lux S protein expression according to the binding energy,solubility of components,application,commercial price and other factors.The antibacterial test showed that 32 mg/m L of the GA could effectively inhibit the growth of Salmonella isolates SP129 in vitro,indicating that GA has a considerable antibacterial capacity.The transcription of lux S gene and the expression of protein Lux S,and the activity of AI-2 and the efflux pump genes acr A and acr B were significantly inhibited by the GA with subinhibitory concentration in a dose-dependent manner.In addition,the GA,combined with AMC,CTX,SIZ and CIP antibiotics,showed synergistic antibacterial effect.The results of the study showed that GA,as a Lux S protein expression inhibitor,can be combined with antibiotics to produce a synergistic antibacterial effect,and can effectively reduce the antimicrobial resistance of SP,and has the potential to become an antibacterial synergist.In conclusion,the results of the study demonstrated that the AMR of Salmonella isolates during 2018-2020 from the chickens of local breeds was not all mediated by the antibiotic-specific ARGs,that might be related to the non-specific resistant mechanism mediated by the efflux pump.It was proved that protein Lux S,a key molecule in the QS system,can enhance the transcription level of sox S gene and the expression level of protein Sox S through the binding of its downstream signal molecule AI-2 with the efflux pump regulatory protein Sox S,thus promoting the expression of the efflux pump genes acr A and acr B to enhance the resistance of SP.In addition,GA can be used as a QSI by binding to protein Lux S and inhibiting its expression and the activity of AI-2,so as to produce synergistic antibacterial effect combined with the antibiotics.And GA can effectively reduce the antimicrobial resistance of SP,which has the potential to become an antibacterial synergistic agent. |
