| Klebsiella pneumoniae is a pathogen that can cause serious community infections and nosocomial infections.It is a gram-negative bacterium,gas-producing,capsular,and immobile.Klebsiella pneumoniae can colonize on the mucosal surface of the human body easily,including the nasopharynx and gastrointestinal tract.When the immune capacity of the human body is reduced,it can enter other tissues from these sites,causing pneumonia,bacteremia,etc.Klebsiella pneumoniae needs to overcome multiple intestinal environmental pressures,including bile salts that is synthesized by perihepatic cells and secreted into the small intestine.Bile salts is an important component of bile,as an antibacterial agent,bile salts can cause multiple damage to bacteria.In order to growth and persistence in the intestinal,intestinal pathogens have developed certain bile salts tolerance mechanisms.At present,the mechanism of resistance to bile salts of klebsiella pneumoniae is still unclear.Studying the mechanism of resistance to bile salts of pathogenic bacteria is helpful to understand the factors affecting bacterial intestinal colonization and provide a theoretical basis for the treatment of pathogenic bacteria.In this paper,the tolerance of 30 strains of-klebsiella pneumoniae to crude bile salt was tested,and the tolerance concentration of crude bile salt was found to be 4%or above in 28 strains.To study the mechanism of bile salt resistance,the mutation library of Klebsiella pneumoniae transposon mutations was constructed,four mutants sensitive to crude bile salts were screened from about 10.000 mutants in five libraries.It was found by sequencing that transposons were inserted into the four genes of acrA,acrB,nlpD and ruvB respectively.among which the multi-drug efflux pump acrA and acrB hav e the function of transporting a varietv of antibacterial substances.Therefore,acrA and acrB were selected to futher study the influence of bile salts resistance of klebsiella pneumoniae.In this paper,the inactivated inserted strains of ΔacrA and ΔacrB were constructed by homologous recombination.Under in vitro experimental conditions,the study found that in different growth period of the mutant strains ΔacrA and ΔacrB incubated with 0.5%of the crude bile salts in an hour,the highest survial rate is 9.9%and 3.4%respectively,the growth of Δ acrA and Δ acrB in cholate sodium,sodium taurocholate,deoxidization cholate was inhibited,indicating that the efflux pump can evacuate different types of bile salts.In addition,since AcrA and AcrB can mediate the efflux of various substances,the resistance of ΔacrA and ΔacrB to SDS and some antibiotics were also tested in this paper.It was found that the lack of efflux pumps acrA or acrB caused by Klebsiella pneumoniae resulted in SDS,Tetracycline,Chloramphenicol,Rifampicin,Spectinomycin increased sensitivity.In order to further explore the key sites of AcrB-mediated bile salt efflux,we constructed a complete complement plasmid and a complement plasmid lacking amino acids 615,616,617,and 618,and found that,the complete complement strain had a bile salt tolerance was able to recovery to wild type levels,while the complemented plants lacking amino acids 615,616,617,618 could only restore part of the bile salt tolerance.This indicates that there are key sites in the 615,616,617,618 amino acids of AcrB that mediate bile salt resistance.Finally,in order to study the effect of the efflux pumps acrA and acrB on the colonization ability of Klebsiella pneumoniae in mice,this article uses two models of adult mouse and Murine Ex vivo Anaerobic Tissue（M-E-A-T）for research.Competitive colonization experiments in the intestine of adult rats found that the colonization ability ofΔacrA and AacrB in the intestine was significantly lower than that of the wild type.With the extension of the colonization days,the difference between the mutant strains and WT can reach 10.000 times.In the "M-E-A-T" model,the mutants of Δ acrA and ΔacrB ability of survival in the small intestine segment was significantly lower than the wild type,but there is no difference in the large intestine,speculated that this phenomenon is due to the high concentration of bile salt in the small intestine and the low concentration of bile salt in the large intestine.In addition,the virulence test of the Galleria mellonella model showed that the virulence of ΔacrA and ΔacrB was weakened compared with the wild type.Since the biofilm formation ability can reflect the virulence of the strain to a certain extent,the biofilm of the strain was further tested and it was found that the absence of acrA or acrB did not affect the biofilm formation.so virulence reduction of mutants ΔacrA and ΔacrB is not caused by biofilms..The resistance of Klebsiella pneumoniae to bile salts is the key to its colonization in the intestine.This study shows that the efflux pumps acrA and acrB play an important role in the resistance of Klebsiella pneumoniae to bile salts and colonization in the small intestine. |
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