| Staphylococcus aureus,referred to as S.aureus,is one of the pathogenic bacteria capable of forming biofilms.It can attach to built-in medical devices(such as artificial heart valves,catheters,and joint prostheses)to form biofilms that increase resistance to antibiotics and often cause chronic or persistent infections.In recent years,more and more studies have shown that efflux pump plays a key role in the formation of bacterial biofilm.In this study,we investigated the role of nor B,a major facilitator superfamily efflux pump,in biofilm formation of S.aureus,compared the expression levels of nor B in biofilm and planktonic bacteria,and investigated its effect on the biofilm formation ability of the strain by knocking out the efflux pump gene nor B,and investigated the mechanism of the effect of the efflux pump gene nor B on biofilm by transcriptomic sequencing and analysis.Objective1.The effect of nor B knockout on biofilm formation in S.aureus was analyzed by measuring biofilm formation under different p H conditions,biofilm formation at different time points and biofilm growth curves in S.aureus before and after the knockout of the efflux pump gene nor B.To understand the biological characteristics of the strain before and after nor B knockout,including morphology,biochemical responses,growth curves and antimicrobial susceptibility tests,and to explore the role of the efflux pump gene nor B in S.aureus.2.Transcriptomic sequencing of nor B knockout and wild strains was performed using RNA-Seq technology to screen for differentially expressed genes associated with biofilm before and after nor B knockout and to perform KEGG pathway enrichment analysis,and to analyze the mechanism by which knockout of the efflux pump gene nor B affects biofilm formation in conjunction with the regulatory network of biofilm formation.Methods1.PCR to detect the prevalence of efflux pump gene in S.aureus.Crystalline violet staining to detect the biofilm forming ability of S.aureus.The qRT-PCR technique was used to detect the expression level of efflux pump gene nor B in S.aureus with different biofilm forming ability and the expression level of nor B in planktonic bacteria and biofilm.2.The nor B gene knockout strain USA300Δnor B was constructed by homologous recombination technology.3.Determination of biofilm formation ability of nor B knockout and wild strains under different p H conditions by crystalline violet staining.Determination of biofilm formation at different time points of nor B knockout and wild strains by laser confocal microscopy.Determination of changes in biofilm formation process of nor B knockout and wild strains by crystalline violet staining and plotting of biofilm growth curve.4.Observation of morphological changes of nor B knockout strains.Determination of the biochemical reactions of nor B knockout and wild strains using Staphylococcus spp.bacterial biochemical coding identification tube TH-16 S.Growth curves of nor B knockout and wild strains at different p H conditions were measured using a real-time microbial growth analysis system.Antibiotic susceptibility of nor B knockout and wild strains was measured and analyzed using the BD automated microbial identification and drug sensitivity analysis system.5.RNA-Seq technology was used to sequence the transcriptomes of nor B knockout strains and wild strains to screen for differentially expressed genes,and to perform GO functional annotation and KEGG pathway enrichment analysis.Differentially expressed genes associated with biofilms were validated by qRT-PCR and explore the mechanism of nor B knockout affecting biofilm formation.Results1.The efflux pump genes nor A,nor B,nor C,mep A and mde A were detected in96 strains of S.aureus,and the prevalence of the efflux pump genes sdr M and qac AB was 92.7% and 15.6%,respectively.The expression levels of nor B were significantly upregulated in the strains with strong biofilm formation.The expression levels of nor B were significantly upregulated in biofilms in comparison to planktonic bacteria.2.The nor B gene knockout strain USA300Δnor B of S.aureus,successfully constructed after verification by PCR and sequencing.3.The results of crystalline violet staining for biofilm formation under different p H conditions showed that nor B gene knockout reduced the biofilm formation ability of the strain under acidic and neutral conditions.At p H=4.5,the biofilm formation grade of both nor B knockout and wild strains was moderate,and the wild strain was slightly larger than the nor B knockout strain(P>0.05).At p H=5.5,p H=6.5 and p H=7.5,the biofilm formation grade of wild strain was strong,and the biofilm formation grade of nor B knockout strain was moderate,and the membrane formation ability of nor B knockout strain was significantly weakened(P<0.05).At p H=8.5 and p H=9.5,the biofilm formation grade of both wild strain and nor B knockout strain was strong,and wild strain was slightly higher than nor B knockout strain(P>0.05).The biofilm formation at 6 h,12 h,24 h,and 48 h was observed by laser confocal microscopy,and it was found that the biofilm formation of nor B knockout strains was significantly reduced and the structure was looser.Biofilm growth curves showed that nor B knockout affected the proliferative maturation stage of biofilm formation which in turn led to reduced biofilm formation.4.The color of the colonies of nor B knockout strain changed significantly to white.The biochemical reactions of the strain before and after nor B knockout changed significantly from positive to negative for urea,and from negative to positive for xylose,honey disaccharide and sorbitol.The results of the growth curves under different p H conditions showed that nor B knockout had a greater effect on the growth of the strain under acidic conditions,and the ability to adapt to acidic conditions and grow was diminished.The antimicrobial susceptibility tests results showed that the nor B knockout strain was less susceptible to ciprofloxacin,amoxicillin-clavulanic acid,Oxacillin and penicillin G.5.Transcriptomic sequencing and analysis showed that compared with the wild strain,the nor B knockout strain had significant changes in the expression of 1358 genes,of which 657 and 701 were significantly up-regulated and down-regulated,respectively.Knockout of the efflux pump gene nor B resulted in significant differential expression of biofilm-associated genes,including sae S,lyt S,lrg A,agr A,agr B,agr C,agr D,which are enriched in the two-component systemic pathway;some surface proteins fnb B,sdr C,sdr D,sdr E,clf A;the extracellular thermonuclease nuc;the co-regulator sar A family sar A and rot genes.The nor B knockout resulted in upregulation of sae S expression levels,and sae S upregulated the expression levels of the downstream gene nuc,which cleaves e DNA in the biofilm matrix.At the same time,nor B knockout led to upregulation of lyt S expression levels and thus positively regulated lrg A expression,and upregulation of lrg A gene expression inhibited the activity of extracellular wall protein hydrolase.Both together inhibit the formation of e DNA in the biofilm matrix.Conclusions1.Knockout of the efflux pump gene nor B affects the proliferation and maturation phases of biofilm formation leading to reduced biofilm formation or even defective biofilm formation in S.aureus.2.The nor B knockout may lead to reduced biofilm formation in S.aureus by inhibiting e DNA production. |
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