| Staphylococcus aureus is an important pathogen acquired from both community and hospital environments,and is responsible for illnesses ranging from superficial skin infections to deep-seated and life-threatening diseases.Infections caused by methicillin-resistant S.aureus(MRSA)are associated with increased mortality and morbidity and multi-drug resistance.Vancomycin is the first choice for treating MRSA;however,with the rapid rise in clinical use of vancomycin,S.aureus strains with reduced vancomycin susceptibility(SA-RVS)have emerged.SA-RVS groups include vancomycin-resistant S.aureus(VRSA),vancomycin-intermediate S.aureus(VISA),and heterogeneous VISA(hVISA).Unlike VRSA,the molecular changes associated with VISA/hVISA remain unclear.Several recent studies have reported that the acquisition of vancomycin resistance in VISA/hVISA strains is often accompanied with a decreased in virulence,and reduced autolytic activity.These phenotypic changes can assist bacteria in evading the killing effects of vancomycin and prolong the length of infection.Therefore VISA/hVISA is commonly associated with increased treatment failure.Biofilm formation is one of the most common mechanisms of antibiotic resistance in S.aureus.A biofilm can assist bacteria in evading the host immune system,and represents a diffusion barrier for antibiotics.It’s unclear whether the biofilm-forming ability of VISA/hVISA change at the same time.Some studies have examined the biofilm-forming ability of VISA/hVISA,but the results are controversial.Objective:The aim of this study was to determine the biofilm-forming ability in VISA/hVISA and the relationship between biofilm formation and vancomycin resistance.Also we explored the regulation mechanism of hVISA/VISA biofilm formation.These results will conduce to further understand survival and pathogenesis of hVISA/ VISA.Methods: In this study,(1)brain heart infusion agar containing 3 mg/l vancomycin(BHI-V3)was used as a screening tool for hVISA/VISA.Genetic background and biofilm formation were compared between two groups(2)One randomly selected strain that grew on BHI-V3(GV3)isolate(0534)was induced to obtain a series of vancomycin-resistant strains.Biofilm formation was quantified by the addition of 100 μl 0.1%(w/v)crystal violet and absorbance was determined at 570 nm.Confocal laser scanning microscopy was also used to measure biofilm structure and thickness.(3)Expression of biofilm formation and regulation related genes(icaA,icaR,fnbA,fnbB,SarA,agr,atlA and lrgAB)and VraSR were investigated by real-time quantitative reverse-transcriptase PCR.(4)Polysaccharide intercellular adhesin(PIA)production was detected by immunochemiluminescence to explore the mechanisms involved in enhanced biofilm formation.(5)Electrophoretic mobility shift assays(EMSA)was used to detect whether vraR can directly or indirectly regulate biofilm formation and DNase I footprinting were used to identify precisely localise the position that vraR binds to promoters.Results :(1)176 clinical isolates of S.aureus were screened on BHI-V3 medium.16 strains that grew on BHI-V3(GV3)were selected as the test group,and a control group of 16 MRSA strains was randomly selected from among those that did not grow on BHI-V3(NGV3).No significant difference in genetic background was found between two groups.Most GV3 and NGV3 isolates were agr group Ⅱ,SCCmec type Ⅲ and t30 type.Quantification of crystal violet staining at OD570 showed a marked increase in biofilm formation of GV3 isolates compared with NGV3 isolates.The mean absorbance for the GV3 and NGV3 isolates was 0.336 ± 0.088 and 0.109 ± 0.036,respectively.(2)A series of vancomycin-resistant strains were acquired by acquired by inducing with increasing concentrations of vancomycin.Isolates that grew on BHI supplemented with 8,16 and 32 mg/l vancomycin were named 0534-V8,0534-V16,and 0534-V32,respectively.The mean absorbance for the 0534-V8(1.64 ± 0.03),0534-V16(2.13 ± 0.10),and 0534-V32(2.49 ± 0.06)strains was higher than that of strain 0534(0.43 ± 0.03),and that the biofilm formation abilities of resistant strains increased significantly with increasing vancomycin MIC.A thin layer of strain 0534 was visible,and all of the induced vancomycin-resistant strains formed a compact,thick biofilm on the surface.The average biofilm thickness of 0534-V32 was greater than that of 0534(12±0.7 μm Vs 2±0.4 μm,n=3).(3)The expression levels of icaA and fnbA were 1.67-and 3.44-fold higher,respectively,in the GV3 group than in the NGV3 group.In contrast,the transcript levels of icaR and agr in the GV3 group were 1.88-fold and 2.18 fold lower than in the NGV3 group.Overall analysis of the four biofilm-associated genes in the series of induced strains showed upregulation of icaA,fnbA,sarA and atlA and downregulation of icaR and agr compared with the parent isolate.Moreover,the increase in gene expression level was associated with the degree of MIC elevation.(4)PIA production was significantly higher in the series of induced strains than in the parent isolate.PIA production by 0534-32 strain was 5.31 times higher than 0534 strain.Moreover,the increase in PIA expression level was associated with the degree of MIC elevation.(5)The expression levels of vraSR were 2.20-fold higher,respectively,in the GV3 group than in the NGV3 group.Transcription of vraSR was up-regulated in the vancomycin-resistant strains 0534-V32(3.11-fold)compared to their parental isolates.Increased expression of vraSR was associated with increased expression of biofilm formation genes(icaA,fnbA,sarA and atlA)and decreased expression of formation-suppressor genes(icaR and agr).In electrophoretic mobility shift assays,VraR did not bind to promoter sequences of icaADBC,fnbA,sarA and atlA genes,but it did bind to the agr promoter region.In DNase I footprinting assays,VraR protected a 15-nt sequence in the intergenic region between the agr P2 and P3 promoters.Conclution:(1)A marked increase in the biofilm formation ability of hVISA/VISA was found.In addition,the biofilm formation ability increased along with the vancomycin MIC.(2)The increased in biofilm formation by hVISA strains may proceed through FnbA-and PIA-dependent pathways.(3)VraR can bind to the agr promoter,regulated biofilm formation indirectly. |
