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<p><b>OBJECTIVE</b>To investigate the predominant contribution of methyl-metabolism pathway to the regulation of LuxS of Strecptococcus mutans.</p><p><b>METHODS</b>The differences in biofilm formation and aciduricity of Strecptococcus mutans among the methyl-metabolism-complementation strain (KO-S), the parental wide-type strain (WT) and the luxS null strain (KO) were observed by real-time PCR for monitoring the transcriptional level of genes related to biofilm formation (smu.238, gtfD) and aciduricity (smu.44, smu.46) of the studied strains, methyl thiazolyl tetrazolium (MTT) for quantifying the biofilm of the exhibited strains and confocal laser scanning microscopy for estimating the structure of the biofilm.</p><p><b>RESULTS</b>The transcriptional level of smu.44, smu.46, smu.238, gtfD in WT were 1.289 ± 0.051, 1.694 ± 0.140, 1.565 ± 0.107, 1.667 ± 0.196 respectively; in KO were 1.001 ± 0.045, 1.007 ± 0.151, 1.000 ± 0.021, 1.012 ± 0.196 respectively, downregulated compared with WT (P < 0.05); in KO-S were 4.662 ± 0.091, 5.019 ± 0.258, 3.462±0.029, 3.071 ± 0.136 respectively, upregulated compared both with KO and with WT (P < 0.05). The quantity of biofilms formed by the studied strains were WT (1.592 ± 0.213), KO (0.939 ± 0.029), KO- S (2.177 ± 0.226), KO- P (1.020 ± 0.093), respectively, representing a less quantity by KO and KO-P than WT (P < 0.05) and a more quantity by KO-S than other three stains (P < 0.05). According to the observation of biofilms texture by confocal laser scanning microscopy, the WT biofilm was condensed and even. In contrast, fissures and gaps were found scattered in biofilms of KO, KO-P while lessened in that of KO-S, in which high-density bacterial aggregates were observed. The acid assay indicated a smaller biofilm decrease by WT and KO-S than that by KO and KO- P(P < 0.05).</p><p><b>CONCLUSIONS</b>The methyl- metabolism pathway contributes to LuxS regulation on biofilm formation and auiduricity of Strecptococcus mutans.</p>
Subject(s)
Bacterial Proteins , Metabolism , Biofilms , Carbon-Sulfur Lyases , Metabolism , Glucosyltransferases , Microscopy, Confocal , Real-Time Polymerase Chain Reaction , Streptococcus mutans , MetabolismABSTRACT
Objective To investigate the influence of abaI expression on acinetobacter baumannii biofilm formation.Methods Acinetobacter baumannii strain S isolated from bums patients was collected for the study,while the standard strain ATCC19606 was served as the control.At 6,24 and 48 hours,the gene expressions of abaI,pgaA,pgaB and pgaC were detected by real-time fluorescent quantitative PT-PCR,secretion of N-acyl-homoserine lactones (AHLs) by biological sensor and biofilm formation by MTT method.Results (1) Gene expressions of abaI,pgaA,pgaB and pgaC at 6 hours were 8.63 ±5.93,1.98 ± 1.93,1.01 ± 1.32 and 2.67 ± 3.46 respectively,which showed a quick increase at 24 hours (22.81 ± 17.60,5.13 ± 4.32,5.66 ± 3.97,11.97 ± 7.75 respectively),followed by a rapid decline in 48 hours (3.43 ± 0.88,1.30 ± 0.24,3.01 ± 3.00,3.02 ± 3.29 respectively).Gene expressions of pgaB and pgaC at 6 hours and that of pgaA and pgaC at 48 hours revealed statistically significant differences from those at 24 hours (P ＜ 0.05).(2) AHLs showed a level of 18.49 ± 11.03 at 6 hours,reached a peak of 52.23 ± 15.95 at 24 hours,then descended to 5.53 ± 0.94 at 48 hours.AHLs level at 24 hours showed statistically significant difference from that at 6 hours and 48 hours (P ＜ 0.05).(3)Biofilm formation at 24 hours and 48 hours was 2.83 ±0.44 and 2.71 ±0.15 respectively,far higher than that at 6 hours (0.49 ± 0.11,P ＜ 0.05).(4) In the correlation analysis among AHLs,biofilm formation and gene abaI,pgaA,pgaB and pgaC expressions,significant positive correlation was found between abaI and pgaA and between AHLs and pgaC expression (P ＜ 0.05).Conclusion Acinetobacter baumannii may regulate gene expressions of pgaA and pgaC responsible for biofilm formation to adjust to the external environment by means of changing abaI gene expression and AHLs secretion.
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Objective To observe the expressions of efflux pump gene cluster adeAB in acinetobacter baumannii isolated from the burn patients and the expression changes of its upstream regulatory genes adeR and adeS and determine the influence of those genes on drug resistance of acinetobacter baumannii.Methods Nine drug-resistant strains and nine sensitive strains of acinetobacter baumannii isolated from the burn patients treated between June 2012 and March 2013 were used.After strain identification using 16SrDNA sequencing,acinetobacter baumannii standard strain ATCC19606 was employed as the control.mRNA expressions of efllux pump genes adeA and adeB and their upstream regulatory genes adeR and adeS were detected by real-time quantitative PCR.Results (1) adeA and adeB genes presented higher expressions in drug-resistant strains (3.71 ±0.95,76.16 ± 8.75) than in sensitive strains (0.92 ± 0.94,0.72 ± 0.78) (F =38.71,663.65 respectively,both P ＜ 0.05).(2) adeS and adeR genes showed higher expressions in drug resistant strains (18.02 ± 6.71,3.02 ± 2.69) than in sensitive strains (1.64±1.51,0.76±0.61) (F =51.04,5.57 respectively,bothP＜0.05).Conclusions The over-expression of efflux pump gene cluster adeAB induced by the expression alteration of its upstream regulatory genes adeR and adeS is closely associated with the drug resistance of acinetobacter baumannii in the burn patient.Besides,the regulatory genes may depend on adeS to sense the nosocomial environment condition,activate or inactivate adeR and hence regulate efflux pump expression.
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Objective To investigate the construction of Streptococcus mutans luxS gene knockout mutant which can act as the technical platform for following researches on luxS quorum sensing function in oral ecosystem.Methods Erythromycin resistance gene was inserted between two 1 kb fragments containing regions of DNA immediately upstream and downstream of the luxS translational start and stop codons.The resuhing construct was linearized and electro-transformed into Streptococcus mutans cells.After allelic exchange,the luxS gene knockout mutant strains were selected on 10μg/ml erythromycin plates,and compared the growth and biofilms formation of luxS knockout mutant with wild type strains.Results The luxSknockout mutant was confirmed by PCR,and it was also confirmed that this gene mutant could be stably passed through in vitro.The growth mode of luxS knockout mutant showed obvious difierences against that of wild type at stationary phase,the knockout mutant gained more bacteria cells growth.Conclusion Streptococcus mutans luxS gene has been successfully disrupted with allelic exchange.This mutant strains showed higher growth abilitv which could be the consequence of quorum sensing mutant.
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砄bjective:To observe streptococcus sanguis (S.s) and Actinomycetes viscosus (A.v) in oral plaque biofilm formation.Methods:20 ml of saliva obtained from a health adult was centrifuged at 4 ℃ and 10 000 r/min for 10 min.The supernatant was disinfected in 60 ℃ water bath for 30 min.Glass coverslips in the size of 24 mm?24 mm were immersed into the saliva supernatant for 2 h to obtain biofilm.100 ?l of S.s ATCC 34 and A.v ATCC 19246 mixture cultured in TSB at the density of 10 5～6 CFU/ml was added into 20 ml of TSB,and then,the coverslips with biofilm were put into the mixture.The biofilm and bacteria were observed by scanning confocal laser microscopy at various times.Resuts:The biofilm reached the thickness of 15.4 ?m in 8 h and the clumps of the bacteria were mostly in the midle layer of the biofilm.The biofilm increased to 34.3 ?m in 16 h and became tassle like in 48 h.Conclusion: S.s and A.v may play some roles in the oral biofilm formation.
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Objective: To develop a new antimicrobial sensitivity test model for oral products in vitro. Methods: An artificial biofilm mouth model for antimicrobial sensitivity test was established by modified LKB chromatography chamber. Using sodium fluoride and tea polyphenol as antimicrobial agents and Streptococcus mutans as target, various methodologies on the sensitivity tests were compared. Results: Agar diffusion, agar dilution and broth macro-dilution were more sensitive than modeling biofilms method. The modeling biofilm assay resulted in MIC values of fluoride or tea polyphenol against S. mutans 32 and 12 times higher respectively than the MIC values generated by broth macro-dilution method. Conclusion :The biofilm artificial mouth model may simulate the environment for oral products test.