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문제 정의
- butyricum exhibits antibacterial and antibiofilm activities against ETBF carrying the toxigenic bft-2 gene by regulating the virulence- and efflux pump-related genes (ompA and bmeB3). This study demonstrates the effectiveness of CFS obtained from C. butyricum against biofilm formation and provides scientific evidence for the development of new antibacterial and antibiofilm agents. Furthermore, these results suggest the potential of C.
제안 방법
- Furthermore, this study analyzed the expression levels of ompA and bmeB3 genes according to the concentration of C. butyricum CFS by qRT-PCR. More specifically, the ompA, the most abundant outer membrane protein in B.
- butyricum against the growth and biofilm of ETBF secreting Bft has yet to be reported . In order to investigate the effect and the possible application of metabolites formed by C. butyricum, this study evaluated the antagonistic activity of cell-free supernatant (CFS) extracted from C. butyricum against the growth, biofilm formation and gene expression of B. fragilis. Our work widens our intellectual horizon on the potential antibacterial and antibiofilm activities of CFS derived from C.
- All steps were conducted in a dark room. The biofilms of B. fragilis strains were visualized at excitation wavelengths of 495, 590, and 358 nm for FITC, Con A and DAPI, respectively, using Zeiss LSM-710 confocal laser microscope (Carl Zeiss, USA) and were imaged using ZEN software (Carl Zeiss).
- , UK), and 1 µg of template was reverse-transcribed into cDNA in a 20 µl reaction volume, using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Japan). The qRTPCR was performed to investigate the relative gene expression of B. fragilis outer membrane protein (ompA) associated with virulence factor and RND-type efflux pump-related bmeB3. Power SYBR Green PCR Master Mix (Applied Biosystems, USA) was employed to analyze the PCR amplification products.
- According to a study conducted by Pierce and Bernstein[38], ETBF carrying the bft gene was formed via mutations in non-toxigenic (NTBF) strain through evolutionary changes. Therefore, this study was conducted using WTETBF (bft-2), rETBF (bft-2), and WT-NTBF to confirm that CFS extracted from C. butyricum was strain-specific.
대상 데이터
- The wild-type enterotoxigenic Bacteroides fragilis 86-5443-2-2 (WT-ETBF; bft-2), a wild-type non-enterotoxigenic Bacteroides fragilis NCTC 9343 (WT-NTBF), and a recombinant strain transformed via insertion of bft-2 gene into WT-NTBF (rETBF; bft-2) and C. butyricum NCTC 7423 (ATCC 19398), a nontoxigenic strain, were used in this study [19]. B.
데이터처리
- Student’s t-test was conducted to analyze the gene expression between treated and control groups.
- The results of qRT-PCR were analyzed using the Student’s t-test.
- The results were analyzed via one-way analysis of variance (ANOVA) followed by Dunnett’s test to evaluate the significant differences between the treated groups and the control group.
이론/모형
- The results of qRT-PCR were analyzed using the Student’s t-test. All statistical analyses were conducted using the GraphPad Prism version 5 (GraphPad Software, USA). Statistical significance was considered at *p < 0.
- The metabolic activity of B. fragilis biofilms was analyzed using the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium5-carboxanilide] reduction assay as described previously [27], with slight modifications. Briefly, the 96-well microtiter plate was used to establish the biofilms of three B.
- butyricum CFS. The metabolic activity of biofilm cells was assessed using XTT reduction assay. Specific absorbance was calculated as A475 (Test) – A475 (Blank) – A650 (Test).
- butyricum CFS. The specific absorbance was calculated using an XTT reduction assay. Even though the viability of WT-ETBF (bft-2) biofilm was less than 78.
성능/효과
- fragilis strains was determined by measuring growth inhibition. A 50% C. butyricum CFS completely inhibited the growth of WT-ETBF (bft-2), rETBF (bft-2) and WT-NTBF by 99.48%, 98.38% and 100%, respectively (Figs. 1A-1C). A 25% C.
- The specific absorbance was calculated using an XTT reduction assay. Even though the viability of WT-ETBF (bft-2) biofilm was less than 78.65% compared with other strains upon treatment with 25% C. butyricum CFS, the viability of all B. fragilis strains used in this study declined by more than 90% with 50% C. butyricum CFS (Fig. 4). As shown in Fig.
- fragilis. Our results indicated that C. butyricum CFS suppressed ompA and bmeB3 genes only for B. fragilis strains with the bft gene.
- butyricum CFS, respectively. The results showed that the biofilm formation of all B. fragilis strains was inhibited by more than 90% by treatment with 50% C. butyricum CFS.
- Thus, our findings showed that CFS derived from C. butyricum exhibits antibacterial and antibiofilm activities against ETBF carrying the toxigenic bft-2 gene by regulating the virulence- and efflux pump-related genes (ompA and bmeB3). This study demonstrates the effectiveness of CFS obtained from C.
후속연구
- butyricum CFS against the growth and biofilm of ETBF. Although further studies are needed to determine its clinical application, the results suggest that C. butyricum acts as an effective probiotic against ETBF secreting Bft by reducing growth and biofilm formation.
- butyricum CFS as biotherapeutic agent to prevent and treat IBD and/or CRC caused by ETBF. Since this study does not analyze the chemical composition of C. butyricum CFS, further study needs to thoroughly investigate the effects of the one major component in C. butyricum CFS against the growth and biofilm of ETBF. Although further studies are needed to determine its clinical application, the results suggest that C.
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