| [Objective] Listeria monocytogenes is an important gram-positive foodborne pathogen. In recent years reports of antibiotic resistance of its isolates in many countries have increased. The alternative sigma factor aB contributes to the transcription of stress response factors in diverse gram-positive bacterial species and antibiotic resistance in Bacillus subtilis and Staphylococcus aureus. This work was aimed to investigate the role of σB in antibiotic resistance in Listeria monocytogenes.[Methods]①In order to set up a rapid, sensitive and simple assay to measure the ability of bacteria against antibiotics,3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) has been used and optimized.②Susceptibilities (or resistance) of Listeria monocytogenes wild type strain EGDe and its sigB deletion mutant EGDeAsigB to six antibiotics (penicillin G, ampicillin, rifampicin, gentamicin sulphate, tetracyclin HCl and erythromycin) were compared by measure of their MICs (minimal inhibitory concentration);②Their growth activities on the media with1×MIC,2×MIC or8×MIC of three antibiotics (ampicillin, erythromycin and rifampicin) were measured by MTT reduction assay and displayed by the growth curves of overcoming the antibiotic.④The transcription of four genetic loci (known to be induced by rifampin in Bacillus subtili) kat,fri, ropB and rsbU in EGDe and EGDe△sigB in the absence or presence of rifampin had been analyzed with reverse transcriptase PCR.[Results]①The MICs of EGDe against penicillin G, tetracyclin HCl and gentamicin sulphate were higher than those of EGDeAsigB, with0.16μg/mL vs0.08μg/mL,0.25μg/mL vs0.125μg/mL and0.5μg/mL vs0.125μg/mL, respectively; The Minimum Inhibitory Concentrations (MICs) of tetracyclin HCl and gentamicin sulphate against EGDeAsigB were two and four folds less than those for EGDe, respectively; the MICs of two strains for ampicillin, erythromycin and rifampicin has no different, being0.19μg/mL,0.125μg/mL and0.032p.g/mL, respectively.②However, the growth activities of EGDe and EGDeAsigB on the media with three antibiotics were easily distinguishable. The resumption of EGDeAsigB from tested antibiotics inhibition was longer than that of EGDe, and this difference became more accentuated at higher antibiotic concentrations. The ability of EGDeAsigB to overcome the growth arrest caused by ampicillin, erythromycin and rifampicin was also weaker than that of EGDe.③The transcription analysis of four genetic loci in EGDe and EGDeAsigB in the absence or presence of rifampin revealed that:1)the transcriptional level of rpoB gene was stable under all the experimental conditions, being not σB-dependent or rifampin stress inducible;2) expression of kat and fri genes is σB-dependent, and inducible by rifampin stress;3) rsbU in EGDeAsigB was remarkably higher in the absence of rifampin and increased significantly in both the EGDe and EGDeAsigB after rifampin application, when compared to that in EGDe control without antibiotic. It suggests that the expression of the rsbU not only with rifampin stress related, and compensation the function of σB.[Conclusion] The alternative stress sigma factor, sigma B, identified in Gram-positive bacteria such as Bacillus subtilis and Staphylococcus aureus is stimulated in response to many adverse environmental conditions. Our results show that σB not only plays an important role in resistance of cell envelope-acting antibiotics but also in protein synthesis-inhibiting antibiotics. The mechanism of σB in bacterial survival from the antibiotics acting on protein synthesis is not similar to that in Bacillus subtilis, suggesting that bacteria against antibiotic is species specificity. An in-depth understanding of the mechanism of bacteria against antibiotic will facilitate to develop new drugs, and reasonable use of the existing antimicrobial agents. |
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