Lactococcin G-a

Objectives: Undecaprenyl pyrophosphate phosphatases (UppPs) have been implicated in bacitracin resistance in some bacterial genera and the aim of this study was to determine the role of UppPs in mediating low-level bacitracin resistance in Enterococcus faecalis. Methods: The uppP gene was identified in the genomes of laboratory (JH2-2) and clinical (V583) strains of E. faecalis. Gene fusions (uppP-lacZ) and 5'-RACE were used to study uppP expression. The uppP gene in both strains was inactivated and mutants were studied for antimicrobial susceptibility and their susceptibilities to various stress agents. Results: The UppP protein from E. faecalis showed high sequence identity to the Escherichia coli BacA-type UppP and was predicted to be a hydrophobic protein with eight transmembrane helices. The expression of uppP-lacZ was constitutive and not affected by bacitracin or cell wall-active antimicrobials. E. faecalis uppP mutants showed no significant changes in growth rate, colony morphology and biofilm formation. The uppP mutants exhibited increased susceptibility to bacitracin (MICs=3-6 mg/L) relative to the isogenic wild-type (MICs=32-48 mg/L). When uppP was expressed in a wild-type background, the MIC of bacitracin increased to 128-≥256 mg/L. The MICs of cefoxitin, teicoplanin, vancomycin, gentamicin, enrofloxacin and d-cycloserine were unaltered in the uppP mutant relative to the wild-type, as were susceptibilities to other stress agents (glycine, lysozyme, NaCl, SDS, low and high pH, oxidative stress and ethanol). Conclusions: The results demonstrate that low-level bacitracin resistance in E. faecalis is mediated by a BacA-type UppP.

Streptococcus mutans, a major etiological agent of dental caries, is resistant to bacitracin. Microarray analysis revealed that mbrA and mbrB, encoding a putative ATP-binding cassette transporter, are prominently induced in the presence of bacitracin. On the basis of the latest report that MbrC, a putative response regulator in a two-component signaling system, binds the promoter region of mbrA and thus regulates its transcription, we cut into the mechanism by generating a mutant MbrC (D(54) N-MbrC) that substituted asparagine for aspartate at position 54, the predicted phosphorylation site. MbrC, but not the mutant D(54) N-MbrC, showed affinity for a DNA probe that contained the hypothetical mbrA promoter sequence. Furthermore, we introduced a point mutation (D(54) N-MbrC) into UA159; this mutant strain exhibited neither mbrA induction nor resistance in the presence of bacitracin. These data suggest that the aspartate residue at position 54 of MbrC is a promising candidate for phosphorylation in a bacitracin-sensing system and indispensable for S. mutans bacitracin resistance.

Antibiotics are used to treat or prevent some types of bacterial infection. The inappropriate use of antibiotics unnecessarily promotes antibiotic resistance and increases resistant bacteria, and controlling these bacteria is difficult. While the emergence of drug-resistant bacteria is a serious problem, the behavior of drug-resistant bacteria is not fully understood. In this study, we investigated the behavior of Streptococcus mutans, a major etiological agent of dental caries that is resistant to bacitracin, which is a cell wall-targeting antibiotic, and focused on biofilm formation in the presence of bacitracin. S. mutans UA159 most strongly induced extracellular DNA (eDNA)-dependent biofilm formation in the presence of bacitracin at 1/8× MIC. The ΔmbrC and ΔmbrD mutant strains, which lack bacitracin resistance, also formed biofilms in the presence of bacitracin at 1/2× MIC. This difference between the wild type and the mutants was caused by the induction of atlA expression in the mid-log phase. We also revealed that certain rgp genes involved in the synthesis of rhamnose-glucose polysaccharide related to cell wall synthesis were downregulated by bacitracin. In addition, glucosyltransferase-I was also involved in eDNA-dependent biofilm formation. The biofilm led to increased transformation efficiencies and promoted horizontal gene transfer. Biofilms were also induced by ampicillin and vancomycin, antibiotics targeting cell wall synthesis, suggesting that cell envelope stress triggers biofilm formation. Therefore, the expression of the atlA and rgp genes is regulated by S. mutans, which forms eDNA-dependent biofilms, promoting horizontal gene transfer in response to cell envelope stress induced by sub-MICs of antibiotics.IMPORTANCE Antibiotics have been reported to induce biofilm formation in many bacteria at subinhibitory concentrations. Accordingly, it is conceivable that the MIC against drug-sensitive bacteria may promote biofilm formation of resistant bacteria. Since drug-resistant bacteria have spread, it is important to understand the behavior of resistant bacteria. Streptococcus mutans is bacitracin resistant, and the 1/8× MIC of bacitracin, which is a cell wall-targeted antibiotic, induced eDNA-dependent biofilm formation. The ΔmbrC and ΔmbrD strains, which are not resistant to bacitracin, also formed biofilms in the presence of bacitracin at 1/2× MIC, and biofilms of both the wild type and mutants promoted horizontal gene transfer. Another cell wall-targeted antibiotic, vancomycin, showed effects on biofilms and gene transfer similar to those of bacitracin. Thus, treatment with cell wall-targeted antibiotics may promote the spread of drug-resistant genes in biofilms. Therefore, the behavior of resistant bacteria in the presence of antibiotics at sub-MICs should be investigated when using antibiotics.