Department of Biochemical Science and Technology, National Taiwan University, Taipei, Taiwan.
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.
Appl Environ Microbiol. 2021 Jun 11;87(13):e0044221. doi: 10.1128/AEM.00442-21.
Enterococcus faecalis, a member of the commensal flora in the human gastrointestinal tract, has become a threatening nosocomial pathogen because it has developed resistance to many known antibiotics. More concerningly, resistance gene-carrying E. faecalis cells may transfer antibiotic resistance to resistance-free E. faecalis cells through their unique quorum sensing-mediated plasmid transfer system. Therefore, we investigated the role of probiotic bacteria in the transfer frequency of the antibiotic resistance plasmid pCF10 in E. faecalis populations to mitigate the spread of antibiotic resistance. Bacillus subtilis subsp. is a probiotic strain isolated from Japanese fermented soybean foods, and its culture fluid potently inhibited pCF10 transfer by suppressing peptide pheromone activity from chromosomally encoded CF10 (cCF10) without inhibiting E. faecalis growth. The inhibitory effect was attributed to at least one 30- to 50-kDa extracellular protease present in B. subtilis subsp. . Nattokinase of B. subtilis subsp. was involved in the inhibition of pCF10 transfer and cleaved cCF10 (LVTLVFV) into LVTL plus VFV fragments. Moreover, the cleavage product LVTL (L peptide) interfered with the conjugative transfer of pCF10. In addition to cCF10, faecalis-cAM373 and gordonii-cAM373, which are mating inducers of vancomycin-resistant E. faecalis, were also cleaved by nattokinase, indicating that B. subtilis subsp. can likely interfere with vancomycin resistance transfer in E. faecalis. Our work shows the feasibility of applying fermentation products of B. subtilis subsp. and L peptide to mitigate E. faecalis antibiotic resistance transfer. Enterococcus faecalis is considered a leading cause of hospital-acquired infections. Treatment of these infections has become a major challenge for clinicians because some E. faecalis strains are resistant to multiple clinically used antibiotics. Moreover, antibiotic resistance genes can undergo efficient intra- and interspecies transfer via E. faecalis peptide pheromone-mediated plasmid transfer systems. Therefore, this study provided the first experimental demonstration that probiotics are a feasible approach for interfering with conjugative plasmid transfer between E. faecalis strains to stop the transfer of antibiotic resistance. We found that the extracellular protease(s) of Bacillus subtilis subsp. cleaved peptide pheromones without affecting the growth of E. faecalis, thereby reducing the frequency of conjugative plasmid transfer. In addition, a specific cleaved pheromone fragment interfered with conjugative plasmid transfer. These findings provide a potential probiotic-based method for interfering with the transfer of antibiotic resistance between E. faecalis strains.
屎肠球菌是人类胃肠道共生菌群的一员，已成为一种具有威胁性的医院获得性病原体，因为它对许多已知的抗生素产生了耐药性。更令人担忧的是，携带耐药基因的屎肠球菌细胞可能通过其独特的群体感应介导的质粒转移系统将抗生素耐药性转移给无耐药性的屎肠球菌细胞。因此，我们研究了益生菌在屎肠球菌种群中抗生素耐药质粒 pCF10 转移频率中的作用，以减轻抗生素耐药性的传播。枯草芽孢杆菌亚种是从日本发酵大豆食品中分离出来的益生菌菌株，其培养液通过抑制染色体编码的 CF10（cCF10）的肽信息素活性而强烈抑制 pCF10 转移，而不抑制屎肠球菌的生长。抑制作用归因于枯草芽孢杆菌亚种中至少一种 30 到 50 kDa 的细胞外蛋白酶。枯草芽孢杆菌亚种的纳豆激酶参与了 pCF10 转移的抑制，并将 cCF10（LVTLVFV）切割成 LVTL 加 VFV 片段。此外，切割产物 LVTL（L 肽）干扰了 pCF10 的共轭转移。除了 cCF10 之外，粪肠球菌-cAM373 和戈登氏菌-cAM373，它们是万古霉素耐药屎肠球菌的交配诱导物，也被纳豆激酶切割，表明枯草芽孢杆菌亚种可能干扰屎肠球菌中万古霉素耐药性的转移。我们的工作表明，应用枯草芽孢杆菌亚种的发酵产物和 L 肽来减轻屎肠球菌抗生素耐药性转移是可行的。屎肠球菌被认为是医院获得性感染的主要原因。这些感染的治疗已成为临床医生的主要挑战，因为一些屎肠球菌菌株对多种临床使用的抗生素具有耐药性。此外，抗生素耐药基因可以通过屎肠球菌肽信息素介导的质粒转移系统在种内和种间进行有效的转移。因此，本研究首次实验证明，益生菌是一种可行的方法，可以干扰屎肠球菌菌株之间的共轭质粒转移，从而阻止抗生素耐药性的转移。我们发现枯草芽孢杆菌亚种的细胞外蛋白酶（s）在不影响屎肠球菌生长的情况下切割肽信息素，从而降低了共轭质粒转移的频率。此外，特定的切割信息素片段干扰了共轭质粒转移。这些发现为干扰屎肠球菌菌株之间抗生素耐药性的转移提供了一种潜在的基于益生菌的方法。
