Faculty of Biology, Department of Genetics, Sofia University "St. Kliment Ohridski", Sofia, Bulgaria.
Faculty of Medicine, Department of Medical Microbiology, Medical University - Sofia, Sofia, Bulgaria.
Mol Biol Rep. 2020 Aug;47(8):6445-6449. doi: 10.1007/s11033-020-05663-5. Epub 2020 Jul 22.
Nowadays, due to their potential application as probiotics for humans or animals, many beneficial lactic acid bacteria have been isolated from different natural environments. These include members of the genus Enterococcus - quite specific due to their ambiguous nature, varying from pathogens to probiotics. In our work we present a whole-genome sequencing (WGS)-based approach for assessing the potential of bacteriocin-producing Enterococcus isolates from beehives to serve as natural preserving agents against bacterial infections associated with honeybees. Potential Enterococcus spp. isolates from pollen granules were tested with the well diffusion assay for bacteriocin activity against Paenibacillus larvae, the causative agent of the American foulbrood disease (AFB). Two of them gave positive results and were determined at species level by 16S rRNA genes sequencing. They were then subjected to WGS using the Illumina HiSeq platform. The resulting raw data reads were processed and further analyzed by using only freely available web-based tools (the Shovill pipeline, QUAST, BAGEL4, ResFinder, VirulenceFinder and PlasmidFinder). The analysis revealed that both of them represent clonally identical isolates of the same strain. This specific strain was named Enterococcus faecium EFD, and was genotyped by the MLST-2.0 Server. Five bacteriocin genes were found in the assembled genome, providing a possible explanation for the antimicrobial properties of the isolate. The protein nature of the inhibitory agent/s was confirmed by treatment with proteinase K. No resistance determinants for clinically important antibiotics and functional virulence factor genes were detected. The bioinformatic analyses of the draft genome sequence suggest that E. faecium EFD is not pathogenic.The observation that E. faecium EFD was present within more than one of the beehives in the apiary proposes the idea that E. faecium EFD is there as a part of the normal beehive microbiota. This finding, in combination with its antibacterial activity against P. larvae, highlights this novel isolate as a potential natural preserving agent against AFB. Furthermore, the WGS-based approach reported here proved to be very cost- and time- efficient, for screening the applicability of new pro- and prebiotic Enterococcus strains as beehive protection agents.
如今，由于其在人类或动物益生菌方面的潜在应用，许多有益的乳酸菌已从不同的自然环境中分离出来。这些包括肠球菌属的成员 - 由于其性质模糊，从病原体到益生菌各不相同，因此相当特殊。在我们的工作中，我们提出了一种基于全基因组测序（WGS）的方法，用于评估来自蜂箱的产细菌素肠球菌分离物作为天然防腐剂的潜力，以抵抗与蜜蜂相关的细菌感染。从花粉粒中测试潜在的肠球菌分离物对导致美洲幼虫腐臭病（AFB）的蜡状芽孢杆菌的细菌素活性，用平板扩散法进行。其中两个结果为阳性，并通过 16S rRNA 基因测序确定为种水平。然后使用 Illumina HiSeq 平台对它们进行 WGS。使用仅可免费获得的基于网络的工具（Shovill 管道、QUAST、BAGEL4、ResFinder、VirulenceFinder 和 PlasmidFinder）对所得原始数据读取进行处理和进一步分析。分析表明，它们都是同一菌株的克隆相同分离物。这种特定的菌株被命名为粪肠球菌 EFD，并通过 MLST-2.0 服务器进行基因分型。在组装的基因组中发现了五个细菌素基因，为分离物的抗菌特性提供了可能的解释。用蛋白酶 K 处理证实了抑制剂/的蛋白质性质。未检测到临床重要抗生素的耐药决定因素和功能性毒力因子基因。草案基因组序列的生物信息学分析表明，粪肠球菌 EFD 没有致病性。观察到粪肠球菌 EFD 存在于养蜂场中的一个以上蜂箱中，这表明粪肠球菌 EFD 是正常蜂箱微生物群的一部分。这一发现，结合其对蜡状芽孢杆菌的抗菌活性，突出了这种新型分离物作为抗 AFB 的潜在天然防腐剂。此外，这里报告的基于 WGS 的方法被证明非常具有成本效益和时间效益，用于筛选新的益生菌和预益生菌肠球菌菌株作为蜂箱保护剂的适用性。
