Rapid Proteomic Characterization of Bacteriocin-Producing Enterococcus faecium Strains from Foodstuffs

Enterococcus belongs to a group of microorganisms known as lactic acid bacteria (LAB), which constitute a broad heterogeneous group of generally food-grade microorganisms historically used in food preservation. Enterococci live as commensals of the gastrointestinal tract of warm-blooded animals, although they also are present in food of animal origin (milk, cheese, fermented sausages), vegetables, and plant materials because of their ability to survive heat treatments and adverse environmental conditions. The biotechnological traits of enterococci can be applied in the food industry; however, the emergence of enterococci as a cause of nosocomial infections makes their food status uncertain. Recent advances in high-throughput sequencing allow the subtyping of bacterial pathogens, but it cannot reflect the temporal dynamics and functional activities of microbiomes or bacterial isolates. Moreover, genetic analysis is based on sequence homologies, inferring functions from databases. Here, we used an end-to-end proteomic workflow to rapidly characterize two bacteriocin-producing Enterococcus faecium (Efm) strains. The proteome analysis was performed with liquid chromatography coupled to a trapped ion mobility spectrometry-time-of-flight mass spectrometry instrument (TimsTOF) for high-throughput and high-resolution characterization of bacterial proteins. Thus, we identified almost half of the proteins predicted in the bacterial genomes (>1100 unique proteins per isolate), including quantifying proteins conferring resistance to antibiotics, heavy metals, virulence factors, and bacteriocins. The obtained proteomes were annotated according to function, resulting in 22 complete KEGG metabolic pathway modules for both strains. The workflow used here successfully characterized these bacterial isolates and showed great promise for determining and optimizing the bioengineering and biotechnology properties of other LAB strains in the food industry.     

Proteomic Characterization of Virulence Factors and Related Proteins in Enterococcus Strains from Dairy and Fermented Food Products

Enterococcus species are Gram-positive bacteria that are normal gastrointestinal tract inhabitants that play a beneficial role in the dairy and meat industry. However, Enterococcus species are also the causative agents of health care-associated infections that can be found in dairy and fermented food products. Enterococcal infections are led by strains of Enterococcus faecalis and Enterococcus faecium, which are often resistant to antibiotics and biofilm formation. Enterococci virulence factors attach to host cells and are also involved in immune evasion. LC-MS/MS-based methods offer several advantages compared with other approaches because one can directly identify microbial peptides without the necessity of inferring conclusions based on other approaches such as genomics tools. The present study describes the use of liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) to perform a global shotgun proteomics characterization for opportunistic pathogenic Enterococcus from different dairy and fermented food products. This method allowed the identification of a total of 1403 nonredundant peptides, representing 1327 proteins. Furthermore, 310 of those peptides corresponded to proteins playing a direct role as virulence factors for Enterococcus pathogenicity. Virulence factors, antibiotic sensitivity, and proper identification of the enterococcal strain are required to propose an effective therapy. Data are available via ProteomeXchange with identifier PXD036435. Label-free quantification (LFQ) demonstrated that the majority of the high-abundance proteins corresponded to E. faecalis species. Therefore, the global proteomic repository obtained here can be the basis for further research into pathogenic Enterococcus species, thus facilitating the development of novel therapeutics.     

一株粪肠球菌8-1所产抑菌物质的生物学特性研究

为获得具有应用潜力的抑菌活性菌株,本实验采用双层琼脂平板扩散法对内蒙古传统乳制品酸马奶中分离的一株粪肠球菌（Enterococcus faecalis）8-1进行研究。排除有机酸和过氧化氢的干扰后,其无细胞发酵上清液对指示菌单核增生李斯特氏菌（Listeria monocytogenes）有明显的抑菌作用。且经过胰蛋白酶、蛋白酶K、胃蛋白酶和木瓜蛋白酶处理后,抑菌活性几乎完全丧失,表明该抑菌物质是蛋白类物质。该抑菌物质在酸性条件下抑菌活性明显,且具有良好的热稳定性。对有机溶剂、金属离子、紫外线不敏感,与SDS和EDTA共同作用抑菌效果增强。有较广的抑菌谱,能抑制多种革兰氏阳性菌和革兰氏阴性菌,初步推断属于类细菌素。      

产细菌素屎肠球菌E6的特性分析及发酵条件优化

研究了屎肠球菌E6抑菌活性的生物学特性,并通过响应面法优化其发酵条件。结果表明,屎肠球菌E6产蛋白质类细菌素,能够抑制单增李斯特氏菌、大肠杆菌、金黄色葡萄球菌和枯草芽孢杆菌生长,在pH3.0～7.0条件下有明显抑菌活性,60～121℃热处理20min后仍具有抑菌活性。通过Box-Behnken实验设计优化屎肠球菌E6发酵条件为培养时间36.0h,培养温度31.0℃,培养基pH5.1。在此条件下,发酵上清液抑菌圈直径可达20.17mm,较优化前提高了27.2%。      

大曲中产阿魏酸酯酶菌株的筛选及其特性分析

目的 从浓香型白酒大曲中分离筛选出一株产阿魏酸酯酶的菌株。方法 将大曲样品中筛选出的11株菌进行划线纯化培养,进一步挑选出产阿魏酸酯酶酶活较强的菌株并鉴定,分析其安全性及生物学特性,最后对发酵产酶条件进行优化。结果 得到一株产阿魏酸酯酶酶活较强的菌株为L-7,经鉴定为屎肠球菌(Enterococcus faecium)。经毒力基因检测、有害代谢产物实验结果均为阴性;生物学特性研究实验结果为:在16 h时生物量达到最大,耐最低pH为4,最高为11, NaCl质量分数为8%的条件下仍能生长,耐受最高的乙醇体积分数为13%;具有较好的安全性且有耐乙醇、耐盐、广泛的pH耐受性等优良性能。通过发酵产酶条件优化,该菌株产阿魏酸酯酶最佳发酵条件为:接种量为6%体积分数、培养温度32℃、转速160r/min、培养时间96 h;此时该菌株产阿魏酸酯酶的酶活为(44.68±0.05) U/L,较优化前提高了57.05%。结论 本研究结果可为产阿魏酸酯酶的菌株筛选提供参考,且筛选所得屎肠球菌在提高浓香型白酒及其他发酵食品中阿魏酸含量及风味具有应用潜力。     

Effects of Enterococcus faecium (SF68) on immune function in mice

Probiotics exert health benefits on human and animals when administered in adequate amounts. The objective of this study was to assess the effects of Enterococcus faecium (SF68) on intestinal colonisation and immune function of BALB/c mice. Six-week-old female BALB/c mice were orally administered with E. faecium (SF68). Results showed that the total anaerobe and lactobacilli in the faeces increased (P < 0.05), while the number of faecal enterobacteria decreased (P < 0.05) in E. faecium-fed mice. Furthermore, supplementation of E. faecium (SF68) increased the percentage of double positive (DP) cells in peripheral blood, the concentration of plasma IgG, and the levels of interleukin-4 (IL-4), interleukin-6 (IL-6) and interferon-γ (IFN-γ) in splenocytes of the mice (P < 0.05). This study demonstrated that E. faecium SF68 affects the intestinal microbial flora and modulates the immune responses, which indicates a viable probiotic characteristic of E. faecium SF68 in modification of immune function.     

两株降胆固醇球菌生长条件的研究

本实验对粪肠球菌的基础培养基碳、氮源进行筛选,同时对其生长所需的温度、时间和接菌量进行研究,优化出适合粪肠球菌生长最佳培养基,以提高活菌数量。在优化后的培养基中菌数分别达5.6×109CFU/mL、8.0×109CFU/mL。     

自然发酵乳中粪肠球菌和屎肠球菌的4种鉴定方法的比较

采用传统生理生化鉴定方法,16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术,变性梯度凝胶电泳技术(DGGE),对分离于自然发酵乳中的9株粪肠球菌和6株屎肠球菌进行鉴定,并对4种鉴定方法进行比较和评价。结果表明,16S-23S rRNA间区序列多态性分析技术和DGGE技术不但可以快速、精确地区分粪肠球菌和屎肠球菌,而且能够将粪肠球菌和屎肠球菌种内的不同基因亚型区分开,而传统生理生化鉴定方法和16S rRNA基因序列分析技术较以上两种方法区分效果略差。     

Physicochemical and Theoretical Characterization of a New Small Non-Metal Schiff Base with a Differential Antimicrobial Effect against Gram-Positive Bacteria

Searching for adequate and effective compounds displaying antimicrobial activities, especially against Gram-positive bacteria, is an important research area due to the high hospitalization and mortality rates of these bacterial infections in both the human and veterinary fields. In this work, we explored (E)-4-amino-3-((3,5-di-tert-butyl-2-hydroxybenzylidene)amino) benzoic acid (SB-1, harboring an intramolecular hydrogen bond) and (E)-2-((4-nitrobenzilidene)amino)aniline (SB-2), two Schiff bases derivatives. Results demonstrated that SB-1 showed an antibacterial activity determined by the minimal inhibitory concentration (MIC) against Staphylococcus aureus, Enterococcus faecalis, and Bacillus cereus (Gram-positive bacteria involved in human and animal diseases such as skin infections, pneumonia, diarrheal syndrome, and urinary tract infections, among others), which was similar to that shown by the classical antibiotic chloramphenicol. By contrast, this compound showed no effect against Gram-negative bacteria (Klebsiella pneumoniae, Escherichia coli, and Salmonella enterica). Furthermore, we provide a comprehensive physicochemical and theoretical characterization of SB-1 (as well as several analyses for SB-2), including elemental analysis, ESMS, ¹H and ¹³C NMR (assigned by 1D and 2D techniques), DEPT, UV-Vis, FTIR, and cyclic voltammetry. We also performed a computational study through the DFT theory level, including geometry optimization, TD-DFT, NBO, and global and local reactivity analyses.     

纯培养条件下细菌需氯量和存活性的试验研究

以埃希氏大肠杆菌和粪肠球菌作为研究对象,在纯培养条件下考察了自由氯、氯胺和二氧化氯的投加量以及细菌初始浓度对需氯量的影响.结果表明:灭活率达到99.9％时,两种细菌的需氯量均随初始菌浓度和消毒剂投加量的提高而增大,粪肠球菌的需氯量大于埃希氏大肠杆菌;投加自由氯时两种细菌的需氯量差别最大,其次是二氧化氯,氯胺的差别最小.