Enterocin 012, a bacteriocin produced by Enterococcus gallinarum isolated from the intestinal tract of ostrich

Enterococcus gallinarum strain 012, isolated from the duodenum of ostrich, produced enterocin 012 which is active against Ent. faecalis, Lactobacillus acidophilus, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp., Clostridium perfringens, Pseudomonas aeruginosa and Salmonella typhimurium. One of the four pathogenic strains of Escherichia coli isolated from the intestinal tract of ostrich was inhibited by enterocin 012. No antimicrobial activity was recorded against Bacillus cereus, Cl. sporogenes, Cl. tyrobutyricum, Leuconostoc cremoris, Pediococcus pentosaceus, Staphylococcus carnosus and Streptococcus thermophilus. Enterocin 012 was resistant to treatment with lysozyme, catalase, lipase and papain, but sensitive to Proteinase K, alpha-chymotrypsin, trypsin and pepsin. Treatment of enterocin 012 with gastric juice from the duodenum resulted in a 50% loss of antibacterial activity. Half of the activity was lost when incubated at 80 degrees C for 30 min, or when kept overnight at a pH of 1.0-5.0 and pH 11.0 and 12.0, respectively. Enterocin 012 production started in mid-logarithmic growth and reached a maximum of 800 AU ml-1, but increased further to 1600 AU ml-1 in the stationary growth phase. The peptide is approximately 3.4 kDa in size, as determined after partial purification with Amberlite XAD-1180 and ammonium sulphate precipitation, followed by tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The mechanism of antimicrobial activity against Lact. sake LMG 13558 is bactericidal and caused cell lysis of active growing cells.     

小菌虫肠道可培养细菌的分离鉴定及产消化酶活性分析

目的 分离小菌虫肠道可培养细菌,并研究其产消化酶活性,探讨肠道细菌对小菌虫消化食物的影响。方法 采用传统细菌分离培养方法分离小菌虫肠道细菌,利用16S rDNA序列进行细菌分子鉴定;利用筛选培养基鉴别各细菌的产蛋白酶、脂肪酶、淀粉酶和纤维素酶活性。结果 在小菌虫肠道中分离到4种可培养细菌,分别是枯草芽胞杆菌（Bacillus subtilis）、肺炎克雷伯菌（Klebsiella pneumoniae）、Pseudocitrobacter faecalis和芽胞杆菌（Bacillus sp.）。其中,2种芽胞杆菌属细菌有产消化酶活性。枯草芽胞杆菌有产纤维素酶、淀粉酶和蛋白酶活性;芽胞杆菌仅有产蛋白酶活性,但产酶能力低于枯草芽胞杆菌。结论 小菌虫肠道细菌中可培养细菌结构简单,但其中的芽胞杆菌属细菌有产纤维素酶、淀粉酶和蛋白酶能力,说明小菌虫肠道中的2种芽胞杆菌属细菌可能有协助小菌虫进行食物消化的功能。     

Dissimilatory Fe(III) reduction by an electrochemically active lactic acid bacterium phylogenetically related to Enterococcus gallinarum isolated from submerged soil

AIMS: The isolation and identification of a glucose-oxidizing Fe(III)-reducing bacteria (FRB) with electrochemical activity from an anoxic environment, and characterization of the role of Fe(III) in its metabolism. METHODS AND RESULTS: A Gram-positive (Firmicutes), nonmotile, coccoid and facultative anaerobic FRB was isolated based on its ability to reduce Fe(III). Using the Vitek Gram-positive identification card kit and 16S rRNA gene sequence analysis, the isolate was identified as Enterococcus gallinarum, designated strain MG25. On glucose this isolate produced lactate plus small amounts of acetate, formate and CO2 and its growth rates were similar in the presence and absence of Fe(O)OH. These results suggest that MG25 can couple glucose oxidation to Fe(III) reduction, but without conservation of energy to support growth. Cyclic voltammetry showed that strain MG25 was electrochemically active. CONCLUSIONS: An electrochemically active and FRB, E. gallinarum MG25, was isolated from submerged soil. Fe(III) is used in the bacterial metabolism as an electron sink. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report concerning the electrochemical activity of glucose-oxidizing FRB, E. gallinarum. This organism and others like it could be used as new biocatalysts to improve the performance of a mediator-less microbial fuel cell.     

Effect of pheromone induction on transfer of the Enterococcus faecalis plasmid pCF10 in intestinal mucus ex vivo

The effect of synthetic sex pheromone on pheromone-inducible conjugation between the isogenic Enterococcus faecalis strains OG1RF and OG1SS was investigated in (i) Todd-Hewitt broth medium and (ii) intestinal mucus isolated from germ-free rats. In broth, the presence of synthetic pheromone cCF10 had no detectable effect on the transfer kinetics observed for the tetracycline resistance encoding plasmid pCF10. In mucus, presence of the same pheromone significantly increased the transfer efficiency observed during the first 2 h of conjugation, while the effect was less pronounced later in the experiment. We suggest that due to differences in diffusion rates and medium-binding of the pheromones, the effect of the synthetic cCF10 was immediately dominated by the effect of pheromones produced by the recipient E. faecalis strain in broth, while this happened later in mucus.     

Characterization of vanA-type Enterococcus faecium isolates from urban and hospital wastewater and pigs

Aims:  In this study we analysed urban, hospital wastewater and pig faeces samples to investigate the presence of vancomycin-resistant Enterococcus faecium strains (VREF) and to determine potential links among the strains originating from the above sources and VREF strains causing clinical infections.
Methods and Results:  Urban, hospital wastewater and pig faeces exhibited high VREF prevalence of 52%, 87% and 85%, respectively. Pulsed field gel electrophoresis (PFGE) clustering of VREF genotypes as well as discriminant analysis of antibiotic resistance patterns of VREF strains revealed their source specificity while strains isolated from hospitalized humans were genetically distinct.
Conclusions:  PFGE genotypes and antimicrobial resistance patterns in VREF isolates are distinguishable by each sample origin. The observed high genetic diversity of VREF suggests horizontal transfer of genetic elements among VREF. Phenotypic and genotypic data indicate that VREF isolates of hospital-treated wastewater might pass to the urban wastewater system.
Significance and Impact of the Study:  This study provides information to understand the origin and the mechanism of circulation of vancomycin resistance in food animals and wastewater treatment plants for minimizing the risk of transmission of VRE in human population.     

Characterization of new insertion-like sequences of Enterococcus hirae and their dissemination among clinical Enterococcus faecium isolates

Sequence analysis of different fragments that hybridized with a 4.5-kb EcoRI fragment originally cloned from Enterococcus hirae ATCC 9790 showed 66% homology to IS-like sequences found in staphylococci and lactococci. We tested several enterococcal ATCC strains and found that only E. hirae ATCC 9790 and Enterococcus faecium ATCC 19434 hybridized with the IS-like sequence. Moreover, we wanted to investigate the dissemination of this new IS among E. faecium strains. We analyzed 131 clinical E. faecium isolated in Italy and the USA for the presence of the IS and we found its presence in more than 63% of the isolates. The hybridization patterns obtained vary considerably between unrelated strains and allow further classification among ribotype-grouped species.     

Occurrence of virulence factors among human intestinal enterococcal isolates

AIMS: The aim of this study was to investigate the frequency of enterococcal virulence factors among human intestinal Enterococcus faecalis strains and to find out whether the pattern differs from that seen in published reports on food and clinical isolates. METHODS AND RESULTS: The E. faecalis isolates were cultured from human faecal samples obtained from five ulcerative colitis patients in remission phase. The species identification was based on API120 strips and species-specific PCR primers. The isolates were further characterized using the pulsed-field gel electrophoresis. The presence of seven different known enterococcal virulence factors among the confirmed E. faecalis isolates were screened using PCR techniques and published primers. CONCLUSIONS: Among the 35 isolates representing nine different pulsotypes the most frequent virulence factors were cpd (33 isolates), agg (25 isolates), gelE (22 isolates) and esp (15 isolates). No complete sets of genes associated for the production of functional cytolysin were encountered indicating that intestinal enterococci may differ in this respect from clinical strains. SIGNIFICANCE AND IMPACT OF THE STUDY: According to the results, the commensal enterococcal strains appear to differ from clinical isolates in their complement of presumed virulence factors.     

Anaerobic transformation of quercetin-3-glucoside by bacteria from the human intestinal tract

From human feces two phenotypically different types of bacteria were isolated on quercetin-3-glucoside as carbon and energy source. Isolates of one type were identified as strains of Enterococcus casseliflavus. They utilized the sugar moiety of the glycoside, but did not degrade the aglycon further. The sugar moiety (4 mM) was fermented to 5.5 ± 2.1 mM formate, 2.1 ± 0.7 mM acetate, 1.6 ± 0.3 mM l-lactate, and 1.3 ± 0.4 mM ethanol. The second type of isolate was identified as Eubacterium ramulus. This organism was capable of degrading the aromatic ring system. Growing cultures of Eubacterium ramulus converted 5 mM quercetin-3-glucoside to 1.7 ± 0.6 mM 3,4-dihydroxyphenylacetic acid, 7.6 ± 1.0 mM acetate, and 4.0 ± 0.4 mM butyrate. Molecular hydrogen, 3,4-dihydroxybenzaldehyde, and ethanol were detected in small amounts. Phloroglucinol was a transient intermediate in the breakdown of quercetin-3-glucoside. Eubacterium ramulus did not grow on the aglycon quercetin or the ring-fission intermediate phloroglucinol, but cleaved the flavonoid ring system when glucose was present as a cosubstrate. The most probable number of quercetin-3-glucoside-degrading bacteria determined in nine human fecal samples was 10⁷–10⁹/g dry mass. Isolates from these experiments were all identified as Eubacterium ramulus. Received: 9 July 1998 / Accepted: 10 November 1998     

Adherence to host extracellular matrix and serum components by Enterococcus faecium isolates of diverse origin

Enterococcus faecium has emerged as an important cause of nosocomial infections over the last two decades. We recently demonstrated collagen type I (CI) as a common adherence target for some E. faecium isolates and a significant correlation was found to exist between acm -mediated CI adherence and clinical origin. Here, we evaluated 60 diverse E. faecium isolates for their adherence to up to 15 immobilized host extracellular matrix and serum components. Adherence phenotypes were most commonly observed to fibronectin (Fn) (20% of the 60 isolates), fibrinogen (17%) and laminin (Ln) (13%), while only one or two of the isolates adhered to collagen type V (CV), transferrin or lactoferrin and none to the other host components tested. Adherence to Fn and Ln was almost exclusively restricted to clinical isolates, especially the endocarditis-enriched nosocomial genogroup clonal complex 17 (CC17). Thus, the ability to adhere to Fn and Ln, in addition to CI, may have contributed to the emergence and adaptation of E. faecium , in particular CC17, as a nosocomial pathogen.     

RNA-Seq探索低水平利奈唑胺耐药粪肠球菌机制

为了探索低水平利奈唑胺耐药粪肠球菌机制,本研究采用Illumina HiSeq 4000高通量转录组测序技术对低水平利奈唑胺耐药粪肠球菌(P10748 MIC:8 mg/L)和利奈唑胺敏感粪肠球菌(3138 MIC:2 mg/L)进行测序及生物信息学分析,以标准菌株ATCC29212作为质量评估参考。利用GO和KEGG数据库对差异表达基因进行注释与富集分析,并利用荧光定量PCR对测序中的差异表达基因进行验证。结果显示,测序共获得了3.57 Gb有效数据,通过De novo拼接获得1 920条unigenes,总长度为2 122 210 bp,平均长度为1 105 bp。差异基因模式聚类分析显示共有150个显著性差异表达基因(FDR≤0.001,|log2 Ratio|≥1),其中141个上调,9个下调。其中生物膜形成和外排泵相关基因esp、optrA、fexA在耐药菌中显著上调。GO功能注释结果显示,催化活性、代谢过程和细胞是注释最多的条目;Pathway富集分析发现,肽聚糖生物合成、缬氨酸亮氨酸和异亮氨酸的降解和硫代谢为显著性富集通路(p<0.05)。荧光定量PCR结果与测序结果基本一致。推测膜转运蛋白和生物膜形成可能在耐药机制中具有重要作用,为低水平利奈唑胺耐药肠球菌机制提供了可参考的耐药靶标。     

纳米党参合剂调整肠道微生态失衡防治多器官功能障碍综合征的研究

目的研究纳米党参合剂通过调整菌群失调,修复肠屏障功能,控制细菌、内毒素易位,对多器官功能障碍大鼠肠源性感染及内毒素血症(IETM)进行防治。方法采用肠缺血再灌注的方法制造多器官功能障碍综合征(MODS)模型,分别以纳米中药、常态中药对动物进行灌胃治疗。取肠内容物做无菌培养,内毒素、sIgA的测定,CD4+、CD8+T细胞计数。结果各治疗组通过扶植肠道有益菌、抑制有害菌的生长繁殖并促进其排泄,保护肠黏膜屏障,明显降低细菌易位,控制内毒素血症,改善相关免疫指标。结论党参合剂从调整微生态失调的角度来防治MODS取得较好效果,纳米中药效果更佳。     

Immunomodulatory properties of Enterococcus faecium JWS 833 isolated from duck intestinal tract and suppression of Listeria monocytogenes infection

The aim of this study was to evaluate the immunomodulatory properties of Enterococcus faecium JWS 833 (JWS 833) isolated from duck intestine and compare them to those of Lactobacillus rhamnosus GG (LGG), a proven immunity-enhancing probiotic. To investigate the immune-enhancing properties of JWS 833, production of nitric oxide (NO) and cytokines was measured in mouse peritoneal macrophages. In addition, a Listeria monocytogenes challenge model was used in the assessment. It was found that heat-killed JWS 833 stimulates mouse peritoneal macrophages to produce NO, interleukin-1 β (IL-1β) and tumor necrosis factor-α (TNF-α) and that oral administration of viable JWS833 enhances NO, IL-1β and TNF-α synthesis upon L. monocytogenes challenge. Moreover, mice fed with JWS 833 were partially protected against lethal challenge with L. monocytogenes. JWS 833 strain has significantly greater immunostimulatory properties than LGG. Moreover, JWS 833 strain partially protects mice against lethal challenge with L. monocytogenes. JWS 833, a novel strain of E. faecium isolated from duck intestine, is potentially a useful feed supplement for controlling pathogens and enhancing host immune responses.     

Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean

AIMS: Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. METHODS AND RESULTS: The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca(2+) (CaCO(3) or CaCl(2)). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6.0, whereas the highest cell growth was obtained at pH 7.0. CONCLUSIONS: Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca(2+) and pH) influenced the bacteriocin production. SIGNIFICANCE AND IMPACT OF THE STUDY: A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains.     

牙鲆肠道内产蛋白酶菌的分离与筛选

利用酪蛋白培养基和脱脂乳培养基,从健康牙鲆肠道筛选出25株产蛋白酶的益生菌,占健康牙鲆肠道菌群的17.7%,并利用福林-酚试剂法测定其酶活力.结果表明,25株菌具有产蛋白酶酶活力,其中E14、F1、G7和Y2-2株产酶活力特别强,有望成为益生菌在水产养殖上应用.     

羊源肠球菌溶血性的检测

[目的]了解羊源肠球菌溶血素的特性.[方法]以平板法、接触法、培养法、上清法及PCR法,对11株肠球菌临床分离株、30株健康羊分离株、肠球菌参考株和G群链球菌参考株进行了溶血性检测.[结果]接触法和上清法均不能检测到11株肠球菌临床分离株对兔血和羊血的溶血;平板法和培养法测得11株肠球菌临床分离株中,63.6%对兔血呈现β溶血,36.4%对羊血平板呈现α[溶血;基于检测cylA基因的PCR法,63.6%溶兔血菌能扩增出特异性条带,扩增产物序列与GenBank(L37110)中肠球菌同源性达99.3%.平板法测定30株健康羊分离株,初次分离培养53.3%对兔血β溶血,53.3%对羊血α溶血,43.3%对羊血β溶血,但二次传代后只有6%对兔血仍有溶血能力,且30株均不能检测到cylA.标准肠球菌对羊血平板有α溶血,而对兔血没有溶血性.[结论]提示肠球菌溶血性具有一定的溶血谱,不同检测方法检测的溶血情况不同;并且肠球菌溶血素必须在红细胞诱导下,通过细菌的生长繁殖产生;溶血素表型和基因型的检测不完全一致,对二者同时检测能提高肠球菌溶血素检测的准确性.     

党参合剂对多器官功能障碍综合征菌群失调及免疫功能的调整

目的研究党参合剂通过调整菌群失调,修复肠屏障功能,控制细菌、内毒素易位,提高机体免疫力,对多器官功能障碍大鼠肠源性感染及内毒素血症(IETM)进行防治。方法采用肠缺血再灌注的方法制造多器官功能障碍综合征(MODS)模型,分别以党参合剂、丽珠肠乐对动物进行灌胃治疗。取肠内容物、肝、脾、肠系膜淋巴结,门静脉血,分别做无菌培养,内毒素及sIgA的测定。结果各治疗组通过扶植肠道有益菌、抑制有害菌的生长繁殖并促进其排泄,保护肠黏膜屏障,明显降低了细菌易位,控制内毒素血症,改善了相关免疫指标。结论党参合剂从调整微生态失调的角度来防治MODS取得较好效果,优于丽珠肠乐。     

Study of two bacteriocins produced by Enterococcus casseliflavus and Ent. faecalis

AIMS: The antimicrobial activity of two plasmid-borne bacteriocins produced by Enterococcus casseliflavus IM 416K1 and Ent. faecalis IM 388C and their mating transferability were studied. METHODS AND RESULTS: Both bacteriocins showed antibacterial activity against taxonomically related micro-organisms and Listeria monocytogenes but differ for heat sensitivity, antimicrobial titre, molecular size and class of affiliation. The transferability by mating of the antibacterial properties from producers to Enterococcus faecalis JH2-2 revealed that the bacteriocin-phenotype was linked in both strains to genes located on a 34 MDa plasmid. This result was confirmed by loss of antibacterial activity and immunity after curing treatment. CONCLUSIONS: Restriction analysis has shown a different profile of the two conjugative plasmids. Enterocin 416K1 and Enterocin 388C could represent natural antilisterial agents to use in food technology. SIGNIFICANCE AND IMPACT OF THE STUDY: The transferability of the 34 MDa conjugative plasmids might be considered a possibility for the study of bacteriocins expression in bacterial hosts different from the native strains.     

神农架川金丝猴源肠道粪肠球菌的分离与鉴定

通过培养特征、形态观察、抑菌性实验,从神农架健康野生金丝猴肠道分离到8株抑菌效果明显的菌株;生理生化鉴定和16S rRNA基因序列同源性分析,该8株菌为粪肠球菌。毒力因子检测和动物急性毒性实验筛选出2株安全性良好的菌株,研究两菌株对胃肠道环境（低pH值、高胆盐）的耐受特性和生长情况。结果表明,粪肠球菌dlt7a和dlt7b株繁殖能力很强,无迟缓期,具有较强的耐受胃酸及肠道高胆盐环境的能力,且安全性好,具有较好的益生特性,可作为金丝猴微生态制剂的候选菌株。