阿魏酸对粪肠球菌和屎肠球菌产酪胺机制的影响

摘 要：研究阿魏酸对高产酪胺的粪肠球菌XL-M66和屎肠球菌XL-M76生长、基因表达以及产酪胺的影响。利用反转录实时荧光定量聚合酶链式反应技术分析2 株菌在阿魏酸作用下的酪氨酸脱羧途径相关基因表达情况,并使用高效液相色谱法检测2 株肠球菌培养48 h期间酪胺积累量。结果表明：未添加酪氨酸底物时,阿魏酸对酪氨酸脱羧酶（tyrosine decarboxylase,tyrDC）和酪氨酸/酪胺透性酶（tyrosine/tyramine permease,tyrP）基因的转录影响不大（P＞0.05）,但能促进酪氨酰-tRNA合成酶（tyrosyl-tRNA synthetase,tyrS）基因的转录（P＜0.05）。反之存在酪氨酸时,阿魏酸对tyrS基因表达的影响不大（P＞0.05）,却能显著抑制tyrDC和tyrP基因的表达（P＜0.05）。同时,阿魏酸能显著抑制粪肠球菌XL-M66和屎肠球菌XL-M76的生长（P＜0.05）,最终使得酪胺产量分别降低27.0%和19.9%。     

粪肠球菌(Enterococcus faecalis)和屎肠球菌(Enterococcus faecium)产生物胺交互作用研究

粪肠球菌和屎肠球菌是发酵香肠中常检出的2种主要的产酪胺和苯乙胺微生物。将粪肠球菌和屎肠球菌按照不同比例进行混合接种培养,发现在48h连续培养过程中,当粪肠球菌和屎肠球菌以1∶9比例混和接种培养时,体系pH值、细菌数量和酪胺生成量均显著低于其他各处理组;粪肠球菌有很强的产苯乙胺能力而屎肠球菌产苯乙胺能力较弱,当两者混合接种培养时,各混合体系的产苯乙胺水平相当,屎肠球菌产苯乙胺能力不受影响,而粪肠球菌产苯乙胺能力显著降低。     

干酪中肠球菌种群的遗传差异及万古霉素抗性基因分析

通过种、属引物特异性扩增、重复序列PCR（rep-PCR）技术和万古霉素抗性基因检测对新疆北疆地区干酪样品中球菌的遗传结构差异进行分析。结果表明,15份样品共分离52株肠球菌,包括31株耐久肠球菌(Enterococcus durans)、18株粪肠球菌(Enterococcus faecalis)和3株屎肠球菌(Enterococcus faecium)。依据rep-PCR遗传指纹带谱分析,52株菌可以聚类成7个群,其中4个由E. durans构成。24株肠球菌检测到了万古霉素抗性基因,17株E. durans为VanC2/C3型,4株E. faecalis为VanC1型,2株E. faecium为VanB型,只有1株E. faecium为VanA型。新疆北疆地区干酪中肠球菌种群分布较为广泛,地域之间优势种群和基因型不同,同种肠球菌菌株之间存在广泛的遗传差异。     

环境因素对屎肠球菌产酪胺的影响

酪胺是发酵食品中广泛存在的、毒性较强的一种生物胺。屎肠球菌作为发酵剂或污染菌广泛存在于发酵食品中,大多数具有产生酪胺的能力,成为发酵食品的潜在安全隐患。该研究以一株黄酒酒曲中分离的产酪胺屎肠球菌为对象,选取葡萄糖、酪氨酸、温度、pH值和酒精含量5个因素,研究这些因素对该菌生长和酪胺合成的影响。结果表明,葡萄糖或酪氨酸添加量对菌体的生长影响不大,温度、环境pH值和酒精含量对菌体生长的影响较显著。酪氨酸对菌体产酪胺促进作用明显,温度＜20 ℃,环境pH值＞5,酒精含量＞10%vol时都会减少酪胺的产生。     

食源性肠球菌荧光定量PCR检测方法的建立与评价

利用基因组序列比对分析等生物信息学方法发掘肠球菌新的属特异性靶点,根据42个候选靶点序列设计50对引物,结合普通PCR初筛和荧光定量PCR复筛,挑选特异性和灵敏度等检测性能最佳的引物,建立相应的荧光定量PCR检测方法,并对该方法应用于食品中肠球菌检测时的效果作出评价。分析结果显示,特异性最强的引物为EF1902,利用该引物建立的荧光定量体系检测肠球菌时均产生特异性扩增信号,而检测非肠球菌菌株时均无特异性扩增信号形成。经优化PCR体系后,该方法的基因组DNA检测灵敏度为13.78拷贝/PCR,纯培养物灵敏度为38.4 cfu/PCR。以肠球菌人工污染牛奶,当初始接菌量为2.63 cfu/mL时,只需增菌6 h即可用该方法检出肠球菌。对52份食品样品进行检测准确率为94.23%,证实了该方法可应用于食源性肠球菌的快速检测。综上所述,作者建立的肠球菌荧光定量PCR方法,特异性强且灵敏度高,可应用于食品中肠球菌的快速检测。     

传统香肠中产生物胺肠细菌和乳酸菌分离方法的研究

研究建立了单层培养,双层显色分离产生物胺肠细菌和乳酸菌的方法,并分离到96株产生物胺肠细菌和58株产生物胺乳酸菌。经变性凝胶梯度电泳分析(PCR-DGGE)和PCR扩增测序后与Genebank数据库比对得知,这154株产胺菌属于5种菌,分别为屎肠球菌、粪肠球菌、阴沟肠杆菌、大肠埃希杆菌和产气肠杆菌。并采用特异性引物证明了产生物胺基因的存在。     

粪肠球菌Enterococcus faecalis EC-12的万古霉素耐药基因检测

对粪肠球菌Enterococcus faecalis EC-12的万古霉素耐药基因进行了筛查。通过PCR方法,以7种典型肠球菌万古霉素耐药基因(vanA、vanB、vanC1、vanC2/C3、vanD、vanE和vanG)进行特异性扩增,结果均为阴性。采用PCR方法检测万古霉素耐药肠球菌简便、高效、准确。     

聚合酶链反应检测乳酸菌酪氨酸脱羧酶基因

酪氨酸脱羧酶与乳酸菌发酵食品中酪胺的产生密切相关。根据从GenBank中检索到的酪氨酸脱羧酶基因序列设计一对特异性引物，采用PCR技术对乳酸菌的基因组DNA片段进行扩增，以此建立以酪氨酸脱羧酶基因为靶的产酪胺乳酸菌的分子生物学检测方法。结果表明，供试12株乳酸菌中有9株菌扩增出1133bp DNA片段对其中3株菌的扩增产物进行DNA序列测定，测序结果采用国际互联网上NCBI的BLAST工具进行同源性检索分析，发现它与已知的Enteroeoeeus faecalis，Enterococcus faecium，Canmbacterium divergens的酪氨酸脱羧酶基因序列均高度同源，其中PLP(5’-磷酸吡哆醛)的结合位点高度保守，证明该扩增产物是酪氨酸脱羧酶的基因片段。应用该法与常规平板检测法比较显示，二者检测结果基本一致，表明本研究建立的PCR方法可作为一种快速、高度特异的检测产酪胺乳酸菌菌株的新方法。     

大连地区水产企业加工用水肠球菌检测和分析

根据水质检测ISO标准（ISO7899-2:2000）薄膜过滤法,对大连地区水产企业生产加工用水进行肠球菌分离培养和生化反应鉴定,并根据肠球菌的高度保守的特异性tuf基因,合成了特异性引物进行PCR扩增,并对扩增产物进行分析。在41份检测的样品中,肠球菌的检出率为14.6%,主要为群Ⅱ菌株。     

雪菊精油对希氏肠球菌产酪胺及相关基因表达的影响

探讨不同质量浓度雪菊精油对希氏肠球菌(Enterococcus hirae)N47产酪胺的影响机制.利用反转录实时定量聚合酶链式反应分析E.hirae在雪菊精油作用下酪氨酸脱羧途径相关基因的表达情况;利用高效液相色谱法检测不同质量浓度雪菊精油对E.hirae产酪胺的影响.并将E.hirae接入到含不同质量浓度雪菊精油的...     

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。     

大蒜精油对屎肠球菌和粪肠球菌产苯乙胺和酪胺的影响

本试验采用双倍试管稀释法,测定大蒜精油分别对高产苯乙胺和酪胺的E.faecium和E.faecalis的最低抑菌浓度(MIC)和最低杀菌浓度(MBC);并利用高效液相色谱法测定不同浓度的大蒜精油对两株高产苯乙胺和酪胺菌株的影响,从而明确大蒜精油对其产生物胺能力的抑制作用。结果表明:大蒜精油对供试菌株都具有较强的抑菌活性,大蒜精油对E.faecium的最大抑制率可达48.20%,对E.faecalis的最大抑制率为52.41%,且抑菌效果随大蒜精油浓度的增大而逐渐增强;在大蒜精油的添加量为1/2 MIC时,大蒜精油对E.faecium和E.faecalis的生长具有抑制作用;当大蒜精油浓度为0.025%时,能够显著(p0.05)降低供试菌株产苯乙胺和酪胺的含量,苯乙胺含量与空白组相比降低了26.61%,酪胺的降低了15.54%。说明大蒜精油对高产酪胺和苯乙胺的菌株具有显著(p0.05)抑制效果,从而减少了酪胺和苯乙胺的生成。     

Enterococcus populations in Pecorino Abruzzese cheese: biodiversity and safety aspects

The presence of enterococci in Pecorino Abruzzese cheese during ripening was evaluated. Counts were high, especially in fully ripened summer batches. Seventy strains were isolated and identified based on phenotypical and genotypical features as Enterococcus faecium (48.5%), Enterococcus faecalis (40%), and Enterococcus durans (11.5%), with the first species predominant in spring batches and the second predominant in summer batches. High biodiversity was revealed by random amplification of polymorphic DNA and a PCR assay, suggesting the presence of autochthonous strains. E. faecium isolates were the most resistant to the tested antibiotics, especially to erythromycin, chloramphenicol, and penicillin, but all strains were susceptible to vancomycin, as confirmed by the absence of vanA and vanB genes. The presence of some virulence determinants was investigated, revealing the diffusion of aggregation substance (asal) and gelatinase (gelE) genes in 37.5% of E. faecalis strains. However, none of the isolates produced gelatinase in vitro, suggesting the presence of silent genes. The virulence genes were absent in E. durans. Among E. faecium strains, only Lab 41/1 possessed gelE and asal, whose presence previously has been reported only in E. faecalis. Decarboxylating activity was revealed for phenylalanine (27% of the strains) and tyrosine (96%) but not histidine. The presence of a tyrosine decarboxylase-encoding gene was observed for all strains. A comparison of these results with those of previous studies of clinical and food isolates indicates that enterococci from Pecorino Abruzzese cheese have low pathogenic potential.     

Antibiotic resistance and incidence of Enterococcus species in Turkish white cheese

Thirty samples of Turkish white cheese were analysed for the presence of Enterococcus spp., and presumptive isolates were identified by morphological, cultural and biochemical tests and confirmed by the API 20 Strep System. Among the 101 isolates of Enterococcus spp., 62 were E. faecalis , 25 were E. faecium , seven were E. durans , five were E. mundtii and two were E. hirae/dispar . The resistance of the isolates to 13 different antibiotics was determined by the Kirby–Bauer disc diffusion test. Resistance to streptomycin, erythromycin, oxacillin and vancomycin was frequently found in these enterococci, and resistance to vancomycin was found in 96.8% of E. faecalis isolates and 76% E. faecium . The most effective antimicrobials were ampicillin (69.3% of isolates inhibited) and imipenem (76.3% of isolates inhibited). This examination confirmed the presence of enterococci, especially vancomycin-resistant strains, in Turkish white cheese, indicating poor sanitary conditions during production and processing and a significant health risk for consumers.     

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety.     

产细菌素屎肠球菌TRS5特性分析及其编码基因的扩增

屎肠球菌TRS5在37℃、p H 6.5的MRS培养基中经过24 h的培养后,其细菌素生成量达到最大。培养基中添加胰蛋白胨或葡萄糖有利于促进TRS5细菌素的生成,而添加麦芽糖、乳糖或甘露糖(20 g/L)后细菌素活性减少50%。外源添加5 g/L的甘油和吐温-80会抑制TRS5细菌素的产生,而添加K_2HPO_4或VB_1、VB_2、VB_6、VC则对细菌素的生成没有影响。药敏实验证实屎肠球菌TRS5对红霉素、氯霉素、万古霉素、替考拉宁、四环素、青霉素敏感。聚合酶链式反应及测序结果证实屎肠球菌TRS5含有肠球菌素enterocin P和类L50的结构基因。     

Relation of Enterococcus faecalis and Enterococcus faecium isolates from foods and clinical specimens

Clinical Enterococcus faecalis (n=65) and Enterococcus faecium (n=12) blood isolates from three Swiss hospitals were characterized with testing for resistance to antimicrobial agents, pulsed-field gel electrophoresis (PFGE), and the occurrence of virulence factors. Phenotypic determination of resistance to antimicrobial agents resulted in 20% of E. faecalis isolates showing a triple resistance against chloramphenicol, tetracycline, erythromycin, and seven isolates (two E. faecalis and five E. faecium) exhibiting a multiresistance against five or more antimicrobials. One isolate each of E. faecalis and E. faecium showed vancomycin resistance. All isolates contained at least two of the nine tested virulence genes (agg, gelE, cyl, esp, efaAfs, efaAfm, cpd, cob, and ccf). Phylogenetic analysis of the PFGE profiles identified several small clusters within E. faecalis isolates, one of which included isolates of all three hospitals. Fifty-six (73%) isolates occurred as unique, patient-specific clones. Several PFGE types were associated with shared features in their resistance patterns, indicating spread between and within wards. Finally, enterococci from this study and previous isolates from cheeses were examined by PFGE typing. The comparison of PFGE profiles from human and food isolates resulted in clusters of genetically strong related strains, which suggests high similarities of the enterococcal community composition of these two environments. A possible spread of the enterococcal isolates through the food supply cannot be excluded.     

Multiple-antibiotic resistance of Enterococcus faecalis and Enterococcus faecium from cecal contents in broiler chicken and turkey flocks slaughtered in Canada and plasmid colocalization of tetO and ermB genes

This study was conducted to characterize the antimicrobial resistance determinants and investigate plasmid colocalization of tetracycline and macrolide genes in Enterococcus faecalis and Enterococcus faecium from broiler chicken and turkey flocks in Canada. A total of 387 E. faecalis and E. faecium isolates were recovered from poultry cecal contents from five processing plants. The percentages of resistant E. faecalis and E. faecium isolates, respectively, were 88.1 and 94% to bacitracin, 0 and 0.9% to chloramphenicol, 0.7 and 14.5% to ciprofloxacin, 72.6 and 80.3% to erythromycin, 3.7 and 41% to flavomycin, 9.6 and 4.3% (high-level resistance) to gentamicin, 25.2 and 17.1% (high-level resistance) to kanamycin, 100 and 94% to lincomycin, 0 and 0% to linezolid, 2.6 and 20.5% to nitrofurantoin, 3 and 27.4% to penicillin, 98.5 and 89.7% to quinupristin-dalfopristin, 7 and 12.8% to salinomycin, 46.7 and 38.5% (high-level resistance) to streptomycin, 95.6 and 89.7% to tetracycline, 73 and 75.2% to tylosin, and 0 and 0% to vancomycin. One predominant multidrug-resistant phenotypic pattern was identified in both E. faecalis and E. faecium (bacitracin, erythromycin, lincomycin, quinupristin-dalfopristin, tetracycline, and tylosin). These isolates were further examined by PCR and sequencing for the genes encoding their antimicrobial resistance. Various combinations of vatD, vatE, bcrR, bcrA, bcrB, bcrD, ermB, msrC, linB, tetM, and tetO genes were detected, and ermB, tetM, and bcrB were the most common antimicrobial resistance genes identified. For the first time, plasmid extraction and hybridization revealed colocalization of tetO and ermB genes on a ca. 11-kb plasmid in E. faecalis isolates, and filter mating experiments demonstrated its transferability. Results indicate that the intestinal enterococci of healthy poultry, which can contaminate poultry meat at slaughter, could be a reservoir for quinupristin-dalfopristin, bacitracin, tetracycline, and macrolide resistance genes.