Enterococcus faecalis and Enterococcus faecium isolates from milk,beef, and chicken and their antibiotic resistance

The occurrence and antibiotic resistance of enterococci, especially Enterococcus faecalis and Enterococcus faecium, in milk, beef, and chicken in Gaborone, Botswana, were studied. Enterococci were isolated from these sources with the use of bile esculin agar and identified with API 20 Strep kits. Antibiotic resistance was determined by the disk diffusion method. The antibiotics tested were vancomycin, teicoplanin, ampicillin, tetracycline, and cephalothin. Among the 1,467 enterococci isolated from the samples, E. faecalis (46.1%) and E. faecium (29.0%) were found to be the predominant species. Other enterococcal species made up 25% of the isolates. More than 96 and 97% of the E. faecalis and E. faecium isolates, respectively, were found to be resistant to ampicillin. Almost 34, 27.3, and 22.4% of the E. faecalis isolates from milk, beef, and chicken, respectively, were also resistant to cephalothin. The percentages of E. faecium isolates that were found to be resistant to cephalothin were 32.8, 16.9, and 17.3% for milk, beef, and chicken, respectively. Resistance to vancomycin was widespread. It was found that 18.8, 7.8. and 13.1% of the E. faecalis isolates from milk, beef, and chicken samples, respectively, were resistant to vancomycin. In contrast, 32.8, 24.7, and 30.7% of the E. faecium isolates from milk, beef, and chicken samples, respectively, were resistant to vancomycin. Isolates that were resistant to multiple drugs were found in relatively large numbers.     

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。     

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.     

屎肠球菌源谷氨酸脱羧酶的制备及其酶学性质研究

为了开发谷氨酸脱羧酶(glutamate decarboxylase,GAD),以Enterococcus faecium为gadB基因供体、纤维素结合域(cellulose-binding domain,CBD)为亲和标签,利用内含肽DnaB自剪切作用分离GAD,对融合酶CBD-DnaB-GAD的构建、表达、GAD纯...     

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。     

产酪胺粪肠球菌和屎肠球菌PCR检测方法的建立

目的：建立快速简便地检测产酪胺粪肠球菌(Enterococcus faecalis)和屎肠球菌(Enterococcus faecium)的PCR方法。方法：将粪肠球菌和屎肠球菌的酪氨酸脱羧酶基因与GenBank数据库中已公布的细菌的酪氨酸脱羧酶基因进行比对,根据它们的非保守序列,分别设计粪肠球菌和屎肠球菌的特异性引物,建立检测产酪胺粪肠球菌和屎肠球菌的PCR方法。结果：根据非保守序列,分别设计粪肠球菌和屎肠球菌的特异性引物,用27株细菌对这两对引物分别进行反复验证,结果显示,所设计的两对引物都只对其目的菌株产生特异性扩增,对其他菌株没有扩增,方法的检测限可达到1.0×102CFU/mL。结论：本方法具有良好的特异性、稳定性和灵敏性,可用作食品中产酪胺粪肠球菌和屎肠球菌的检测。     

Comparison of the incidence of virulence determinants and antibiotic resistance between Enterococcus faecium strains of dairy, animal and clinical origin

Enterococci are part of the dominant microbiota of several dairy products. They are also present in the gut of humans and animals. Their presence in traditional raw milk cheeses is probably due to faecal contamination of milk during milking. Due to their importance as a cause of nosocomial infections, enterococci are acquiring increased significance. Such infections are becoming more and more difficult to treat as resistance to antibiotics increases. The aim of this investigation was to compare the potential virulence of Enterococcus faecium isolated from different ecological habitats and to establish if strains isolated from dairy products should really be considered as potential pathogens. In the present work, the antibiotic resistance pattern of 40 E. faecium strains isolated from dairy products, 26 E. faecium isolated from ewes' faeces and 28 clinical isolates of the same species was studied, and checks were made to see if known virulence determinants were present. Resistance to 12 different antibiotics commonly used in the treatment of human infections was tested using the broth microdilution method as described by the NCCLS. In addition, polymerase chain reaction (PCR) tests were carried out to see if genes for vancomycin resistance were present. The presence of the aggregation substance (AS) gene, the surface protein gene esp, the accessory colonisation factor ace, the Enterococcus faecalis endocarditis antigen efaA and the gelatinase gelE gene, which are involved in the virulence of enterococci, were also tested by PCR. The results of this study clearly indicate that E. faecium strains isolated from both cheese and sheep faeces are less pathogenic than those isolated from clinical samples. A similar pattern of resistance to antibiotics was observed in both dairy and animal strains. It was also found that there was difference in the kind of virulence determinants present in dairy and clinical isolates, while no virulence traits were found in sheep faeces strains. The results of this study suggest that E. faecium from traditional Sardinian raw milk cheeses should not be considered to be the main source of untreatable nosocomial enterococcal infections in humans in the island of Sardinia.     

屎肠球菌和枯草芽孢杆菌混合培养条件的优化

以屎肠球菌和枯草芽孢杆菌为研究对象,以MRS培养基为基础培养基,采用单因素试验、Box-Behnken实验设计对屎肠球菌和枯草芽孢杆菌的混合培养进行优化。结果表明:初始p H、装液量和氮源对活菌数影响显著。最优条件为:初始p H 6.5、装液量50/500 m L、氮源用量35 g/L、接种比1∶12、转速180 r/min,在此条件下活菌数达到6.99×1010 CFU/m L,比优化前提高了5倍,屎肠球菌菌株和枯草芽孢杆菌菌株的活菌数分别达到4.58×1010 CFU/m L和2.41×1010CFU/m L。     

屎肠球菌冷冻干燥保护剂筛选及稳定性比较研究

屎肠球菌在微生态产品中的应用效果受产品活菌量、贮存稳定性影响明显,为寻找适于屎肠球菌规模化生产的冻干保护剂,本研究通过正交试验设计,及屎肠球菌冷冻干燥保护剂4℃、37℃贮存稳定性比较,优化适合屎肠球菌的冻干保护剂配方为:脱脂乳5g/100mL、蔗糖1g/100mL、麦芽糊精5g/100mL。采用此保护剂配方制备的屎肠球菌冻干样品,在铝箔袋密封包装、温度37℃、相对湿度75%条件下存放4周,菌存活率为97.90%。     

屎肠球菌对广式腊肠蛋白质水解程度的影响

研究了分离自广式腊肠中的两株屎肠球菌AD4和AD8对广式腊肠中蛋白质组成的影响。结果表明,广式腊肠烘烤过程中可溶性氮和氨基酸态氮含量总体呈下降趋势,同时大分子肽发生了降解,小分子肽所占比例上升;但受工艺影响,广式腊肠蛋白质降解比较弱;接种屎肠球菌对广式腊肠的可溶性氮和氨态氮的影响不显著,对肽的分布有一定影响。烘烤结束时接种AD4和AD8,腊肠中小于10000Da的肽段占总肽比例分别增加了9·6%和8·2%。     

Genetic diversity of Enterococcus faecium isolated from Bryndza cheese

One hundred and seventy-six Enterococcus faecium isolates from Slovak dairy product Bryndza were tested for the presence of plasmid DNA. Eighty-two isolates were positive and their plasmid DNA was isolated and digested by EcoRI and HindIII restriction endonucleases. The patterns obtained were compared with those obtained after pulsed-field gel electrophoresis of macrorestriction fragments (PFGE), (GTG)(5)-PCR and ERIC-PCR. All these molecular approaches were applied for the study of genetic variability and determination of strain relatednesses among plasmid-positive isolates of E. faecium. In general, all methods revealed a considerable genetic diversity of E. faecium isolates. Plasmid profiling and ERIC-PCR have offered a higher resolution than PFGE and (GTG)(5)-PCR.     

屎肠球菌纤维素结合域谷氨酸脱羧酶构建及其酶学性质

为了开发可用于高效固定化的优质谷氨酸脱羧酶(glutamate decarboxylase,GAD,EC 4. 1. 1. 15),以Enterococcus faecium GDMCC60203为gad B基因供体,对纤维素结合域(cellulose-binding domain,CBD) GAD融合酶(CBD-GAD)的构建及其酶学性质进行了探讨。结果显示:构建的含pRPOCB-Efagad B重组质粒的Escherichia coli GDMCC 60445可高效表达CBD-GAD,120 m L发酵液制备的纤维素固定化CBD-GAD的活力为(347. 93±27. 63) U/g;在无氨苄青霉素LB培养基中连续培养5批,E. coli GDMCC 60445仍可稳定表达CBD-GAD;经一步纯化,CBD-GAD在SDS-PAGE电泳上呈单一条带,分子质量约为74. 02 k Da; CBD-GAD最适反应p H和温度分别为4. 6和60℃,在p H 4. 8～5. 6和4～40℃较稳定,仅对L-谷氨酸具有催化活性,Km和Vmax分别为10. 58 mmol/L和3. 83μmol/(m L·min),其催化反应不受底物和产物抑制。重组菌株稳定,CBD-GAD酶学性质优良,有望应用于γ-氨基丁酸和D-谷氨酸工业化生产。     

Isolation and characterization of tyramine-producing Enterococcus faecium strains from red wine

Enterococcus faecium strains were isolated from red wines undergoing malolactic fermentation and identified by comparison of their 16S rDNA gene sequences with those included in the GenEMBL Databases. The tyrosine decarboxylase gene was identified in all the strains analysed by PCR using gene-specific primers and the ability to produce tyramine in a synthetic media was analysed by RP-HPLC. Survival of an E. faecium strain was also evaluated in microvinification assays using two different musts with different ethanol concentrations (10% and 12% (v/v)). Tyramine production was monitored during the vinification trials. Our results suggest that E. faecium strains isolated from wine are able to produce tyramine and tolerate wine conditions following a pre-acidic stress.     

饲用屎肠球菌的筛选、鉴定及其生物学特性

分别采用肠球菌选择性培养基(EC)从健康婴儿的粪便、健康仔猪的肠道及粪便中分离筛选了4株屎肠球菌,分别命名为E fB、E f1-2、E f3-4、E f4-1,利用B iolog自动微生物分析系统鉴定结果表明,它们都是屎肠球菌,而且经16S rRNA基因序列测序比较分析,表明这4株菌与数据库中已发表的屎肠球菌16S rRNA序列(AccessionNo.AY675247)同源性均为99%,两者鉴定结果一致。试验表明,所筛得菌株生长速度较快,且对致病型大肠杆菌K88,K99,金黄色葡萄球菌,巴氏杆菌及沙门氏菌都有较强的抑制作用,其中对大肠杆菌K99的抑菌效果最好。屎肠球菌新菌种的鉴定与筛选为开发新型微生态类饲料添加剂提供了帮助。     

屎肠球菌扩大发酵的培养基优化

通过正交试验对扩大发酵中的碳源、氮源、番茄汁以及无机盐进行优化,得到最佳培养基为:蔗糖0.5%,豆粕4%,麸皮2.5%,番茄汁15mL/L,K2HPO4 0.2%,NaAC 0.3%,MgSO.47H2O0.08%,MnSO.44H2O 0.03%,结果表明,发酵液的菌体量和抑菌效果明显优于原培养基。     

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%。     

Dissemination of Enterococcus faecalis and Enterococcus faecium in a Ricotta Processing Plant and Evaluation of Pathogenic and Antibiotic Resistance Profiles

In this work, the sources of contamination by Enterococcus spp. in a ricotta processing line were evaluated. The isolated strains were tested for virulence genes (gelE, cylA,B, M, esp, agg, ace, efaA, vanB), expression of virulence factors (hemolysin and gelatinase), and the resistance to 10 different antibiotics. Enterococcus faecium and Enterococcus faecalis were subjected to discriminatory identification by intergenic spacer region (ITS)‐polymerase chain reaction and sequencing of the ITS region. The results showed that Enterococcus spp. was detected in the raw materials, environment samples and the final product. None of the 107 Enterococcus isolates were completely free from all virulence genes considered. A fraction of 21.5% of the isolates containing all of the genes of the cylA, B, M operon also expressed β‐hemolysis. Most of the isolates showed the gelE gene, but only 9.3% were able to hydrolyze gelatin. In addition, 23.5% of the observed Enterococcus isolates had the vanB gene but were susceptible to vancomycin in vitro. The dissemination of antibiotic‐resistant enterococci was revealed in this study: 19.3% of the E. faecium samples and 78.0% of the E. faecalis samples were resistant to at least one of the antibiotics tested. Sequencing of region discriminated 5 and 7 distinct groups among E. faecalis and E. faecium, respectively. Although some similarity was observed among some of the isolates, all E. faecalis and E. faecium isolates had genetic differences both in the ITS region and in the virulence profile, which makes them different from each other.     

酵母浸出物对屎肠球菌活菌数的影响

屎肠球菌(Enterococcus faecium)发酵饲料中的活菌数是保证其功能特性的关键因素,为了提高屎肠球菌发酵过程中的活菌总数,本研究以屎肠球菌为试验菌株,选用4种不同特性的酵母浸出物来考察其对屎肠球菌活菌数的影响。通过单因素试验,确定以酵母浸出物为唯一氮源培养屎肠球菌的活菌数,根据酵母浸出物检测指标找出影响屎肠球菌活菌的关键因子。试验结果表明,培养基中主要提供生长因子和微量元素的酵母浸出物对屎肠球菌的活性有明显的影响,其中游离核苷酸的含量是影响屎肠球菌发酵活菌数重要因子,通过外源添加一定量的游离核苷酸,厌氧培养12 h,获得的最终活菌数为176×10~8 CFU/m L,与未添加游离核苷酸相比,活菌数提高了将近40%。结果显示,在培养基中添加一定量的游离核苷酸可提高屎肠球菌在发酵液中活菌数量,为工业化生产高活菌数的饲用屎肠球菌提供参考和借鉴。     

屎肠球菌R2发酵豆腐黄浆水的代谢作用

为研究屎肠球菌R2对豆腐黄浆水的代谢作用,采用高效液相色谱法测定发酵过程中豆腐黄浆水中的低聚糖、有机酸及大豆异黄酮的组分和含量,对α-半乳糖苷酶和β-葡萄糖苷酶进行初步定位及活性测定,并测定发酵黄浆水对DPPH自由基的清除能力.结果表明,发酵过程中,豆腐黄浆水中的水苏糖、棉籽糖、葡萄糖、蔗糖和果糖均被屎肠球菌R2利用,...