典型菌落计数对金黄色葡萄球菌检测结果的影响

本文分析总结了盲样考核样品中金黄色葡萄球菌在Baird-Parker琼脂平板上的典型菌落计数对检验结果准确性的影响。结果显示当实验人员挑取典型菌落不正确或没有对典型菌落进行分类鉴定时结果将存在很大的差异。这表明金黄色葡萄球菌在Baird-Parker琼脂平板上出现不同类型典型菌落应通过分类计数鉴定,以减少典型菌落计数干扰,增加检测结果的准确性。     

食品中金黄色葡萄球菌计数方法的比较研究

比较食品中金黄色葡萄球菌计数的两种方法,寻找再现性较好的金黄色葡萄球菌计数方法。采用Baird-Parker琼脂倾注法与涂布法,对含一定浓度的金黄色葡萄球菌标准菌液、加菌样液进行计数,通过结果分析两种方法哪一种稳定性、重复性更好。金黄色葡萄球菌计数方法,利用Baird-Parker琼脂倾注法操作简便、结果稳定、再现性好。金黄色葡萄球菌计数方法,采用Baird-Parker琼脂倾注法,更加简便,便于推广。     

直接发酵法生产L-丝氨酸高产菌株的选育

为了解除高浓度L-丝氨酸对3-磷酸甘油酸脱氢酶的反馈抑制,进一步提高L-丝氨酸的产量,以一株能直接利用糖质原料发酵生产L-丝氨酸的谷氨酸棒杆菌SYPS-062-36为出发菌,经硫酸二乙酯逐级诱变处理,在D-丝氨酸抗性梯度平板上挑取正突变株,获得一株遗传性能稳定的高产菌株SYPS-062-33a-18,其可积累L-丝氨酸(11.0±0.25)g/L,较出发菌L-丝氨酸产量(6.65±0.23)g/L提高了65.4%。说明本研究不仅以D-丝氨酸作为抗性平板筛选得到高产L-丝氨酸的突变株,而且为下一步进行反向代谢工程的研究提供了良好的实验材料。     

TTC在乳及乳制品菌落总数检测中的应用

检测乳及乳制品菌落总数,为达到菌落在培养基中清晰可辨,在平板计数琼脂培养基中添加2,3,5-三苯基氯化四氮唑(TTC)。以灭菌乳、发酵乳、冰淇淋为样品基质,分别加入适当菌含量的大肠埃希氏菌、金黄色葡萄球菌、粪肠球菌、枯草芽胞杆菌工作菌悬液,制备适当浓度的TTC平板计数琼脂,同时以不添加TTC的平板计数琼脂作为对照,对同一样品进行菌落计数并同国标方法结果进行对比。TTC平板计数琼脂终浓度在0.001%~0.002%时能够使菌落显色,且与不添加TTC的对照组对比结果一致。在检测成分复杂的乳及乳制品样品时添加适宜浓度的TTC,有助于菌落的辨认,能够提高检验人员菌落计数的准确性,可用于检测乳及乳制品中菌落总数。     

不同品牌乳酸菌国标检验MRS琼脂培养基质量比对研究

目的 考察市售5个国产品牌和2个进口品牌的MRS琼脂培养基对乳酸菌的培养效果,比较国内外不同品牌培养基的产品质量。方法 采用螺旋涂布计数的方法,对3株GB 4789.28—2013中规定的乳酸菌和9株非国标规定乳酸菌(包括标准菌株和实验室分离菌株)在5个国产品牌和2个进口品牌的MRS琼脂培养基上的生长情况进行定量测定。通过比较不同品牌培养基对不同菌株的生长率,对各品牌培养基质量进行评价。结果 GB 4789.28标准菌株在不同品牌MRS琼脂培养基上的生长率存在明显差异,部分培养基存在质量问题。从益生菌粉中分离的嗜热链球菌ST-21在参比培养基生长能力良好,且不同品牌MRS琼脂培养基上的生长率存在显著差异。结论 本研究结果表明目前市售的国内外不同品牌MRS琼脂培养基质量存在较大差异;嗜热链球菌ST-21可有效评价不同品牌培养基质量差异,适宜用于MRS琼脂培养基的质量控制。     

快速测试片与国标方法测试脱盐乳清粉中金黄色葡萄球菌的对比

使用快速测试片法和国标法对脱盐乳清粉中金黄色葡萄球菌进行计数比对,对比两种方法的差异。结果表明,经过对脱盐乳清粉的本底试验确定,Baird-Parker平板和快速测试片在10?1的稀释度下均无菌落生长,菌落数均小于10 CFU/g,表明本实验中选用的脱盐乳清粉不对标准菌株的添加量产生影响。此外,对选取的金黄色葡萄球菌标准菌株进行的适应性试验结果表明,该脱盐乳清粉不会对本实验选取的标准菌株生长产生抑制。在此基础上,对18个经人工污染的（加入定量金黄色葡萄球菌标准菌株）脱盐乳清粉分别进行了快速测试片法和国标法的计数对比,重复性试验中重复性限在r≤0.25的偏差范围内,结果符合ISO 4833-1:2013对于重复性偏差的要求;经t检验验证两种方法无显著性差异。因此,快速测试片法可快速、准确、有效地应用于脱盐乳清粉的金黄色葡萄球菌计数的检测。     

金黄色葡萄球菌定性定量检测中显色培养基的效果评价

目的 评估显色培养基对金黄色葡萄球菌定性定量的检测效果。方法 对金黄色葡萄球菌标准菌株、食品样本分离菌株：30株金黄色葡萄球菌、30株其他葡萄球菌,5株常见食源性致病菌分别用金黄色葡萄球菌显色培养基和Baird-Parker培养基进行培养试验,并用国标法和全自动微生物鉴定药敏分析系统对培养结果进行确证鉴定。结果 试验定性结果显示金黄色葡萄球菌在显色培养基上呈紫红色,30株其他葡萄球菌呈蓝色或乳白色,5株常见食源性致病菌其中单核细胞增生李斯特氏菌呈蓝绿色、表皮葡萄球菌呈乳白色、其余3株不生长,可在直观上对几种食品中常见葡萄球菌进行有效区别,而且目标菌在显色培养基和Baird-Parker培养基上计数值无显著差异(P＞0.05),确证结果显示全自动微生物鉴定药敏分析系统结果与GB 4789.10-2016方法结果一致。结论 显色培养基能快速锁定疑似目标菌落,为溯源调查指明方向,定量计数直观有效,配合全自动微生物鉴定药敏分析系统更提高了对目标菌的鉴定效率,更适用于金黄色葡萄球菌定性定量检测。     

膜过滤法微生物检验简介——快速微生物检验法与检测

<正>快速微生物检验法1、从传统微生物检验技术的基础上发展而来的快速微生物检验方法已经使用了20多年,在微生物检验技术上并没有本质上的提高,只是在微生物的样品准备上和微生物的计数上相对于传统微生物检验方法有所提高A:SpirlPlating方法这种方法是将带有浓度梯度的菌悬液涂布在预先倾注好的选择性和非选择性的琼脂平板上,菌悬液从琼脂平板中心向周围浓度递减,将琼脂平板在合适的温度下经过适当时间的培养后通过激光计数器对琼脂平板上的菌落进行扫描计数,样品的准备和计数时间相对于传统的微生物检验法节省了时间,利用这种原理的快速微生物检验产品主要有AUTOPLATER(SpiralBiotech,Bethesda,Md.,U.S.A)。     

纳他霉素产生菌的诱变育种研究

采用硫酸二乙酯(DES)对纳他霉素产生菌恰塔努加链霉菌(Streptomyces chattanoogensis)SC21进行诱变处理,以添加了硫酸链霉素的平板进行筛选,选育出一株正突变株SC216,在同等发酵条件下,纳他霉素产量达到1.262g/L,比出发菌株提高了1.31倍,连续5次传代,其产量稳定在1.2g/L左右,性状无大突变,表明该菌株遗传性状稳定.     

GB 4789.28食品微生物学检验用培养基和试剂中沙门氏菌检验用培养基质控菌株的筛选

该研究根据《GB 4789.28-2013 食品安全国家标准 食品微生物学检验 培养基和试剂的质量控制》（以下简称GB 4789.28-2013）的检测要求,从我国菌种库中筛选出1株鼠伤寒沙门氏菌可等效于GB 4789.28-2013中的标准菌株（ATCC14028）,用于评价培养基的质量。该研究联合15家验证机构用7株不同分离源的鼠伤寒沙门氏菌,依据GB 4789.28-2013的检验方法验证不同品牌的培养基,将生长率、选择性与ATCC41028一致性最高的菌株选为标准的等效质控菌株。为确保结果的可重复性和一致性,各实验均采用同品牌同批次的培养基进行验证,每株菌株均由至少5家单位进行验证。综合生长率与选择性的结果,ATCC41028在各品牌培养基上均可良好生长,CMCC(B)50976与ATCC41028结果的一致性最高,而CMCC(B)50976最难进行培养或分离。同时,结果显示,不同品牌、不同人员、不同实验室环境下进行的培养基验证结果均有差异。工作组从7株鼠伤寒沙门氏菌中筛选出CMCC(B)50976作为GB 4789.28的质控菌株,可等效于国外的标准菌株（ATCC14028）,新菌株更具有代表性,且减少了国外菌株运输过程中的生物风险。     

Evaluation of molecular methods to determine enterotoxigenic status and molecular genotype of bovine, ovine, human and food isolates of Staphylococcus aureus

This study evaluated the use of PFGE and single enzyme AFLP techniques for the determination of the genetic relationships between Staphyloccocus aureus isolates from human, bovine, ovine and food related sources and reports the prevalence of 'classic' (sea to see) and 'new' (seg, seh, sei, sej, sem, sen and seo) staphylococcal enterotoxin (se) genes in 92 S. aureus strains. A sub-set of the se genotyping results was confirmed by ELISA and the presence of SE toxin determined in isolates from different sources. A 100% correlation was observed, between detection of enterotoxin genes sea-see and expression of corresponding enterotoxin proteins in vitro. The se genotyping data generated from 90 of the S. aureus isolates showed that many of the S. aureus strains producing identical se genotypes correlated with both AFLP and PFGE pattern types. However, single enzyme AFLP technique did not possess the discriminatory power of the PFGE method, but similar clonal relationships were observed by both techniques in many of the isolates tested. Results reported here include the first comprehensive study using a single enzyme AFLP technique to investigate the genetic background of S. aureus isolates from a wide distribution including animal, human and food related sources.     

Comparison of the Baird-Parker agar and 3M Petrifilm Staph Express Count plate methods for enumeration of Staphylococcus aureus in naturally and artificially contaminated foods

The recently developed 3M Petrifilm Staph Express Count plate (PFSE) method was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual's Baird-Parker agar spread plate (B-P) method for enumeration of Staphylococcus aureus in naturally contaminated, mechanically separated poultry (MSP; n = 92) and raw milk (n = 12). In addition, mozzarella and Parmesan cheeses and hot-smoked rainbow trout and chub were surface inoculated with a three-strain mixture of S. aureus, stored at 5 degrees C, and periodically analyzed with both methods for numbers of S. aureus. For naturally contaminated raw milk and MSP samples, the PFSE method yielded counts that were not significantly different (P > 0.05) from counts obtained using the B-P method. From raw milk and MSP samples, 60% (21 of 35) and 55% (124 of 226), respectively, of confirmed (DNAse-positive) isolates from PFSE plates were identified by further testing as S. aureus. Corresponding S. aureus identification rates for isolates forming typical colonies on B-P plates were 53% (19 of 36) and 50% (125 of 248). For both methods, other staphylococci composed the vast majority of tested isolates that were not identified as S. aureus. For inoculated hot-smoked fish, S. aureus counts from the PFSE method were not significantly different from counts from the B-P method. Compared to the B-P method, significantly lower numbers of inoculated S. aureus were recovered using the PFSE method in analyses of mozzarella cheese stored 28 and 42 days at 4 degrees C. The PFSE and B-P methods were not significantly different for inoculated cheeses at all other sampling times. DNAse-positive isolates from PFSE analyses of inoculated cheeses and smoked fish were identified as S. aureus 98% (51 of 52) and 86% (36 of 42) of the time, respectively, as compared with 100% (58 of 58) and 95% (40 of 42) of the time for typical B-P isolates. Overall, the PFSE and B-P methods appeared to perform similarly in enumeration of S. aureus in animal-derived foods.     

北京市食源性金黄色葡萄球菌耐药及分子分型研究

分析北京市食源性金黄色葡萄球菌对常用抗生素的耐药情况及其分子分型特征,预防和控制由该菌引起的食源性疾病,为临床合理用药提供依据。方法 收集2010—2012年北京地区食源性致病菌监测网分离到的金黄色葡萄球菌187株,采用微量肉汤稀释法检测金黄色葡萄球菌对8种常用抗生素的敏感性。采用脉冲场凝胶电泳(PFGE)进行分子分型。结果 116株金黄色葡萄球菌表现出对抗生素耐药,耐药率为62.03%,对抗生素的耐药率为红霉素(ERY)45.45%、苯唑西林(OXA)27.81%、克林霉素(CLI)20.86%、四环素(TET)13.90%、氯霉素(CHL)11.76%、环丙沙星(CIP)6.42%、甲氧苄啶/磺胺甲恶唑(TPM/SMZ)5.88%。其中耐甲氧西林金黄色葡萄球菌(MRSA)52株、对甲氧西林敏感的金黄色葡萄球菌(MSSA)135株。分离到的MRSA中34株菌表现出多重耐药。全部菌株共分为129种PFGE带型,优势带型不明显。结论 食源性金黄色葡萄球菌的耐药和多重耐药普遍存在,PFGE带型分布分散与耐药谱的关联性较差。     

一起食物中毒事件中产独特肠毒素表型的金黄色葡萄球菌病原学分析

目的对一起疑似为金黄色葡萄球菌所导致的食物中毒事件进行葡萄球菌肠毒素检测,结合金黄色葡萄球菌病原学分析,为明确食物中毒诊断提供依据。方法根据流行病学调查,采用ELISA方法对可疑食物进行葡萄球菌肠毒素检测,同时对可疑食物和患者呕吐物进行金黄色葡萄球菌分离,运用Vitek2 Compact全自动细菌鉴定仪和血浆凝固酶试验鉴定为金黄色葡萄球菌,采用脉冲场凝胶电泳(PFGE)对病原菌进行同源性分析,以ELISA方法对检出的金黄色葡萄球菌菌株进行肠毒素检测,用PCR方法对肠毒素基因进行分型。结果食物和患者样品中分别分离出2和11株金黄色葡萄球菌,PCR方法及ELISA方法对肠毒素分型结果显示,其中12株同时存在SEA、SEB、SED、SEE 4种肠毒素及相关基因,PFGE聚类分析显示,其中12株产肠毒素金黄色葡萄球菌具有高度同源性。结论本起食物中毒事件为具有独特肠毒素表型的金黄色葡萄球菌导致,在金黄色葡萄球菌中毒实验室调查过程中,肠毒素检测结合病原菌溯源分析可以为相关公共卫生事件提供科学依据。     

金黄色葡萄球菌食品分离株肠毒素基因分布及分型研究

目的 对2006-2009年上海市Staphylococcus aureus(S.aureus)食品分离株进行肠毒素基因检测和基因分型研究,以了解肠毒素基因的分布规律及S.aureus的流行特点.方法 利用PCR方法检测食品中金黄色葡萄球菌肠毒素基因,包括5种传统肠毒素基因(SEA-SEE)和4种新型肠毒素基因(SEG-SEJ);利用脉冲场凝胶电泳法对49株食品分离株进行基因分型.结果 本研究发现49株食品分离株中有19株含有肠毒素基因,16株含有传统肠毒素SEA和SEC,且SEC占93.8％,并检测到新型肠毒素SEG、SEI、SEJ和SEH.PFGE法基因分型显示5株菌不能被分型,其余44株可分为28个基因型,表现为基因型的多样性,且分离自不同时间的菌株具有相同的带型.结论 应加强食品中S.aureus的监测分析,为其引起的食物中毒的预防和控制提供科学依据.     

Characterization of Staphylococcus aureus isolated from powdered infant formula milk and infant rice cereal in China

Dry infant foods are not sterile and could be contaminated with various bacteria including certain pathogens. The aim of this study was to investigate the prevalence of Staphylococcus aureus in infant foods and to characterize these strains. A total of 367 infant food samples, including 143 samples of powdered infant formula milk (PIF) and 224 samples of infant rice cereal (IRC), were collected in the Shaanxi Province of China during the period of July to August 2010 and screened for S. aureus. All S. aureus isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and detection of genes encoding enterotoxins, exfoliative toxins, Panton-Valentine leukocidin (PVL), and toxic shock syndrome toxin 1. Among all the samples examined, sixteen of 143 PIF samples (11.2%) and 14 of 224 IRC samples (6.3%) were positive for S. aureus. From these positive samples, 29 S. aureus strains were isolated from PIF and 25 from IRC. Of these S. aureus isolates, 83.3% were resistant to at least one antimicrobial, 35.2% to three or more antimicrobials. Resistance was most frequently observed to erythromycin (75.9%), followed by ciprofloxacin (51.9%) and trimethoprim/sulfamethoxazole (27.8%), while significantly fewer isolates were resistant to gentamicin (22.2%), tetracycline (18.5%), or cefoxitin (3.7%). In addition, 63.0% of isolates were positive for one or more toxin genes tested. The three most predominant toxin genes were pvl (40.7%), seg (38.9%), and sec (18.5%), followed by sea (7.4%), seb (7.4%), sed (5.6%), and see (5.6%). The ets, tsst-1, seh, sei, and sej genes were not detected. A total of 39 PFGE patterns were generated among 51 selected food isolates. Our findings indicate that PIF and IRC in the Shaanxi province were contaminated with S. aureus, and many S. aureus isolates harbored multiple toxin genes and exhibited multiple antimicrobial resistance. In addition, these S. aureus isolates were genetically diverse. The presence of S. aureus strains in these infant foods poses a potential threat to infant health.     

Genotypic and phenotypic diversity of Lactococcus lactis isolates from Batzos, a Greek PDO raw goat milk cheese

The genotypic and phenotypic variability of 40 Lactococcus lactis isolates obtained from three cheese-making trials of Batzos cheese made one in each, winter, spring and summer was investigated. RAPD-PCR, plasmid profiling and PFGE were used to study the genetic variability and distinguish closely related isolates. Results showed a high degree of heterogeneity among strains. According to PFGE data, all strains except one were clustered together (at a similarity level of approximately 50%) with the L. lactis subsp. lactis reference strain and eleven groups of isolates consisting of 2-8 strains each were distinguished. Plasmid profiling results revealed that there were eight isolates lacking plasmids and nine having unique plasmids. Twenty-three isolates were allocated into six groups. There was an interesting similarity between the plasmid profiling groups and those formed according to PFGE. Clustering of strains according to RAPD-PCR was in agreement with results obtained by both plasmid profiling and PFGE for the majority of the strains. In addition, results obtained by molecular methods indicate a grouping of most of the strains according to the season of cheese production. All strains inhibited the growth of Escherichia coli O157:H7. Their ability to affect the growth of Yersinia enterocolitica, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Enterococcus faecalis was strain dependent. In 42.5% of the isolates high acidifying ability in milk after 24 h was recorded and these were isolates, mainly, from fresh cheese. The 75% of the isolates from winter cheese exhibited higher Lys- than Leu-aminopeptidase activity while the approximately 67% of the isolates from summer cheese showed higher Leu- than Lys-aminopeptidase activity. Their caseinolytic activity after growth in milk for 24 h was significant with preference for alpha(s)-casein degradation. The majority (90%) of the strains formed methanethiol from methionine and this ability was strain dependent. These results suggest that among the wild lactococcal population from Batzos cheese there are interesting strains appropriate to be used as starters for the dairy industry.     

一起由金黄色葡萄球菌引起食物中毒的实验室诊断

目的对一起食物中毒进行相关样品的实验室检测,针对分离病原菌进行分子分型溯源和耐药分析。方法对可疑食品(冰激凌、牛奶)样品、患者呕吐物和粪便标本使用国家标准或其他方法进行沙门菌、志贺菌、致泻大肠埃希菌、金黄色葡萄球菌、单核细胞增生李斯特菌、蜡样芽胞杆菌和诺如病毒检测。对检出的金黄色葡萄球菌进行肠毒素检测、脉冲场凝胶电泳(PFGE)分型和抗生素敏感性检测。结果从生产冰激凌剩余牛奶及冰激凌样品中检出金黄色葡萄球菌12株,患者呕吐物和粪便标本中检出4株,菌株均产生A+C+E混合型肠毒素,PFGE显示患者呕吐物、粪便、中毒同批冰激凌样品、生产冰激凌用牛奶中检出的金黄色葡萄球菌菌株同源性为100.0%;药敏结果显示,这16株菌株均对青霉素和红霉素耐药。结论本起事件是由冰激凌加工点使用了被金黄色葡萄球菌污染且产生大量肠毒素的牛奶生产冰激凌所致。建议监管部门加强对农村食品小作坊、牛奶供应站等生产经营场所的监管,预防类似事件的发生。     

2007—2016年四川省德尔卑沙门菌耐药与分子分型分析

目的 研究2007—2016年四川省德尔卑沙门菌分子分型和耐药趋势,掌握四川省德尔卑沙门菌污染状况,为暴发预警、溯源调查及抗生素使用策略提供参考数据。方法 运用脉冲场凝胶电泳(PFGE)和微量肉汤稀释法对2007—2016年四川省自临床病例、食品从业人员、食物中毒样品、养殖动物及零售食品中分离的106株德尔卑沙门菌进行分子分型及14种抗生素的敏感性测试。结果 106株德尔卑沙门菌对8类14种抗生素均有不同程度耐药,人源性和动物源性菌株对四环素、萘啶酸、氯霉素、复方磺胺的耐药率均大于20%。其中人源性菌株对头孢噻肟、环丙沙星、氨苄西林、氨苄西林/舒巴坦、头孢唑啉的耐药率均明显高于动物源性菌株;动物源性菌株对四环素、萘啶酸、氯霉素耐药率均明显高于人源性菌株。经Xba I酶切后106株菌共分为67个PFGE带型,不同年份的临床病例、食品从业人员及零售食品生猪肉分离株具有相同PFGE带型。结论 四川省德尔卑沙门菌分离株耐药率较高,并有逐年上升趋势。PFGE型别呈多样性,部分临床病例、食品从业人员分离株与生猪肉分离株PFGE具有相同带型。     

An outbreak of food poisoning due to egg yolk reaction-negative Staphylococcus aureus

An outbreak of staphylococcal food poisoning due to an egg yolk (EY) reaction-negative strain occurred in Japan. Twenty-one of 53 dam construction workers who ate boxed lunches prepared at their company cafeteria became ill, and eight required hospital treatment. The outbreak showed a typical incubation time (1.5-4 h with a median time of 2.7 h) and symptoms (vomiting and diarrhea) of staphylococcal food poisoning. Staphylococcus aureus, which produces staphylococcal enterotoxin (SE) A, was isolated from four fecal specimens of eight patients tested. Scrambled egg in the boxed lunches contained 20-40 ng/g of SEA, and 3.0 x 10(9)/g of viable S. aureus cells that produced this toxin. All isolates from patients and the food were EY reaction-negative, coagulase type II, and showed the same restriction fragment length polymorphism (RFLP) pattern. We concluded that the outbreak was caused by scrambled egg contaminated with EY reaction-negative S. aureus. In Japan, outbreaks of staphylococcal food poisoning are mainly caused by EY reaction-positive S. aureus, and EY reaction-negative colonies grown on agar plates containing EY are usually not analyzed further for detection of S. aureus. The present outbreak suggested that EY reaction-negative isolates should be subjected to further analysis to detect the causative agents of staphylococcal food poisoning.