校园周边熟食中金黄色葡萄球的分离及肠毒素基因检测

了解校园周边熟食中金黄色葡萄球菌的污染及其肠毒素基因的分布情况。采集校园周边农贸市场及其路边小摊的熟食样品89份,采用国家标准GB4789.10-2016和PCR方法进行金黄色葡萄球菌分离鉴定;采用PCR方法对分离菌株进行21种肠毒素基因检测。结果表明共分离出71株金黄色葡萄球菌,样品中金黄色葡萄球菌的污染率为79.78%,其中,农贸市场和路边小摊的熟食污染率分别为80.70%(46/57)和78.12%(25/32);检测的21种肠毒素基因中,sec、see、seh、sel、sep和ses未检出,其他15种基因型被检出,包括3种传统肠毒素基因和12种新型肠毒素基因。71株金黄色葡萄球菌中,46株菌株检出携带肠毒素基因,检出率为64.78%(46/71)。46株携带肠毒素菌株中,sex检出率最高为86.96%(40/46),sei和sem为32.61%(15/46),sen和seo为30.43%(14/46),set为21.74%,seg为13.04%,sea、seb和ser为10.87%,sej和seu为6.52%,sek和seq为4.34%,sed为2.17%。通过本研究发现校园周边农贸市场及路边小摊的熟食中金黄色葡萄球菌污染率和肠毒素基因携带率均较高,新型肠毒素基因检测率高于传统肠毒素基因型,研究结果为金黄色葡萄球菌食品安全提供参考依据。     

PCR检测食品中金黄色葡萄球菌肠毒素基因

目的:检测食品中金黄色葡萄球菌肠毒素基因谱携带情况,探讨与食物中毒发病相关的金黄色葡萄球菌基因类型。方法:取食品中检出的金黄色葡萄球菌进行分离培养鉴定,通过PCR技术检测样本18种不同型的肠毒素基因的携带情况,并进行统计学分析。结果:食品中检出的金黄色葡萄球菌肠毒素基因有较高的阳性检出率,主要检出sea、seb、sei、seg、sel、sem、sen等基因,其中sea基因检出率最高,达到77.27%;sei、seg、sem、sen基因具有相关性,检出率为9.09%;seb、sel基因的检出率为4.55%。结论:在食品中主要检出金黄色葡萄球菌肠毒素sea、sei、seg、sem、sen、seb、sel等基因,其中,sei、seg、sem、sen基因的相关性还有待进一步深入研究。     

食源性金黄色葡萄球菌肠毒素基因型分布研究

目的 采用PCR法扩增食源性金黄色葡萄球菌中肠毒素基因以了解该菌肠毒素基因携带情况,比较食物中毒和食品监测来源菌株中肠毒素基因检出率差异.方法 合成sea、seb、sec、sed和see五种肠毒素基因特异性引物,用常规PCR方法扩增食物中毒和食品监测来源菌株中各自肠毒素基因,同时采用mini-VIDAS检测食物中毒来源菌株中肠毒素.结果 110株菌株中有30株检出肠毒素基因,检出率为27.3％,肠毒素基因阳性菌株均只检出1种肠毒素基因.其中来自2起食物中毒的14株菌株均检出seb型肠毒素和相关基因,检出率为100％.来源于食品监测样本的96株菌株中有16株检出肠毒素基因,检出率为16.7％,包括sea型4株、seb型2株、sec型4株、sed型6株.结论 在宁波市食品监测中所分离的金黄色葡萄球菌所携带的肠毒素基因主要有sea、seb、sec和sed四型,而seb型肠毒素是引起金黄色葡萄球菌肠毒素所致食物中毒的主要因素.     

婴幼儿奶粉和米粉中金黄色葡萄球菌的分离鉴定及其毒素基因检测

目的：了解婴幼儿奶粉和米粉中金黄色葡萄球菌的污染及其毒素基因的携带情况。方法：采集陕西省18个地区不同品牌的婴幼儿奶粉143份和米粉224份,按国标法和PCR方法进行金黄色葡萄球菌分离鉴定;采用PCR方法检测ses基因、ets基因、tsst-1基因和pvl基因。结果：367份样品中检测出30个金黄色葡萄球菌污染样品,检出率为8.17%,其中奶粉11.19%(16/143)、米粉6.25%(14/224),分别从中分离鉴定出29株和25株金黄色葡萄球菌;从54株金黄色葡萄球菌中共检测到有64.8%(35/54)的菌株携带有毒素基因,其中奶粉72.4%(21/29)、米粉56.0%(14/25);奶粉分离株中检测到的毒素基因型有pvl、sea、seb、sed、seg、sea＋pvl、seb＋seg、seb＋sed＋seg、sec＋pvl、sec＋seg＋pvl和seg＋pvl,其检出率分别为10.3%(3/29)、3.4%(1/29)、3.4%(1/29)、3.4%(1/29)、6.9%(2/29)、6.9%(2/29)、6.9%(2/29)、3.4%(1/29)、3.4%(1/29)、13.8%(4/29)和10.3%(3/29);米粉分离株中检测到的毒素基因型有pvl、sed、seg、sea＋seg＋pvl、sec＋pvl、sec＋seg、sec＋see＋seg、sec＋see＋seg＋pvl、see＋pvl和seg＋pvl,其检出率分别为8%(2/25)、4%(1/25)、4%(1/25)、4%(1/25)、4%(1/25)、8%(2/25)、4%(1/25)、4%(1/25)、4%(1/25)和12%(3/25)。奶粉和米粉中都没有检测到ets、tsst-1和seh、sei、sej基因。结论：婴幼儿奶粉和米粉中均存在一定程度的金黄色葡萄球菌的污染,且多数菌株都携带一定的毒素基因,这对消费这些产品的婴幼儿身体健康构成潜在的危险。     

葡萄球菌肠毒素基因分型PCR检测技术的研究

通过生物信息学分析,设计了不同基因型肠毒素检测引物;应用含溶菌酶的碱裂解法提取葡萄球菌DNA及梯度PCR方法提高了肠毒素检出率和特异性.从国内28株葡萄球菌分离株中共检测出14种不同型的肠毒素基因(sea-see、tsst-1、seg-sel、sek-sel、sen-seo、seq-ser和seu),未检测到sei和sem基因.毒素基因携带率为16.67%,同时携带4种及以上毒素基因的菌株有11株,占39.29%,其中sek、seq和sea毒素基因在所研究菌株中分布最广,分别占到15.50%、14.30%和10.70%.结果表明不同葡萄球菌菌株间毒素型的关联度不同,sek与seq,seb和sed,sec、sei、sen和seu基因型呈现密切相关,各型肠毒素的分布还与菌株分离来源有着密切联系.所建立的PCR方法可用于金黄色葡萄球菌肠毒素基因分型和分布的研究.     

2005—2012年宁波市食源性金黄色葡萄球菌肠毒素基因和多位点序列分型研究

目的了解宁波市食源性金黄色葡萄球菌(S.aureus)肠毒素基因分布和分子分型特征。方法收集2005—2012年宁波市食品中金黄色葡萄球菌菌株,利用聚合酶链式反应(PCR)检测肠毒素A、B、C和D基因(sea,seb,sec和sed),对肠毒素基因阳性菌株利用多位点序列分型(MLST)进行分子分型。结果 2005—2012年共分离菌株190株,肠毒素基因阳性菌株为41株,阳性率为21.58%。4种肠毒素基因阳性率分别为7.37%(14/190)、5.26%(10/190)、8.95%(17/190)和5.79%(11/190),其中13株菌株具有两个及两个以上肠毒素基因。41株菌株可分为12个序列型(ST),以ST5、ST6、ST188和ST1为主,共占75.61%(31/41),在进化树上主要形成4个分支。结论 2005—2012年宁波市食品中金黄色葡萄球菌携带肠毒素基因频率较高,需加强监测。肠毒素基因阳性菌株与中国其他地区食品株在分子分型上存在较大差异。     

鸡肉生产中分离金黄色葡萄球菌的基因分型与耐药性分析

为探究鸡肉制品在加工过程中金黄色葡萄球菌的肠毒素基因分布、分子分型和耐药性情况,本研究对某鸡肉制品加工厂的生产加工过程进行取样,通过DNA提取、PCR扩增nuc基因对金黄色葡萄球菌进行分离鉴定,然后分析肠毒素基因分布情况,对获得带有毒素基因的菌株进行多位点序列分型（MLST）和耐药性研究。结果表明,260份样本中分离到38株金黄色葡萄球菌（其中携带肠毒素基因的菌株有24株）,检出率为63.16%;共检测到7种毒素基因型,其中sed携带率最高（60.53%）,依次为seg（26.32%）、see（21.05%）、sei（18.42%）、sec（10.53%）、sea（7.89%）、seh（5.26%）,携带两种及以上肠毒素基因的菌株有14株（36.84%）。24株携带毒素基因的菌株MLST分型都为ST型,分为3种ST分型,16株为ST7序列型（66.67%）;5株为ST5序列型（20.83%）;3株为ST464序列型（12.5%）。耐药性分析实验结果表明,抑菌效果最好的抗生素是万古霉素,38株菌株都对其敏感（100%）;绝大多数菌株都对抗生素有耐药性,依次为青霉素G（86.84%）、环丙沙星（60.53%）、克林霉素（55.26%）、四环素（52.63%）;对3-6种抗生素耐药的金黄色葡萄球菌菌株分别占18.42%、15.79%、23.68%、7.89%。研究结果表明鸡肉产品中存在金黄色葡萄球菌污染的情况,并且其中存在携带多种类型肠毒素基因的菌株,也存在具有多重耐药性的菌株。     

上海市闵行区初级农产品中金黄色葡萄球菌污染状况及毒力基因分布特征研究

了解上海市闵行区初级农产品中金黄色葡萄球菌的污染状况及毒力基因的携带情况。方法 按国标方法,采用科玛嘉显色培养基,对初级农产品中的金黄色葡萄球菌进行分离、生化鉴定,收集的菌株采用实时荧光PCR方法检测血浆凝固酶基因coa,耐热核酸酶基因nuc,粘附素基因clfA和肠毒素基因sea、seb、sec、sed、see。结果 206份食品中检出金黄色葡萄球菌43株,检出率为20.87%;43株金黄色葡萄球菌均含有coa、nuc和clfA基因,而see基因全部缺失,其余4个毒力基因则表现为部分缺失。结论 本地区初级农产品中存在金黄色葡萄球菌的污染,生肉禽类食品中金黄色葡萄球菌污染比较严重,应加强监督管理;金黄色葡萄球菌菌株均包含有coa毒力基因,可以作为一项检测其致病力的指标。     

2006—2011年西安市食品及食物中毒中金黄色葡萄球菌毒素基因分布及分型研究

了解2006—2011年西安市污染食品及食物中毒中金黄色葡萄球菌(S.aureus)分离株肠毒素(SEs)、杀白细胞素(PVL)、表皮剥落毒素(ETs)、毒性休克综合征毒素-1(TSST-1)等毒素基因的分布状况,并比较两种分离株在基因分布及分型上的差异。方法 采用多重PCR 法检测61株S.aureus(包括40株食品分离株和21株食物中毒分离株)sea、seb、sec、sed、pvl、eta、etb、tsst-1基因,其中sea、seb、sec、sed基因引物加入同一反应体系,剩余4对基因引物加入另一反应体系。结果 40株食品分离株中17株检出毒素基因(42.50%);21株食物中毒分离株中18株检出毒素基因(85.71%),食物中毒分离株毒素基因的检出率明显高于食品分离株(P<0.01)。食品分离株中主要流行的毒素基因为sea(25%)、eta(12.5%),未检测到携带etb、tsst-1基因的菌株;同时得到8种毒素基因型,主要流行的基因型为sea(10.00%)、sea+eta(7.50%)。食物中毒分离株中主要流行的毒素基因为sea(76.19%)、sec(28.57%),未检测到携带pvl基因的菌株;同时得到6种毒素基因型,主要流行的基因型为sea(42.86%)、sea+sec+tsst-1(14.29%)。结论 S.aureus食品分离株和食物中毒分离株在毒素基因的分布及分型上存在较大差异。     

市售凉皮中金黄色葡萄球菌的肠毒素基因、耐药特征检测及SPA分型

为监测并研究凉皮中金黄色葡萄球菌的污染情况,选取连锁超市、流动摊点、小餐馆和学校餐厅4?种销售渠道,在陕西杨凌地区进行为期一年的凉皮跟踪采样,共得样本432?个,金黄色葡萄球菌检出率为24.3%（105/432）。检测sea、seb、sec、sed、see、seg、seh、sei、sej?9?种肠毒素基因,sea携带率最高为96.2%（101/105）,see次之为64.8%（68/105）,还有部分seb和sec检出,检出率分别为54.3%（57/105）和49.5%（52/105）,seh检出率为1.0%（1/105）,sed、seg、sei、sej的检出率则均为0.0%（0/105）。研究针对青霉素、氨苄西林和苯唑西林等14?种常见药物进行耐药性实验,105?株来自阳性样本的金黄色葡萄菌全部具有耐药性,对青霉素和甲氧苄啶/磺胺甲恶唑耐受率为100.0%（105/105）,对头孢哌酮、环丙沙星、万古霉素和阿米卡星均敏感。此外,多重（n≥3）耐药率高达90.5%（95/105）,表型为青霉素-氨苄西林-甲氧苄啶/磺胺甲恶唑-阿莫西林/克拉维酸的耐药率最高,为61.9%（65/105）。检测105?株金黄色葡萄球菌的SPA型别,共包括4?种结果,优势型别为t701（81.9%,86/105）,其次是t441（16.2%,17/105）,t127和t796占比最少均为1.0%（1/105）。最终,发现该地区所售凉皮除学校餐厅外,其余3?种销售渠道的检出率均较高,且菌株具有较高的肠毒素基因携带率和耐药性,优势SPA型别为t701和t441,为相应监管部门提供一定理论指导。     

Molecular typing of Staphylococcus aureus isolated from Italian dairy products on the basis of coagulase gene polymorphism, multiple-locus variable-number tandem-repeat and toxin genes

Coagulase gene restriction fragment length polymorphism (RFLP), six-locus variable-number tandem-repeat analysis patterns (MLVA) and detection of enterotoxin genes (se) (sea, sec, sed, seg, seh, sei, sej and sel) were used to determine the phylogenetic relationship among isolates of Staphylococcus aureus isolated from dairy products from different regions of Italy. A total of 25 Staph. aureus were subtyped into 16 coagulase genotypes by RFLP, and MLVA revealed marked genomic variability. Furthermore, 17 of the isolates harboured at least one toxin gene, with the predominance of sea, sed and sej among cow isolates and sec-sel among the goat and sheep strains. Combined RFLP, MLVA polymorphism and se genes were found to be useful techniques for discriminating several genetic variants in Staph. aureus isolates.     

Genotype analysis of enterotoxin H-positive Staphylococcus aureus strains isolated from food samples in the Czech Republic

Twenty-eight enterotoxin H-positive Staphylococcus aureus strains isolated from food samples collected in eleven districts of the Czech Republic between 2000 and 2005 were genotypically characterized by pulsed-field gel electrophoresis (PFGE) profiling, spa gene polymorphism analysis, enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) fingerprinting and prophage carriage detection. These strains accounted for about 21% of the food-derived, staphylococcal enterotoxin (SE)-positive isolates. One strain, detected in feta cheese, was implicated in a case of enterotoxinosis. Sixteen of the twenty-eight isolates carried the seh gene alone. The remaining twelve strains harbored the seh gene in combination with other enterotoxin genes, most often the seg and sei genes, followed by the sea, seb, sec and sed genes. Comparison of various genomic profiles resulted in the determination of twenty genotypes designated G-1 to G-20. Two new, to date not defined, spa types (t2000 and t2002) were identified in one strain isolated from raw meat and two strains obtained from prepacked pizza. Evidence has been given that the seh-positive S. aureus isolates from foodstuffs did not originate from a single source or a common ancestor.     

Novel multiplex PCR for the detection of the Staphylococcus aureus superantigen and its application to raw meat isolates in Korea

A multiplex PCR assay that allows for the rapid screening of the 19 genes that encode staphylococcal enterotoxins (SEs) (sea to see, and seg to sei), SE-like (SEl) toxins (sej to ser, and seu), and toxic shock syndrome toxin-1 (TSST-1) (tst) was developed in this study. These toxins are included in the pyrogenic toxin superantigen (PTSAg) family and are responsible for many diseases such as staphylococcal food poisoning (SFP) and TSS. The primers were designed based on dual priming oligonucleotide (DPO) technology to detect all of the 19 SAg genes in three sets of PCR. The developed multiplex PCR was applied to 143 Staphylococcus aureus strains isolated from pork and chicken meat in Korea. Almost 50% of the strains possessed at least one of the 19 SAg genes. The most frequently found genes were seg, sei, sem, and sen (53 isolates, 37%), which were often found simultaneously in the same isolate. In those isolates, the seo (39 isolates, 27%) or seu (6 isolates, 4%) genes were frequently found together and this combination (seg, sei, sem, sen, and seo or seu) was considered to be a part of the enterotoxin gene cluster (egc). The sea gene (10 isolates, 7%) was the gene most frequently detected out of all the classical SE genes (sea to see). Although these classical SEs are considered to be major etiological factors in SFP, newly described SE or SEl genes (seg to ser, and seu) were more frequently detected than the classical SE genes in this study. There was no isolate detected containing the seb, sec, sek, sel, or seq genes. S. aureus possessing mobile genetic elements known to encode these SAg genes, such as egc, were presumed to be widely distributed among pork and chicken meats in Korea. The multiplex PCR developed in this study could be applied to the investigation of SAg genes in S. aureus strains isolated from various sources.     

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.     

Characterization of Staphylococcus aureus strains isolated from bovine milk in Hungary

Staphylococcus aureus is a major foodborne pathogen due to its capability to produce a wide range of heat-stable enterotoxins. The primary purpose of this research was to characterize S. aureus isolates recovered from mammary quarter milk of mastitic cows and from bulk tank milk produced on Hungarian dairy farms of different sizes. Macrorestriction analysis of chromosomal DNA from S. aureus isolates was performed using the restriction enzyme SmaI followed by pulsed-field gel electrophoresis (PFGE). The prevalence rates of nine S. aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and of the toxic shock syndrome toxin 1 gene (tst) were determined by multiplex polymerase chain reaction (PCR). The bulk tank milks of 14 out of 20 farms were contaminated with S. aureus at levels of up to 6.0x10(3 )CFU/ml. Farm size had no significant effect (P>0.05) on the S. aureus counts in bulk milk. The prevalence rates of penicillin resistance were 88.9% and 20.0% among the S. aureus recovered from mastitic quarter milk and bulk tank milk, respectively. After phenotypic characterization, a total of 59 S. aureus isolates were selected for genotyping. PFGE analysis revealed 22 distinct pulsotypes, including 14 main types and 8 subtypes, at a similarity level of 86%. Only one or two main types were observed on each of the farms tested, indicating a lack of genetic diversity among S. aureus isolates within farms, and there were only two pulsotypes which occurred on more than one farm. The PFGE patterns showed genetic relatedness between the S. aureus strains recovered from quarter milk and bulk milk on two large farms, implying that on farms having a high number of mastitic cows, S. aureus from infected udders may contaminate bulk milk and, subsequently, raw milk products. Sixteen (27.1%) of the S. aureus isolates tested by multiplex PCR were found to be positive for enterotoxin genes, with 15 of them carrying just one gene and one strain carrying two genes (seg and sei). The most commonly detected toxin genes were seb, sea, and sec, whereas none of our isolates possessed the see, seh, sej, or tst genes. On 75% of the dairy farms surveyed, no enterotoxigenic staphylococci were recovered from either mastitic quarter milk or bulk tank milk.     

Toxigenic status of Staphylococcus aureus isolated from bovine raw milk and Minas frescal cheese in Brazil

A group of 291 Staphylococcus aureus isolates from mastitic cow's milk (n = 125), bulk tank milk (n = 96), and Minas frescal cheese (n = 70) were screened for staphylococcal enterotoxin (SE) genes (sea, seb, sec, sed, see, seg, seh, sei, selj, and sell) and for the tst-1 gene encoding staphylococcal toxic shock syndrome toxin 1 by PCR assay. A total of 109 (37.5%) of the isolates were positive for at least one of these 11 genes, and 23 distinct genotypes of toxin genes were observed. Of the S. aureus isolates bearing SE genes, 17 (13.6%) were from mastitic cow's milk, 41 (41.7%) were from bulk tank milk, and 51 (72.9%) were from Minas frescal cheese. The occurrence of exclusively more recently described SE genes (seg through sell) was considerably higher (87 of 109 PCR-positive strains) than that of classical SE genes (sea through see, 15 strains). The SE genes most commonly detected were seg and sei; they were found alone or in different combinations with other toxin genes, but in 60.8% of the cases they were codetected. No strain possessed see. The tst-1 gene was found in eight isolates but none from mastitic cow's milk. Macrorestriction analysis of chromosomal DNA from 89 S. aureus isolates positive for SE gene(s) was conducted with the enzyme SmaI. Fifty-five distinct pulsed-field gel electrophoresis patterns were found, demonstrating a lack of predominance of any specific clone. A second enzyme, Apa I, used for some isolates was less discriminating than Sma I. The high genotype diversity of potential toxigenic S. aureus strains found in this study, especially from Minas frescal cheese, suggests various sources of contamination. Efforts from the entire production chain are required to improve consumer safety.     

Use of novel PCR primers specific to the genes of staphylococcal enterotoxin G, H, I for the survey of Staphylococcus aureus strains isolated from food-poisoning cases and food samples in Taiwan

Data regarding the incidence of the newly found enterotoxigenic Staphylococcus aureus strains in food poisoning cases and in food samples were to date not available in Taiwan. In this study, PCR primers specific for the detection of SEG, H and I genes, i.e., seg, seh and sei, were used for the assay of 55 human isolates of S. aureus negative to the classical enterotoxins (SEA-->SEE) detection. These isolates were from the fecal specimens of the patients suffering from food poisoning outbreaks. Only eight strains were found to have the seg, seh and sei. The presence of other bacterial pathogens, such as Vibrio parahaemolyticus, Bacillus cereus and Salmonella spp. and perhaps, strains producing other new staphylococcal enterotoxins, in the fecal specimens of these patients, may account for these food poisoning cases. For 139 strains from food samples, such as frozen Chinese foods, Chinese sausages and lunch meals, sea strains accounted for the major portion and it seemed to be the most common SE type to coexist with seg, seh and sei. Only two strains had sec and none of them had seg, seh or sei. For strains without the classical SE genes, only 13 strains had seg, seh and/or sei. The above results imply that seg, seh and sei S. aureus strains play only a minor role in food-borne outbreaks in Taiwan.