Prevalence of enterotoxin and toxic shock syndrome toxin genes in Staphylococcus aureus isolated from milk of cows with mastitis

Staphylococcus aureus isolated from milk of cows with mastitis were evaluated for the prevalence of 16 enterotoxin genes (sea-see and seg-seq) and toxic shock syndrome toxin gene (tsst-1). Of 78 S. aureus examined, 73 (93.6%) were positive for one or more enterotoxin genes and these were divided into 36 groups by the presence of different enterotoxin genes. Enterotoxin genes including sen (84.6%), sem (71.8%), sei (60.3%) and sed (52.6%) were found frequently, while seg (24.4%), seq (16.7%), seo (12.8%), and seb (1.3%) were found at lower frequencies. Toxic shock syndrome toxin (tsst-1) gene was detected in 20 (25.6%) isolates and was always found in combination with other enterotoxin genes. The majority (88.5%) harbored more than one enterotoxin gene in different combinations. Eight S. aureus isolates (10.3%) were positive for sed, sei, sem, and sen; six (7.7%) possessed sed, seg, sei, sem, sen, and tsst-1; five (6.4%) had sei, sem, and sen; and four (5.1%) had sei, and sen. One isolate was positive for seb along with other SE genes including sed, seh, sem, sen, seq, and tsst-1. None of the isolates carried other enterotoxin genes (sea, sec, see, sej, sek, sel, and sep). PFGE profiles revealed 15 distinct pulsotypes among the 78 S. aureus isolates evaluated. PFGE and enterotoxin gene profiles did not match with each other because a single pulsotype carried different combinations of enterotoxin genes. The majority of S. aureus isolated from milk of mastitic cows carried newly described SE genes sem, sen and sei along with classical SE genes, sed and tsst-1. This is the first report describing the high prevalence of newly described enterotoxin genes, sem and sen in S. aureus from bovine mastitis. The high prevalence of enterotoxin genes and tsst-1 in S. aureus may be important as it is relevant to udder pathogenicity and food hygiene.     

Characterization of toxin genes and antimicrobial susceptibility of Staphylococcus aureus isolates from Louisiana retail meats

Staphylococcus aureus is a leading cause of food poisoning worldwide due to the production of heat-stable enterotoxins. Recently, the isolation of methicillin-resistant S. aureus (MRSA) from food animals and retail meats raised additional food safety concerns. In this study, we characterized 152 S. aureus isolates, including 22 MRSA recovered from Louisiana retail pork and beef meats, for the prevalence of nine enterotoxin and four other exotoxin genes by polymerase chain reaction and antimicrobial susceptibility testing by broth microdilution. Overall, 85% of S. aureus isolates were positive for at least one of six enterotoxin genes identified and 66% harbored two to four enterotoxin genes. The two most predominant ones were seg and sei (66% each), followed by seh (20%), sed (15%), sej (13%), and sea (1%). No isolates harbored enterotoxin genes seb, sec, or see, the toxic shock syndrome toxin 1 gene tst, or the exfoliative toxin genes eta or etb. Three MRSA isolates were the only ones harboring Panton-Valentine leucocidin. Resistances were common to penicillin (71%), ampicillin (68%), and tetracycline (67%), followed by erythromycin (30%), clindamycin (18%), oxacillin with 2% NaCl (14%), ciprofloxacin (13%), levofloxacin (13%), gentamicin (3%), quinupristin/dapfopristin (3%), chloramphenicol (2%), and moxifloxacin (1%). Multidrug resistance was commonly observed among MRSA isolates and S. aureus isolates from pork. This study demonstrated that S. aureus isolates found in Louisiana retail pork and beef meats possessed various enterotoxin genes and antimicrobial resistance profiles. Therefore, vigilant food safety practice needs to be implemented for people who handle raw meat products to prevent foodborne infections and intoxications due to S. aureus contamination.     

2013-2017年西安市食物中毒中金黄色葡萄球菌分型分析

  目的  了解2013-2017年西安市食物中毒中分离的金黄色葡萄球菌（staphylococcus aureus,S.aureus）分子流行特点。  方法  通过脉冲场凝胶电泳（pulsed field gel electrophoresis,PFGE）、多位点序列分型（multilocus sequence typing,MLST）和spa分型方法对八起食物中毒标本的26株金黄色葡萄球菌进行分子分型,运用聚合酶链式反应（polymerase chain reaction,PCR）检测相关肠毒素基因。  结果  26株S.aureus PFGE可分为7种型别（A-G）;MLST可分为3种序列型（ST6,ST5和ST59）。其中,ST6占65.38%,为主要型别;spa可分为4种型别（t437,t701,t11363和t5923）,优势型别为t701,占65.38%。肠毒素基因检测中,sea基因占76.92%（20/26）;sed-seg基因占23.08%（6/26）;其它肠毒素基因未检出。  结论  2013-2017年西安市食物中毒标本中分离培养的S.aureus的主要流行型别为ST6-t701-sea,并首次检出含有seg新型肠毒素基因。PFGE、MLST和spa分型三种方法相结合全面的了解菌株的分子流行特点。     

一起食物中毒金黄色葡萄球菌肠毒素基因的检测

目的：检测从食物中毒中分离的金黄色葡萄球菌的肠毒素基因，明确肠毒素的基因型。对所分离的金黄色葡萄球菌进行药敏试验，检测其耐药性。方法：应用PCR扩增金黄色葡萄球菌SEA—SEJ基因，电泳检测扩增结果；用VITEK32检测药敏结果。结果：检测到金黄色葡萄球菌的肠毒素基因SEG和SEI，菌株的耐药性不强。结论：金黄色葡萄球菌的肠毒索SEG和SEI可能是这起食物中毒的因子。     

金黄色葡萄球菌食品和病人分离株肠毒素基因和耐药性比较

目的：对金黄色葡萄球菌食品（不含生牛奶）分离株和病人分离株的肠毒素基因进行分型和耐药性检测,比较两种分离菌株的肠毒素基因分布和耐药性差异。方法：用国标方法分离鉴定金黄色葡萄球菌,VITEK32生化验证,PCR检测金黄色葡萄球菌肠毒素基因（肠毒素SEA-SEJ基因）,VITEK32测定分离菌株抗生素的耐药性。结果：从病人分离的80株金黄色葡萄球菌检测到9种基因。肠毒素基因携带率为85.0%,同时携带2种及以上毒素基因的菌株占76.3%,含传统肠毒素基因（SEA-SEE）的菌株占43.8%,有新发现肠毒素基因（SEG-SEJ）的菌株占71.3%。从食品中分离的51株金黄色葡萄球菌检测到7种肠毒素基因。肠毒素基因携带率为68.7%,同时携带2种及以上毒素基因的菌株占21.6%。有SEA基因的占27.5%,含其它传统的毒素基因类型（SEB-SEE）基因的菌株占23.5%,携带新发现的毒素基因SEG、SEH、SEI的菌株只占9.8%。金黄色葡萄球菌病人分离株与食品分离株的耐药谱和耐药率有较大差异。结论：金黄色葡萄球菌食品分离株和病人分离株的肠毒素基因分布不同,其耐药性有明显区别。     

Nasal carriage of enterotoxin-producing Staphylococcus aureus among restaurant workers in Kuwait City.

Enterotoxin-producing Staphylococcus aureus is a common cause of staphylococcal food poisoning. To determine the incidence of carriage of enterotoxin-producing S. aureus in a sample of the healthy population in Kuwait city, restaurant workers in the city were screened for nasal carriage of S. aureus. 26.6% of 500 workers studied carried S. aureus and 86.6% of the S. aureus produced staphylococcal enterotoxins. 28% produced enterotoxin A, 28.5% produced enterotoxin B, 16.4% produced enterotoxin C and 3.5% produced enterotoxin D. Ten isolates produced both enterotoxins A and B or A and C. 73% of the isolates were untypeable with standard phages. However, 17.1%, 3% and 6% belonged to phage groups I, II and III respectively. The results demonstrated a high level of enterotoxigenic S. aureus carriage among restaurant workers which although lower than that reported for the general population and hospital workers may be important in the restaurant industry.     

金黄色葡萄球菌食物中毒菌株肠毒素检测及基因分析

目的:了解一起食物中毒中分离到的金黄色葡萄球菌(Staphylococcus aureus,SA)的生物学特性及其肠毒素基因检测。方法:采用VITEK-32全自动微生物鉴定/药敏分析系统对分离到的金黄色葡萄球菌菌株进行生化鉴定和药敏试验,并采用mini-VIDAS全自动荧光酶标免疫仪对菌株进行肠毒素检测,再采用PCR技术对产肠毒素的菌株进行基因分型。结果:总共分离到的19株金黄色葡萄球菌,所有分离株均对青霉素G耐药,均分泌β-内酰胺酶,肠毒素基因分型检测均为SEA与SEE。结论:19株分离株均携带肠毒素,经鉴定均为SEA与SEE肠毒素混合型,由此对食物中毒的原因有一个较圆满的解释。     

基于WGS的学生与环境MRSA菌株间毒素基因的相互关联分析

目的 探究学生鼻腔和学校环境耐甲氧西林金黄色葡萄球菌(methicillin-resistant staphylococcus aureus, MRSA)的毒素基因情况及遗传相似性。方法 利用全基因组测序技术,比较广州市8所小学学生和环境中MRSA的毒素基因情况。采用χ²检验或Fisher确切概率法分析毒素基因种类和数目的差异,构建系统进化树和聚类分析比较学生与环境的毒素基因谱。结果 检出的135株MRSA中,学生与环境均携带以溶血毒素(hlgA、hlgB、hlgC)(96.88%～100.00%)和胞外酶基因(aur)(98.06%～100.00%)为主的毒素基因。在种类上,34种毒素基因除部分肠毒素基因(sec3、seg、sei、sem、sen、seo、seu)外,其余均无统计学差异(P>0.05);在携带基因数目上,除携带12种毒素基因在二者之间有差异(χ²=12.35,P<0.001),其余均无差异(P>0.05)。此外,hlgA-hlgB-hlgC-aur-scn-sak和seg-sei-sem-sen-seo-s...     

日常食品及食物中毒样本中金黄色葡萄球菌肠毒素的分型检测分析

目的通过对北京市海淀区日常食品及食物中毒样本检出的金黄色葡萄球菌进行肠毒素分型检测,比较两种分离菌株的肠毒素分布差异。方法实验所用的金黄色葡萄球菌为2007-2012年海淀区日常食品和食物中毒样本中分离检出的菌株,依据GB 4789.10-2010采用ELISA方法测定金黄色葡萄球菌肠毒素(SEA-SEE)。结果 127株金黄色葡萄球菌中有108株肠毒素阳性,产肠毒素阳性率为85.0%。日常食品检出金黄色葡萄球菌93株,76株菌产肠毒素,产肠毒素阳性率为81.7%;食物中毒样本检出金黄色葡萄球菌34株,32株菌产肠毒素,产肠毒素阳性率为94.1%。产1种肠毒素和同时产3种肠毒素的菌株分别是47、37株,在产毒株中分别占43.5%和34.3%。食物中毒分离株产SEA和SED的比例(65.6%、65.6%)大于日常食品分离株(32.9%、30.3%).共有98株菌产SEE,在产毒株中占90.7%,肠毒素类型分布由高到低依次为E、A、D、C、B。结论食源性金黄色葡萄球菌产毒素能力较强,金黄色葡萄球菌食物中毒分离株和日常食品分离株在肠毒素分布上有差异。     

食源性金黄色葡萄球菌流行特征、产肠毒素特性及耐药性研究

目的：了解食源性金黄色葡萄球菌流行特点及产肠毒素特性及其耐药性，为制定HACCP以防止金黄色葡萄球菌的食源性疾病提供依据。方法：采集8类食品1243件分离金黄色葡萄球菌并进行产肠毒素A—E试验及药物敏感试验。结果：从生肉、熟食、生奶、及水产品中分别分离出40株（10．96％）、12株（3．13％）、34株（16．27％）及1株（1．12％）金黄色葡萄球菌，相应的产肠毒素率分别达到52．50％、58．33％、61．76％、0．oo％；对青霉素、四环素、凝固酶阴性苯唑西林耐药率分别高达93．10％、49．43％、37．93％；79．31％菌株对2种以上的药物多重耐药，最多耐药种类达7种，表现出30种耐药谱。结论：扬州市食品中存在着较严重的金黄色葡萄球菌潜在的危害，主要来源于生肉、生奶及熟食；食源性金黄色葡萄球菌产毒素能力较强。金黄色葡萄球菌对多种药物的耐药性提示安全应用治疗药物的重要性。     

金黄色葡萄球菌污染冰淇淋的调查

目的 通过冰淇淋及其各种原料和生产线样本分离、培养金黄色葡萄球菌,分析分离株的分子分型和表型特征,对金黄色葡萄球菌污染冰淇淋进行调查.方法 采集冰淇淋及其各种原料和生产线投料口、切片机出口、成品托盘等样本,用国标方法分离、培养、鉴定金黄色葡萄球菌,VITEK32生化验证,Mini-Vidas检测分离株肠毒素(多克隆肠毒素SEA-SEE),PCR检测分离株肠毒素基因(肠毒素SEA-SE:J基因),VITEK32测定分离菌株抗生素的耐药性,脉冲凝胶电泳(PFGE)分析菌株间的基因指纹图谱特征.结果 分别从冰淇淋和切片机出口分离到2株金黄色葡萄球菌.它们都含有肠毒素(多克隆肠毒素SEA-SEE),都有SEC、SED、SEG、SEH、SEI和SEJ肠毒素基因,耐药谱相同,脉冲凝胶电泳(PFGE)的指纹图谱相同.结论 污染冰淇淋和切片机出口的金黄色葡萄球菌是同一克隆株,切片机出口的污染导致冰淇淋污染.     

Molecular subtyping of Staphylococcus aureus from an outbreak associated with a food handler

On 6 May 2000, a staphylococcal food poisoning outbreak occurred at a high school, affecting 10 of the 356 students who attended the breakfast. Twenty-seven Staphylococcus aureus isolates, producing enterotoxin A (SEA), SEB-, or non-SEA-E, were recovered from 7 patients, 2 food handlers and left-overs. To investigate the outbreak, we genotyped the isolates by using pulsed-field gel electrophoresis (PFGE) and three PCR-based techniques: inter-IS256 PCR typing, protein A gene (spa) typing, and coagulase gene restriction profile (CRP) analysis. Our results show that PFGE was the most discriminatory technique, whereas the three PCR-based techniques were insufficient in the discriminatory power to distinguish the S. aureus isolates from the outbreak. Based on the enterotoxin-producing types and the results of genotyping, three distinct types of strains (A1111, B2221 and N3221) were designated. Both the A1111 and B2221 strains were found in the specimens from the patients and a hand lesion of a food handler, suggesting that the source of contamination for the outbreak was most likely originated from a food handler.     

Staphylococcal food poisoning in the United Kingdom, 1969-90.

Between 1969 and 1990 strains of Staphylococcus aureus from 359 outbreaks and sporadic cases of staphylococcal food poisoning in the United Kingdom were examined in the PHLS Food Hygiene Laboratory for the production of enterotoxin. In a number of instances the incriminated foods were also examined for the presence of enterotoxin. Strains from 79% of incidents produced enterotoxin A alone or together with another enterotoxin. The level of S. aureus present in the foods ranged from no viable S. aureus detected to 1.5 x 10(10) c.f.u./g with a median of 3.0 x 10(7) c.f.u./g. Enterotoxin was detected in foods in the absence of viable S. aureus in only two outbreaks and in both cheese was the implicated food. Meat, poultry or their products were the vehicle in 75% of incidents with ham and chicken most frequently implicated. Other foods included fish and shellfish (7%) and milk and milk products (8%). Most contamination took place in the home followed by restaurants and shops. Seventy-one percent of the incident strains were lysed by phages of group III or I/III.     

一起金黄色葡萄球菌食物中毒病原学检测及耐药分析

目的实验室检测分析福州市一起食物中毒的病原菌及耐药情况。方法参照WS/T 81-1996、WS/T 13-1996、WS/T 80-1996、WS 271-2007附录B2和GB/T 4789. 5-2012标准。结果该起食物中毒标本共检出3株金黄色葡萄球菌。这些菌株均产肠毒素,其中ZD2017-13和ZD2017-14 2株菌产肠毒素C,ZD2017-16产肠毒素B。这3株菌均为耐甲氧西林金黄色葡萄球菌(MARS),均对头孢西丁、氨苄西林、苯唑西林和青霉素耐药。结论从该起食物中毒标本中分离得到的金黄色葡萄球菌均产肠毒素,为耐甲氧西林金黄色葡萄球菌,且对多种抗生素耐药。     

应用实时荧光PCR技术检测金黄色葡萄球菌肠毒素基因

目的:建立一种简便、特异的荧光PCR检测方法,用于金黄色葡萄球菌肠毒素的检测。方法:按金黄色葡萄球菌SEA～SEE型肠毒素基因序列设计引物,在普通PCR检测体系中,加入SYBR Green I荧光染料,建立荧光PCR检测体系。结果:46株金黄色葡萄球菌中检出22株携带肠毒素基因,阳性率为47.83%。以SEA、SEB检出率较高,分别为31.82%和27.27%;不同来源的分离株携带肠毒素基因的比例不同,同时携带2种及以上毒素基因的菌株占27.27%。结论:荧光PCR检测金黄色葡萄球菌肠毒素的方法具有快速、敏感、特异性高的特点,适用于肠毒素基因的分型与分布的研究,适合基层疾控部门使用。     

The use of a sandwich ELISA for the detection of staphylococcal enterotoxin A in foods from outbreaks of food poisoning.

Foods from outbreaks of food poisoning were examined for the presence of staphylococcal enterotoxin A (SEA) by a sandwich ELISA using microtitre trays as the solid phase and SEA antibodies raised in sheep. The presence of SEA was confirmed by neutralization tests. The toxin was detected in 12 of 15 foods from separate outbreaks of staphylococcal food poisoning; all 15 foods contained a strain of Staphylococcus aureus which produced SEA. For most foods a simple extraction procedure without a concentration step was sufficient to detect the toxin. The method was semi-quantitative and recoveries of SEA added to control foods varied from 30 to 80%. The foods from outbreaks contained between 1 and 10 micrograms of SEA/100 g. SEA was not found in foods from 21 outbreaks in which an SEA-producing strain of Staph. aureus was not isolated.     

The use of a sandwich ELISA for the detection of staphylococcal enterotoxin A in foods from outbreaks of food poisoning

Foods from outbreaks of food poisoning were examined for the presence of staphylococcal enterotoxin A (SEA) by a sandwich ELISA using microtitre trays as the solid phase and SEA antibodies raised in sheep. The presence of SEA was confirmed by neutralization tests. The toxin was detected in 12 of 15 foods from separate outbreaks of staphylococcal food poisoning; all 15 foods contained a strain of Staphylococcus aureus which produced SEA. For most foods a simple extraction procedure without a concentration step was sufficient to detect the toxin. The method was semi-quantitative and recoveries of SEA added to control foods varied from 30 to 80%. The foods from outbreaks contained between 1 and 10 micrograms of SEA/100 g. SEA was not found in foods from 21 outbreaks in which an SEA-producing strain of Staph. aureus was not isolated.     

Genotypes, antibiotic resistance, and virulence factors of staphylococci from ready-to-eat food

Sixty-seven staphylococcal isolates belonging to 12 species were obtained from 70 ready-to-eat food products. Staphylococcus aureus (n=25), and Staphylococcus epidermidis (n=13) were dominant. Susceptibility to penicillin, oxacillin, tetracycline, clindamycin, gentamicin, erythromycin, ciprofloxacin, and vancomycin was determined. All investigated S. aureus isolates were resistant to at least one antibiotic, and fifteen isolates were resistant to four and more antibiotics. Thirty-eight coagulase-negative staphylococci (CNS) isolates were resistant to at least one antibiotic, and seventeen to four and more antibiotics. Fifteen CNS isolates were mecA positive, and grew in the presence of 6 μg/mL oxacillin. All S. aureus isolates were mecA-negative. Arginine catabolic mobile element (ACME) was found in seven S. epidermidis isolates. Five S. epidermidis isolates harbored ica operon, ACME and were able to form biofilm. Three of them also possessed IS256 element and were mecA-positive. The expression of icaA gene was comparable in five ica-positive S. epidermidis isolates. One of six mecA positive S. epidermidis isolates was classified as sequence type (ST)155, one as ST110, and two as ST88. Two methicillin-resistant Staphylococcus epidermis (MRSE) belonged to new STs, that is, ST362, and ST363. Enterotoxin genes were found in 92% of S. aureus isolates. No enterotoxin gene was detected in analyzed CNS population. We show that ready-to-eat products are an important source of antibiotic-resistant CNS and potentially virulent strains of S. epidermidis, including genotypes undistinguishable from hospital-adapted clones.     

一起食物中毒事件的金黄色葡萄球菌分子分型研究

目的 运用分子分型方法分析一起在广州发生的由金黄色葡萄球菌所致的重大食物中毒事件,并进行溯源.方法 在常规分离的基础上,采用荧光定量PCR检测所获得的金黄色葡萄球菌特异耐热核酸酶基因(nuc)、耐甲氧西林决定基因(mecA)和其他5种肠毒素基因(sea,seb,sec,sed,see),并对16 S rRNA核苷酸进行序列扩增和使用DNAStar MegAlign 5.0软件分析,同时应用脉冲场凝胶电泳(PFGE)和BioNumerics Version 4.0软件进行聚类分析.结果 10株金黄色葡萄球菌特异性nuc基因均为阳性,其中7株分离菌的肠毒素sea,seb基因为阳性,分离菌可以分为4个16 SrRNA型别和5个PFGE型别.结论 本次食物中毒事件至少由3株以上亲缘关系相近的产毒金黄色葡萄球菌污染食物所致,分子分型方法可以为疫情溯源提供分子流行病学证据和支持.     

贵州省即食米面制品食源性致病菌污染及病原特征分析

目的 了解贵州省即食米面制品中食源性致病菌污染状况和病原学特征,为细菌性食源性疾病的防治提供参考依据。方法 2014—2019年在贵州省的88个县（市、区）采集即食米面制品1128份,依据食品安全国家标准（GB4789）进行五种食源性致病菌的检测;采用荧光定量PCR法和ELISA法分别检测金黄色葡萄球菌肠毒素基因及蛋白,普通PCR法检测蜡样芽胞杆菌毒力基因。结果 1128份即食米面制品有130份样品检出食源性致病菌,检出率为11.52%,分别是蜡样芽胞杆菌7.71%,金黄色葡萄球菌3.10%,沙门氏菌0.27%,单核细胞增生李斯特氏菌0.44%,未检出致泻大肠埃希氏菌。分离的35株金黄色葡萄球菌有65.71%携带肠毒素基因,54.29%呈肠毒素蛋白阳性。87株蜡样芽胞杆菌都至少携带两种以上毒力基因,同时携带hblACD及nheABC的有29.88%。结论 贵州省即食米面制品存在多种食源性致病菌污染,蜡样芽胞杆菌和金黄色葡萄球菌污染率较高且携带多种毒力基因,存在较大食源性疾病风险,监管部门应加强对即食米面制品的加工制作管理。