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。     

Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal origin product in Italy

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a global health concern. The present study regarded 160 S. aureus strains that had been isolated from 1634 foodstuff samples of animal origin in a previous survey conducted in Italy during 2003-2005. The strains were characterized by detecting the mecA gene, the production of type A to D staphylococcal enterotoxins (SEs), and studying their resistance properties against several antibiotics; their ecological origin was determined by biotyping. Of the 160 analyzed S. aureus strains six (3.75%) were mecA positive and derived from six different samples; four isolates were from bovine milk and two from dairy products (pecorino cheese and mozzarella cheese). Two strains isolated from milk belonged to the non-host-specific biovar while the others to the ovine biovar. The strain isolated from mozzarella cheese belonged to the non-host-specific biovar and the strain isolated from pecorino cheese to the ovine biovar. All the MRSA strains isolated were enterotoxigenic; two strains synthesized SEA/SED two SED and one SEC. All the strains showed resistance to at least one of the antibiotics tested but none was resistant to glycopeptides.     

The detection of enterotoxins and toxic shock syndrome toxin genes in Staphylococcus aureus by polymerase chain reaction

A simple polymerase chain reaction (PCR)-based procedure was developed for the detection of fragments of staphylococcal enterotoxins (SEs) SEA, SEB, SEC, SED, SEE, SEG, SEH, and SEI together with the toxic shock syndrome toxin (TSST-1) genes of Staphylococcus aureus. One hundred and twenty-nine cultures of S. aureus were selected, 39 of which were recovered from 38 suspected staphylococcal food-poisoning incidents. The method was reproducible, and 32 different toxin genotypes were recognized. The presence of SE genes was associated with S. aureus strains reacting with phages in group III, and the TSST-1 gene with phages in group I. There was a 96% agreement between the PCR results for detection of SEA-D and TSST-1 as compared with a commercial reverse passive latex agglutination assay for the detection of SEs from cultures grown in vitro. Enterotoxin gene fragments were detected in S. aureus cultures recovered from 32 of the 38 suspected staphylococcal food poisoning incidents, and of these, 17 were associated with SEE, SEG, SEH, and SEI in the absence of SEA-D. Simple PCR procedures were also developed for the detection of SE directly in spiked food samples, and this was most successfully achieved in mushroom soup and ham. Detection was less successful in three types of cheese and in cream. SEA or SEB were detected by enzyme-linked immunosorbent assay in three food samples (two of which were associated with food poisoning incidents) naturally heavily contaminated with S. aureus: the appropriate SEA or SEB gene fragments were detected directly in these three foods by PCR.     

Development of two multiplex polymerase chain reactions for the detection of enterotoxigenic strains of Staphylococcus aureus isolated from foods

Two multiplex polymerase chain reactions were developed for the detection of enterotoxigenic strains of Staphylococcus aureus: one multiplex reaction for the simultaneous detection of enterotoxigenic strains type A (entA), type B (entB), and type E (entE) and another for the simultaneous detection of enterotoxigenic strains type C (entC) and type D (entD). Both reactions were standardized with the use of the reference enterotoxigenic strains of S. aureus: FRI 722, producer of staphylococcal enterotoxin (SE) type A (SEA); FRI 1007, producer of SEB; FRI 137, producer of SEC1; FRI 472, producer of SED; and FRI 326, producer of SEE. Optimized methods were used to determine the presence of enterotoxigenic types for 51 S. aureus strains isolated from meat (sausage, ham, and chorizo) and dairy (powdered milk and cheese) products by the Baird-Parker technique. The enterotoxigenic capacities of the strains were determined by the indirect enzyme-linked immunosorbent assay (ELISA) with the use of reference staphylococcal toxins and antitoxins. Fifty of the 51 strains isolated were enterotoxigenic and produced one to four enterotoxin types, with the most frequently produced types being SEA and SED. Levels of correlation between the presence of genes that code for the production of SE (as determined by polymerase chain reaction) and the expression of these genes (as determined by the indirect ELISA) were 100% for SEA and SEE, 86% for SEC, 89% for SED, and 47% for SEB.     

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.     

Classical enterotoxigenic characteristics of Staphylococcus aureus strains isolated from bovine subclinical mastitis in Van, Turkey

The aim of the present study was to investigate classical enterotoxigenic properties of Staphylococcus aureus strains isolated from cows with subclinical mastitis. For this purpose, 480 milk samples from cows with subclinical mastitis raised in different villages neighbouring Van city Center were collected. A total of 106 S. aureus strains were isolated. Twenty seven isolates (25.5%) were found to be enterotoxigenic by reverse passive latex agglutination (RPLA). Of these, 25 (23.6%) were positive for staphylococcal enterotoxin A (SEA), 2 (1.9%) for SEB. None of the isolates was positive for SEC or SED. This study showed that most S. aureus strains isolated from bovine subclinical mastitis produced SEA compared to other SEs.     

Application of extended single-reaction multiplex polymerase chain reaction for toxin typing of Staphylococcus aureus isolates in South Korea

The extended single-reaction multiplex PCR (esr-mPCR) developed in this study to detect staphylococcal enterotoxins (SEs), including SEA, SEB, SEC, SED, SEE, SEH, SEI, and SEJ, requires fewer sets of primers than other conventional multiplex PCRs and can be used to detect newly identified staphylococcal enterotoxins SEs more readily. Esr-mPCR analysis of 141 isolates of Staphylococcus aureus obtained from abattoir and livestock product samples revealed that 27 of the S. aureus isolates were toxigenic, and two were 2 multitoxigenic isolates. The most prevalent SE type was SEI followed by SEA and SEH. In addition, we investigated the clonal relatedness of toxigenic S. aureus isolates by arbitrarily primed PCR (AP-PCR). AP-PCR analysis of toxigenic S. aureus isolates revealed that the discriminatory power of AP-PCR was 9 (D=0.81), 8 (D=0.77), and 10 types (D=0.83) with primers AP1, ERIC2, and AP7, respectively. The combination of three each AP-PCR result could rearrange toxigenic S. aureus isolates into 10 types and five subtypes, with the D-value of 0.92. Interestingly, our data showed that toxigenic S. aureus isolates from different sources had different fingerprinting patterns although some of them carried the same types of SE genes. These data suggest that combinations of esr-mPCR and AP-PCR can provide a powerful approach for epidemiological investigation of toxigenic S. aureus isolates.     

Detection of Enterotoxigenic Staphylococcus aureus in Raw Milk and Dairy Products by Multiplex PCR

Abstract: The aim of this study was to investigate the prevalence of enterotoxigenic Staphylococcus aureus in 122 samples, including 60 raw milk, 32 white cheese, 10 kashar cheese, 10 butter, and 10 ice cream samples obtained from Samsun province, Turkey. In this study, S. aureus was detected in 64 samples, including raw milk (45/60; 75%), white cheese (12/32; 37.5%), kashar cheese (3/10; 30%), butter (3/10; 30%), and ice cream (1/10; 10%) samples. A total of 81 isolates were identified as S. aureus by PCR with the presence of 16S rRNA and nuc genes. The presence of genes encoding the staphylococcal enterotoxins (SEs) SEA, SEB, SEC, and SED was detected by multiplex PCR. According to the analysis, seven isolates from the raw milk samples (7/51; 13.7%) were enterotoxigenic; five of them produced SEA (5/7; 71.4%), one produced SEB (1/7; 14.2%), and one produced SEA+SEB (1/7; 14.2%). Four isolates from the white cheese samples (4/21; 19%) produced the SEA (1/4; 25%), SEC (1/4; 25%), SED (1/4; 25%), and SEA+SED (1/4; 25%) toxins. Two isolates from the kashar cheese samples (2/4; 50%) were found to be enterotoxigenic; one produced SEA (1/2; 50%) and the other produced SED (1/2; 50%). One isolate from the butter samples (1/4; 25%) showed enterotoxigenic character (SEB, 1/1; 100%). The products were found to be potentially hazardous to public health because of the fact that levels of contamination were higher than 105–106 cfu/g ml in 39% (25/64, 17 raw milk, 7 white cheese, and 1 butter) of the analyzed samples.     

PCR detection of Staphylococcal enterotoxins (SEs) N, O, P, Q, R, U, and survey of SE types in Staphylococcus aureus isolates from food-poisoning cases in Taiwan

Staphylococcal enterotoxins (SEs) are superantigenic toxins. They are five major classical types, i.e., SEA, SEB, SEC, SED, SEE, and new SEs or SE-like superantigens, such as SEG to SEU. Only the staphylococcal superantigens (SAgs) that induce emesis following oral administration in a monkey model are designated as SEs while other related toxins are called SE-like (SEl) superantigens. To survey the enterotoxin genotypes for S. aureus strains isolated from food-poisoning cases in Taiwan, we developed PCR primers specific for SEN, SEO, SEP, SEQ, SER, and SEU genes. The complete SE sequences and their expression potential for strains positive to sen, seo, sep, seq, ser, and seu specific primers were also determined. These strains were used as reference strains. With the PCR primers specific for all SEs or SAgs, including toxic shock syndrome toxin I (TSST-1), we assayed the genotypes of 147 S. aureus strains isolated from patients associated with staphylococcal food-poisoning outbreaks occurred during 2001-2003. For these 147 strains, 135 (91.8%) were found positive for one or more SE or SAg genes. For classical enterotoxin and TSST-1 types, the major one was tsst-1 (59.1%) following by sea (29.2%), seb (19.7%), sec (6.8%), and sed (2.0%). For new SE and SAg types, the major one was sei (29.9%) and sep (27.9%) followed by, sek (16.3%), seo (14.3%), seu (14.2%), sem (11.6%), sen (10.9%), seq (10.9%), seh (8.2%), sel (6.8%), and ser (5.4%) etc. This report reveals the whole SE and SAg genotypes for S. aureus strains isolated from staphylococcal food-poisoning cases in Taiwan.     

Characterization of coagulase-positive staphylococci isolated from tank and silo ewe milk

The prevalence of coagulase-positive staphylococci (CPS) was studied among 390 samples of ewe milk. Fifty-seven (14.85%) samples of tank milk and all samples (6) of silo milk gave a positive result on Baird-Parker agar base supplemented with rabbit plasma fibrinogen, whereas amplification of the coagulase (coa) gene was successful in 6 (1.56%) samples of tank milk and in all silo samples. Phenotypic and genetic analysis of 153 isolates identified 151 (98.69%) as Staphylococcus aureus. Amplification of the coa gene was positive for 149 isolates (97.39%). The staphylococcal enterotoxin (SE) C gene was detected in 116 strains (75.82%), whereas more than one SE gene was carried in 5 strains (3.26%). None of the isolates harbored the genes for SEE or for methicillin resistance. A high prevalence of CPS carrying enterotoxin genes should be of concern because ewe milk is mainly processed into raw milk cheeses. The detection of the coa gene from milk samples could help to assess the microbiological safety of raw milk intended for direct use in the dairy industry.     

Benefits of the Combined Use of Immunological- and PCR-Based Methods for Determination of Staphylococcal Enterotoxin Food Safety Criteria in Cheeses

The aim of this study, performed under official controls, was to assess the relationship between the content of staphylococcal enterotoxins SEA to SEE in cheese samples and the enterotoxigenic potential of Staphylococcus aureus isolates. The level of correlation may further underline the benefit of using complementary approaches based on immunological tests and the PCR method to improve the diagnosis for determination of staphylococcal enterotoxin food safety criteria in cheeses submitted to official controls and own checks.     

多重PCR检测金黄色葡萄球菌六型肠毒素基因的研究

目的:为了建立一种简单、快速检测金黄色葡萄球菌六型肠毒素基因的多重PCR方法。方法:根据相关文献和Genebank报道的编码金黄色葡萄球菌肠毒素A、B、C、D、E、H的基因序列,选择合成了6对特异性引物,建立多重PCR体系,并对反应条件进行了优化。结果:6对引物能同时特异地扩增出120、478、257、319、170、375bp的目的片段,表明6对引物具有良好的特异性。结论:成功地建立了一种同时检测金黄色葡萄球菌六型肠毒素基因的多重PCR方法,在金黄色葡萄球菌肠毒素快速筛查方面具有良好的应用前景。     

Prevalence of Staphylococcus aureus and staphylococcal enterotoxins in raw pork and uncooked smoked ham--a comparison of classical culturing detection and RFLP-PCR.

In many countries Staphylococcus aureus is considered to be the second or third most common pathogen causing outbreaks of food poisoning, only outnumbered by Salmonella spp. and in competition with Clostridium perfringens. Often the consumption of ham or meat containing staphylococcal enterotoxins (SE) is identified as cause of the illness. Thus, to gain an insight into the prevalence of S. aureus and its emetic enterotoxins in raw pork and uncooked smoked ham and to investigate how the prevalence of the pathogen is influenced during the fabrication process, a total of 135 samples of raw pork, salted meat and ready-for-sale uncooked smoked ham were examined for the prevalence of S. aureus and staphylococcal enterotoxins A to D (SEA-SED). To this means classical cultural methods were employed as well as molecular biological techniques (PCR) and the results were compared. In 25.9% of all samples S. aureus was detected by culture whereas 51.1% of the samples showed a positive result when PCR was used for the detection of the pathogen. Fresh meat was contaminated most often. By PCR, 62.2% were identified as being S. aureus positive compared to 57.7% positive samples using the cultural technique. The detection rate during the fabrication process declined significantly. The pathogen was cultivated from 8.9% of the salted meat samples. Here, 55.6% of the samples reacted positively in the PCR, and finally, in approximately a third of the ready-for-sale smoked hams, S. aureus genes were found. From 11.1% of these samples, the pathogen could be isolated by culture. From these results, we conclude that the PCR used in this study is more sensitive than the classical cultural method. By PCR, one or more staphylococcal enterotoxin genes were found in 24 of the 135 examined samples. This means that 34.8% of the staphylococcal strains identified using the PCR technique were enterotoxigenic. Using the SET-RPLA, a percentage of 28.6% enterotoxigenic isolates was ascertained. No staphylococcal enterotoxin formation was detected by the SET-RPLA in ready-for-sale ham, although SE-genes were found by PCR. The detection of SE-genes by PCR is faster and easier to perform than the SET-RPLA.     

Significance of the detection of staphylococcal enterotoxin A gene in low fat milk which caused a serious outbreak of food poisoning

In June 2000, there was a large-scale outbreak of food poisoning after consumption of Snow Brand low fat milk. In the evening of a day the incident made public, some cartons of low fat milk were brought to our laboratory for examination. Next day, we detected only staphylococcal enterotoxin (SE) A gene among SE (A-E) genes by PCR in left-over milk samples or samples from the same lots that patients had consumed. We presumed that the outbreak was caused by the intake of SEA. We subsequently confirmed the presence of SEA in these samples. To investigate the existence of SE (A-E) genes in milk, we examined 100 samples of commercial low fat milk and milk by PCR, but none of the genes was detected. We estimated the detection limit of SEA gene in low fat milk by PCR. Four strains of SEA-producing Staphylococcus aureus cultures were serially diluted in low fat milk. The SEA gene was detected at levels of 5.5 x 10(2) to 1.6 x 10(4) cfu/mL of S. aureus. These amounts of S. aureus are higher than the values in raw milk reported previously. Therefore we consider that SE genes in low fat milk should usually be undetectable by our PCR. This study shows that quick detection of SE genes by PCR is very helpful to analyze outbreaks, especially if no significant bacterium can be cultured.     

Comparison of the enterotoxigenic types,toxic shock syndrome toxin I (TSST-1) strains and antibiotic susceptibilities for enterotoxigenicStaphylococcus aureusstrains isolated from food and clinical samples

The distribution of enterotoxin types and toxic shock syndrome toxin I (TSST-1) strains for 176Staphylococcus aureusstrains obtained from food samples and 62S. aureusstrains isolated from clinical samples were compared. It was found that for both the food and clinical isolates, staphylococcal enterotoxin A (SEA) strains accounted for the major part (75% and 45% of the total enterotoxigenic strains for food and clinical isolates, respectively) followed by SEB or SEAB, SEC and SED strains. For food isolates, none of theS. aureusstrains was TSST-1 strain while for clinical isolates, three strains (1 SEC, 1 SED and 1 SEAB strain) were found to be TSST-1 strains. When susceptibilities for these enterotoxigenicS. aureusstrains to antibiotics, such as penicillin, oxacillin, vancomycin, methicillin, streptomycin, tetracycline, gentamycin and kanamycin were compared, results showed that 51.6% of the food isolates were resistant to penicillin G only but sensitive to the other antibiotics tested. Also, 8 of the 64 enterotoxigenic strains isolated from food samples were all sensitive to antibiotics while none of the enterotoxigenic strains from clinical samples showed this antibiotic susceptibility pattern. No methicillin resistantS. aureus(MRSA) strains could be found among the food isolates. On the other hand, the majority (42.4%) of the enterotoxigenicS. aureusstrains from clinical samples were penicillin and/or other antibiotics resistant MRSA strains. Since MRSA strains often posed the therapeutic problem, the MRSA strains were further confirmed by PCR assay using the primers specific formecA gene. It was found that results obtained from the disc agar diffusion method and the PCR method were the same.     

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.     

Enterotoxin production by Staphylococcus aureus isolated from mastitic cows

Staphylococcus aureus is an important cause of mastitis in cows. The ability of S. aureus strains to produce one or more enterotoxins in milk and dairy products is linked to staphylococcal food poisoning. To determine whether staphylococci causing bovine mastitis could cause human foodborne intoxication, the production of staphylococcal enterotoxins A through D (SEA, SEB, SEC, and SED) by 160 S. aureus isolates was evaluated with the use of a reverse passive latex agglutination enterotoxin kit. All S. aureus strains were isolated over a 9-month period from 2,343 routine submissions of a composite quarter collection of individual mastitic cows at 18 dairy farms in the San Joaquin Valley in California. Prior to enterotoxin detection, isolates were grown by a method that enhances the in vitro synthesis of enterotoxin. Twenty-two of 160 S. aureus isolates produced enterotoxin. Seven produced SEC, 12 produced SED, and 3 produced both SEC and SED. None of the isolates produced SEA or SEB.     

Enterotoxin production and DNA fingerprinting in Staphylococcus aureus isolated from human and food samples. Relations between genetic types and enterotoxins

A total of 224 Staphylococcus aureus strains from human carriers (110 strains) and manually handled foods (114 strains) collected in the Principality of Asturias, Spain over 1995-1999 were analysed for the production of enterotoxins (SEs) A, B, C, and D by a reversed passive latex agglutination test and by amplification of ent genes (A, B, C, D, E, and J) using PCR. Sixty-two strains were enterotoxigenic and a good relation between detection of SEs and their ent genes was found. No strain carried entE and all strains producing SED carried entD and entJ genes. Among the enterotoxigenic strains the percentages registered were 29, 8, 35, 18, 2, 2, and 6 for SEA, SEB, SEC, SEDJ, SEAC, SEADJ and SECDJ, respectively. DNA fingerprinting of 77 strains (the SE prototypes, 62 enterotoxigenic and 10 non-enterotoxigenic [NE]) was carried out by randomly amplified polymorphic DNA using two selected primers independently. Combining results from both primers, 10 genetic types were defined, which showed a different degree of relationship (similarity coefficient: 0.9-0.36) and were clustered into three lineages. One lineage clustered five genetic types and a wide diversity of strains, mainly SEA, SEB, SEDJ, and NE. Another lineage clustered only SEC, SECDJ and NE strains. These two lineages showed a low genetic relationship and appeared as endemic in healthy humans living in the Principality of Asturias. The third lineage included only the prototype strains for SEA and SEE.