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.     

Distribution of newly described enterotoxin-like genes in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from ready-to-eat foods in Korea

To investigate the distribution of staphylococcal enterotoxin-like (SEl) genes in Staphylococcus aureus from food, a total of 154 S. aureus isolates from ready-to-eat (RTE) foods in Korea were analyzed by mutiplex PCR for the detection of the following 9 staphylococcal enterotoxin-like genes; sek, sel, sem, sen, seo, sep, seq, ser, and seu. Seventy-nine isolates (51.3%) were found to have at least one of SEl genes. The major SEl genes were sek, sem, sen, and seq. Other SEl genes found in the isolates were seo (21 isolates, 13.6%), seu (12 isolates, 7.8%), sep (8 isolates, 5.2%), sel (7 isolates, 4.5%), and ser (2 isolates, 1.3%). Most (95%) of the isolates with staphylococcal enterotoxin (SE) genes were also carried SEl genes. The genes seg, sei, sem, and sen were all detected in the same isolates. Ninety-seven % of isolates with seg+sei+sem+sen also contained seo or seu. Ninety-four % of isolates with sea+seh were found to coexist with sek+seq. The toxic shock syndrome toxin gene, tst-1, was found in all isolates with egc-2, including seg, sei, sem, sen, and seu. The coexistence of SE and SEl genes in S. aureus isolates from RTE foods can be explained by the mobile genetic elements. Because of the mobile genetic element, SE and SEl genes of S. aureus in foods may be transferable to nontoxigenic S. aureus and other food pathogens. Additional studies must be conducted to prevent spread of pathogenic genes such as enterotoxin gene.     

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.     

Distribution of newly described enterotoxin-like genes in Staphylococcus aureus from food

Extensive analysis of the Staphylococcus aureus genome has allowed the identification of new genes encoding enterotoxin-like superantigens (SEls). Some of these are thought to be involved in staphylococcal food poisoning, while others do not elicit any emetic effect. The potential impact of these members of the enterotoxin-like family on the human organism seems to rely mainly on their superantigenic activity. In this paper the distribution of the genes coding for enterotoxin-like superantigens in S. aureus isolated from food was studied. Fifty isolates of S. aureus were examined and 27 were shown to be enterotoxigenic. Only 9 of the 27 strains carried genes encoding enterotoxins SEA-SEE. In 18 SEA-SEE-negative strains the presence of newly described enterotoxin genes was detected. All SEA-SEE-positive strains simultaneously carried genes of new SEls. We show that the gene encoding SElH (staphylococcal enterotoxin-like enterotoxin H) was the most frequently detected (n=14), while genes encoding SElI together with SElG accompanied by the other genes of the egc locus were detected in three strains. We also detected the presence of three less investigated genes: sep, sel, and sek. These genes were present in eight, two, and one isolate, respectively. In one strain, sep was accompanied by genes of other SEls, while in the remaining seven it was the only enterotoxin-like gene detected. The high prevalence of newly discovered enterotoxin genes, including the genes encoding emetic toxins, was demonstrated in food-derived strains. This supports the need for additional work on its role in food poisoning and, alternatively, to monitor its presence in S. aureus isolated from food. Our results suggest that yet unknown genetic elements encoding enterotoxin genes may exist.     

Staphylococcus aureus isolates from Irish domestic refrigerators possess novel enterotoxin and enterotoxin-like genes and are clonal in nature

A previous study carried out by the National Food Centre in Dublin on bacterial contamination of Irish domestic refrigeration systems revealed that 41% were contaminated with Staphylococcus aureus. One hundred fifty-seven S. aureus isolates were screened by multiplex PCR analysis for the presence of 15 staphylococcal enterotoxin and enterotoxin-like genes (sea-see, seg-sei, selj-selo, and selq) and the toxic shock toxin superantigen tst gene. Of the refrigerator isolates, 64.3% possessed more than one staphylococcal enterotoxin or staphylococcal enterotoxin-like gene. All bar one of the 101 staphylococcal enterotoxin or staphylococcal enterotoxin-like gene-positive strains possessed the egc locus bearing the seg, sei, selm, seln, and selo genes. Twelve random amplified polymorphic DNA (RAPD) types accounted for 119 (75.8%) of the strains, two of these types accounting for 25 (RAPD type 1, 15.9%) and 52 (RAPD type 5, 33.1%), respectively. All of the RAPD type 5 isolates possessed the egc gene cluster only. The RAPD type 5 amplicon profile was identical to that of S. aureus isolates associated with osteomyelitis in broiler chickens in Northern Ireland that also possessed the egc locus only. However, the RAPD type 5 domestic refrigerator and chicken isolates differed in penicillin G sensitivity, production of Protein A and staphylokinase, and crystal violet agar growth type. These findings highlight that the average Irish household refrigerator harbors potential enterotoxin-producing S. aureus that may or may not be of animal origin and, accordingly, is a potential reservoir for staphylococcal food poisoning.     

肉鸡屠宰环节中金黄色葡萄球菌的流行及分子特征和耐药性

为探究肉鸡屠宰加工环节中金黄色葡萄球菌的污染关键环节及菌株的分子特征和耐药性,对陕西省某肉鸡屠宰场5个屠宰环节（活鸡肛拭、浸烫、预冷、分割和贮存）进行4次样本收集。通过选择培养和聚合酶链式反应（polymerase chain reaction,PCR）扩增nuc基因对金黄色葡萄球菌进行分离鉴定。然后对分离株进行23种毒素编码基因、27种耐药基因、16种抗生素耐药性及葡萄球菌蛋白A(staphylococcal protein A,SPA)、多位点序列分型（multilocus sequence typing,MLST）和肠杆菌科基因间重复序列PCR(enterobacterial repetitive intergenic consensus-PCR,ERIC-PCR)分型检测。结果表明,收集的144份样本中有32份样本被污染（22.2%,32/144）。除活鸡肛拭样本无检出外,其余环节样本均有检出。其中浸烫褪毛环节污染率最高（40.0%,12/30）,其次为分割环节（26.7%,8/30）、预冷环节（21.9%,7/32）和贮存环节（15.6%,5/32）。23种被检毒素编码基因中...     

Virulence factors and genetic variability of Staphylococcus aureus strains isolated from raw sheep's milk cheese

Contamination of dairy products with Staphylococcus aureus can be of animal or human origin. The host pathogen relationship is an important factor determining genetic polymorphism of the strains and their potential virulence. The aim of the present study was to carry out an extensive characterization of virulence factors and to study the genetic variability of S. aureus strains isolated from raw ewe's milk cheese. A total of 100 S. aureus strains isolated from cheese samples produced in 10 artisan cheese factories were analyzed for the presence of enterotoxins (sea-see) and enterotoxins-like genes (seh, sek, sel, sem, seo, sep), leukocidins, exfoliatins, haemolysins, toxic shock syndrome toxin 1 (TSST-1) and the accessory gene regulator alleles (agr). Strains were also typed using pulsed-field gel electrophoresis (PFGE). AMOVA analysis carried out on PFGE and PCR data showed that the major component explaining genetic distance between strains was the dairy of origin. Of the total isolates 81% had a pathogenicity profile ascribable to "animal" biovar while 16% could be related to "human" biovar. The biovar allowed to estimate the most likely origin of the contamination. Minimum inhibitory concentrations (MICs) of nine antimicrobial agents and the presence of the corresponding genes coding for antibiotic resistance was also investigated. 18 strains carrying blaZ gene showed resistance to ampicillin and penicillin and 6 strains carrying tetM gene were resistant to tetracycline. The presence of mecA gene and methicillin resistance, typical of strains of human origin, was never detected. The results obtained in the present study confirm that S. aureus contamination in artisan cheese production is mainly of animal origin.     

2009-2018年广州市即食食品中金黄色葡萄球菌污染情况和菌型特征

目的 分析2009-2018年广州市零售即食食品中金黄色葡萄球菌污染情况,以及菌株肠毒素基因、耐药表型特征.方法 从广州市辖11个区的农贸市场、超市随机购买零售即食食品,增菌后分离金黄色葡萄球菌.对所有菌株进行多位点序列分型(MLST)、抗生素敏感性试验,并检测24种肠毒素基因.对耐甲氧西林金黄色葡萄球菌(MRSA)进...     

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.     

Antimicrobial resistance and heat sensitivity of oxacillin-resistant, mecA-positive Staphylococcus spp. from unpasteurized milk

Eight Staphylococcus spp. carrying the mecA gene were isolated from oxacillin enrichments of 70 unpasteurized milk samples. The isolates were identified as five Staphylococcus epidermidis, two Staphylococcus lentus, and one Staphylococcus haemolyticus. No mecA-positive Staphylococcus aureus were isolated. All isolates carried genes for other antibiotic resistances in addition to mecA. The results establish that mecA-carrying coagulase-negative Staphylococcus spp. in unpasteurized milk have the potential to be a reservoir of other genes encoding antimicrobial resistance. Two S. epidermidis isolates with qacA/B genes were resistant to benzalkonium chloride. Decimal reduction times (D-values) for the mecA-Staphylococcus spp. at 56 degrees C in whole milk ranged from 1.46 to 2.82 min. D-values at 56 degrees C for nine S. aureus milk isolates ranged from 10.8 to 20.1 min. Heat treatments intended to control S. aureus may be an effective means to protect consumers of milk and dairy products. Contact with or consumption of milk and dairy products that have not been heat treated may lead to the spread of antimicrobial resistance genes in Staphylococcus spp. to animals and humans.     

羊奶粉生产中金黄色葡萄球菌分离鉴定及其分子特性和耐药性检测

为探究羊奶粉在加工过程中金黄色葡萄球菌污染的关键环节及菌株的分子特征和耐药性,对某羊奶粉加工厂不同生产环节进行样本收集,通过采用选择培养和聚合酶链式反应扩增nuc基因对金黄色葡萄球菌进行分离鉴定,然后对分离的菌株进行毒素基因（21 种）、耐药性（16 种常用抗生素）及葡萄球菌A蛋白（staphylococcal protein A,SPA）、多位点序列分型（multilocus sequence typing,MLST）和脉冲场凝胶电泳（pulsed field gel electrophpresis,PFGE）分型研究。结果表明,收集的112 份样本中有6 份样本被污染（5.4%,6/112）,其中包括加工设备（2 份）、加工人员（2 份）、罐奶（1 份）和车间落地粉（1 份）。所有的分离株至少携带1 种毒素基因,其中以pvl基因的携带率最高（100.0%,6/6）,其次为sea、sec、see、seh、sek和seq（50.0%,3/6）,seg、sej和ser（33.3%,2/6）,sed、sei、sem和seo（16.7%,1/6）。所有分离株至少耐受4 种抗生素,其中对氨苄西林、头孢哌酮、甲氧苄啶/磺胺甲恶唑和青霉素的耐药性最为普遍（100.0%,6/6）,其次为红霉素和阿莫西林/克拉维酸（83.3%,5/6）、四环素（50.0%,3/6）和庆大霉素（16.7%,1/6）。此外,所有菌株对苯唑西林、头孢西丁、万古霉素、利奈唑胺等抗生素敏感。所有分离株共有4 种spa分型和3 种ST分型,其中以ST1-t127（50.0%,3/6）为主,其次为ST5-t002、ST5-t548和ST188-t189（16.7%,1/6）。对于PFGE分型,可分为3 个大簇（I、II和III簇）和4 个基因型（A、B、C和D）,其中加工设备和落地粉存在相同的PFGE分型。研究结果表明在羊奶粉加工过程中存在金黄色葡萄球菌污染现象,罐奶、加工设备、加工人员和落地粉是受污染的关键环节,且在不同的加工环节中存在一定的交叉污染。虽然污染率较低,有必要对奶厂不同加工环节的污染情况进行长期调查,确认关键污染环节,可以有效控制金黄色葡萄球菌在奶粉制品中的扩散。     

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.     

Antibiotic resistance genes and identification of staphylococci collected from the production chain of swine meat commodities

Staphylococci harbouring antibiotic resistance (AR) genes may represent a hazard for human health and, as other resistant food-related bacteria, they contribute to the spread of AR. In this study, we isolated resistant staphylococci from an entire swine production chain and investigated the occurrence of 11 genes [aac(6')Ie-aph(2')Ia, blaZ, mecA, vanA, vanB, ermA, ermB, ermC, tet(M), tet(O) and tet(K)] encoding resistance to some antibiotics largely used in clinical practice. The 66 resistant staphylococcal isolates were identified as Staphylococcus epidermidis (27 isolates), Staphylococcus aureus (12), Staphylococcus xylosus (12), Staphylococcus simulans (5), Staphylococcus pasteuri (4), Staphylococcus carnosus (3), Staphylococcus lentus (2) and Staphylococcus sciuri (1). Specific-PCR detection of AR genes showed the prevalence of the tet(K) gene in most of the isolates (89.4%), followed by tet(M) and ermC (about 75%); mecA was detected in more than half of S. aureus and S. epidermidis isolates. The genes vanA and vanB were not retrieved. It was found that a high proportion of coagulase-positive and -negative isolates are multidrug-resistant and some of them carry up to six AR genes. Our findings show that the swine production chain is a source of antibiotic-resistant staphylococci suggesting the importance of resistance surveillance in the food production environment.     

成都市生鲜猪肉MRSA分离株的流行特征分析

目的:分析成都市各地区生鲜猪肉样品中耐甲氧西林金黄色葡萄球菌（Methicillin-resistant Staphylococcus aureus,MRSA）的流行特征。方法:对鉴定出的MRSA菌株进行SCCmec分型、spa分型以及多位点序列（MLST）分型分析,同时运用PCR技术对MRSA菌株携带的多种毒力基因、生物被膜形成相关基因、耐消毒剂基因以及耐药基因进行检测,并采用K-B纸片扩散法调查MRSA菌株的耐药表型。结果:本研究从297株猪肉源金黄色葡萄球菌中筛选出24株MRSA菌株,分离率为8.08%。MRSA菌株主要以ST88-IVa-t1376、ST59-IVa-t437和ST9-IVb-t3433型为主,携带耐药基因3~7个;所有菌株均表现出对氨苄西林和青霉素G耐药,并且存在多重耐药情况;分离菌株携带多种肠毒素基因,其中MRSA8和MRSA13菌株携带了完整的egc基因簇;MRSA分离菌株均携带clfB、eno、icaBC和sasG等生物被膜形成相关基因;携带耐消毒剂基因（qacA/B、qacC和qacG）的菌株占比为62.5%,其中以qacG为主要携带基因类型。另外,79.17%的MRSA菌株携带溶血素基因（hla、hlb）,所有菌株中杀白细胞素基因（pvl）均未检出。结论:成都市猪肉源MRSA的流行可能存在一定的交叉污染,应加强动物源食品携带MRSA菌株的监测。     

The distribution of enterotoxin and enterotoxin-like genes in Staphylococcus aureus strains isolated from nasal carriers and food samples

Staphylococcus aureus strains were examined for the presence of 18 enterotoxin (se) and enterotoxin-like (sel) genes by PCR and four classic enterotoxins SEA to SED by reverse passive latex agglutination. We screened three groups of isolates: 53 recovered from food samples collected in years 2004-2005, 18--from food samples collected in the 1960s and 1970s and 30--from nasal carrier samples collected in the years 2000-2002. Eighty percent of all tested strains were se and sel positive, but the frequency of these genes was higher among nasal carrier strains (93%) than among food samples isolates (circa 76%). The enterotoxin genes cluster (egc) was the most prevalent among carrier strains (18/30-60%) and the least--among food strains isolated in the 1960s and 1970s (6/18 strains--33%). sea coding enterotoxin A, which was recognised as the major cause of staphylococcal food poisoning so far, was more often found among carrier strains than among the years 2004-2005 food strains (27% versus 11%), but it was the most frequent se/sel gene among food strains isolated in the 1960s and 1970s (10/18 strains). Moreover in our results certain se and sel genes coexisted, which was in accordance with current knowledge about movable genetic elements carrying those genes. The exception was for only one strain, which harboured the sole selr gene on a possibly new genetic element not yet described. As we found both types of egc, as well as seh (especially food samples strains) occurred alone in a majority of positive strains for each of those genes. The analysis of the results obtained by SET-RPLA method for the productivities of classical enterotoxins A-D and the results obtained by PCR for the presence of sea-sed genes revealed the correlation between each other. Only three of all sea-sed positive strains had silent genes--sed.     

牛奶源金黄色葡萄球菌血清型、毒力基因及PFGE分型

目的：研究生鲜牛奶中金黄色葡萄球菌分离、荚膜多糖血清型分布、毒力基因携带及脉冲场凝胶电泳（pulsed field gel electrophoresis,PFGE）分型情况。方法：从河南省4?个地区奶牛养殖场采集生鲜牛奶样品按照国标法进行分离,扩增耐热核酸酶基因nuc鉴定金黄色葡萄球菌,利用聚合酶链式反应方法测定荚膜多糖血清型和毒力基因携带情况,采用PFGE分析菌株间的相关性和遗传关系。结果：从350?份生鲜牛奶样品中分离鉴定到80?株金黄色葡萄球菌,分离率为22.86%。荚膜多糖血清型测定发现,cap5（60%）是流行血清型。从这些阳性菌株中,发现有62?株（77.5%）携带有毒力基因,毒力基因set、hlb、hld、lukED、ebp、clfA和clfB,检出率分别为40.00%、51.25%、57.50%、60.00%、58.75%、57.50%和58.75%。此外,47?株（58.75%）菌携带不少于6?个毒力基因,流行的毒力基因谱型为set-hla-hlb-hld-lukED-cna-ebp-clfA-clfB。PFGE结果显示,获得72?株菌的PFGE图谱,按90%的相似性可分为12?个簇和46?种PFGE型。D簇（3?种PFGE型）、G簇（3?种PFGE型）和J簇（5?种PFGE型）菌株中均检出一定基因类型的毒力基因,表明河南地区生鲜牛奶中金黄色葡萄球菌毒力基因广泛存在于多种PFGE型别中。结论：生鲜牛奶均有一定程度的金黄色葡萄球菌污染,多数菌株携带毒力基因,且毒力基因的类型较为复杂,这对消费这些牛奶的人群构成潜在的健康威胁。PFGE分型菌株主要以克隆形式进行传播,且克隆型具有多样性和差异性,故临床应加强生奶及乳品血清型、毒力基因检测及分子分型研究。     

金黄色葡萄球菌新型肠毒素SEK原核表达、纯化及溶液构象分析

金黄色葡萄球菌K型肠毒素（Staphylococcus aureus enterotoxin K,SEK）由金黄色葡萄球菌肠毒素基因簇编码,流行病学显示,编码SEK蛋白的sek基因在食品源金黄色葡萄球菌菌株中具有较高的检出率,说明SEK也可能是一种重要的食品中毒潜在致病因子。本研究将截去N端信号肽的SEK蛋白编码基因与原核表达载体pET-28a（＋）连接,构建重组表达质粒pET-28a（＋）-ΔNspsek;通过对sek基因在不同原核表达宿主菌中的表达及表达条件优化分析,确立SEK蛋白的最优表达条件;利用镍亲合层析纯化含组氨酸（His）标签的SEK融合蛋白,将凝血酶切除His标签后的SEK蛋白进行聚合状态及热稳定分析、荧光发射谱和圆二色谱分析。结果表明,重组蛋白SEK表达成功,热处理导致蛋白部分降解;荧光光谱揭示SEK蛋白在278?nm和295?nm波长处具有相同的最大色氨酸发射峰;344?nm波长处表明SEK蛋白处于紧密折叠的天然状态;圆二色谱分析表明,ΔNspSEK重组蛋白富含β-折叠（23.6%）、β-转角（28%）以及α-螺旋（29.1%）等二级结构。为深入研究SEK蛋白的结构与功能提供基础,也对改进食品加工工艺和提高食品安全具有指导意义。     

Characterization of Staphylococcus aureus isolates from white-brined Urfa cheese

The aim of this study was to investigate the presence of Staphylococcus aureus and staphylococcal enterotoxin (SE) genes in Urfa cheese samples and to characterize the enterotoxigenic potential of these isolates. From a total of 127 Urfa cheese samples, 53 isolates (from 41.7% of the samples) were identified by a species-specific PCR assay as S. aureus. Of these isolates, 40 (75.5%) gave positive PCR results for the 3' end of the coa gene. The coa-positive S. aureus strains were characterized for their population levels and enterotoxigenic properties, including slime factor, β-lactamase, antibiotic susceptibilities, production of the classical SEs (SEA through SEE), in both cheese and liquid cultures by enzyme-linked immunosorbent assay (ELISA) and for the presence of specific genes, including classical SE genes (sea through see), mecA, femA, and spa, by PCR. The genetic relatedness among the coa-positive S. aureus isolates was investigated by PCR-based restriction fragment length polymorphism (RFLP) analysis and the 23S rRNA gene spacer. The 23S rRNA gene spacer and coa RFLP analysis using AluI and Hin6I revealed 14 different patterns. SEB, SEC, and SEA and SEE were detected by ELISA in three cheese samples. Fourteen S. aureus strains harbored enterotoxin genes sea through see, and three strains carried multiple toxin genes. The most commonly detected toxin gene was sec (25% of tested strains). Of the 40 analyzed S. aureus strains, 3 (7.5%) were mecA positive. Based on tandem repeats, four coa and spa types were identified. The results of this study indicate that S. aureus and SEs are present at significant levels in Urfa cheese. These toxins can cause staphylococcal food poisoning, creating a serious hazard for public health.     

Occurrence and characteristics of methicillin-resistant and -susceptible Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci from Japanese retail ready-to-eat raw fish

Staphylococci are not part of the normal fish microflora. The presence of staphylococci on fish is an indication of (a) post-harvest contamination due to poor personnel hygiene, or (b) disease in fish. The aim of this study was to determine the prevalence, molecular genetic characteristics, antibiotic resistance and virulence factors of methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant S. aureus (MRSA) and methicillin-resistant coagulase-negative staphylococci (MR-CoNS) isolated from 200 samples of retail ready-to-eat raw fish (sashimi) collected from the Japanese prefecture of Hiroshima. We characterized 180 staphylococcal strains. A majority of the grocery stores surveyed (92%, 23/25) contained fish contaminated with Staphylococcus species. We recovered 175 S. aureus isolates from 174 (87%, 174/200) samples, with 170 isolates of MSSA. For the MRSA and MR-CoNS, 10 isolates were obtained from 10 samples (5%, 10/200) collected from 10 shops (40%, 10/25) belonging to four supermarket chains. SCCmec typing revealed the presence of a type IV.1 SCCmec cassette in S. warneri isolates, a type II.1 SCCmec cassette in S. haemolyticus isolates and a cassette in methicillin-resistant S. aureus (MRSA) isolates that could not be typed. Molecular typing of two MRSA isolates by spa sequencing and multilocus sequence typing (MLST) identified t1767 and ST8, respectively. Antibiotic resistance genes that confer resistance to aminoglycosides, tetracyclines, β-lactams, macrolides, lincosamides and streptogramin B (MLS(B)) antibiotics were detected. Genes encoding one or more of the following virulence factors: staphylococcal enterotoxins (seb, and sed), toxic shock syndrome toxin 1 (tst), exfoliative toxin (etaA) were detected in 14.2% (25/175) of S. aureus isolates. The accessory gene regulator (agr) typing of S. aureus isolates revealed that agr type 1 was most prevalent (96.5%, 169/175) followed by type 2 (2.2%, 4/175) and type 3 (1.1%, 2/175). None of the S. aureus isolates carried the Panton-Valentine leucocidin (PVL) encoding genes, lukF-PV and lukS-PV. To the best of our knowledge, this is the first report to show MRSA and MR-CoNS isolated from retail ready-to-eat food in Japan. Our results showed that sashimi is a likely vehicle for transmission of multidrug-resistant and toxigenic staphylococci.