Systematic survey on the prevalence of genes coding for staphylococcal enterotoxins SElM, SElO, and SElN

Staphylococcus aureus remains a leading cause of food-poisoning with substantial impact on public health. Using a multiplex polymerase chain reaction-DNA enzyme immunoassay (PCR-DEIA), we studied the presence of genes encoding staphylococcal enterotoxin-like (SEl) superantigens sem, sen, and seo, associated with the enterotoxin gene cluster (egc), in 429 clinical Staphylococcus aureus isolates. 294 (68.5%) isolates tested positive for at least one of the three SEl genes. In contrast to the fixed gene combination seg/sei also located on egc, a substantial number of isolates (n = 108) were found to bear only one or two of the genes encoding SElM, SElN, and SElO. Regarding the origin of the S. aureus isolates, a significant difference (P = 0.022) was found for the possession of seo (61.2% of blood isolates versus 42.9% of nasal strains). Also sem (not significantly) was found more common in blood isolates (52.1% versus 40.5%). The survey of the newly described SEl genes sem-seo supports the concept that most clinical S. aureus isolates harbor subsets of pyrogenic toxin superantigens. The potential contribution of seo and sem to the pathogenic potential of S. aureus has to be further evaluated.     

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 <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.     

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.     

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.     

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.     

食源性金黄色葡萄球菌肠毒素基因及其表达检测

金黄色葡萄球菌作为一种常见人类病原菌,可通过各种途径污染食品,引发食物中毒。其中,金黄色葡萄球菌肠毒素(SE)的分泌会大大增加其侵袭性和致病力。通过对比肠毒素基因的携带及其表达情况,有助于进一步分析肠毒素表达的环境条件,为产肠毒素金黄色葡萄球菌的防范和控制提供理论和数据支持。本研究采用PCR和3M^TMTecra^TM微孔板法分别检测33株食源性金黄色葡萄球菌中5种传统肠毒素SEA^SEE基因的携带及表达情况,结果显示,该批金黄色葡萄球菌中SE基因的检出率为100%,携带率最高和最低的分别为seb(48.48%,16/33)和see(9.09%,3/33)。而其中SE蛋白得到表达的菌株仅为63.64%(21/33),这表明不是有SE基因携带就一定可以有相应毒素蛋白的表达,可能还需相应的系统调控和适宜的环境因素。     

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。     

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.     

Staphylococcal enterotoxin genes of classical and new types detected by PCR in France

The PCR detection of the genes coding for the newly described staphylococcal enterotoxins (SE) G, H, I and J was carried out for 332 foodborne staphylococci, isolated from a variety of foods in France. The frequency of the Staphylococcus aureus strains harboring these genes was found to be very high (57%) and greater than that of the strains harboring "classical" SE genes as previously established. If one takes into account the newly described SE genes, in addition to the classical SE genes, the percentage of foodborne enterotoxigenic S. aureus strains doubles. The S. aureus biovars that were rarely or never enterotoxigenic (i.e., the poultry and bovine biovars) frequently become more potentially toxigenic, if taking into account the seg. seh, sei and sej genes. Furthermore, this work indicated a systematic association between the seg and sei genes and their high incidence among the S. aureus strains, which suggests that these two SE's could be an important phylogenetic link among the staphylococcal enterotoxins.     

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...     

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...     

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.     

广州市白云口岸航空食品中金黄色葡萄球菌凝固酶基因分型及肠毒素基因研究

目的对2013—2015年从广州市白云口岸航空食品中分离的金黄色葡萄球菌进行基因分型研究,为食源性金黄色葡萄球菌分子溯源提供基础数据。方法以血浆凝固酶和肠毒素为目标基因,采用聚合酶链式反应(PCR)方法对9株金黄色葡萄球菌进行基因分型,其中6株为航空食品分离株,1株为配餐车间大门手拭分离株,2株为标准菌株。肠毒素基因检测包括5种传统肠毒素基因(sea、seb、sec、sed、see)和6种新型肠毒素基因(ser、seg、seh、sei、sej、sep)。结果 6株航空食品分离株的血浆凝固酶基因扩增分型结果为2个PCR型,酶切后得3种亚型;肠毒素基因检测结果显示有2株航空食品分离株含有肠毒素基因,检出率为33.3%(2/6),检出的基因为2种传统肠毒素基因(sec、sed)和4种新型肠毒素基因(ser、seg、sei、sej),均同时携带2种以上肠毒素基因。结论血浆凝固酶基因扩增分型结果显示,不同时间、不同采集地点存在相同的基因型,提示金黄色葡萄球菌存在交叉污染的可能性;航空食品分离株共检出6种肠毒素基因,提示金黄色葡萄球菌基因型多样性,应加强其他新型肠毒素基因检测。     

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.     

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.     

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.     

Relationships among superantigen toxin gene profiles,genotypes, and pathogenic characteristics of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is one of the major etiological agents of bovine mastitis, harboring a wide variety of staphylococcal superantigen (SAg) toxin genes. The SAg toxin genes are reported to be closely associated with the pathogenicity of the Staph. aureus causing the bovine mastitis. This study was conducted to investigate SAg toxin gene profiles and to assess the relationships among SAg toxin genes, genotypes of Staph. aureus, and their pathogenic properties. A total of 327 quarter milk samples were collected from bovine mastitis cases for isolation and identification of pathogens. In total, 35 isolates were identified as Staph. aureus, and the prevalence of Staph. aureus in milk samples was 13.6% (35/256). Polymerase chain reaction (PCR) and randomly amplified polymorphic DNA (RAPD) assays were used to detect the SAg toxin genes and to genotype Staph. aureus strains isolated from milk samples of bovine mastitis in 10 dairy herds located in Ningxia, China, respectively. The results showed that among the Staph. aureus isolates (n = 35), 71.4% (n = 25) of isolates carried at least one SAg toxin gene. In total, 18 SAg genes and 21 different gene combination patterns were detected among these isolates. The most common SAg genes in Staph. aureus isolates were sei, sen, and seu (44.0% each), followed by seo, tst, and etB (28.0% each), etA (24.0%), sem and sep (16.0% each), seb, sec, sed, and sek (12.0% each), and sea and seh genes (8.0% each); the seg, sej, and ser genes were present in 4.0% of the isolates. Three gene combinations were found to be related to mobile genetic elements that carried 2 or more genes. The egc-cluster of the seg-sei-sem-sen-seo genes, located on the pathogenicity island Type I υSaβ, was detected in 16% of isolates. Interestingly, we observed 6 RAPD genotypes (I to VI) in Staph. aureus isolates, and 2 of these genotypes were strongly associated with the severity of bovine mastitis; there was a close relationship between the RAPD genotypes and SAg genes. Isolates of RAPD type III were more frequently associated with clinical and subclinical mastitis, whereas strains of type VI were mostly related to subclinical mastitis. In addition, SAg genes were related to severity of bovine mastitis. We conclude that an obvious relationship exists among RAPD genotypes, SAg toxin genes, and severity of bovine mastitis.