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.     

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。     

Evaluation of molecular methods to determine enterotoxigenic status and molecular genotype of bovine, ovine, human and food isolates of Staphylococcus aureus

This study evaluated the use of PFGE and single enzyme AFLP techniques for the determination of the genetic relationships between Staphyloccocus aureus isolates from human, bovine, ovine and food related sources and reports the prevalence of 'classic' (sea to see) and 'new' (seg, seh, sei, sej, sem, sen and seo) staphylococcal enterotoxin (se) genes in 92 S. aureus strains. A sub-set of the se genotyping results was confirmed by ELISA and the presence of SE toxin determined in isolates from different sources. A 100% correlation was observed, between detection of enterotoxin genes sea-see and expression of corresponding enterotoxin proteins in vitro. The se genotyping data generated from 90 of the S. aureus isolates showed that many of the S. aureus strains producing identical se genotypes correlated with both AFLP and PFGE pattern types. However, single enzyme AFLP technique did not possess the discriminatory power of the PFGE method, but similar clonal relationships were observed by both techniques in many of the isolates tested. Results reported here include the first comprehensive study using a single enzyme AFLP technique to investigate the genetic background of S. aureus isolates from a wide distribution including animal, human and food related sources.     

Characterization of Staphylococcus aureus isolated from powdered infant formula milk and infant rice cereal in China

Dry infant foods are not sterile and could be contaminated with various bacteria including certain pathogens. The aim of this study was to investigate the prevalence of Staphylococcus aureus in infant foods and to characterize these strains. A total of 367 infant food samples, including 143 samples of powdered infant formula milk (PIF) and 224 samples of infant rice cereal (IRC), were collected in the Shaanxi Province of China during the period of July to August 2010 and screened for S. aureus. All S. aureus isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and detection of genes encoding enterotoxins, exfoliative toxins, Panton-Valentine leukocidin (PVL), and toxic shock syndrome toxin 1. Among all the samples examined, sixteen of 143 PIF samples (11.2%) and 14 of 224 IRC samples (6.3%) were positive for S. aureus. From these positive samples, 29 S. aureus strains were isolated from PIF and 25 from IRC. Of these S. aureus isolates, 83.3% were resistant to at least one antimicrobial, 35.2% to three or more antimicrobials. Resistance was most frequently observed to erythromycin (75.9%), followed by ciprofloxacin (51.9%) and trimethoprim/sulfamethoxazole (27.8%), while significantly fewer isolates were resistant to gentamicin (22.2%), tetracycline (18.5%), or cefoxitin (3.7%). In addition, 63.0% of isolates were positive for one or more toxin genes tested. The three most predominant toxin genes were pvl (40.7%), seg (38.9%), and sec (18.5%), followed by sea (7.4%), seb (7.4%), sed (5.6%), and see (5.6%). The ets, tsst-1, seh, sei, and sej genes were not detected. A total of 39 PFGE patterns were generated among 51 selected food isolates. Our findings indicate that PIF and IRC in the Shaanxi province were contaminated with S. aureus, and many S. aureus isolates harbored multiple toxin genes and exhibited multiple antimicrobial resistance. In addition, these S. aureus isolates were genetically diverse. The presence of S. aureus strains in these infant foods poses a potential threat to infant health.     

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.     

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。     

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.     

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.     

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

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

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.     

A polyphasic approach to detect enterotoxigenic Staphylococcus aureus and diarrheagenic Escherichia coli in raw milk Italian cheeses by multiplex PCR

A polyphasic approach was evaluated for the detection of eight staphylococcal enterotoxin (SE)-encoding genes (sea, sec, sed, seg, seh, sei, sej, sel) and the Escherichia coli genes most commonly associated with virulence factors (eae, elt, ipaH, stx) in traditional soft cheeses, manufactured artisanally from whole raw milk in the Lombardy region (northern Italy). To determine the presence of the target genes, two multiplex PCRs were performed on DNA extracted both directly from cheese samples (culture-independent approach) and from whole cultivable cells, formed by harvesting colonies from the first serial dilution agar plates of selective media, as representative of cultivable community ("bulk"). Genes associated with enteroinvasive E. coli, ipaH, and Shiga toxin-producing E. coli, stx, were detected in two of the bulk samples analyzed; no virulence genes were found by amplifying DNA directly extracted from cheeses. SE-encoding genes were found in three cheeses (sea in all three samples, associated with sed and sej in two of these). More SE-encoding genes were detected by amplifying DNA obtained from bulk samples: sea, sed, sej, sec, seg, sel, and sei. No samples harbored the gene encoding for SE type H. The polyphasic approach followed has been useful in enhancing detection of target genes. Our results indicate that some of the artisanal cheeses examined may constitute a potential hazard for consumer health.     

市售凉皮中金黄色葡萄球菌的肠毒素基因、耐药特征检测及SPA分型

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

Characterization of Staphylococcus aureus strains associated with food poisoning outbreaks in France

Enterotoxins produced by Staphylococcus aureus are responsible for staphylococcal food-poisoning outbreaks (SFPO). In France, SFPO are the second cause of food-borne diseases after Salmonella. However, very little is known about the strains involved. The objective of this study was to characterize the staphylococcal strains related to these SFPO through phenotypic and genotypic analyses. A total of 178 coagulase-positive staphylococcal isolates recovered from 31 SFPO (1981-2002) were screened through biotyping. Thirty-three strains representative of the different biotypes in each SFPO were further examined for SmaI macrorestriction-type, phage-type, resistance to various antimicrobial drugs, presence of staphylococcal enterotoxin (se) genes sea to sei, and production of enterotoxins SEA to SED. All these 33 strains were identified as S. aureus species: 27 were of human biotypes and six ovine or non-host-specific biotypes. Most (74.1%) strains reacted with group III phages. Eleven strains were resistant to at least two classes of antibiotics and among them, two were resistant to methicillin. Twenty-nine strains carried one or several of the eight se genes tested; the gene sea was most common (n=23), and often linked to sed (n=12) or seh (n=5). The novel se genes seg-i were in all cases associated with se genes sea to sed except for one strain which carried only seg and sei. Pulsed-Field Gel Electrophoresis (PFGE) of SmaI macrorestriction digests of the 33 strains discriminated 32 PFGE patterns grouped into nine biotype-specific clusters. All five strains carrying sea and seh were grouped together into the same sub-cluster. Three of the four se-gene-negative strains were in one PFGE cluster: all four should be tested for se genes not included in this study and, if negative, be further investigated for the presence of unidentified SEs.     

Comparison of antimicrobial susceptibility of Staphylococcus aureus isolated from bulk tank milk in organic and conventional dairy herds in the midwestern United States and Denmark

An observational study was conducted to compare the antimicrobial susceptibility patterns of Staphylococcus aureus isolated from bulk tank milk in organic and conventional dairy farms in Wisconsin, United States, and southern Jutland, Denmark. Bulk tank milk samples and data regarding management and production were collected from 30 organic and 30 conventional dairy farms in Wisconsin and 20 organic and 20 conventional dairy farms in Denmark. S. aureus isolates were tested for resistance against 15 antimicrobial agents by semiautomatic microbroth dilution methods in each country. Of the 118 bulk tank milk samples in Wisconsin, 71 samples (60%) yielded at least one S. aureus isolate, and a total of 331 isolates were collected. Of the 40 bulk tank milk samples from Denmark, 27 samples (55%) yielded at least one S. aureus isolate, and a total of 152 isolates were collected. Significant differences between organic and conventional dairies were detected only to ciprofloxacin in Wisconsin and avilamycin in Denmark. Significant differences (P < 0.05) between the two countries were detected in nine antimicrobials. Denmark had a higher probability of having reduced susceptibility to ciprofloxacin and streptomycin (P = 0.015 and 0.003, respectively). Wisconsin isolates had a higher probability of having reduced susceptibility to seven other antimicrobial agents (bacitracin, gentamicin, kanamycin, penicillin, sulphamethoxazole, tetracycline, and trimethoprim). We found small differences between organic and conventional farm types in each country and larger differences between the two national agricultural systems.     

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.     

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。     

Occurrence of toxigenic Staphylococcus aureus in ready-to-eat food in Korea

Toxigenic Staphylococcus aureus contamination in ready-to-eat (RTE) food is a leading cause of foodborne illness in Korea. To monitor food contamination by S. aureus, a total of 3332 RTE food samples were selected from nationwide wholesale marts between 2003 and 2004 and examined. A total of 285 (8.6%) of the overall samples were contaminated by S. aureus. According to the analysis, 31.6% of the tested cream-cakes, 19.8% of the raw fish, and 19.3% of the rice cakes with filling were contaminated with S. aureus. Forty-seven percent of the strains isolated from the contaminated food were enterotoxigenic S. aureus. The phenotypic result of the strain isolated from food showed that 48% of the strains produced one or more toxins, such as staphylococcal enterotoxins A, B, and C (SEA, SEB, and SEC). At least one SEA was produced by over 90% of the toxigenic strains. Other toxins, such as SEB, SEC, SED, SEA+SEC, and SEC+SED, were each detected. Toxic shock syndrome toxin 1 (TSST-1), a causative agent of toxic shock syndrome, was detected in 13 strains of the toxigenic isolates from the food. As the result of genotyping, 22 strains with a toxin gene that was not detected in the phenotypic analysis were also detected. Sixty-nine percent of the toxigenic strains had at least one sea gene, and the most prevalent genotype was sea+seh (34.4%), followed by sea (18.8%) and sea+seg+sei (15.6%). The tst gene encoding TSST-1 was found in 13 strains (13.5%). The genes (eta and etb) encoding exfoliative toxins A and B were not detected in any of the samples.     

Growth of gram-positive mastogenic bacteria in normal, simulated bulk tank, and mastitic milk held at simulated fluctuating temperatures of farm bulk tank

Growth of Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus bovis, and Streptococcus uberis was studied in normal milk, simulated bulk tank milk, and aseptic mastitic milk held at simulated fluctuating temperatures of farm bulk tank for 48 h. With the exception of S. bovis, growth rates of the other five bacteria were similar in both normal and simulated bulk tank milk. Mastitic milk inhibited growth of all bacteria studied. A 24-h adjustment period occurred before most of the bacteria started growing. The mastitis level in a dairy herd may be monitored by cultures of bulk tank milk samples and by calculations as discussed in this study.     

Study of Staphylococcus aureus collected at slaughter from dairy cows with chronic mastitis

Staphylococcus aureus is one of the most important pathogens associated with bovine mastitis. Recent studies have shown that Staph. aureus strains may differ in virulence, and in their ability to disseminate across commercial dairy herds. The goal of this study was to determine whether Staph. aureus isolates differed in their ability to colonize mammary tissue, and whether such differences could be related to molecular characteristics. Quarter milk and mammary tissues of 22 cows from two dairy herds, were collected at slaughter and bacteriological analysis was performed. All Staph. aureus isolates were characterized by Pulsed Field Gel Electrophoresis (PFGE) and microarray. Overall 45 mammary quarters were infected and 20 Staph. aureus isolates were identified. The bacteria were mostly recovered from both milk and tissue of the same quarter in significantly higher numbers from herd A cows compared with herd B. Molecular characterization of the isolates showed distinct PFGE profiles for isolates from each herd. Differences in virulence factors between herds A and B isolates were evidenced The genes for enterotoxin D, J and R were present in herd A, those for G, I, N, M, O and U were shown in herd B, whilst both components of the leukocidin lukD/E genes were only carried by herd A isolates. Furthermore, all herd A isolates showed β-haemolysin activity, which was absent in all but one isolate from herd B. Therefore our data indicate that Staph. aureus isolates showing differences in their ability to disseminate and colonize across quarters, also have significantly different virulence characteristics.