Staphylococcal enterotoxin A: a chromosomal gene product.

Physical and genetic analysis of the plasmid make-up of Staphylococcus aureus strains S6 and FRI-100 has shown that staphylococcal enterotoxin A is a chromosomal gene product.     

Identification of a new putative enterotoxin SEU encoded by the egc cluster of Staphylococcus aureus

AIMS: This paper reports on a new putative enterotoxin SEU encoded by the enterotoxin gene cluster egc from Staphylococcus aureus and on a real-time polymerase chain reaction (PCR) assay for detecting the seu gene. METHODS AND RESULTS: PCR and sequencing revealed a new putative enterotoxin SEU encoded by some egc clusters. The seu gene resulted from sequence divergence in the psient1 and psient2 pseudogenes previously described in the egc cluster (Jarraud et al. [2001] Journal of Immunology166, 669). The presence of the seu gene was investigated in a collection of S. aureus strains by conventional PCR and by a specific 5'-nuclease PCR assay. Among the 24 strains harbouring the egc cluster, four tested positive for the seu gene. CONCLUSIONS: The existence of the seu gene adds to the number of newly described se genes and underlines the need for a better understanding of their role in the pathogenesis of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: A thorough study of the seu gene should provide further insight into the phylogenetics of the staphylococcal enterotoxins.     

Identification of a chromosomal determinant of enterotoxin A production in Staphylococcus aureus.

Chromosomal mapping of the determinants for enterotoxin A and enterotoxin B production in three strains of Staphylococcus aureus was attempted by using conventional transformation procedures and a series of multiply marked derivatives of NCTC 8325 as recipients. A gene governing enterotixin A production (entA+) in strain S-6 was located on the chromosome between the pur-110 and ilv-129 markers, very close to a determinant of alpha-hemolysin production, hla+. The entA+ gene of strain FRI-196E was shown not to be located at the same position; its location could not be determined. The entB+ genes of strains S-6 and C243 were not located within the known linkage groups examined. Recombinants were screened for enterotoxin production by a new procedure that combined characteristics of immune serum plate and optimal sensitivity plate procedures. The strains and methods used in this study of enterotoxin determinants should prove useful in genetic studies to locate other chromosomal determinants of S. aureus whose phenotypes are difficult to score or select for.     

奶牛源MRSA肠毒素基因、耐药基因及分子分型分析

为了解宁夏地区奶牛源耐甲氧西林金黄色葡萄球菌的肠毒素基因和耐药基因分布及其分子流行病学特征,本研究通过聚合酶链式反应(polymerase chain reaction, PCR)技术对前期分离于宁夏地区的9株奶牛源耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus, MRSA)进行了18种肠毒素基因和16种耐药基因的检测,同时采用脉冲场凝胶电泳(pulsed-field gel electrophoresis, PFGE)、正向重复序列(direct-repeat unit, dru)和辅助基因调节因子(accessory gene regulator, agr)分子分型技术对MRSA菌株进行分型研究。结果显示所有MRSA菌株均携带经典型肠毒素基因和新型肠毒素基因,共检出12种肠毒素基因,其中selk基因的检出率最高,达到了100%,未检出see、selj、selo、selp、ser和selu基因;11种耐药基因被检出,其中norA、gyrA、grlA和blaZ 4种基因的检出率均达到了100%,未检出tet (O)、optrA、Lin (A)、fexA和cfr基因。PFGE分型结果显示受试菌株间亲缘关系较近;dru分型检出dt11v和dt10a两种型,其中以dt11v(77.8%, 7/9)为主;agr分型主要为agr-Ⅰ型(88.9%, 8/9),agr-Ⅱ型仅有1株。研究表明宁夏地区奶牛源耐甲氧西林金黄色葡萄球菌(MRSA)中的肠毒素基因和耐药基因分布广泛,菌株间亲缘关系较近,agr-Ⅰ-dt11v为MRSA菌株中的流行基因型。这为以后宁夏地区奶牛源MRSA的产毒性、耐药性和分子流行病学特征的进一步研究提供理论依据。     

Development of a single-reaction multiplex PCR toxin typing assay for Staphylococcus aureus strains

We describe here the development of a single-reaction multiplex PCR assay for the enterotoxin genes from Staphylococcus aureus that utilizes a universal toxin gene primer in combination with toxin-specific primers to amplify characteristic toxin gene products. In combination with a new DNA purification method, the assay can detect enterotoxin genes A to E from a pure culture within 3 to 4 h. The test was used to characterize a diverse set of environmental S. aureus isolates, and a 99% correlation with toxin typing using standard immunological tests was found. The design of the assay allows it to be extended to include both newly characterized and as-yet-unknown toxin genes.     

Cloning and nucleotide sequence of the type E staphylococcal enterotoxin gene.

The gene for staphylococcal enterotoxin type E (entE) was cloned from Staphylococcus aureus into plasmid vector pBR322 and introduced into Escherichia coli. A staphylococcal enterotoxin type E-producing E. coli strain was isolated. The complete nucleotide sequence of the cloned structural entE gene and the N-terminal amino acid sequence of mature staphylococcal enterotoxin type E were determined. The entE gene contained 771 base pairs that encoded a protein with a molecular weight of 29,358 which was apparently processed to a mature extracellular form with a molecular weight of 26,425. DNA sequence comparisons indicated that staphylococcal enterotoxins type E and A are closely related. There was 84% nucleotide sequence homology between entE and the gene for staphylococcal enterotoxin type A; these genes encoded protein products that had 214 (83%) homologous amino acid residues (mature forms had 188 [82%] homologous amino acid residues).     

Enterotoxigenicity of Staphylococcus strains isolated from Spanish dry-cured hams.

The ability of 135 Staphylococcus strains isolated from Spanish dry-cured hams to produce enterotoxins in culture was investigated by the reversed passive latex agglutination method. A high percentage of enterotoxigenic Staphylococcus aureus strains (85.9%) was recorded, and 54.3% of these produced enterotoxin A. One of the two Staphylococcus epidermidis strains produced enterotoxin C. The reversed passive latex agglutination method yielded satisfactory results.     

Enterotoxigenicity of Staphylococcus strains isolated from Spanish dry-cured hams.

The ability of 135 Staphylococcus strains isolated from Spanish dry-cured hams to produce enterotoxins in culture was investigated by the reversed passive latex agglutination method. A high percentage of enterotoxigenic Staphylococcus aureus strains (85.9%) was recorded, and 54.3% of these produced enterotoxin A. One of the two Staphylococcus epidermidis strains produced enterotoxin C. The reversed passive latex agglutination method yielded satisfactory results.     

Detection of Staphylococcus aureus and enterotoxin genotype diversity in Monte Veronese, a Protected Designation of Origin Italian cheese

AIMS: To evaluate the risk associated with the load and enterotoxigenicity of Staphylococcus aureus in Monte Veronese, a PDO (Protected Designation of Origin) cheese of the Lessinia area (Verona, Italy). METHODS AND RESULTS: Staphylococcus aureus was quantified by a conventional culture method and by a nucA targeted real-time PCR assay developed in this study. Staphylococcus aureus numbers in cheese were higher than the limit tolerated by the Italian food legislation in 78% instances, according to both detection methods. Multiplex PCR tests for 17 Staph. aureus enterotoxin (SE) genes were applied to nucleic acids extracted from curds, cheeses and Staph. aureus isolates. The SE gene diversity appeared reduced after ripening. The gene encoding SED was found most frequently in dairy samples and the enterotoxin genes ser, sed, seg and sem predominated in the isolates. CONCLUSIONS: The occurrence of enterotoxigenic Staph. aureus strains with complex SE genotypes in this PDO cheese at numbers often exceeding the Italian tolerance threshold represents an important risk factor. SIGNIFICANCE AND IMPACT OF THE STUDY: The high frequency of contamination of Monte Veronese PDO cheese and, expectedly, similar typical productions from raw milk, by enterotoxigenic Staph. aureus imposes a tighter hygienic control in the earlier manufacturing phases.     

Modified method for production and purification of Staphylococcus aureus enterotoxin B.

A medium containing 4% bio-trypcase and 1% yeast extract was used for the production of Staphylococcus aureus enterotoxin B. The yield obtained was estimated at 200 micrograms of enterotoxin per ml of S. aureus S-6 culture supernatant. The purification method involves chromatography on Biorex 70 resin, isoelectric focusing, and gel filtration on Sephadex G-100. The purified enterotoxin (isoionic point, pH 8.55) was shown to be homogenous protein with a molecular weight of 29,000 when tested by gel electrophoresis.     

The profiles of enterotoxin genes in Staphylococcus aureus from nasal carriers

AIMS: To evaluate the occurrence of enterotoxin genes in Staphylococcus aureus recovered from nasal carriers. METHODS AND RESULTS: Eighty S. aureus strains were tested for the presence of 17 new enterotoxin genes using multiplex-PCR. Sixty-one isolates were found to carry enterotoxin genes. The majority of the enterotoxigenic isolates carried enterotoxin gene cluster (egc) genes, namely seg, sei, sem, sen and seo. The egc type containing the seu gene was found in 19 of the 47 isolates with egc-like genes. Interestingly, no seu-containing egc coexisted with sec and sel, as was the case for a considerable portion of the isolates carrying a seu-negative egc. The tst gene was detected in two isolates carrying sec and sel only and in eight isolates carrying seu, but not in the isolates containing the seu-negative egc type. CONCLUSIONS: The genes forming an egc were found to be predominant in S. aureus from nasal carriers. The coexistence of a seu-positive egc with tst in contrast to an egc lacking the seu gene apparently is not associated with the presence of tst and can reflect a difference between these gene groupings. SIGNIFICANCE AND IMPACT OF THE STUDY: The egc types carried by the analysed isolates seem to have an influence on the distribution of other genes located on staphylococcal pathogenicity islands, which may modulate the repertoire of virulence factors carried by a single S. aureus strain.     

耐甲氧西林金黄色葡萄球菌及其肠毒素的检测和耐药性分析

目的评价乳胶结合试验检测耐甲氧西林金黄色葡萄球菌(MRSA)及其肠毒素(SE),并进行耐药性分析.方法收集130株金黄色葡萄球菌临床分离株,通过药敏试验将其分为耐甲氧西林金黄色葡萄球菌和甲氧西林敏感金黄色葡萄球菌(MSSA),用反向间接血凝试验(RPHA)检测金黄色葡萄球菌肠毒素.结果67株MR-SA产肠毒素,19株MSSA产肠毒素,MRSA产肠毒素率为100%,MSSA产肠毒素率为30%.结论实验室应重视金黄色葡萄球菌肠毒素的检测.     

Staphylococcal enterotoxin B gene is associated with a discrete genetic element.

The chromosomal location of the enterotoxin B gene in Staphylococcus aureus is unknown. Southern hybridization analysis of the chromosomal DNA from several enterotoxin B (SEB)-producing strains has shown that at least 26.8 kilobases (kb) of DNA is associated with the enterotoxin B gene (entB). We have found that one end of the entB element is located approximately 1.5 kb downstream of the entB gene. The chromosomal region adjacent to this end of the entB element was found to be homologous in several SEB-producing (SEB+) and SEB-nonproducing (SEB-) S. aureus strains. The chromosomes of all the SEB+ strains studied were homologous for at least 24 kb upstream of the entB gene. Some naturally occurring SEB- strains lacked the entire entB element, while others showed variable homology to the region upstream of the entB gene. These data suggest that the entB gene is part of a discrete genetic element that is at least 26.8 kb in size.     

安徽不同地区奶站牛奶中金黄色葡萄球菌及其肠毒素污染状况评估

目的评定安徽地区各奶站牛奶中金黄色葡萄球菌以及肠毒素的污染情况。方法通过从安徽省不同地区30所奶站采集乳样,进行乳源性金黄色葡萄球菌的分离与生化鉴定,并采用PCR技术对分离出的菌株进行金黄色葡萄球菌肠毒素血清型鉴定。结果安徽省30个奶站中有4个地区奶站的牛奶中污染金黄色葡萄球菌;从污染牛奶中共分离出5株金黄色葡萄球菌,检出率为16.7%。经鉴定,所分离出的金黄色葡萄球菌中2株为肠毒素A型,1株为肠毒素C型,2株为同时产肠毒素A和肠毒素C。结论安徽省不同地区奶站中的牛奶污染的金黄色葡萄球菌产肠毒素类型以肠毒素A为主。     

Staphylococcus aureus in rural drinking water.

Coagulase-positive Staphylococcus aureus was isolated from over 6% of 320 rural drinking water specimens. Well water was the most common source examined. The presence of S. aureus was not found to correlate with the presence of coliform bacteria. Strains of Staphylococcus that produced enterotoxin A were found in 40% of the samples containing S. aureus. Additional studies showed that faucet aerator screens were common sources of high cell densities of S. aureus.     

Transport and processing of staphylococcal enterotoxin A

A larger-molecular-weight precursor of enterotoxin A was found in membranes of Staphylococcus aureus and was shown to be the kinetic precursor to the extracellular form of the toxin. Subcellular fractionation revealed that mature enterotoxin A was transiently associated with the cell wall before being released to the extracellular environment.     

Characterization of Staphylococcus aureus strains isolated from raw milk of bovine subclinical mastitis in Tehran and Mashhad

Staphylococcus aureus is considered one of the most important food borne pathogens. A total of 111 isolates of S. aureus were cultured from raw milk samples during January 2009 to June 2009 from Tehran and Mashhad. The coagulase gene polymorphism and the prevalence of classical enterotoxin genes of S. aureus strains were determined by PCR-RFLP (restriction fragment length polymorphism) and Multiplex-PCR. Disk diffusion method was used to determine the susceptibility of isolates to antimicrobial agents as instructed by Clinical and Laboratory Standards Institute. Sixty-seven % of the isolates harboured one or more enterotoxin genes. The most prevalent gene was sec, found in 59 % of the isolates. Approximately 8% of the isolates were positive for sea, seb and sed genes. Only one isolate had see gene. The rate of coexistence of enterotoxin genes was 14%. All S. aureus isolates were susceptible to ciprofloxacin, gentamicin, imipenem, minocycline, oxacillin and vancomycin. They were resistant to ampicillin (64%), penicillin (56%), clindamycin (22%), tetracycline (22%), doxycycline (19%), teicoplanin (13%), rifampin (2%) and trimethoprim-sulfamethoxazole (2%). On the basis of coagulase gene analysis of 111 S. aureus isolates, the PCR products of 56 isolates were digested with Alu I that produced three distinct patterns. These data indicate the high prevalence of enterotoxigenic S. aureus in raw bovine milk in Tehran and Mashhad, and highlight the importance of proper quality control of dairy products for public health.