Staphylococcus aureus and Staphylococcal Enterotoxin A in Breaded Chicken Products: Detection and Behavior during the Cooking Process

In this study we examined the presence of Staphylococcus aureus and staphylococcal enterotoxin A (SEA) in 20 industrial breaded chicken products obtained from different retail butchers and supermarket stores in Italy. The levels of contamination in the products analyzed were quite low, although the pH values and water activities (a_w) in the samples considered were in ranges favorable for S. aureus growth. As demonstrated by phenotypic and molecular characterization, in spite of the high percentage of coagulase-positive Staphylococcus strains, only three strains could be referred to the species S. aureus. Moreover, all the strains were negative in PCR assays targeting staphylococcal enterotoxin genes (seA to seE, seG to seJ, and seM to seO), as well as the toxic shock syndrome toxin 1 gene, and no SEA was detected in the retail breaded chicken samples analyzed by a reversed passive latex agglutination assay or by Western blotting. Hence, we evaluated the thermal resistance of two strains of SEA-producing S. aureus in a laboratory-scale preparation of precooked breaded chicken cutlets. The heat treatment employed in the manufacture determined the inactivation of S. aureus cells, but the preformed SEA remained active during product storage at 4°C. The presence of the staphylococci and, in particular, of S. aureus in the retail breaded chicken products analyzed is a potential health risk for consumers since the pH and a_w values of these kinds of products are favorable for S. aureus growth. The thermal process used during their manufacture can limit staphylococcal contamination but cannot eliminate preformed toxins.     

Detection rate of enterotoxigenic Staphylococcus aureus isolated from children with intestinal disbacteriosis

Detection rate of enterotoxigenic Staphylococcus aureus isolated from faeces of 62 children aged from 3 months old to 7 years old with intestinal dysbacteriosis was studied by indirect hemagglutination assay and enzume immunoassay. It was shown that strains of S.aureus producing staphylococcal enterotoxin A (SEA) are prevailed (40.3%) in children with disturbances of intestinal microflora while staphylococcal enterotoxin B (SEB)-producing strains were detected in 20.9% of children. Amount of produced enterotoxin varied for SEA from 125 ng/ml to 2000 ng/ml and for SEB--from 125 ng/ml to 250 ng/ml. Inverse dependence of detection rate of enterotoxin-producing strains in faeces on age of children was established. The most number of enterotoxigenic strains of S.aureus was detected in infants. These data point to expediency of determination of enterotoxin-producing ability of S. aureus strains isolated from children with dysbacteriosis as measure of danger of this microorganism for children's health and indication for adequate actions for its elimination.     

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。     

Survey and properties of Staphylococcus aureus isolated from Japanese-style desserts

We surveyed the contamination of 315 Japanese- and western-style desserts and 247 human hands by Staphylococcus aureus and other staphylococcal bacteria. The most frequently isolated staphylococcal bacterium was S. warneri, followed by S. aureus. Only 1.9% of western-style desserts were contaminated by S. aureus strains, while 19.4% and 13.0% of Japanese-style desserts and human hands respectively were contaminated. Ninety-four isolates of S. aureus were characterized as to their biological properties and enterotoxigenicity. Although staphylococcal enterotoxins (SEs) were detected by enzyme-linked immunosorbent assay in the cultured broth of all S. aureus isolates, the reversed passive latex agglutination method and the polymerase chain reaction showed only 39 (41.5%) and 40 (42.6%) samples respectively as SE-positive. The predominant type of SE was SEB (67.5%), and eight strains produced SEA. None of the S. aureus strains had penicillin-binding protein 2', showing that methicillin-resistant S. aureus was not present in the samples.     

Pulsed-field gel electrophoretic analysis and some characteristics of Staphylococcus aureus isolated from retail foods and human hands

This study investigates whether there is a predominant Staphylococcus aureus strain in retail foods and healthy human hands, and examines the relationship between pulsed-field gel electrophoresis (PFGE) banding patterns and the S. aureus characteristics of staphylococcal enterotoxin (SE) type, coagulase type, and β-lactamase activity. Ninety-four strains of S. aureus isolated from retail foods and healthy human hands were analyzed by PFGE. Several strains isolated from the same shop or a chain store showed identical patterns, indicating that the origins of these strains were identical. After excluding these strains showing identical patterns, 54 strains were used for the PFGE analysis. No spread of a particular clone in the environment surrounding the food was apparent. The PFGE analysis of these 54 strains was classified in 6 lineages (L1-L6). There was no relationship between the PFGE banding pattern and coagulase type or SE type. Eleven (84.6%) of the 13 isolates in PFGE banding pattern L5 did not produce β-lactamase, suggesting that the production of β-lactamase influenced a specific PFGE banding pattern.     

Comparison of type A enterotoxigenic Staphylococcus aureus strains isolated from geographically far distant locations by pulsed field gel electrophoresis

Staphylococcal enterotoxin A (SEA) is one of the major staphylococcal enterotoxins which may cause food-borne outbreaks. In order to investigate the difference in genomic types and to elucidate the most disseminated strains for enterotoxin A-producing strains of Staphylococcus aureus , a total of 60 SEA Staph. aureus strains isolated from food and clinical samples in Taiwan and 30 strains of the same enterotoxigenic type of strains obtained from geographically far distant locations were compared for their pulsed field gel electrophoresis (PFGE) patterns. The rare cutting endonuclease Sma I generated 10 distinct genome patterns for the 60 local SEA isolates and 15 and eight genome patterns, respectively, for the 20 and 10 SEA strains originally isolated from the USA and other countries. The local isolates are less diverse in genome patterns as compared to the US isolates. Of all these PFGE patterns, a certain pattern, such as pattern 3, is shared by the food and clinical isolates and the local and foreign isolates. Thus, although SEA Staph. aureus strains from geographically far distant locations showed considerable genetic diversity, PFGE pattern 3 strain might be one of the most disseminated strains.     

Staphylococcus aureus and its food poisoning toxins: characterization and outbreak investigation

Staphylococcal food poisoning (SFP) is one of the most common food-borne diseases and results from the ingestion of staphylococcal enterotoxins (SEs) preformed in food by enterotoxigenic strains of Staphylococcus aureus. To date, more than 20 SEs have been described: SEA to SElV. All of them have superantigenic activity whereas half of them have been proved to be emetic, representing a potential hazard for consumers. This review, divided into four parts, will focus on the following: (1) the worldwide story of SFP outbreaks, (2) the characteristics and behaviour of S. aureus in food environment, (3) the toxinogenic conditions and characteristics of SEs, and (4) SFP outbreaks including symptomatology, occurrence in the European Union and currently available methods used to characterize staphylococcal outbreaks.     

Staphylococcus aureus and food poisoning

Food-borne diseases are of major concern worldwide. To date, around 250 different food-borne diseases have been described, and bacteria are the causative agents of two thirds of food-borne disease outbreaks. Among the predominant bacteria involved in these diseases, Staphylococcus aureus is a leading cause of gastroenteritis resulting from the consumption of contaminated food. Staphylococcal food poisoning is due to the absorption of staphylococcal enterotoxins preformed in the food. Here, we briefly review the latest data on staphylococcal enterotoxins and some papers exemplifying the interactions between S. aureus and the food matrix; environmental factors affecting staphylococcal enterotoxin production are discussed.     

The Incidence of Enterotoxin Production in Strains of Staphylococcus aureus Isolated from Foods

S ummary . The production of staphylococcal enterotoxins A, B, C, D and E among 200 strains of Staphylococcus aureus was surveyed using a double diffusion immunoprecipitation technique. Enterotoxin A was found to occur most frequently and enterotoxin B least frequently. The distribution of enterotoxigenicity in strains isolated from meat products differed from that of strains isolated from dairy products. The correlations of porcine plasma coagulation and of bacteriophage pattern with enterotoxigenicity were determined.     

Lowering of bactericidal activity of mouse peritoneal macrophages under combined use of staphylococcal enterotoxin type A and endotoxin

The present study investigated the effect of staphylococcal enterotoxin type A (SEA) and endotoxin Serratia marcescens (LPS) on the phagocytosis and killing of Staphylococcus aureus by mouse peritoneal macrophages. Two hours after enterotoxin intraperitoneal injection phagocytic and bactericidal activity were depressed. 24 hours later there was increased functional activity of macrophages by SEA and LPS, apart. But when two toxins were administered together (LPS four hours later enterotoxin) marked inhibition of bacterial killing was observed. When peritoneal macrophages were treated in vitro for 24 hours with the same toxins they were also markedly suppressed in bactericidal activity.     

PCR detection of staphylococcal enterotoxin genes in Staphylococcus spp. strains isolated from meat and dairy products. Evidence for new variants of seG and seI in S. aureus AB-8802

AIMS: Evaluation of the occurrence of most known staphylococcal enterotoxin (SE) genes, egc (enterotoxin gene cluster) and TSST1 (toxic shock syndrome toxin 1) gene in both coagulase-positive (CPS) and coagulase-negative (CNS) staphylococcal strains isolated from meat and dairy products. METHODS AND RESULTS: Specificity and reliability of the PCR detection methods used were ascertained by using nine reference strains of Staphylococcus (S. aureus) harbouring SE genes (seA to seE; seG, seH, seI, seM, seJ, seN and seO) and egc (containing the following sequence of genes: seO, seM, seI, phient1, phient2, seN and seG). Of 109 wild Staphylococcus spp. strains analysed, only 11 S. aureus strains were SE and/or TSST1 PCR-positive. The last 11 strains also appeared to harbour the egc. Restriction endonuclease analysis of part of the egc of both reference and wild strains showed that different variants of the egc exist. Moreover, nucleotide sequences of seG and seI indicate that the egc of the strain AB-8802 is characterized by the presence of variants of these enterotoxins (seGv and seIv). CONCLUSIONS: The occurrence of SE genes in CNS and other non-S. aureus species isolated from Napoli-type salami, raw water buffalo milk and natural whey cultures used for mozzarella cheese manufacturing is very rare. SIGNIFICANCE AND IMPACT OF THE STUDY: During this study it was shown that at least five different egc may exist in S. aureus. A thorough study of egc polymorphism should provide further insight into the phylogenetics of the egc.     

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.     

Enterotoxin A synthesis in Staphylococcus aureus: inhibition by glycerol and maltose

Studies indicated that prior growth of Staphylococcus aureus 196E on glycerol or maltose led to cells with repressed ability to produce staphylococcal enterotoxin A (SEA). A PTS- mutant (196E-MA) lacking the phosphoenolpyruvate phosphotransferase system (PTS), derived from strain 196E, showed considerably less repression of SEA synthesis when cells were grown in glycerol or maltose. Since SEA synthesis is not repressed in the PTS- mutant, repression of toxin synthesis by glycerol, maltose or glucose in S. aureus 196E appears to be related to the presence of a functional PTS irrespective of whether the carbohydrate requires the PTS for cell entry. With lactose as an inducer, glucose, glycerol, maltose or 2-deoxyglucose repressed the synthesis of beta-galactosidase in S. aureus 196E. It is postulated that these compounds repress enzyme synthesis by an inducer exclusion mechanism involving phosphorylated sugar intermediates. However, inducer exclusion probably does not explain the mechanism of repression of SEA synthesis by carbohydrates.     

Enterotoxins and phage typing of Staphylococcus aureus isolated from clinical material and food in Libya

Enterotoxin was detected in 22 (61.1%) of the 36 S. aureus strains isolated from clinical materials and in 3 (13%) of the 23 S. aureus strains from food samples (P < 0.05). On the basis of individual types of enterotoxin, staphylococcal enterotoxin A (SEA) was produced by 11.1%, SEB by 38.9% and SEC by 22.2% of SS. aureus strains from clinical material. Of the food S. aureus strains, SEC and SED produced by 8.7% and 4.3% respectively. Of the clinical and food S. aureus strains, 52.8% and 39.1%, respectively, were typeable by the 23 phages of International Phage Set. The majority of the typeable S. aureus strains from clinical and food sources belonged to group II being at 22.2% and 17.4% respectively. Furthermore, of the 14 SEB-producing S. aureus, 42.9% were of phage group II. In conclusion, the results obtained indicate that enterotoxin-producing S. aureus strains from clinical materials in Libya are not uncommon; however, certain foods appear not to be the source of such strains. Because of the low susceptibility to bacteriophages shown by S. aureus isolated in Libya, compared to reports from several countries, other methods of typing should be used in conjunction with phage typing in epidemiological investigations concerning this organism.     

The innate immune modulators staphylococcal complement inhibitor and chemotaxis inhibitory protein of Staphylococcus aureus are located on beta-hemolysin-converting bacteriophages

Two newly discovered immune modulators, chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) and staphylococcal complement inhibitor (SCIN), cluster on the conserved 3' end of beta-hemolysin (hlb)-converting bacteriophages (betaC-phis). Since these betaC-phis also carry the genes for the immune evasion molecules staphylokinase (sak) and enterotoxin A (sea), this 8-kb region at the 3' end of betaC-phi represents an innate immune evasion cluster (IEC). By PCR and Southern analyses of 85 clinical Staphylococcus aureus strains and 5 classical laboratory strains, we show that 90% of S. aureus strains carry a betaC-phi with an IEC. Seven IEC variants were discovered, carrying different combinations of chp, sak, or sea (or sep), always in the same 5'-to-3' orientation and on the 3' end of a betaC-phi. From most IEC variants we could isolate active bacteriophages by mitomycin C treatment, of which lysogens were generated in S. aureus R5 (broad phage host). All IEC-carrying bacteriophages integrated into hlb, as was measured by Southern blotting of R5 lysogens. Large quantities of the different bacteriophages were obtained by mitomycin C treatment of the lysogens, and bacteriophages were collected and used to reinfect all lysogenic R5 strains. In total, five lytic families were found. Furthermore, phage DNA was isolated and digested with EcoR1, revealing that one IEC variant can be found on different betaI-phis. In conclusion, the four human-specific innate immune modulators SCIN, CHIPS, SAK, and SEA form an IEC that is easily transferred among S. aureus strains by a diverse group of beta-hemolysin-converting bacteriophages.     

Enterotoxigenic Staphylococcus aureus in bulk milk in Norway

AIMS: To investigate the presence of enterotoxigenic Staphylococcus aureus in bulk milk and in a selection of raw milk products. METHODS AND RESULTS: Samples of bovine (n = 220) and caprine (n = 213) bulk milk, and raw milk products (n = 82) were analysed for S. aureus. Isolates were tested for staphylococcal enterotoxin (SE) production (SEA-SED) by reversed passive latex agglutination and for SE genes (sea-see, seg-sej) by multiplex PCR. Staphylococcus aureus was detected in 165 (75%) bovine and 205 (96.2%) caprine bulk milk samples and in 31 (37.8%) raw milk product samples. Enterotoxin production was observed in 22.1% and 57.3% of S. aureus isolates from bovine and caprine bulk milk, respectively, while SE genes were detected in 52.5% of the bovine and 55.8% of the caprine bulk milk isolates. SEC and sec were most commonly detected. A greater diversity of SE genes were observed in bovine vs caprine isolates. CONCLUSIONS: Staphylococcus aureus seems highly prevalent in Norwegian bulk milk and isolates frequently produce SEs and contain SE genes. Enterotoxigenic S. aureus were also found in raw milk products. SIGNIFICANCE AND IMPACT OF THE STUDY: Staphylococcus aureus in Norwegian bovine and caprine bulk milk may constitute a risk with respect to staphylococcal food poisoning from raw milk products.     

Diversity of Staphylococcus aureus enterotoxin types within single samples of raw milk and raw milk products

AIM: To find out if testing of up to 10 Staphylococcus aureus isolates from each sample from raw milk and raw milk products for staphylococcal enterotoxin (SE) might increase the chances of identifying potential sources of food intoxication. METHODS AND RESULTS: Altogether 386 S. aureus isolates were tested for the presence of SE by reversed passive latex agglutination (SET-RPLA), and SE genes (se) by a multiplex polymerase chain reaction (PCR). In 18 of 34 (53%) S. aureus positive samples a mixture of SE and/or se positive and negative isolates were identified. Multiplex PCR increased the number of potential SE producing strains, i.e. isolates that harboured se, with 51% among the product and 48% among the raw bovine milk isolates. Examination by pulsed-field gel electrophoresis mostly confirmed clonal similarity among isolates sharing SE/se profile, but did not further differentiate between them. CONCLUSIONS: Isolates of S. aureus collected from one sample may show great diversity in SE production and different plating media seem to suppress or favour different strains of S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: Several isolates of S. aureus from each sample should be tested for enterotoxin production in cases with typical SE intoxication symptoms with methods that are able to reveal new SE/se.     

Enterotoxigenicity of Staphylococcus aureus Cultures Isolated from Acute Cases of Bovine Mastitis

To determine whether staphylococci causing bovine mastitis are potential causes of human intoxications, 142 cultures identified as etiological agents of acute cases and 18 cultures causing chronic cases of staphylococcal mastitis were obtained from investigators in the United States and Canada, examined microscopically, and tested for carbohydrate utilization, terminal pH, catalase, coagulase, egg yolk hydrolysis, gelatin hydrolysis, cytochrome oxidase, urease production, nitrate reduction, micrococcal nuclease, phage type, and enterotoxin production. Three cultures were not confirmed as Staphylococcus aureus. Of the 157 S. aureus cultures, 23 produced staphylococcal enterotoxins. Although a direct relationship between staphylococcal mastitis and outbreaks of staphylococcal food poisoning was not proved, results indicated that staphylococcal infections of the bovine mammary gland represent a significant reservoir of enterotoxigenic strains of S. aureus.