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.     

Rapid detection of enterotoxigenic Staphylococcus aureus harbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay

AIM: The aim of this study was to develop a loop-mediated isothermal amplification (LAMP) assay targeting the genes for the four classical enterotoxins, SEA, SEB, SEC and SED, in Staphylococcus aureus. METHODS AND RESULTS: Specific primers were designed which target each specific sequence of the enterotoxin genes. With 30 strains of Staph. aureus, the results of the LAMP assay to each enterotoxin, SEA, SEB, SEC and SED, completely accorded with the results of polymerase chain reaction (PCR) assay. Enterotoxin production, determined by a reverse passive latex agglutination assay, strongly correlated with the presence of the corresponding genes. Amplification was not observed when 14 strains of nonenterotoxigenic Staph. aureus and 20 strains consisting of 19 bacterial species other than Staph. aureus were tested. In addition, the sensitivity of the LAMP assay was generally higher than that of conventional PCR assay and it rapidly detected enterotoxigenic Staph. aureus strains within 60 min. CONCLUSIONS: The LAMP assay developed in this study is rapid, specific and sensitive for the detection of enterotoxigenic Staph. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: The method is suitable for clinical diagnosis and food safety applications.     

Detection of enterotoxigenic Staphylococcus aureus isolates in raw milk cheese

AIM: To develop an easy, rapid and efficient DNA extraction procedure for Staphylococcus aureus detection with a low number of steps and removing completely the PCR inhibitors, applicable to raw milk cheese samples, and to compare phenotypical and genotypical method to detect Staph. aureus isolates and staphylococcal enterotoxins (SEs) production. METHODS AND RESULTS: A total of 33 bovine and caprine raw milk cheese samples were analysed by means of both classic microbiological and molecular techniques. All samples were positive for Staph. aureus contamination. The DNA extraction protocol optimized was found to achieve a detection limit of 100 CFU g(-1) for Staph. aureus. None of the samples tested with immunological assays contained SEs but in 14 of 33 samples a mixture of se positive (sea, sec, sed, seg, sel, sej) isolates were identified. CONCLUSIONS: Staphylococcus aureus is a food-borne pathogen mainly detected in finished dairy products. The rapid and efficient detection of Staph. aureus isolates from dairy products is essential for consumer safety. The direct detection of pathogens from food is possible with careful attention to sample preparation and nucleic acid amplification optimization. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that raw milk cheese samples can be tested for Staph. aureus contamination with a rapid, simple and reproducible procedure.     

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.     

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.     

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.     

Occurrence and clonal relatedness of sec/tst-gene positive Staphylococcus aureus isolates of quartermilk samples of cows suffering from mastitis

AIMS: To investigate the prevalence of sec/tst-gene positive Staphylococcus aureus in bovine mastitis and to get information about the clonal relatedness of these clinical isolates. METHODS AND RESULTS: A total of 533 Staph. aureus strains isolated from bovine mastitic quartermilk samples at 493 randomized dairy farms in Hessia, Germany, from January 1997 until June 1998 were examined for enterotoxin C (sec) gene and toxic shock syndrome toxin (tst) gene by multiplex polymerase chain reaction. Fifty-three (9.3%) of the strains were sec/tst-gene positive. Phenotypic TSST-1 production was found in all positive strains by reversed passive latex agglutination test. With DNA macrorestriction analysis, sec/tst-gene positive strains were divided into five different macrorestriction types. Type I (10 isolates) and III (40 isolates) were found to be the predominant types in terms of frequency of isolation in the investigated area. These DNA macrorestriction types differed in only two bands in the 500 and 270 bp region. CONCLUSIONS: Closely related Staph. aureus strains seem to be responsible for an unusual large proportion of bovine mastitis cases in geographically widely distinct locations. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first reports about the relatedness of sec/tst-gene positive Staph. aureus clinical isolates from bovine mastitis.     

PCR-based detection of enterotoxigenic Staphylococcus aureus in the early stages of raw milk cheese making

AIMS: To define PCR-based detectability of Staphylococcus aureus in raw milk and intermediate products of raw milk cheese making in the presence of a complex background microflora by targetting different specific genes harboured by a single strain. METHODS AND RESULTS: The strain Staph. aureus FRI 137 harbouring nuc, sec, seg, seh and sei genes was used in this study. Raw milk artificially contaminated by different concentrations of Staph. aureus FRI 137 was employed in dairy processing resembling traditional raw milk cheese making. Samples of milk and curds were PCR-analysed after DNA extraction by targetting all the above genes. The pathogen was detected when the initial contamination was 10(4) CFU ml(-1) by amplification of nuc and seh genes. 10(5) and 10(7) CFU ml(-1) were needed when seg or sei and sec genes were targetted, respectively. Enrichment cultures from raw milk and curd samples proved to increase the detection limit of 1 log on average. CONCLUSIONS: The direct detection of the pathogen in the raw material and dairy intermediates of production can provide rapid results and highlight the presence of loads of Staph. aureus potentially representing the risk of intoxication. However, every target gene to be used in the analysis has to be studied in advance in a system similar to the real case in order to determine the level of contamination potentially predictable. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection in real dairy systems of significant loads of Staph. aureus by multiple targets PCR can be more accurate.     

Potential problems in the use of oligonucleotide probes for staphylococcal enterotoxin genes

Oligonucleotide probes unique to the five major enterotoxin genes of Staphylococcus aureus were synthesized and used to detect DNA sequences homologous to these genes in 27 non-clinical isolates of Staph. aureus isolated from nasal swabs of 74 healthy human volunteers. Genomic DNA from all 27 isolates reacted with at least one of the probes. In a phenotypic assay for toxin production by a reverse passive latex agglutination test however, only 15 of the 27 isolates produced enterotoxin in culture. The results raise the possibility that a number of Staph. aureus isolates harbour DNA sequences that are apparently silent or mutant copies of the enterotoxin genes. This complicates the identification of enterotoxin producers by tests which depend on oligonucleotide or DNA hybridization.     

PCR-based procedures for detection and quantification of Staphylococcus aureus and their application in food

AIMS: To evaluate the specificity of nuc targeted primers for PCR detection of Staphylococcus aureus in different food matrices and to establish a RTQ-PCR procedure suitable for the routine detection and quantification of this pathogen in food. METHODS AND RESULTS: Specificity of nuc targeted primers (Pri1-Pri2 and the newly designed RTQ-PCR primers) was tested on a total of 157 strains of genetically confirmed identity, including reference and food isolates. PCR detection on artificially inoculated beef samples by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) showed a sensitivity value around 10(3) CFU g(-1). The two RTQ-PCR systems, incorporating SYBR-Green I and TaqMan, respectively, applied in the present work improved the sensitivity of conventional PCR by lowering the detection level to 10 and 100 cells, respectively. Out of 164 naturally contaminated foods tested for the presence of Staph. aureus, 74 were positive by conventional PCR and 69 by the traditional culture method with a high degree of result agreement between both methodologies (93.3%). CONCLUSIONS: PCR approaches, using nuc targeted primers, have proved specific and combined with growth techniques may improve detection of Staph. aureus in different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The SYBR-Green I real-time PCR approach established allows the sensitive, automated and quantitative detection of Staph. aureus for routine analysis at a reasonable cost.     

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.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2–3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10⁷ cfu/g.     

Staphylococcus aureus, thermostable nuclease and staphylococcal enterotoxins in raw ewes' milk Manchego cheese

Growth and survival of two enterotoxigenic strains of Staphylococcus aureus were studied during manufacture and ripening of eight batches of raw ewes' milk Manchego cheese. Only 2-3 generations of Staph. aureus occurred in the vat and during pressing. The death rate of Staph. aureus (mean decrease in log cfu/g/week of ripening) from day 1 to day 60 was 0.421 in cheese made with 1% Streptococcus lactis starter and 0.404 in cheese made without starter. Thermostable nuclease was produced in the vat by growing Staph. aureus cells; it was inactivated by rennet during the first 24 h and synthesized again by surviving cells of Staph. aureus from day 1 to day 60. Staphylococcal enterotoxins A, B, C and D were not detected in any batches of cheese, even though Staph. aureus counts exceeded 10(7) cfu/g.     

A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus

AIMS: To develop a multiplex PCR for detection of genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD from Staphylococcus hyicus and to estimate the prevalence of exfoliative toxins among Staph. hyicus isolates from Danish pig herds with exudative epidermitis (EE). METHODS AND RESULTS: A multiplex PCR employing specific primers for each of the genes encoding four different exfoliative toxins was developed and evaluated using a collection of Staph. hyicus with known toxin type and a number of other staphylococcal species. A total of 314 Staph. hyicus isolates from pigs with EE were screened by multiplex PCR and the combined results of the present and previous investigations showed that ExhA, ExhB, ExhC and ExhD was found in 20, 33, 18 and 22%, respectively, of 60 cases of EE investigated. CONCLUSIONS: This study has provided a new tool for detection of toxigenic Staph. hyicus and a more comprehensive picture of the prevalence of the Staph. hyicus exfoliative toxins in Danish pig herds. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex PCR can be used in studies on the prevalence of toxigenic Staph. hyicus elucidating the epidemiology of EE in pigs. The multiplex PCR is currently being used for selection of Staph. hyicus isolates for production of autogenous vaccine.     

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.     

Detection of heat-stable and heat-labile enterotoxin genes of Escherichia coli in diarrhoeic faecal samples of mithun (Bos frontalis) calves by polymerase chain reaction

Aims:  To find out the prevalence of different serogroups of Escherichia coli ( E. coli ) and to detect heat-stable (ST) and heat-labile (LT) enterotoxin genes of enterotoxigenic E. coli (ETEC) from the faeces of mithun calves with diarrhoea.
Methods and Results:  Faecal samples obtained from 65 diarrhoeic mithun calves of under 2 months of age were examined for E. coli using polymerase chain reaction (PCR). Fifty-four E. coli isolates were obtained from those samples, which belonged to 38 different serogroups. Out of 54 isolates tested by PCR, two isolates (3·70%) belonging to serogroups O26 and O55 were found to possess gene that code for ST enterotoxin and one isolate (1·85%) belonging to serogroup O125 was found to carry LT enterotoxin gene.
Conclusions:  Escherichia coli isolates from diarrhoeic mithun calves were found to possess ST and LT enterotoxin genes, which are designated as ETEC, and these isolates can be detected through PCR using specific primers.
Significance and Impact of the Study:  This study reports the isolation of ETEC possessing ST and LT enterotoxin genes for the first time and ETEC could be a cause of diarrhoea in mithun calves leading to calf mortality.     

Growth and enterotoxin production of Staphylococcus aureus during the manufacture and ripening of Camembert-type cheeses from raw goats' milk

Tests were carried out to determine the effect of manufacturing procedures for a Camembert-type cheese from raw goats' milk on the growth and survival of Staphylococcus aureus organisms added to milk at the start of the process, and to study the possible presence of staphylococcal enterotoxin A in these cheeses. The initial staphylococcal counts were, respectively, 2, 3, 4, 5 and 6 log cfu ml⁻¹. Cheese was prepared following the industrial specifications and ripened for 41 d. Detection of enterotoxins was done by the Vidas SET test and by an indirect double-sandwich ELISA technique using antienterotoxin monoclonal antibodies. Generally, numbers of microbes increased at a similar rate during manufacture in all cheeses until salting. During the ripening period, the aerobic plate count population and Staph. aureus levels remained stable and high. There was an approximately 1 log reduction of Staph. aureus in cheeses made with an initial inoculum of Staph. aureus greater than 10³ cfu ml⁻¹ at the end of the ripening period (41 d) compared with the count at 22 h. The level of staphylococcal enterotoxin A recovered varied from 1 to 3·2 ng g⁻¹ of cheese made with an initial population of 10³–10⁶ cfu ml⁻¹. No trace of enterotoxin A was detected in cheeses made with the lowest Staph. aureus inoculum used in this study.     

Enterotoxigenic potential of Staphylococcus intermedius.

Staphylococcal food poisoning (SFP) caused by enterotoxigenic staphylococci is one of the main food-borne diseases. In contrast to Staphylococcus aureus, a systematic screening for the enterotoxins has not yet been performed on the genomic level for the coagulase-positive species S. intermedius. Therefore, the enterotoxigenic potential of 281 different veterinary (canine, n = 247; equine, n = 23; feline, n = 9; other, n = 2) and 11 human isolates of S. intermedius was tested by using a multiplex PCR DNA-enzyme immunoassay system targeting the staphylococcal enterotoxin genes sea, seb, sec, sed, and see. Molecular results were compared by in vitro testing of enterotoxin production by two immunoassays. A total of 33 (11.3%) S. intermedius isolates, including 31 (12.6%) canine isolates, 1 equine isolate, and 1 human isolate, tested positive for the sec gene. In vitro production of the respective enterotoxins was detected in 30 (90.9%) of these isolates by using immunological tests. In contrast, none of 65 veterinary specimen-derived isolates additionally tested and comprising 13 (sub)species of coagulase-negative staphylococci were found to be enterotoxigenic. This study shows on both molecular and immunological levels that a substantial number of S. intermedius isolates harbor the potential for enterotoxin production. Since evidence for noninvasive zoonotic transmission of S. intermedius from animal hosts to humans has been documented, an enterotoxigenic role of this microorganism in SFP via contamination of food products may be assumed.     

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.