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.     

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.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes' milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10(3) and 10(5) cfu/ml and stored at 4 degrees, 10 degrees and 15 degrees C and at room temperature (10 degrees-15 degrees C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C1 and C2 were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10(8) cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10(7) cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

Behaviour of Staphylococcus aureus strains, producers of enterotoxins C1 or C2, during the manufacture and storage of Burgos cheese

Burgos cheese was manufactured from pasteurized ewes milk inoculated with Staphylococcus aureus strains FRI 137 and FRI 361, at levels of ca 10³ and 10⁵ cfu/ml and stored at 4°, 10° and 15°C and at room temperature (10°-15°C). Populations of Staph. aureus and mesophilic aerobes, pH, and production of thermonuclease and enterotoxins C₁ and C₂ were investigated. Aerobic counts increased during cheese-making and storage. With both test strains, important growth was observed only during the storage period, the larger levels corresponding to the higher temperatures. Although Staph. aureus strains attained populations of over 10⁸ cfu/g, no enterotoxin was detected. Strain FRI 361 reached 10⁷ cfu/g without production of a detectable amount of thermonuclease. With strain FRI 137, the minimal population associated with enzyme activity was influenced by the inoculum size. Staphylococcus aureus counts are better indicators of staphylococcal growth in Burgos cheese than the thermonuclease test.     

An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk

Mashed potato made with raw bovine milk was suspected to have been the source of a food poisoning outbreak. Almost 8 x 10(8)Staphylococcus aureus CFU g(-1) were detected in the mashed potato. S. aureus was also found in bulk milk from the farm that had supplied milk for the mashed potato. Isolates from mashed potato and bulk milk were positive for the gene encoding staphylococcal enterotoxin H (seh), and the corresponding protein toxin, SEH, was detected by ELISA in the mashed potato. Production of SEH by S. aureus isolates from mashed potato (n = 4) and bulk milk (n = 4) was also demonstrated by ELISA. Sequencing of seh from one mashed potato isolate and one bulk milk isolate confirmed that the gene was a variant seh, and that the genes in both isolates were identical. Macrorestriction of chromosomal DNA with Sma1 followed by pulsed-field gel electrophoresis of seh-positive S. aureus from mashed potato and bulk milk revealed indistinguishable banding patterns between isolates from both sources. It seems likely that raw bovine milk used in the preparation of mashed potato contained S. aureus that subsequently produced sufficient SEH in the mashed potato to cause food poisoning.     

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.     

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.     

Inactivation of Staphylococcus aureus in raw milk cheese by combinations of high-pressure treatments and bacteriocin-producing lactic acid bacteria

AIMS: To investigate the combined effect of high-pressure treatments (HPT) and milk inoculation with bacteriocin-producing lactic acid bacteria (BP-LAB) on the survival of Staphylococcus aureus during ripening of raw milk cheese. METHODS AND RESULTS: Cheeses were manufactured from raw milk artificially contaminated with S. aureus at ca 5 log CFU ml(-1), a commercial starter culture and one of seven strains of BP-LAB, added as adjuncts at 0.1%. HPT of cheeses were performed on days 2 or 50 at 300 MPa (10 degrees C, 10 min) or 500 MPa (10 degrees C, 5 min). On day 3, S. aureus counts were 6.46 log CFU g(-1) in control cheese. Milk inoculation with different BP-LAB lowered S. aureus counts on day 3 when compared with control cheese by up to 0.46 log CFU g(-1), HPT at 300 MPa on day 2 by 0.45 log CFU g(-1) and HPT at 500 MPa on day 2 by 2.43 log CFU g(-1). Combinations of BP-LAB with HPT at 300 and 500 MPa on day 2 lowered S. aureus counts on day 3 by up to 1.02 and 4.00 log CFU g(-1) respectively. CONCLUSIONS: The combined effect of milk inoculation with some of the BP-LAB tested and HPT of cheese on S. aureus inactivation was synergistic. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of HPT at lower pressures with BP-LAB inoculation is a feasible system to improve cheese safety in case of deleterious effects on cheese quality caused by HPT at higher pressures.     

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.     

Comparison of different approaches to quantify Staphylococcus aureus cells by real-time quantitative PCR and application of this technique for examination of cheese.

Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/microl) than using a fluorigenic TaqMan probe (6 nuc gene copies/microl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 x 10(2) to 6.4 x 10(2) copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.     

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.     

Assessing and analysing contamination of a dairy products processing plant by Staphylococcus aureus using antibiotic resistance and PFGE

A dairy product processing plant was studied for 2.5 years to examine contamination with Staphylococcus aureus and try to correlate the source of contamination. Cultures were submitted to an antibiotic susceptibility test (AST) and characterised by Pulsed-field Gel Electrophoresis (PFGE) analysis. Results showed that 35.2% (19/51) of food handlers were asymptomatic carriers of S. aureus, and that 90.4% (19/21) of raw milk sampled was contaminated. Staphylococcus aureus was isolated from only 10 samples among more than 3200 investigated dairy products. No S. aureus contamination was found on machinery. The AST analysis demonstrated sensitivity of tested S. aureus to oxacillin, cephalothin, vancomycin, gentamicin, and sulfamethoxazole/trimethoprim. AST analysis generated eight different phenotypic profiles, but did not allow us to identify the source of contamination in seven of ten final products. PFGE analysis proved to be a sensitive method as it generated 42 different DNA banding profiles among the 48 S. aureus investigated, demonstrating a lack of predominance of endemic strains in the plant, contrary to suggestions raised by antibiotic resistance typing. Based on PFGE genotyping, S. aureus strains isolated from four contaminated final products were similar to four S. aureus isolated from raw milk. Five final products contained S. aureus different from all other strains collected, and one showed similarity to a strain isolated from a food handler. These results suggest contamination by raw milk as the main source of contamination of the final dairy products.     

Application of current methods for isolation and identification of staphylococci in raw bovine milk

Samples of raw milk were examined for counts of somatic cells, total viable bacteria, staphylococci (Schleifer & Kramer's medium) and Staphylococcus aureus (Baird-Parker medium, Baird-Parker medium with pig plasma and Baird-Parker medium with additional antibiotics). For the isolation of staphylococci from raw milk, Schleifer & Kramer's medium was found to be very selective and in general performed satisfactorily. From the results obtained with the three remaining media the continued use of Baird-Parker medium for isolation of Staph. aureus from raw milk is recommended with the proviso that colonies selected for identification should include those that clear and do not clear the egg yolk and are not limited to colonies with diameters greater than 1 mm. Staphylococci isolated from raw milk were identified by key tests using a multipoint inoculation procedure. A selected number were also examined by the API STAPH system in conjunction with the API LAB computer programme for identification of staphylococci. Of the staphylococci examined, 90.0% were identified using the multipoint procedure. For strains identified as Staph. aureus, Staph. hyicus subsp. hyicus, Staph. epidermidis, Staph. simulans, Staph. xylosus or members of the Staph. hominis/Staph. warneri/Staph haemolyticus group, the API system provided confirmatory evidence. With strains identified by the multipoint procedure as Staph. hyicus subsp. chromogenes, Staph. sciuri subsp. sciuri and Staph. sciuri subsp. lentus the API system did not always provide concurring results. Several strains which could not be identified by the multipoint procedure could be identified by the API system. Staph. aureus, Staph. hyicus subsp. hyicus and Staph. hyicus subsp. chromogenes strains isolated from milk were examined for production of enterotoxin A-E. Only 3.9% of Staph. aureus strains examined produced detectable enterotoxin (type C). None of the Staph. hyicus subsp. hyicus or Staph. hyicus subsp. chromogenes strains produced any of the known enterotoxins.     

Microbiological quality of Pecorino Siciliano "primosale" cheese on retail sale in the street markets of Palermo, Italy

Pecorino Siciliano (PS) "primosale" is a traditional Sicilian fresh soft cheese made from sheep's milk. Short-ripening time and production from unpasteurized or raw milk can facilitate bacterial contamination of PS "primosale". The microbiological quality of "primosale" on retail sale in the street markets of Palermo, Italy was studied by detecting the common food pathogens Listeria monocytogenes and Staphylococcus aureus and indicator microorganisms, such as Escherichia coli, Enterobacteriaceae and Staphylococcaceae. In our study, 4% and 44% of the samples, respectively, did not comply with the acceptability levels fixed by European regulations for S. aureus and E. coli. A high contamination of bacteria belonging to Enterobacteriaceae and Staphylococcaceae was found in 42% and 50% of the cheeses analyzed, respectively. Such results indicate poor husbandry and poor hygiene practices during milk collection or preservation or during cheese production processes and handling. In addition, the retail sale conditions may have played a role in cheese contamination since a correlation was found between poor microbiological quality and some selling parameters. This study emphasizes the need to improve production hygiene throughout the PS food chain in line with the traditional cheese-making procedures. Labelling of PS with clear information on whether the cheese was prepared from raw milk also requires improvement.     

egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus

The recently described staphylococcal enterotoxins (SE) G and I were originally identified in two separate strains of Staphylococcus aureus. We have previously shown that the corresponding genes seg and sei are present in S. aureus in tandem orientation, on a 3.2-kb DNA fragment (Jarraud, J. et al. 1999. J. Clin. Microbiol. 37:2446-2449). Sequence analysis of seg-sei intergenic DNA and flanking regions revealed three enterotoxin-like open reading frames related to seg and sei, designated sek, sel, and sem, and two pseudogenes, psi ent1 and psi ent2. RT-PCR analysis showed that all these genes, including seg and sei, belong to an operon, designated the enterotoxin gene cluster (egc). Recombinant SEG, SEI, SEK, SEL, and SEM showed superantigen activity, each with a specific V beta pattern. Distribution studies of genes encoding superantigens in clinical S. aureus isolates showed that most strains harbored such genes and in particular the enterotoxin gene cluster, whatever the disease they caused. Phylogenetic analysis of enterotoxin genes indicated that they all potentially derived from this cluster, identifying egc as a putative nursery of enterotoxin genes.     

Improvement in Salmonella detection in milk and dairy products: comparison between the ISO method and the Oxoid SPRINT Salmonella test

The Oxoid SPRINT Salmonella test was compared with the ISO method (ISO 6579: 1993) for the detection of Salmonella in milk and dairy products. Samples were artificially contaminated, in some cases with sublethally injured salmonellas. Experiments with raw milk, soft cheese made from heat-treated milk (mould-ripened and with smear) and soft cheese with smear made from raw milk showed no significant differences between the SPRINT and ISO methods. With dried milk products and mould-ripened soft cheese made from raw milk the reference method gave significantly more positive results. The addition of ferrioxamine E to pre-enrichment (ISO) or pre-enrichment/enrichment broth (SPRINT test) did not improve Salmonella detection.     

Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: use of the polymerase chain reaction for amplification and detection of staphylococcal enterotoxin genes entB and entC1 and the thermonuclease gene nuc

The polymerase chain reaction was used to amplify the staphylococcal enterotoxin B and C genes (entB and entC1) and the staphylococcal nuclease gene (nuc). Two sets of primers ("nested primers") were found to be necessary for the detection of low copy numbers of purified DNA in diluent. These allowed detection of ca. 1 fg of purified target DNA, while 100 pg was required before detection of entB, entC1, and nuc with single primer pairs was possible. With nested primers, enterotoxigenic Staphylococcus aureus cells could be detected in artificially contaminated dried skimmed milk samples at levels of ca. 10(5) CFU ml-1 within 8 h. No cross-reaction was observed between the highly homologous entB and entC1 genes. The method showed total specificity for entC1 when tested against a wide variety of other bacteria.     

Detection of enterotoxigenic Staphylococcus aureus in dried skimmed milk: use of the polymerase chain reaction for amplification and detection of staphylococcal enterotoxin genes entB and entC1 and the thermonuclease gene nuc.

The polymerase chain reaction was used to amplify the staphylococcal enterotoxin B and C genes (entB and entC1) and the staphylococcal nuclease gene (nuc). Two sets of primers ("nested primers") were found to be necessary for the detection of low copy numbers of purified DNA in diluent. These allowed detection of ca. 1 fg of purified target DNA, while 100 pg was required before detection of entB, entC1, and nuc with single primer pairs was possible. With nested primers, enterotoxigenic Staphylococcus aureus cells could be detected in artificially contaminated dried skimmed milk samples at levels of ca. 10(5) CFU ml-1 within 8 h. No cross-reaction was observed between the highly homologous entB and entC1 genes. The method showed total specificity for entC1 when tested against a wide variety of other bacteria.     

Detection of Mycobacterium avium ssp. paratuberculosis in cheese, milk powder and milk using IS900 and f57-based qPCR assays

Aims: To develop a quantitative PCR assay for sensitive and specific detection of Mycobacterium avium ssp. paratuberculosis (Map) in a range of dairy products. Methods and Results: TaqMan^® assays were designed to target the IS900 and f57 genetic elements of Map. Both real‐time PCR assays were integrated with the Adiapure^® Map DNA extraction kit and assessed separately for the detection/quantification of Map in spiked milk, Cheddar cheese and milk powder. Assays were validated against Cheddar cheese samples containing known concentrations of Map. The IS900 qPCR assay was significantly more sensitive than the assay based on the f57 primer/probe. At a threshold cycle value of 38, limits of detection (LOD) for the IS900 qPCR assay were 0·6 CFU ml^?1, 2·8 CFU g^?1 and 30 CFU g^?1 for artificially contaminated pasteurized milk, whole milk powder and Cheddar cheese, respectively. The respective LOD’s for the f57 assay were 6·2 CFU ml^?1, 26·7 CFU g^?1 and 316 CFU g^?1. Conclusion: The integrated Adiapure^® extraction – IS900 real time assay described is a sensitive, quantitative method for the detection of Map in dairy products. This is the first study to consider qPCR as a quantitative estimation of Map‐DNA in cheese and whole milk powder. Significance and Impact of the Study: The assay developed allows sensitive detection and quantification of Map DNA in a range of dairy products which is valuable for the screening and surveillance of this potential zoonotic organism.