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.     

Enzymatic Detection of the Growth of Staphylococcus aureus in Foods

A specific method has been developed for the extraction and measurement of staphylococcal nuclease in foods in which Staphylococcus aureus has grown. The method was used to compare staphylococcal growth with nuclease production in foods under varying conditions of temperature, aerobiosis, and competition from other microorganisms. It was concluded that the nuclease is produced under any conditions that permit growth of S. aureus, and little or no interference with the test was encountered either from mixed, natural populations or from a variety of pure, laboratory cultures. Nuclease and enterotoxin A production were shown to vary in synchrony for the 234 (Casman) strain of S. aureus, and the sensitivity of the enzymatic detection of nuclease was comparable to the sensitivity of serological detection of enterotoxin A. It was found that 15 min at 121 C was required to reduce the nuclease activity in slurries of contaminated ham below the level present in the unheated slurry. The extraordinary heat resistance of the nuclease permits its detection even in foods heated subsequent to the growth of S. aureus. The nuclease analysis requires about 3 hr to complete and requires no unusual equipment or reagents.     

The quantitative determination of the DNAse activity in Staphylococcus aureus isolated from monkeys

A new, cheaper and more sensitive method for the quantitative determination of DNAase produced by S. aureus is described. The method permits the determination of DNAase activity in a wider range of titers. The method is based on the detection of the depolymerizing action of staphylococcal nuclease on DNA dyed with ethidium bromide. In this work 22 S. aureus strains isolated from monkeys and 12 strains isolated from humans have been used. The amount of produced by these strains has been determined. The DNAase results of this determination have shown that among S. aureus strains isolated from monkeys and humans the occurrence of strains with both high and low DNAase activity can be observed.     

Use of hydrostatic pressure for inactivation of microbial contaminants in cheese

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30 degrees C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10 degrees C and by 6-log-unit cycles at temperatures of 20 and 30 degrees C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20 degrees C and >400 MPa at 30 degrees C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20 degrees C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.     

Rapid qualitative method for detecting staphylococcal nuclease in foods.

A rapid method for the detection of heat-stable staphylococcal nuclease in foods is described. The procedure consists of an acid precipitation, boiling, and centrifugation followed by enzyme detection in an agar plate containing deoxyribonucleic acid. To test the efficacy of the procedure, purified Staphylococcus aureus nuclease was added to various foods and recovery experiments were performed. Additionally, foods were inoculated and incubated with S. aureus, and the staphylococcal counts were compared with nuclease activity. The results indicate that the procedure possesses merit for a rapid method that can be incorporated into quality control programs. The procedure requires approximately 2.5 h, and it will detect nuclease levels as low as 10 ng/g of food.     

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.     

金黄色葡萄球菌反向线性杂交探针检测方法的建立

目的建立一种检测金黄色葡萄球菌的简单、快速、灵敏、准确的方法。方法根据金黄色葡萄球菌的耐热核酸酶nuc基因,设计一对通用引物及两条特异性探针,用生物素标记通用引物的5＇端,将两条特异性探针固定于硝酸纤维膜上,使PCR产物与探针杂交。结果建立的反向线性杂交探针方法,其检测限为2 ng/μL,检测特异性和准确性均为100%。结论建立的反向线性杂交检测方法具有较高的敏感性和特异性,可用于实验动物金黄色葡萄球菌的快速检测。     

Staphylococcal Nuclease in Udder Secretions of Cows with Acute Mastitis

High concentrations of nuclease produced by Staphylococcus aureus were demonstrated within a few hours by direct examination of udder secretions from cows with a severe staphylococcal mastitis. A positive correlation between the nuclease concentration and the severity of the mastitis was found. The cows with high nuclease concentrations were generally young individuals and/or in the first 2 weeks of the lactation period. Most had a low titre of antibodies against staphylococcal nuclease. Two-thirds of the cows in which high nuclease concentrations were demonstrated were culled or died because of the mastitis attack. The role which nuclease plays for staphylococcal virulence is discussed, and it is concluded that nuclease contributes to the pathogenicity of S. aureus.     

An automated method for the detection of staphylococcal heat stable deoxyribonuclease in dairy products

An automated sandwich immunoassay with specific polyclonal antibodies for the detection of Staphylococcus aureus thermostable nuclease (DNase) is described. To evaluate this assay, different quantities of purified S. aureus nuclease were added to dairy products. Additionally, staphylococcal counts and nuclease activity of milk samples inoculated with S. aureus were determined. Different extraction procedures were performed and compared. The results indicated that the automated test was a reliable method for detecting DNase activity in milk products. The procedure was completed in 2 h and detected 1 ng of DNase ml-1. Detection of the DNase was especially useful in cheeses and could be used to confirm positive enterotoxin results.     

Sensitive enzyme-amplified electrical immunoassay for protein A-bearing Staphylococcus aureus in foods

An amperometric electrochemical immunoassay specific for protein A-bearing Staphylococcus aureus was developed. The method was based on a sandwich immunosorbent assay and incorporated an enzyme amplification step, using a NAD-specific redox cycle generating NADH (C. H. Stanley, A. Johannsson, and C. H. Self, J. Immunol. Methods 83:89-95, 1985). Reduction of the mediator, ferricyanide, was dependent on the initial concentration of antigen. The final potential was measured by using a Pt disk electrode polarized at +0.8 V to the Ag/AgCl reference electrode. The assay was rapid (4 h) and generated protein A- and cell (S. aureus)-dependent signals. The system was highly sensitive and could detect 10 pg of protein A ml-1 and less than 100 CFU of S. aureus ml-1. Similar sensitivities were observed with S. aureus cultures inoculated into beef and milk, but the sensitivity was reduced slightly (ca. 10(3) g-1) with samples of Cheddar cheese.     

Sensitive enzyme-amplified electrical immunoassay for protein A-bearing Staphylococcus aureus in foods.

An amperometric electrochemical immunoassay specific for protein A-bearing Staphylococcus aureus was developed. The method was based on a sandwich immunosorbent assay and incorporated an enzyme amplification step, using a NAD-specific redox cycle generating NADH (C. H. Stanley, A. Johannsson, and C. H. Self, J. Immunol. Methods 83:89-95, 1985). Reduction of the mediator, ferricyanide, was dependent on the initial concentration of antigen. The final potential was measured by using a Pt disk electrode polarized at +0.8 V to the Ag/AgCl reference electrode. The assay was rapid (4 h) and generated protein A- and cell (S. aureus)-dependent signals. The system was highly sensitive and could detect 10 pg of protein A ml-1 and less than 100 CFU of S. aureus ml-1. Similar sensitivities were observed with S. aureus cultures inoculated into beef and milk, but the sensitivity was reduced slightly (ca. 10(3) g-1) with samples of Cheddar cheese.     

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.     

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.     

Two thermostable nucleases coexisted in Staphylococcus aureus: evidence from mutagenesis and in vitro expression

Thermostable nuclease is known to be an important pathogenic factor unique to Staphylococcus aureus and it is commonly presumed to have had the same genetic origin. However, two ORFs in S. aureus genomes were predicted to encode nucleases. One encoded an unnamed nuclease A (SNase) (termed nuc1 ), and the other encoded a thermonuclease (TNase) named nuc (termed nuc2 ). In order to verify whether the two thermostable nuclease proteins are coexpressed in S. aureus , the nuc1 and nuc2 genes were cloned and expressed in Escherichia coli , and both of the recombinant proteins showed thermostable nuclease activity in a toluidine blue-DNA assay. Furthermore, a nuc1 -deleted mutant of S. aureus strain RN4220 (termed RNΔ nuc1 ) was successfully constructed by homologous recombination. Selection and characterization of this mutant strain revealed that it still exhibited thermostable nuclease activity, but at a relative lower level than that of the parent strain. The nucleases secreted by the parent strain and nuc1 -deleted strain still showed functional activity after 30 min at 121 °C. The findings indicated that two types of thermostable nucleases, encoded by two different genes, coexisted in S. aureus .     

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.     

Design and application of a Staphylococcus-specific single strand conformation polymorphism-PCR analysis to monitor Staphylococcus populations diversity and dynamics during production of raw milk cheese

AIM: Development of a nested-PCR single strand conformation polymorphism (SSCP) assay targeting the 16S rRNA genes of the Staphylococcus genus, to monitor staphylococci in cheese. METHODS AND RESULTS: New primer sets to specifically amplify 16S rDNA of staphylococci were designed to be used in a nested-PCR SSCP assay. The method was efficient in discriminating the staphylococcal species most frequently found in cheese. It was validated by monitoring Staphylococcus populations in three productions of raw milk cheese. Analysis of milk samples revealed dominant SSCP peaks corresponding to Staphylococcus aureus, Staphylococcus equorum and Staphylococcus saprophyticus. After 12 h, the S. aureus peak became dominant. CONCLUSIONS: The combination of specific Staphylococcus nested-PCR and SSCP allows rapid and direct monitoring of staphylococci diversity and dynamics in milk and cheese. In the core of the cheeses studied, S. aureus may have ecological advantages against other Staphylococcus populations. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach is a promising tool to study the ecology of staphylococci in cheeses and in other food samples.     

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.     

COMPARISON OF THREE POLYMERASE CHAIN REACTION-BASED METHODS FOR THE DETECTION OF LISTERIA MONOCYTOGENES IN FOOD

Three PCR-based methods for the detection of Listeria monocytogenes in food (BAX for Screening, Probelia and a method according to Kaclíková et al. (2003) were compared on the basis of the determination of detection limits for 15 artificially contaminated food products. Detection limits of all methods for all samples were 10⁰ cfu per 10 g, with the exception of three cheese samples which did not produce valid results because of the inhibition of Probelia PCR. Detection limits for nonviable L. monocytogenes cells were sufficiently high ( 10⁹ cfu per 10 g) for BAX and the method according to Kaclíková et al. (2003), but considerably low ( 10⁶ cfu per 10 g) for Probelia. The results demonstrate that BAX for Screening as well as the Kaclíková et al. (2001) method fulfill the sensitivity requirements for a rapid alternative method for the detection of L. monocytogenes in food, which would be equivalent to the standard method EN ISO 11290–1.     

Production and Heat Stability of Staphylococcal Nuclease

No correlation existed between numbers of organisms and nuclease activity in laboratory-grown cultures of Staphylococcus aureus. Nuclease production was inhibited by anaerobic incubation and stimulated by aeration. Strains of S. aureus varied in the production of nuclease. The optimum pH for enzyme production was 8.3 and employment of a tris(hydroxymethyl)aminomethane buffer system resulted in increased production of the enzyme as compared with a phosphate buffer. The nuclease was extremely heat-stable and had a D value of 16.6 min at 130 C.