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.     

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.     

Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture andripening of Manchego cheese

The inhibitory effect of enterocin 4, a bacteriocin produced by Enterococcus faecalis INIA 4, on Listeria monocytogenes strains Ohio and Scott A during themanufacture and ripening of Manchego cheese was investigated. Raw ewe's milk wasinoculated with ca 10⁵ cfu ml⁻¹ of L.monocytogenes and with 1% of a commercial lactic starter, 1% of an Ent. faecalis INIA 4 culture, or 1% of each culture. Manchego cheeses were manufactured according tousual procedures. Listeria monocytogenes Ohio counts decreased by 3 log units after8 h and by 6 log units after 7 d in cheese made from milk inoculated with Ent. faecalis INIA 4 or with both cultures, whereas no inhibition was recorded after 60 d in cheese made frommilk inoculated with commercial lactic starter. Listeria monocytogenes Scott A wasnot inhibited by enterocin 4 during cheese manufacture, but decreases of 1 log unit after 7 d andof 2 log units after 60 d were achieved in cheese made from milk inoculated with bothcommercial lactic starter and Ent. faecalis INIA 4.     

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis

The efficacy of high-temperature, short-time (HTST) pasteurization (72 °C/15 s) when low numbers (≤ 10³ cfu ml ⁻¹ ) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10³ cfu ml⁻¹, 10² cfu ml⁻¹, 10 cfu ml⁻¹, and 10 cfu 50 ml⁻¹) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14·8% and 10% of HTST-pasteurized milk samples at the 10³ and 10² cfu ml⁻¹ inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3·7% and 6·7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis . No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml⁻¹ or 10 cfu 50 ml⁻¹.     

A Simple Sensitive Method for Determining Staphylococcal Thermonuclease in Cheese

A simple quantitative method is described for the determination of staphylococcal thermostable nuclease in cheese. The method does not require concentration or purification of the nuclease prior to determination because of the increased sensitivity of the assay (0.5 ng/ml). Assay results of cheddar cheese made from pasteurized milk inoculated with enterotoxin-A producing Staphylococcus aureus strains demonstrated the sensitivity and reliability of the method for indicating the presence of Staph. aureus at concentrations of ≥ 1.4 × 10⁶/g of cheese.     

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.     

Survival of Escherichia coli O157 : H7 in yoghurt during preparation and storage at 4°C

Cow's milk was inoculated with ca 10³ and 10⁷ cfu ml⁻¹ Escherichia coli O157 : H7. After fermentation at 42°C for 0–5 h, the yoghurt was stored at 4°C. Two kinds of yoghurt were used : traditional yoghurt (TY), made with Streptococcus thermophilus and Lactobacillus bulgaricus starter cultures, and 'bifido' yoghurt (BY), made with the two starter cultures plus Bifidobacterium bifidum . After 7 d E. coli O157 : H7 decreased from 3·52 to 2·72 log₁₀ cfu ml⁻¹ and from 7·08 to 5·32 log₁₀ cfu ml⁻¹ in TY, and from 3·49 to 2·73 log₁₀ cfu ml⁻¹ and from 7·38 to 5·41 log₁₀ cfu ml⁻¹ in BY. The pH values of yoghurt dropped from 6·6 to 4·5 and 4·4 in TY (for low and high pathogen inocula, respectively), and from 6·6 to 4·6 and 4·5 in BY (for low and high pathogen inocula, respectively).     

Fate of Listeria monocytogenes during manufacture and ripening of semi-hard cheese

Semi-hard cheeses were experimentally elaborated with pasteurized milk from sheep, goat and cow (15: 35: 50) and inoculated to contain 1.9 times 10⁵ Listeria monocytogenes /ml in cheeses 1 and 2 and 4 times 10³ L. monocytogenes /ml in cheeses 3 and 4. Counts of L. monocytogenes were determined by direct surface plating of samples on listeria selective agar medium. The results show the substantial survival of L. monocytogenes present in milk during manufacture and ripening of this type of cheese.     

The survival of Staphylococcus aureus during the fermentation and storage of yoghurt

The effect of yoghurt culture R _x on the survival of Staphylococcus aureus CCM 5984 added to milk in various concentrations was observed during the fermentation and storage of yoghurt. The end of the fermentation process (3.5 h) was only accompanied by a slight reduction. During the storage of yoghurt at 4°C a 1-2 log reduction was observed. No Staph. aureus was detected in yoghurt produced from milk contaminated by 10³ Staph. aureus cells 1^-1 after 48 h of cold storage. When a concentration of 10² Staph. aureus cells was used for milk contamination, the pathogen was not recovered from yoghurt during the fermentation and storage. The fermentation and storage of yoghurt was accompanied by increases in lactic acid and titrimetric acidity, as well as by a decrease in pH value.     

A rapid sample preparation method for PCR detection of food pathogens based on buoyant density centrifugation

The use of buoyant density centrifugation (BDC) to prepare samples for PCR analysis of food pathogens is described. Blue cheese and milk homogenates were inoculated with Shigella flexneri and layered on top of Percoll® media. After BDC, the food homogenates remained in the upper part of the centrifuge tube, separated from the bacteria, which retained viability and were concentrated below the lighter Percoll® layer. PCR inhibitors stayed in the homogenate and PCR analyses of treated samples consistently detected 10⁴ cfu g⁻¹ of blue cheese and 500 cfu ml⁻¹ of milk, respectively. Differences in the density of live and killed Sh. flexneri and Yersinia enterocolitica were detected by BDC but were dependent on the mechanism of killing.     

The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes

The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6–8°C was determined. Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5·9) and/or calcium chloride addition during heat treatment. Nisin was added in the commercial form of Nisaplin^® pre-production to the milk. Each batch of cheese was inoculated with 10²–10³ cfu g⁻¹ of a five-strain cocktail of L. monocytogenes before storage. Shelf-life analysis demonstrated that incorporation of nisin at a level of 2·5 mg l⁻¹ could effectively inhibit the growth of L. monocytogenes for a period of 8 weeks or more (dependent on cheese type). Cheese made without the addition of nisin contained unsafe levels of the organism within 1–2 weeks of incubation. Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6–8°C, with only 10–32% nisin loss.     

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.     

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw milk

We have developed a quantitative PCR-ELISA for the rapid enumeration of bacteria inrefrigerated raw milk using primers designed from conserved regions in the 16S ribosomal RNAgene (rRNA). The designed primers permitted the amplification of a 147 bp DNA fragment froma wide selection of bacteria which may grow in milk at refrigeration temperatures. Amplified PCRproducts generated using a digoxigenin-labelled primer were heat-denatured before beingquantified by an enzyme-linked immunosorbent assay (ELISA). A biotinylated probe immobilizedonto streptavidin-coated microplates was used to capture the digoxigenin-labelled fragments thatwere detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion ofsubstrate gave distinct absorbence differences when assaying milk samples containing bacteria inthe range 10³–10⁷ cfu ml⁻¹. The detection threshold for thePCR-ELISA assay developed in this work is 103 cfu ml⁻¹.     

Microbiological composition of raw milk from selected farms in the Camembert region of Normandy

Raw milk from 27 farms was sampled over 6 months for listerias, salmonellas, Yersinia enterocolitica and campylobacters. Total bacterial counts and somatic cell counts were measured. Lactococci, lactobacilli, dextran-producing leuconostocs, Brevibacterium linens , yeasts and moulds, Staphylococcus aureus and other Micrococcaceae, Pseudomonas , coliforms, Escherichia coli , enterococci, Clostridium perfringens and spores of anaerobic lactate-fermenting bacteria were also counted. Pseudomonas (2000 cfu ml⁻¹), lactococci (760 cfu ml⁻¹) and Micrococcaceae (720 cfu ml⁻¹) were the most numerous groups. Lactic acid bacteria were detected in all samples. Coliforms were present in most samples, but 84% of samples had counts <100 cfu ml⁻¹. Staphylococcus aureus was detected in 62% of milks, the average count was 410 cfu ml⁻¹. About 80% of supplies had ≤10 E. coli cfu ml⁻¹ and all samples had 1 Cl. perfringens cfu ml⁻¹. Two of the tested milks were positive for salmonellas (2·9%), four were positive for Listeria monocytogenes (5·8%), 25 for Yersinia enterocolitica (36%) and one for campylobacters (1·4%).     

The effect of cryptolepine on the morphology and survival of Escherichia coli, Candida albicans and Saccharomyces cerevisiae

The antimicrobial activity of the indoloquinoline alkaloid, cryptolepine, isolated from Cryptolepis sanguinolenta (Fam. Periplocaceae) was determined against selected micro-organisms. The minimum inhibitory concentration (MIC) ranges obtained, expressed as μg ml⁻¹, were: 5–10 for Saccharomyces cerevisiae NCPF 3139; 10–20 for S. cerevisiae NCPF 3178; 20–40 for Escherichia coli NCTC 10418; 40–80 for E. coli NCTC 11560, Candida albicans ATCC 10231 and C. tropicalis NCPF; and 80–160 for C. albicans NCPF 3242 and NCPF 3262.
Biocidal effects were noted at concentrations 2–4 times those of the MIC of the alkaloid following challenge with 10⁶ cfu ml⁻¹ of micro-organisms. Time-kill studies showed a reduction in viable count from 10⁶ to < 10 cfu ml⁻¹ in 4 h in C. albicans ATCC 10231 exposed to 320 μg ml⁻¹ of the agent; 3 log cycle reductions were recorded for the 6 h counts of E. coli NCTC 10418 and S. cerevisiae NCPF 3139 exposed to 40μg ml⁻¹ and 160 μg ml⁻¹ of the alkaloid respectively.
These results were consistent with findings using scanning electron microscopy. Exposure of cells to biocidal concentrations of cryptolepine produced filamentation prior to lysis in E. coli NCTC 10418 and extreme disturbance of surface structure, including partial and total collapse, followed by lysis in C. albicans ATCC 10231 and S. cerevisiae NCPF 3139.     

Increased ELISA sensitivity using a modified extraction buffer for detection of Xanthomonas campestris pv. vesicatoria in leaf tissue

In vitro and in planta sensitivity of an indirect enzyme-linked immunoassaytechnique, using a monoclonal antibody specific for the lipopolysaccharide (LPS) of Xanthomonas campestris pv. vesicatoria , was increased 10-foldby using a newextraction buffer (gl of : KH₂PO₄, 2; NaHPO₄, 11·5; EDTAdisodium, 0·14; thimerosal, 0·02; and lysozyme, 0·2). The procedure improvedsensitivity without increasing background levels. In vitro , the limit of detection wasbetween 1×10⁷ and 1×10⁸ cells ml⁻¹ with the conventionalextraction buffer phosphate-buffered saline (PBS) and less than 1×10⁶ cells ml⁻¹ when lysozyme extraction buffer was substituted for PBS. In comparing 22 X. c.vesicatoria strains, absorbance readings were increased close to three-fold with the lysozymeextraction buffer as opposed to PBS. When leaf tissue extract was spiked with the bacterium, thelimit of detection was 1×10⁷ cfu ml⁻¹ and 1×10⁸ cfu ml⁻¹ with the lysozyme solution and PBS, respectively, as the extraction buffers. Whenusing the lysozyme extraction buffer in combination with a commercial amplification system, thelimit of detection was decreased to less than 1×10⁵ cfu ml⁻¹ in leaftissue. The addition of the lysozyme and EDTA to the phosphate buffer resulted in release of asignificant quantity of LPS and concomitant dramatic increase in sensitivity. The new procedure,termed lysozyme ELISA (L-ELISA), should increase sensitivity of ELISA reactions where LPS isthe reacting epitope.     

Anti-bacterial activity of synthetic N-heterocyclic oxidizing compounds

Synthetic chlorochromate derivatives of pyridine and quinoline were active in vitro against type cultures of Escherichia coli (ATCC 128), Staphylococcus aureus (ATCC 14775), Pseudomonas aeruginosa (ATCC 10145) and Bacillus subtilis (NCTC 8236). The minimum inhibitory concentrations (MIC) were 125–250 μg ml⁻¹ and 250–500 μg ml⁻¹ for pyridinium chlorochromate and quinolinium chlorochromate, respectively. An established derivative of quinoline (Perfloxacin) had an MIC of 125–250 μg ml⁻¹. The extinction time for 10⁵ cfu in broth was 90 min for pyridinium chlorochromate and 120 min for quinolinium chlorochromate, except for B. subtilis which survived up to about 180 min and 360 min. A combination of the two compounds produced an antagonistic effect. The 50% lethal dose (LD₅₀ toxicity) in mice was estimated at 76 μg g⁻¹ and 33 μg g⁻¹ body weight for the quinolinium and pyridinium chlorochromates. The compounds also exhibited some potential for suppressing a simulated staphylococcal infection in mice at the dosage levels of ca 22 μg g⁻¹ for pyridinium chlorochromate and 45 μg g⁻¹ for quinolinium chlorochromate.     

Effect of heat treatment on Listeria monocytogenes and Gram-negative bacteria in sheep, cow and goat milks

Sheep milk, compared with cow and goat milk, had a protective effect on Gram-negative bacteria and Listeria spp. heated at 65°C in a test-tube method. This effect was not solely due to fat content as cow milk artificially reconstituted to 10% homologous fat was not as protective. Listeria monocytogenes in whole sheep, cow and goat milks at an inoculum level of 1 times 10⁶ cfu ml^-1 was heated at 68°C for 15 s in the plate pasteurizer and survival was only detected in whole sheep milk after heating. Whole sheep, cow and goat milks containing high levels of L. monocytogenes (1 times 10⁶ cfu ml^-1) could not survive the current HTST plate pasteurization protocol.     

Enterotoxigenicity of Staphylococcus aureus strains isolated from poultry: raw poultry carcases as a potential food-poisoning hazard

Staphylococcus aureus strains were isolated from end-of-lay poultry carcases obtained from a plant at two different stages of processing before and after storage at different temperatures. These strains were supplemented with Staph. aureus strains isolated from poultry from a wide range of sources and biotyped, phage typed, and tested for production of enterotoxins A-E. The isolates were found to consist of poultry and human specific strains and each of these groups contained strains able to produce enterotoxin. Poultry strains produced only enterotoxin D whereas human strains produced enterotoxins A, C and D. The hen carcases used in storage experiments were found to be naturally contaminated with enterotoxin D producing staphylococci. No enterotoxin D could be detected on any of the carcases even after storage at temperatures which allowed multiplication of the organisms to occur (final Staph. aureus counts ranged from 10² to 10⁷/16 cm² of breast skin).     

Design and evaluation of malolactic enzyme gene targeted primers for rapid identification and detection of Oenococcus oeni in wine

Rapid identification and detection of Oenococcus oeni was achieved by species-specific PCR. Two primers flanking a 1025 bp region of the O. oeni gene encoding the malolactic enzyme were designed. The expected DNA amplificate was obtained only when purified DNA from O. oeni was used. The identity of PCR product was confirmed by nested PCR and restriction analysis. Within 8 h, 10³ cfu ml⁻¹ of oenococci were detected in fermenting grape must containing 10⁷ yeast cells, whereas the detection limit in wine was 10⁴ cfu ml⁻¹. The rapidity and reliability of the PCR procedure established suggests that the method may be profitably applied in winery laboratories for quality control.