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.     

Detection of Shigella spp. in food with a nested PCR method – sensitivity and performance compared with a conventional culture method

A nested PCR method was developed and its performance evaluated by detection of Shigella flexneri in food. The nested PCR amplifies sequences within an invasion-associated locus (ial) on the invasion plasmid specific for Shigella and enteroinvasive Eschrichia coli (EIEC). The nested PCR detected Sh. flexneri in lettuce inoculated with 2, 20 and 200 cfu g-1 after 1, 7 and 18 d of storage, respectively. In comparison, a culture method (NMKL no. 151) detected 10(5) cfu g-1 after 1 but not after 7 d of storage. The presence of inhibitors in blue cheese and shrimps reduced the sensitivity of the PCR assay. To overcome this inhibition, a sample preparation step based on buoyant density centrifugation was developed. This treatment resulted in a successful recovery of Sh. flexneri in lettuce, milk, shrimp and blue cheese inoculated with 10 cfu g-1. The proposed method, which includes a combination of enrichment, buoyant density centrifugation and a nested PCR, can be completed in less than two working days.     

Detection of Clostridium botulinum types A, B, E and F in foods by PCR and DNA probe

A PCR procedure was developed for the detection of Clostridium botulinum in foods. PCR products were detected in agarose gels and by Southern hybridization. The sensitivity of PCR was tested in broth cultures and in canned asparagus, dry cured ham and honey. The sensitivity of the method in broth was high (2·1–8·1 cfu ml⁻¹) for types A and B, but rather low (10⁴ cfu ml⁻¹) for types E and F. However, after enrichment at 37°C for 18 h, it was possible to detect Cl. botulinum types A, B, E and F in food samples at initial levels of about 1 cfu 10 g⁻¹ of food. This PCR detection protocol provides a sensitive and relatively rapid technique for the routine detection of Cl. botulinum in foods.     

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.     

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.     

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%).     

Survival of κ-carrageenan-encapsulated and unencapsulated Pseudomonas aeruginosa UG2Lr cells in forest soil monitored by polymerase chain reaction and spread plating

Abstract A method was developed for direct extraction, purification and amplification of DNA from forest soil. Eighty-two % of the DNA in Pseudomonas aeruginosa UG2Lr introduced into soil was recovered. The detection limit for the strain was approximately 800 cfu g⁻¹ of dry soil based on the polymerase chain reaction (PCR). Survival of κ-carrageenan-encapsulated and unencapsulated UG2Lr was monitored by antibiotic selective and bioluminescence-based nonselective plating and PCR-amplification of a tnsA fragment. After freeze-thaw treatment of soil samples, the unencapsulated UG2Lr declined from an initial population density of 1 × 10⁹ cfu g⁻¹ of dry soil to below the detection threshold of both selective (14 cfu g⁻¹ of dry soil) and nonselective (1 × 10³ cfu g⁻¹ of dry soil) plating. However, presence of nonculturable UG2Lr cells in the soil was revealed by PCR and resuscitation of the bacteria. Population density of the encapsulated UG2Lr increased from 2.7 × 10⁶ to 2.9 × 10⁸ cfu g⁻¹ of dry soil after a 3-week incubation at 22°C and declined to 6.3 × 10⁶ cfu g⁻¹ of dry soil after the freeze-thaw treatment.     

Microbiological characteristics of anevato: a traditional greek cheese

Nine batches of Anevato, raw goat milk cheese, were examined throughout a 60 day storage time at three different periods within the lactation season of the goat. High mean log counts per gram of cheese for aerobic bacteria (7·92–9·56), lactic acid bacteria (7·78–9·32), Gram-negative organisms 5·64–9·67), psychrotrophs (7·90–11·79) and proteolytic bacteria (7·57–9·36) were found. Enterobacteriaceae, coliforms and yeasts were considerably lower. Enterobacteriaceae and coliforms in the curd of cheese made in May were lower by approximately 3·0 log₁₀ cfu g⁻¹ than counts in curd made in January, and were lower by about 2·5 log₁₀ cfu g⁻¹ than those in cheese made in March. This coincided with lower pH and higher counts of lactic acid bacteria in cheese made in March and May. Yeast populations were affected by the season and were higher in May than March and/or January. Lactococci dominated in the cheese until 15 days, but lactobacilli became predominant after 30 days. Lactococcus lactis was the most abundant species of lactic acid bacteria found in Anevato cheese. Results suggest the need for improving milk quality and/or using heat-treated milk to produce Anevato cheese; the use of L. lactis as a starter would possibly eliminate or suppress the growth of undesirable organisms.     

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.     

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.     

The magnetic immuno-polymerase chain reaction assay for the detection of Campylobacter in milk and poultry

L. DOCHERTY, M.R. ADAMS, P. PATEL AND J. McFADDEN. 1996. A rapid and sensitive technique, based on the magnetic immuno-polymerase chain reaction assay (MIPA), was developed for the detection of Campylobacter jejuni in milk and chicken products. Target bacteria are captured from the food sample by magnetic particles coated with a specific antibody and the bound bacteria then lysed and subjected to PCR. The MIPA could detect 420 cfu g^-1of chicken after 18 h, 42 cfu g^-1after 24 h, and 4.2 cfu g^-1after 36 h enrichment. For artificially contaminated milk 63 cfu ml^-1could be detected after 18 and 24 h and 6.3 cfu ml^-1after 36 h enrichment.     

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.     

Rapid detection of Mycoplasma bovis in milk samples and nasal swabs using the polymerase chain reaction

Two methods are suggested that allow direct detection of Mycoplasma bovis from clinical samples, i.e. milk and nasal swabs, respectively. Milk samples were trypsinized in the presence of Triton X-100 and passed through a DNA-binding filter membrane, from which DNA was extracted and subjected to PCR. The detection limit was 500 cfu ml⁻¹ on agarose gels and 50 cfu ml⁻¹ after Southern hybridization so that the method can be used to monitor low-titre samples from animals at the subclinical stage. Results became available within 24 h, thus rendering the procedure more rapid than ELISA and culture techniques. Six other methods designed for milk or other complex samples were tested in this study, but found unsatisfactory. Rapid and specific detection of the pathogen by PCR from nasal mucus treated with lysis buffer and proteinase K was demonstrated for swabs taken from experimentally infected calves. Both methods represent useful tools for effective livestock monitoring and single-animal diagnosis.     

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.     

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.     

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.     

Evaluation of the TECRA immunocapture ELISA for the detection of Salmonella typhimurium in foods

The TECRA immunocapture ELISA was compared with the standard Food and Drug Administration (FDA) method for the detection of Salmonella typhimurium. A variety of foods including dairy (butter, cheese, milk powder, yoghurt and Fromage frais), meat, vegetable and fish products were examined. There was close agreement between the results using the ELISA and the FDA procedure over 176 tests. The ELISA and FDA methods both detected a lower limit of 0.4 cfu Salmonella typhimurium g^-1 in the original food sample but the ELISA result was obtained in 24 h compared with 96 h for the FDA test.     

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.     

Bacteriological studies on Iraqi milk products

F. ABBAR AND H. KH. KADDAR. 1991. Samples from different types of domestic milk products including cheese, kishfa and gaymer were assessed for bacteriological quality over a 4-month period. A total of 400 samples were randomly selected across Mosul city and tested for faecal coliform counts, enteropathogenic Escherichia coli (EPEC) and enterotoxigenic E. coli (ETEC). Faecal coliforms were present at levels greater than 10² cfu/g in 72.5% and less than 10² cfu/g in 27.5% of samples. Of the 430 E. coli strains isolated from the 400 samples of milk products, 138 were serotypes of EPEC. These were found in 81 (40.5%) samples of cheese, 35 (23.8%) of kishfa and 22 (29.7%) of gaymer. During this period, 26 strains of ETEC were also isolated, all of which demonstrated heat-labile or heat-stable toxins. The high proportion of strains of three groups of E. coli showing resistance to antibiotics is discussed in relation to widespread use of antibiotics and the possible public health implication.     

Quantitative detection of meat spoilage bacteria by using the polymerase chain reaction (PCR) and an enzyme linked immunosorbent assay (ELISA)

A quantitative PCR-ELISA for the rapid enumeration of bacteria in refrigerated raw meat has been developed using primers designed from conserved regions in the 16S ribosomal RNA gene (rRNA). Amplified PCR products generated using a digoxigenin-labelled primer were automatically hybridized to a biotinylated probe included in the PCR reaction. The hybridization was performed as part of the PCR programme. The biotin-digoxigenin hybrids were quantified by an enzyme-linked immunosorbent assay (ELISA). Streptavidin bound to the wells of a microtitre plate was used to capture the biotin-digoxigenin-labelled fragments that were detected with a peroxidase anti-digoxigenin conjugate. Subsequent enzymic conversion of substrate gave distinct absorbance differences when assaying meat samples containing bacteria in the range 10²–10⁷ cfu cm⁻². The detection threshold for the PCR-ELISA assay developed in this work is 10² cfu cm⁻².