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.     

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 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.     

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.     

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%).     

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.     

A one step PCR-based method for the detection of economically important soft rot Erwinia species on micropropagated potato plants

A simple, rapid and sensitive PCR-based method was developed for the detection of all five subspecies of Erwinia carotovora , including subsp. carotovora and subsp. atroseptica , and all pathovars/biovars of Erwinia chrysanthemi , on plant tissue culture material. Primers SR3F and SR1cR, based on a conserved region of the 16S rRNA gene, amplified a DNA fragment of 119 bp from all 65 such strains tested. Detection limits of the method in vitro were 2·0 × 10²–3·4 × 10³ cfu ml⁻¹ (equivalent to 1–17 cfu per PCR) and, following extraction of genomic DNA from plant extract, detection limits were 2·3 × 10²–1·9 × 10⁴ cfu per microplant sample (equivalent to 5 cfu – 3·8 × 10² cfu per PCR). To improve the sensitivity of the method in planta , to obviate the need for complex and laborious DNA extractions, and to remove inhibitory substances present in the plant extract, an enrichment step was included prior to PCR. Following enrichment, the sensitivity of detection was <10 cfu per microplant sample. This method provides the first sensitive means of detecting latent infection caused by several economically important soft rot erwinias simultaneously on potato tissue culture material.     

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).     

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.     

Conductance method for quantitative determination of Photobacterium phosphoreum in fish products

This paper presents the development of a sensitive and selective conductance method for quantitative determination of Photobacterium phosphoreum in fresh fish. A calibration curve with a correlation coefficient of −0.981 was established from conductance detection times (DT) for estimation of cell levels. Less than 50 cells g⁻¹ of fish could be detected in less than 45 h. The selectivity of the method in relation to other Photobacteria or other bacteria isolated from spoiled fish was good. DTs for 10–100 cfu ml⁻¹ of P. phosphoreum were shorter than DTs for 10⁶ cells ml⁻¹ of the other organisms tested. In naturally contaminated fresh fish, P. phosphoreum was specifically enumerated when it made up 0.1% of the total level of micro-organisms. The repeatability (S.D.%) of the conductance assay ranged from 2.9% to 7.3%.     

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.     

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.     

WLIP, a lipodepsipeptide of Pseudomonas 'reactans', as inhibitor of the symptoms of the brown blotch disease of Agaricus bisporus

A cell-free crude extract containing the white line inducing principle (WLIP), a lipodepsipeptide produced by Pseudomonas 'reactans' , could inhibit browning of mushrooms caused by Pseudomonas tolaasii . Mushrooms inoculated with Ps. tolaasii at concentrations of 2·7 × 10⁶ cfu ml⁻¹ or higher showed the symptoms of the disease after 2 d of incubation. Mushroom caps treated with various concentrations of a crude WLIP preparation, and later inoculated with bacterial concentrations higher than the threshold value, did not develop the symptoms of the disease. One milligram of a crude WLIP preparation could block 50% of the symptoms caused by 1·2 × 10⁷ cfu. The inhibition of browning was effective when incubating at low temperatures for 4 d. A suspension containing 1·6 mg ml⁻¹ of pure WLIP was also able to inhibit the symptoms of brown blotch disease induced by 7·6 × 10⁶ cfu ml⁻¹ of Ps. tolaasii .     

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.     

An evaluation of potential interferences in a fluorimetric assay for the rapid detection of thermotolerant coliforms in sewage

C.M. DAVIES and S.C. APTE.2000. A 1-h fluorimetric assay of β- d -galactosidase activity was evaluated for determining thermotolerant coliforms (TTC) in sewage samples. Above TTC concentrations of 2·3 × 10³ colony-forming units (cfu) 100 ml⁻¹, the assay response was related to TTC concentration. However, below this concentration, a large background signal was observed which was independent of TTC concentration. A separation scheme involving various filtration treatments and additions of a β- d -galactosidase inhibitor was devised and used to quantify the sources of this anomalous assay response. The interferences encountered were largely due to the presence in sewage of non-specific cell-free enzymes or other cell-free substances that were capable of hydrolysing the fluorogenic substrate. Despite this apparent limitation, the fluorimetric enzyme assay has potential as an 'early warning' indicator of treatment process failure and gross sewage contamination and leakage in situations where TTC concentrations exceed 2·3 × 10³ cfu 100 ml⁻¹     

Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence

A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719. A dot immunoblot assay was then developed to detect nisin Z in milk and whey. As few as 1·5 10⁻¹ international units per ml (IU ml⁻¹), corresponding to 0·003 μg ml⁻¹ of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence. When milk and whey samples were tested, approximately 0·155 μg ml⁻¹ (7·9 IU ml⁻¹) of nisin Z was detected. The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.     

Improved PCR detection sensitivity of Erwinia carotovora subsp. atroseptica in potato tuber peel extract by prior enrichment on a selective medium

A simple and sensitive method was developed to replace the need for complex and laborious DNA extraction to remove inhibitory substances in potato tuber peel extract before detection of Erwinia carotovora subsp. atroseptica (Eca) by PCR. Eca was enriched by a factor of 10⁵ when peel extract was inoculated onto a selective medium, CVP, and incubated at 27°C for 24 h. Bacterial micro-colonies which developed were suspended in 500 μl of water and the bacteria diluted in water 100-fold, or 10-fold followed by washing by centrifugation, before PCR testing. The sensitivity of detection obtained with the former was ca 10¹–10² cells ml⁻¹ and with the latter ca 10¹ cells ml⁻¹, when different numbers of streptomycin-resistant Eca strain were added to peel extract from three Eca-free potato cultivars. The method was validated and the sensitivity confirmed relative to two different commonly used Eca detection methods using naturally contaminated tubers.     

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.     

Reduction of Brochothrix thermosphacta on beef surfaces following immobilization of nisin in calcium alginate gels

Lean and adipose beef carcass tissues inoculated with Brochothrix thermosphacta (BT) (approx. 4.50 log₁₀ cfu cm⁻²) were left untreated (U) or treated with 100 μg ml⁻¹ nisin (N), calcium alginate (A) or 100 μg ml⁻¹ nisin immobilized in a calcium alginate gel (AN). Tissue samples were refrigerated after treatments and bacterial populations and nisin activity were determined at 0, 1, 2 and 7 d. U, A and N treatments of lean and adipose tissues did not suppress bacterial growth (>6 log₁₀ cfu cm⁻² by day 7) while treatments of lean and adipose tissues with AN suppressed bacteria (>2.42 log₁₀ cfu cm⁻² by day 7). Bacteriocin titres from both tissues were higher in AN vs N samples after the 7 d incubation. This study demonstrates that immobilization of nisin in a gel may be a more effective delivery system of a bacteriocin to the carcass surface than direct application.     

Polymerase chain reaction detection and speciation of Campylobacter upsaliensis and C. helveticus in human faeces and comparison with culture techniques

A polymerase chain reaction (PCR) assay based on the 16S rRNA gene and an improved DNA extraction procedure were developed for the direct detection and differentiation of Campylobacter upsaliensis and C. helveticus in seeded human faeces. The PCR assay was compared with culture detection by a membrane filter (MF) technique and on selective agar (SA) containing 8 mg l⁻¹ cefoperazone. Both MF culture and the PCR assay detected 10⁵ colony-forming units (cfu) g⁻¹ faeces. Selective agar culture of some strains could detect as few as 10³ cfu g⁻¹ faeces. However, some strains were susceptible to cefoperazone and either failed to grow or were detected only with reduced sensitivity in the presence of the antibiotic. Detection by MF and SA both required 48–96 h incubation in a microaerobic atmosphere and did not specifically identify the isolate. By contrast, the PCR assay could be completed within 8 h and accurately identified the two phenotypically similar species, C. upsaliensis and C. helveticus.