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.     

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.     

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.     

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).     

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.     

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.     

Detection of bacterial antigens using immuno-PCR

E. KAKIZAKI, T. YOSHIDA, H. KAWAKAMI, M. OSETO, T. SAKAI AND M. SAKAI. 1996. A new and very sensitive antigen detection technique, immuno-polymerase chain reaction (immuno-PCR), was developed. This method is basically similar to the enzyme-linked immunosorbent assay which detects an antigen-antibody reaction, but instead of an enzyme being conjugated to an antibody, a DNA fragment is used and this DNA can be amplified by PCR. We applied this method to the detection of the fish pathogen, Pasteurella piscicida , in naturally infected yellowtail. Using immuno-PCR, 3.4 cfu ml⁻¹ of bacteria could be detected. In comparison, ELISA detected only 3.4 × 10⁴ cfu ml⁻¹. Immuno-PCR is a powerful method for detection of pathogens in host tissues.     

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%).     

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.     

Development and use of a multiplex PCR system for the rapid screening of heat labile toxin I, heat stable toxin II and shiga-like toxin I and II genes of Escherichia coli in water

Enterotoxigenic Escherichia coli (ETEC) may produce heat-labile toxin (LT) I and LTII and heat-stable toxin (ST) I and STII, while shiga toxin producing E. coli (STEC) strains, including enterohaemorrhagic E. coli (EHEC), may produce shiga-like toxin (SLT) I and/or SLTII. Both ETEC and STEC are pathogenic to humans, pigs and cattle. As contamination of environmental water by any of these pathogenic E. coli cells is possible, a multiplex polymerase chain reaction (PCR) system for the rapid screening of LTI, STII, and SLTI and SLTII genes of E. coli was developed. The PCR primers used were the SLTI and SLTII genes specific primers developed by the present authors and the LTI and STII genes specific primers reported by other laboratories. The detection specificity of this multiplex PCR system was confirmed by PCR assay of ETEC, STEC and other E. coli cells as well as non- E. coli bacteria. Its detection limit was 10²–10³ cfu each of the target cells per assay. When this multiplex PCR system was used for the rapid screening of LTI, STII ETEC and STEC in water samples such as tap, underground and lake waters, it was found that after the enrichment step, as few as 10⁰ cells 100 ml⁻¹ of the water sample could be detected. Therefore, this PCR system could be used for the rapid monitoring of ETEC and/or STEC cells contaminating water samples.     

Interaction between fish spoilage bacteria Pseudomonas sp. and Shewanella putrefaciens in fish extracts and on fish tissue

The interaction between fish spoilage bacteria, Pseudomonas sp. and Shewanella putrefaciens , was investigated using fish extract and fish tissue as model systems. Isolates of Pseudomonas that produced iron chelators, siderophores, inhibited growth of S. putrefaciens in a fish-extract-agar diffusion assay but no, or only weak, antagonistic activity was seen when the medium was supplemented with iron. Sterile-filtered supernatant fluid from a siderophore-producing Pseudomonas grown in fish extract was inhibitory to S. putrefaciens if the number of Pseudomonas was above 10⁸ cfu ml⁻¹. In contrast, supernatant fluids from siderophore-negative Pseudomonas isolates did not inhibit growth of S. putrefaciens. The inhibitory effect was, except for one strain of Pseudomonas , not seen in supernatant fluids from iron-enriched cultures of Pseudomonas sp. Finally, siderophore-producing Pseudomonas sp. lowered the maximum cell level of S. putrefaciens 1–2 log units from 10⁹ to 10¹⁰ cfu g⁻¹ when the strains were grown on fish muscle blocks at 0°C but the growth rate of S. putrefaciens was not affected.     

Production of amylase by the intestinal microflora in cultured freshwater fish

The amylase-producing ability of the intestinal microflora in cultured specimens of ayu, carp, channel catfish, Japanese eel and tilapia was determined. Mean viable counts of aerobes and anaerobes ranged from 1·1×10⁶ to 3·7×10⁸ cfu g⁻¹ and from 1·3×10³ to 1·6×10⁸ cfu g⁻¹, respectively. Aeromonas spp. and Bacteroidaceae were predominant in four to five fish species. Of 206 strains examined, 65 (31·6%) produced ≥0·01 U amylase ml⁻¹. The percentage of producers differed among families and genera of bacteria and fish species. While 56% of the anaerobes produced amylase, only 20% of the aerobes did. More than 50% of Aeromonas , Bacteroidaceae and Clostridium strains produced amylase efficiently while Acinetobacter , coryneforms, Enterobacteriaceae, Moraxella , Plesiomonas and Streptococcus strains did not. High amylase production (≥0·05 U ml⁻¹) was found in 12 strains, 11 from Aeromonas and one Pseudomonas . The percentage of high amylase producers in Japanese eel was lower than the other four fish (2–30%). These results strongly suggest that the amylase produced by the intestinal microflora play an important role in the digestion of starch in freshwater fish to some extent.     

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 .     

Production of amylase by the intestinal bacteria of Japanese coastal animals

H. SUGITA, J. KAWASAKI, J. KUMAZAWA AND Y. DEGUCHI. 1996. The amylase-producing ability of intestinal bacteria in one marine crab and seven fish species was determined. Mean total viable counts ranged from 1.3 × 10⁵ to 1.5 × 10⁸ cfu g⁻¹, and Vibrionaceae were predominant in all specimens. Of 1585 strains examined, 341 (21.5%) produced ≥0.01 U amylase ml⁻¹. Percentage of producers (≥0.01 U ml⁻¹) differed among genera/families. High abilities (≥0.05 U ml⁻¹) were found in 1.4-3.6% of Enterobacteriaceae, Pseudomonas and Vibrionaceae strains. On the other hand, percentage of producers varied with animal species. These results reveal that the amylase producers were widely distributed in the digestive tracts of coastal animals including crabs and fish, irrespective of their food habitats.     

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     

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.     

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.     

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.     

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%.     

Rapid detection of pathogenic Vibrio parahaemolyticus by a sensitive and specific duplex PCR-hybridization probes assay using LightCycler

Aims:  To develop a real-time polymerase chain reaction (PCR) hybridization probe assay for rapid and specific detection of thermostable direct haemolysin-producing Vibrio parahaemolyticus.
Methods and Results:  Primers and hybridization probes were designed to target the toxR and tdh2 genes. Mismatches were introduced in the tdh2 primers for specific amplification of the target. The 3' ends of donor probes for both genes were labelled with fluorescein. The 5' ends of recipient probes for tdh2 and toxR were labelled with LC Red 640 and LC Red 705, respectively. The real-time assay was evaluated against conventional biochemical tests and the KAP-RPLA kit (Kanagawa phenomenon detection kit by reverse passive latex agglutination). toxR and tdh2 were detected in 100% and 91% of clinical V. parahaemolyticus isolates ( n  = 118), respectively. Specificity and sensitivity of the real-time assay for toxR and tdh2 were 100%, respectively. Dynamic range of detection for toxR was 10⁷–10¹ CFU ml⁻¹ and that for tdh2 was 10⁷–10⁴ CFU ml⁻¹.
Conclusions:  The LightCycler assay described is sensitive and highly specific for detection of pathogenic V. parahaemolyticus in a single reaction tube within 80 min.
Significance and Impact of the Study:  The assay developed allows accurate detection of pathogenic V. parahaemolyticus , which is valuable for rapid tracing of infection source during outbreaks.