A decade with nucleic acid-based microbiological methods in safety control of foods.

In the last decade, nucleic acid-based methods gradually started to replace or complement the culture-based methods and immunochemical assays in routine laboratories involved in food control. In particular, real-time polymerase chain reaction (PCR) was technically developed to the stage of good speed, sensitivity and reproducibility, at minimized risk of carry-over contamination. Basic advantages provided by nucleic acid-based methods are higher speed and added information, such as subspecies identification, information on the presence of genes important for virulence or antibiotic resistance. Nucleic acid-based methods are attractive also to detect important foodborne pathogens for which no classical counterparts are available, namely foodborne pathogenic viruses. This review briefly summarizes currently available or developing molecular technologies that may be candidates for involvement in microbiological molecular methods in the next decade. Potential of nonamplification as well as amplification methods is discussed, including fluorescent in situ hybridization, alternative PCR chemistries, alternative amplification technologies, digital PCR and nanotechnologies.

Effective application of multiple locus variable number of tandem repeats analysis to tracing Staphylococcus aureus in food-processing environment.

UNLABELLED: A total of 256 isolates of Staphylococcus aureus were isolated from 98 samples (34 swabs and 64 food samples) obtained from small or medium meat- and cheese-processing plants in Slovakia. The strains were genotypically characterized by multiple locus variable number of tandem repeats analysis (MLVA), involving multiplex polymerase chain reaction (PCR) with subsequent separation of the amplified DNA fragments by an automated flow-through gel electrophoresis. With the panel of isolates, MLVA produced 31 profile types, which was a sufficient discrimination to facilitate the description of spatial and temporal aspects of contamination. Further data on MLVA discrimination were obtained by typing a subpanel of strains by multiple locus sequence typing (MLST). MLVA coupled to automated electrophoresis proved to be an effective, comparatively fast and inexpensive method for tracing S. aureus contamination of food-processing factories. SIGNIFICANCE AND IMPACT OF THE STUDY: Subspecies genotyping of microbial contaminants in food-processing factories may facilitate identification of spatial and temporal aspects of the contamination. This may help to properly manage the process hygiene. With S. aureus, multiple locus variable number of tandem repeats analysis (MLVA) proved to be an effective method for the purpose, being sufficiently discriminative, yet comparatively fast and inexpensive. The application of automated flow-through gel electrophoresis to separation of DNA fragments produced by multiplex PCR helped to improve the accuracy and speed of the method.

Selection and identification of autochthonous yeasts in Slovakian wine samples using a rapid and reliable three-step approach.

AIMS: The investigation of yeast microflora during the must fermentation of two wine varieties (Frankovka modra - Blaufränkisch and Veltlinske zelene - Grüner Veltliner) from two consecutive vintages was performed using a three-step approach. METHODS AND RESULTS: The investigation strategy consisted of the combination of yeast cultivation, selection of the isolated yeasts based on the amplification of internal transcribed spacer 2 using a fluorescence-labelled primer (f-ITS-PCR) and a final identification step based on amplification and sequencing of the ITS1-5.8S rDNA-ITS2 region of the selected yeasts. By this three-step approach, it was possible to screen 433 yeasts isolates that belonged to 13 different species. CONCLUSIONS: The f-ITS-PCR allowed the unambiguous differentiation of all isolated yeast species that produced their typical f-ITS-PCR profile. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of few reports that treat the yeast diversity in Slovakian wines and in two varieties largely cultivated in Central Europe. The three-step approach permitted the rapid and reliable identification of isolated yeasts. The f-ITS-PCR with its good discrimination power can represent a suitable molecular tool for the selection of yeast members recovered from food or other environments.

Randomly-amplified microsatellite polymorphism for preliminary typing of lactic acid bacteria from Bryndza Cheese.

A high-throughput, medium-discrimination method for preliminary typing and selecting non-identical isolates of lactic acid bacteria in cheeses was developed. RAMP, a PCR with one microsatellite-targeted and one random primer, was used for preliminary typing of 1119 isolates of lactic acid bacteria from Slovak Bryndza cheese. A total of 59 genotypes were identified based on RAMP profiles consisting of 12-23 DNA fragments of 150-3000 bp. For example, 18, 17, 13 and 7 different RAMP-types were identified in Lactobacillus brevis, L. plantarum, L. paracasei and L. fermentum, respectively. The method facilitated well reproducible, medium-discrimination typing of Lactobacillus spp. and Pediococcus spp. at a subspecies level and proved to be suitable for preliminary typing of lactic acid bacteria isolated from cheese.

A single-tube nested real-time polymerase chain reaction for sensitive contained detection of Cryptosporidium parvum.

AIM: A new real-time polymerase chain reaction (PCR) was developed for sensitive contained detection of Cryptosporidium parvum. METHODS AND RESULTS: The method is a nested PCR targeting a specific region of rDNA of C. parvum, which takes place in one tube, using different annealing temperatures to control the first and the second rounds of PCR, with real-time fluorogenic probe-based detection of the second round of PCR. The DNA-based detection limit of the method was 2 fg, which corresponds to approx. one genome per reaction. The detection level determined using diluted samples of C. parvum oocysts was ten oocysts per millilitre. CONCLUSIONS: The method facilitates sensitive detection of C. parvum thanks to the nested format, while reducing the risk of laboratory contamination thanks to the single-tube, real-time fluorimetric format. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed method may be useful for sensitive contained detection of C. parvum in environmental and food samples, after appropriate separation of oocysts.

Detection of Salmonella enterica in food using two-step enrichment and real-time polymerase chain reaction.

AIMS: A new real-time polymerase chain reaction-based method was developed for the detection of Salmonella enterica in food. METHODS AND RESULTS: The method consisted of a novel two-step enrichment involving overnight incubation in buffered peptone water and a 5-h subculture in Rappaport-Vassiliadis medium, lysis of bacterial cells and a Salmonella-specific 5'-nuclease real-time PCR with an exogenous internal amplification control. Because a two-step enrichment was used, the detection limit for dead S. enterica cells in artificially contaminated ice cream and salami samples was high at 10(7 )CFU (25 g)(-1), eliminating potential false-positive results. When the method was evaluated with a range of 100 naturally contaminated food samples, three positive samples were detected by both the real-time PCR-based method and by the standard microbiological method, according to EN ISO 6579. When the real-time PCR-based method was evaluated alongside the standard microbiological method according to EN ISO 6579 with 36 food samples artificially contaminated at a level of 10(0 )CFU (25 g)(-1), identical results were obtained from both methods. CONCLUSIONS: The real-time PCR-based method involving a two-step enrichment produced equivalent results to EN ISO 6579 on the day after sample receipt. SIGNIFICANCE AND IMPACT OF THE STUDY: The developed method is suitable for rapid detection of S. enterica in food.

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua.

AIMS: Detectability of Listeria monocytogenes at 10(0) CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real-time PCR-based method. METHODS AND RESULTS: Enrichment in half-Fraser broth followed by subculture in Fraser broth according to EN ISO 11290-1 was used. False-negative detection of 10(0) CFU L. monocytogenes was obtained in the presence of 10(1) CFU L. innocua per sample using the standard detection method in contrast to more than 10(5) CFU L. innocua per sample using real-time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. CONCLUSIONS: Standard microbiological method was insufficient for the reliable detection of 10(0) CFU L. monocytogenes in the presence of more than 10(0) CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.

A novel real-time PCR-based method for the detection of Listeria monocytogenes in food.

AIMS: A new real-time PCR-based method was developed for the detection of Listeria monocytogenes in food. METHODS AND RESULTS: A two-step enrichment involving a 24-h incubation in half-Fraser broth followed by a 6-h subculture in Fraser broth was used, followed by cell lysis and real-time PCR with primers and a TaqMan probe previously developed in our laboratory. When the method was evaluated with 144 naturally contaminated food samples, 44 were detected as positive by the PCR-based method and 42 by the standard method EN ISO 11290-1. With 61 food samples artificially contaminated at a level of 10(0) CFU per 25 g, 61 and 58 positive samples were detected by the respective methods. CONCLUSIONS: The developed real-time PCR-based method facilitated the detection of L. monocytogenes in food on the next day after the sample reception, with a reduction of false-positive results because of dead bacterial cells and false-negative results because of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be used for L. monocytogenes detection in food as a faster alternative to current methods.

Detection and quantification of Listeria monocytogenes by 5'-nuclease polymerase chain reaction targeting the actA gene.

AIMS: The aim of this study was to develop a 5'-nuclease polymerase chain reaction (PCR) for the rapid detection and quantification of Listeria monocytogenes. METHODS AND RESULTS: Specific primers and a fluorogenic probe were designed, which target a specific sequence of the actA gene encoding for a protein involved in the actin filament assembly. The PCR system was highly sensitive and specific for L. monocytogenes (inclusivity, 100%; exclusivity, 100%), which was determined using 46 L. monocytogenes and 28 non-L. monocytogenes strains. Detection limits of 10(4) cfu ml(-1) after 35 cycles and 10(2) cfu ml(-1) after 45 cycles were achieved by PCR in both real-time and end-point fluorescence measurement modes. Linear calibration lines were obtained in the range from 10(2) to 10(9) cfu ml(-1) for three L. monocytogenes strains in real-time PCR with 45 cycles. CONCLUSIONS: The developed 5'-nuclease PCR of the actA gene provides a new target for the rapid detection and quantification of L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: In conjunction with enrichment or with an appropriate quantitative sample preparation technique, the method is suitable for food safety applications.

Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene.

AIMS: A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. METHODS AND RESULTS: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method. CONCLUSIONS: The developed real-time PCR is suitable for rapid quantification of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.

A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model--towards an international standard.

A PCR assay with an internal amplification control was developed for Listeria monocytogenes. The assay has a 99% detection probability of seven cells per reaction. When tested against 38 L. monocytogenes strains and 52 nontarget strains, the PCR assay was 100% inclusive (positive signal from target) and 100% exclusive (no positive signal from nontarget). The assay was then evaluated in a collaborative trial involving 12 European laboratories, where it was tested against an additional 14 target and 14 nontarget strains. In that trial, the inclusivity was 100% and the exclusivity was 99.4%, and both the accordance (repeatability) and the concordance (reproducibility) were 99.4%. The assay was incorporated within a method for the detection of L. monocytogenes in raw milk, which involves 24 h of enrichment in half-Fraser broth followed by 16 h of enrichment in a medium that can be added directly into the PCR. The performance characteristics of the PCR-based method were evaluated in a collaborative trial involving 13 European laboratories. In that trial, a specificity value (percentage of correct identification of blank samples) of 81.8% was obtained; the accordance was 87.9%, and the concordance was 68.1%. The sensitivity (correct identification of milk samples inoculated with 20 to 200 L. monocytogenes cells per 25 ml) was 89.4%, the accordance was 81.2%, and the concordance was 80.7%. This method provides a basis for the application of routine PCR-based analysis to dairy products and other foodstuffs and should be appropriate for international standardization.

Typing of food-borne Listeria monocytogenes by the optimized repetitive extragenic palindrome-based polymerase chain reaction.

The repetitive extragenic palindrome-based polymerase chain reaction was optimized for typing Listeria monocytogenes by 1) using the QlAamp method to increase the reproducibility of DNA isolation, 2) running PCR with three different DNA concentrations in parallel, 3) using antibody-protected therrnostable DNA polymerase to reduce non-specific priming, and 4) using an improved temperature programme to increase the amplification yield. When applied to 42 L. monocytogenes strains isolated from food in the Czech and Slovak republics during 1999-2000, profiles of 7-15 DNA fragments of 330-3,310 bp were amplified. Based on REP-profiles, strains (serotypes 1/2 and 4) could be divided into 12 groups.

Detection of Listeria monocytogenes by polymerase chain reaction oriented to inlB gene.

Primers were designed for the detection of Listeria monocytogenes by the polymerase chain reaction oriented to specific sequences of the inlB gene encoding an internalin. At optimized reaction conditions, 100% sensitivity (on a panel of 33 strains of L. monocytogenes) and 100% specificity (on panels of 15 strains of other Listeria spp. and 41 other bacteria), were determined for the inlB-L/R primers. The detection limit of PCR with these primers was 10(4) cfu/ml and was not affected by up to 10(8) cfu/ml of L. innocua.

Separation of Listeria from cheese and enrichment media using antibody-coated microbeads and centrifugation.

An immunoseparation system for the separation of Listeria from enriched cheese samples was developed. The system utilizes polystyrene microbeads (3.8 microm in diameter) coated with covalently bound anti-Listeria genus-specific antibody. The beads were incubated with cheese enriched in half-Fraser broth and the bead-bacterial complex was separated by centrifugation at 110 g then spread on selective agar plates. Although cross-reactivity with certain Gram-positive bacteria (Staphylococcus saprophyticus and Arthrobacter sp.) was determined, this had no negative effect on capture effectiveness of the beads to Listeria spp. The minimum density of Listeria cells positively detected by immunoseparation with subsequent plating was 10(0) cfu/ml. The application of the separation method facilitates a reduction in the time of Listeria detection in cheese by 2 days without affecting the sensitivity.

Inhibition of germ tube formation, filamentation and ergosterol biosynthesis in Candida albicans treated with 6-amino-2-n-pentylthiobenzothiazole.

Three representative Candida albicans strains were selected out of 26 clinical isolates and strains from culture collections on the basis of their high level of conversion to germ tubes and mycelial form at mycelium-promoting culture conditions, and on their different sensitivity to 6-amino-2-n-pentylthiobenzothiazole (APB). When these strains were treated with APB at mycelium-promoting culture conditions, a concentration-dependent decrease in the proportion of germ tubes and hyphae was observed, while the proportion of the yeast from increased. When non-saponifiable lipids were extracted from these cultures and analyzed, a concentration-dependent decrease in ergosterol and an increase in 4-methylated sterols was observed. However, the sensitivity of sterol biosynthesis did not directly relate to the sensitivity of the morphological conversion, and was exhibited at higher concentrations of APB. On the basis of these results it is suggested that the inhibition of germ tube formation and filamentation is not a consequence of inhibition of ergosterol biosynthesis in APB-treated C. albicans.

Confirmation of presumptive Salmonella colonies by the polymerase chain reaction.

The potential of a genus-specific polymerase chain reaction (PCR) for the confirmation of Salmonella colonies was evaluated on 209 presumptive Salmonella colonies obtained by the standard method ISO 6579. The PCR method employing primers ST11 and ST15 (S. Aabo et al., Mol. Cell. Probes 7:171-178, 1993) gave results identical (100%) to those of the biochemical and serological identification, in terms of discrimination of Salmonella from non-Salmonella strains. PCR could be used directly on the colonies from selective plating media, which allowed a reduction of the time required for confirmation to a maximum of 6 h.

Analysis of hopanoids in bacteria involved in food technology and food contamination.

Hopanoids are pentacyclic triterpenoids which are believed to act as reinforcers of membranes in certain prokaryotic microorganisms. A rapid and sensitive method for their screening in bacteria was elaborated, involving extraction of nonsaponifiable lipids and the analysis by gas chromatography-mass spectrometry, selectively monitoring the ion of m/z = 191. Using the method, hopanoids were detected in strains of Acetobacter pasteurianus, but were found to be absent in lactic acid bacteria (Lactobacillus spp., Lactococcus spp.) and in food-contaminating bacteria (Salmonella spp., Listeria spp., Yersinia spp. and others).

Inhibition of ergosterol biosynthesis is not accompanied by a change in fatty acid composition in Saccharomyces cerevisiae treated with the antifungal agent 6-amino-2-n-pentylthiobenzothiazole.

The antifungal agent 6-amino-2-n-pentylthiobenzothiazole at a concentration of 40 microM lowered the specific growth rate of exponentially growing cultures of Saccharomyces cerevisiae by 36%. Treatment with 6-amino-2-n-pentylthiobenzothiazole inhibited the biosynthesis of ergosterol and caused an accumulation of the methylated sterol precursors ergosta-5,7-dienol and squalene, but had no significant effect on the composition and the rate of biosynthesis of fatty acids. The results indicate that neither the inhibition of ergosterol biosynthesis, nor the slowing-down of culture growth by this antifungal agent, led to a compensatory alteration in the pattern of fatty-acyl chains in membrane lipids. This finding contradicts the accepted wisdom for the action of a number of antifungal agents.

Inhibition of sterol 4-demethylation in Candida albicans by 6-amino-2-n-pentylthiobenzothiazole, a novel mechanism of action for an antifungal agent.

The effects of 6-amino-2-n-pentylthiobenzothiazole (APB), a new antifungal agent, on ergosterol biosynthesis in Candida albicans and Saccharomyces cerevisiae were studied, using [14C]acetate incorporation. In C. albicans, the inhibition of growth was accompanied by a marked inhibition of acetate incorporation in 4-desmethylsterols, with a significant portion of the radiolabel being incorporated in 4,4-dimethylsterols, lanosterol, and 4,4-dimethylzymosterol and minor amounts being incorporated in 4-methylsterols and squalene. The data are interpreted as evidence of a block of the ergosterol biosynthesis pathway at the level of 4-demethylation of 4,4-dimethylzymosterol, with partial inhibition of lanosterol 14-dimethylation and squalene epoxidation also being possible. In 6-amino-2-n-pentylthiobenzothiazole-treated S. cerevisiae, a significant amount of the radiolabel was incorporated also in 4-methylsterols, 4-methylzymosterol, and 4-methylfecosterol, indicating that in this microorganism there are different sensitivities of the two 4-demethylations and that the pathway is blocked at the level of 4-demethylation of 4-methylsterols.