Detection of pandemic Vibrio parahaemolyticus O3:K6 serovar in Gulf of Mexico water and shellfish using real-time PCR with Taqman fluorescent probes

We describe a real-time multiplexed PCR method using Taqman probes for the detection of total and pandemic Vibrio parahaemolyticus O3:K6 serovar in oysters and Gulf of Mexico water (gulf water). The specificity of these primers and probes was tested for amplification of a 450 bp thermolabile hemolysin (tlh) and a 369 bp ORF8 amplicon representing all V. parahaemolyticus and post-1996 clinical isolates of pandemic serovar O3:K6, respectively. The sensitivity of detection was 10 pg purified DNA or 10(3) CFU in 1 mL pure culture. Enrichment of this pathogen in oyster tissue homogenate or gulf water for 5 or 8 h resulted in the detection of an initial inoculum of 1 CFU in 1 mL or 1 g of samples. Application of the Taqman PCR assay on natural oysters exhibited a positive detection of V. parahaemolyticus, ranging from 16% to 100% of the samples collected primarily during the summer months. None of the samples exhibited a positive detection of O3:K6 serovar. Rapid and sensitive detection of this pathogen will help shellfish industry and Interstate Shellfish Sanitation Conference (ISSC) undertake appropriate measures to monitor this pathogen in oysters and oyster-growing waters, thereby preventing disease outbreaks and consequently protecting consumer health.     

Acid adaptation of Listeria monocytogenes can enhance survival in acidic foods and during milk fermentation.

We have previously shown that tolerance to severe acid stress (pH 3.5) can be induced in Listeria monocytogenes following a 1-h adaptation to mild acid (pH 5.5), a phenomenon termed the acid tolerance response (ATR) (B. O'Driscoll, C. G. M. Gahan, and C. Hill, Appl. Environ. Microbiol. 62:1693-1698, 1966). In an attempt to determine the industrial significance of the ATR, we have examined the survival of adapted and nonadapted cells in a variety of acidic foods. Acid adaptation enhanced the survival of L. monocytogenes in acidified dairy products, including cottage cheese, yogurt, and whole-fat cheddar cheese. Acid-adapted L. monocytogenes cultures also demonstrated increased survival during active milk fermentation by a lactic acid culture. Similarly, acid-adapted cells showed greatly improved survival in low-pH foods (orange juice and salad dressing) containing acids other than lactic acid. However, in foods with a marginally higher pH, such as mozzarella cheese, a commercial cottage cheese, or low-fat cheddar cheese, acid adaptation did not appear to enhance survival. We have previously isolated mutants of L. monocytogenes that are constitutively acid tolerant in the absence of an induction step (O'Driscoll et al., Appl. Environ. Microbiol. 62:1693-1698, 1996). In the present study, one such mutant, ATM56, demonstrated an increased ability to survive in low-pH foods and during milk fermentation when compared with the wild-type strain. Significant numbers of ATM56 could be recovered even after 70 days in both whole-fat and low-fat cheddar cheese. Collectively, the data suggest that ATR mechanisms, whether constitutive or induced, can greatly influence the survival of L. monocytogenes in low-pH food environments.     

Assessment of the Accuprobe Listeria monocytogenes culture identification reagent kit for rapid colony confirmation and its application in various enrichment broths.

The Accuprobe Listeria monocytogenes Culture Identification Reagent Kit, a nonradioactive probe, was evaluated as a colony confirmation test and in different selective or nonselective enrichment broths. The probe was 100% sensitive and 100% specific when applied to isolated colonies. The minimal detection limit in physiological saline was established to be about 10(5) CFU of L. monocytogenes. Hybridization done directly in broths seeded with L. monocytogenes showed variable results. Three nonselective broths (Todd-Hewitt broth, brain heart infusion broth, and tryptic soy broth) and one selective broth (FDA) gave positive reactions at an inoculum of 5 x 10(6) CFU, whereas two other selective broths (UVM, and PALCAMY) gave negative reactions with up to 10(8) and 10(9) CFU. In FDA broth, the level of detection of L. monocytogenes was not modified by the presence of other organisms in mixed cultures.     

Assessment of the Accuprobe Listeria monocytogenes culture identification reagent kit for rapid colony confirmation and its application in various enrichment broths.

The Accuprobe Listeria monocytogenes Culture Identification Reagent Kit, a nonradioactive probe, was evaluated as a colony confirmation test and in different selective or nonselective enrichment broths. The probe was 100% sensitive and 100% specific when applied to isolated colonies. The minimal detection limit in physiological saline was established to be about 10(5) CFU of L. monocytogenes. Hybridization done directly in broths seeded with L. monocytogenes showed variable results. Three nonselective broths (Todd-Hewitt broth, brain heart infusion broth, and tryptic soy broth) and one selective broth (FDA) gave positive reactions at an inoculum of 5 x 10(6) CFU, whereas two other selective broths (UVM, and PALCAMY) gave negative reactions with up to 10(8) and 10(9) CFU. In FDA broth, the level of detection of L. monocytogenes was not modified by the presence of other organisms in mixed cultures.     

Comparison of methods of extracting Salmonella enterica serovar Enteritidis DNA from environmental substrates and quantification of organisms by using a general internal procedural control

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 x 10(3) to 1.8 x 10(3) CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.     

A real-time PCR for the detection of Salmonella Enteritidis in poultry meat and consumption eggs

A robust duplex 5' nuclease (TaqMan) real-time PCR was developed and in-house validated for the specific detection of Salmonella enterica subspecies enterica serovar Enteritidis in whole chicken carcass rinses and consumption eggs. The assay uses specifically designed primers and a TaqMan probe to target the Prot6e gene located on the S. Enteritidis specific 60-kb virulence plasmid. As an internal amplification control to monitor Salmonella DNA in the sample, a second primer/TaqMan probe set detects simultaneously the Salmonella specific invA gene. The assay identified correctly 95% of the 79 Salmonella Enteritidis strains tested comprising 19 different phage types. None of the 119 non-Enteritidis strains comprising 54 serovars was positive for the Prot6e gene. The assay detection probability was for 10(2) or more genome equivalents 100% and for 10 equivalents 83%. A pre-PCR sample preparation protocol including a pre-enrichment step in buffered peptone water, followed by DNA extraction was applied on low levels of artificially contaminated whole chicken carcass rinses and eggs from hens as well as 25 potentially naturally contaminated chickens. The detection limit was less than three CFU per 50 ml carcass rinse or 10 ml egg. The sensitivity and specificity compared to the traditional culture-based detection method and serotyping were both 100%. Twenty-five potentially naturally contaminated chickens were compared by the real-time PCR and the traditional cultural isolation method resulting in four Salmonella positive samples of which two were positive for the Prot6e gene and serotyped as S. Enteritidis. We show also that Salmonella isolates which have a rough lipopolysaccharide structure could be assigned to the serovar Enteritidis by the real-time PCR. This methodology can contribute to meet the need of fast identification and detection methods for use in monitoring and control measures programmes.     

Quantitative detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology

We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R(2) values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).     

Comparison of Methods of Extracting Salmonella enterica Serovar Enteritidis DNA from Environmental Substrates and Quantification of Organisms by Using a General Internal Procedural Control

This paper compares five commercially available DNA extraction methods with respect to DNA extraction efficiency of Salmonella enterica serovar Enteritidis from soil, manure, and compost and uses an Escherichia coli strain harboring a plasmid expressing green fluorescent protein as a general internal procedural control. Inclusion of this general internal procedural control permitted more accurate quantification of extraction and amplification of S. enterica serovar Enteritidis in these samples and reduced the possibility of false negatives. With this protocol it was found that the optimal extraction method differed for soil (Mobio soil DNA extraction kit), manure (Bio101 soil DNA extraction kit), and compost (Mobio fecal DNA extraction kit). With each method, as little as 1.2 × 10³ to 1.8 × 10³ CFU of added serovar Enteritidis per 100 mg of substrate could be detected by direct DNA extraction and subsequent S. enterica-specific TaqMan PCR. After bacterial enrichment, as little as 1 CFU/100 mg of original substrate was detected. Finally, the study presents a more accurate molecular analysis for quantification of serovar Enteritidis initially present in soil or manure using DNA extraction and TaqMan PCR.     

Amplification of rfbE and fliC Genes by Polymerase Chain Reaction for Identification and Detection of Salmonella Serovar Enteritidis,Dublin and Gallinarum-Pullorum

Polymerase chain reaction (PCR) primers for O9 antigen (rfbE) and phase 1 flagellin antigen (fliC) were designed for the rapid identification and detection of Salmonella serovar Enteritidis and Dublin. The rfbE primer pairs selectively amplified the rfbE region of group O9 Salmonella serovars. The fliC primer pairs amplified the DNAs of g,m and g,p-type flagellar antigen; Salmonella serovar Enteritidis, Dublin, and Essen. However, DNA from flagellar-negative Salmonella serovar Gallinarum-Pullorum was also amplified. The sensitivity of PCR primer pairs was 10 CFU/assay by boiled DNA preparation and 10² CFU/assay by proteinase K-treated DNA preparation.     

A newly developed assay to study the minimum inhibitory concentration of Satureja spinosa essential oil

AIMS: The minimum inhibitory concentration (MIC) of Satureja spinosa essential oil against Staphylococcus aureus, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enterica, Salmonella serovar Enteritidis PT4 and Bacillus cereus was comparatively assessed with an established optical density method as well as a novel impedimetric method. METHODS AND RESULTS: The impedimetric analysis takes into account information of microbial growth, such as detection time, maximum conductance, and slope of the conductance curve. For each pathogen two levels of inoculation were studied, a high (10(5) CFU ml(-1)) and a low level (10(2) CFU ml(-1)). Non-linear regression analysis was used to fit the data using a modification of a previously published model, from which a more exact value can be obtained for the MIC. Both methods gave similar MICs as shown by t-test statistical analysis. Salm. Enteritidis seems to be the least sensitive to the action of S. spinosa essential oil followed by L. monocytogenes, E. coli, B.cereus and Staph. aureus. The MICs of low inoculum were lower than that of high inoculum. CONCLUSIONS: The new impedimetric assay of MIC of essential oils can be considered a reliable rapid method for screening antimicrobial effectiveness of natural additives. SIGNIFICANCE AND IMPACT OF THE STUDY: Determination of the minimum inhibitory concentration of an essential oil with the simple conductance technique and further study of the mode of action of its components is a good combination for obtaining additional knowledge for industrial application of such natural additives.     

Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR

AIMS: Surface contamination by Listeria monocytogenes of gouda-like cheeses during processing represents a potential public health problem. The aim of this work was to develop novel real-time PCR diagnostics to detect the presence of viable, dead or viable but not culturable (VBNC) cells on gouda-like cheeses. METHODS AND RESULTS: We used ethidium monoazide bromide (EMA)-PCR for direct quantification of viable and dead cells, while semiquantitative detection of culturable cells below the PCR detection limit (c. 100 CFU g(-1)) was obtained by combining growth and real-time PCR. We were able to quantify the fraction of >0.5% viable cells in a background of dead cells by EMA-PCR, given that the viable cell concentration was above the PCR detection limit. The combined growth and real-time PCR complemented the EMA-PCR, and enabled semiquantitative detection of low levels of culturable cells (10 and 100 CFU g(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: The significance of this work is that we have developed a novel concept for detection of viable and potentially infectious L. monocytogenes.     

16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods.

A rapid polymerase chain reaction (PCR) method was developed for detection of Listeria monocytogenes in foods. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L. monocytogenes, which were previously reported by us to yield a specific nucleic acid probe. Our method included use of a shorter denaturing time, a shorter annealing time, a rapid transition, and an increase in the number of cycles, resulting in good sensitivity. Just 3 h for PCR plus 1 h for electrophoresis was required. Additional time for DNA isolation and DNA hybridization was not needed. This method detected as few as 2 to 20 CFU of L. monocytogenes in pure cultures and as few as 4 to 40 CFU of L. monocytogenes in inoculated (10(8) CFU), diluted food samples. Seven of eight foods, including four poultry products, gave positive results. Only one food sample, soft cheese, gave interference. An internal probe hybridization test was used to confirm that the PCR products were from L. monocytogenes. A specificity test indicated that this PCR method was positive for all 13 strains of L. monocytogenes tested but negative for the other 6 species of Listeria, including 6 strains of L. innocua, and negative for 17 other gram-positive and gram-negative bacteria tested.     

Inhibition of Listeria innocua in cheddar cheese by addition of nisin Z in liposomes or by in situ production in mixed culture

The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in the mixed starter on the inhibition of Listeria innocua in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing Lactococcus lactis subsp. lactis biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added L. innocua (10(5) to 10(6) CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese. The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3- and 1.5-log-unit reductions in viable counts of L. innocua were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of L. innocua per g and 90% of the initial nisin activity, compared with 10(4) CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.     

16S rRNA-based probes and polymerase chain reaction method to detect Listeria monocytogenes cells added to foods.

A rapid polymerase chain reaction (PCR) method was developed for detection of Listeria monocytogenes in foods. This method used a pair of primers based on a unique region in the 16S rRNA sequence of L. monocytogenes, which were previously reported by us to yield a specific nucleic acid probe. Our method included use of a shorter denaturing time, a shorter annealing time, a rapid transition, and an increase in the number of cycles, resulting in good sensitivity. Just 3 h for PCR plus 1 h for electrophoresis was required. Additional time for DNA isolation and DNA hybridization was not needed. This method detected as few as 2 to 20 CFU of L. monocytogenes in pure cultures and as few as 4 to 40 CFU of L. monocytogenes in inoculated (10(8) CFU), diluted food samples. Seven of eight foods, including four poultry products, gave positive results. Only one food sample, soft cheese, gave interference. An internal probe hybridization test was used to confirm that the PCR products were from L. monocytogenes. A specificity test indicated that this PCR method was positive for all 13 strains of L. monocytogenes tested but negative for the other 6 species of Listeria, including 6 strains of L. innocua, and negative for 17 other gram-positive and gram-negative bacteria tested.     

Detection of Listeria monocytogenes in cheese with the magnetic immuno-polymerase chain reaction assay.

A new detection system, the magnetic immuno-polymerase chain reaction (PCR) assay (MIPA) has been developed to detect Listeria monocytogenes in food. This method separates Listeria cells from PCR-inhibitory factors present in enrichment broths containing food samples by using magnetic beads coated with specific monoclonal antibodies (MAbs). The separated bacteria were lysed, and the supernatant containing the bacterial DNA was subjected to the PCR. Detection of L. monocytogenes in three naturally contaminated cheese samples with two different MAbs and PCR primers specific for the gene encoding the delayed-hypersensitivity factor showed that with MAb 55 all three samples were positive whereas with MAb A two samples were positive. A further improvement of the method was obtained by using a PCR step based on the listeriolysin O gene. A MIPA employing MAb 55 and the listeriolysin O gene primer set detected L. monocytogenes after 24 h of culture in Listeria Enrichment Broth samples from Port Salut artificially contaminated with 40 CFU/25 g. We could detect 1 CFU of L. monocytogenes per g of cheese after a second enrichment for 24 h in Fraser broth. The analysis time including both enrichments is approximately 55 h.     

Inhibition of Listeria innocua in Cheddar Cheese by Addition of Nisin Z in Liposomes or by In Situ Production in Mixed Culture

The effect of addition of purified nisin Z in liposomes to cheese milk and of in situ production of nisin Z by Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in the mixed starter on the inhibition of Listeria innocua in cheddar cheese was evaluated during 6 months of ripening. A cheese mixed starter culture containing Lactococcus lactis subsp. lactis biovar diacetylactis UL719 was selected for high-level nisin Z and acid production. Experimental cheddar cheeses were produced on a pilot scale, using the selected starter culture, from milk with added L. innocua (10⁵ to 10⁶ CFU/ml). Liposomes with purified nisin Z were prepared from proliposome H and added to cheese milk prior to renneting to give a final concentration of 300 IU/g of cheese. The nisin Z-producing strain and nisin Z-containing liposomes did not significantly affect cheese production and gross chemical composition of the cheeses. Immediately after cheese production, 3- and 1.5-log-unit reductions in viable counts of L. innocua were obtained in cheeses with encapsulated nisin and the nisinogenic starter, respectively. After 6 months, cheeses made with encapsulated nisin contained less than 10 CFU of L. innocua per g and 90% of the initial nisin activity, compared with 10⁴ CFU/g and only 12% of initial activity in cheeses made with the nisinogenic starter. This study showed that encapsulation of nisin Z in liposomes can provide a powerful tool to improve nisin stability and inhibitory action in the cheese matrix while protecting the cheese starter from the detrimental action of nisin during cheese production.     

A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes

Development of a routine detection assay for Listeria monocytogenes in foods that uses the polymerase chain reaction (PCR) and enrichment cultures was investigated. Oligonucleotide primers were chosen to amplify a 3'region of L. monocytogenes hly A gene spanning a conserved Hin dIII site. PCR detection sensitivity for L. monocytogenes in dilutions of pure enrichment cultures was between 50 and 500 colony forming units. A short enrichment period before PCR amplification allowed detection of the organisms in a range of complex foods contaminated with 10⁴ cfu/g. Detection sensitivity for the assay in the presence of chicken skin and soft cheese was determined at 10–100 cfu/g. Utilization of enrichment cultures and PCR allowed identification of the organism within 24 h or 2 days.     

Prevalence and fingerprinting of Listeria monocytogenes strains isolated from raw whole milk in farm bulk tanks and in dairy plant receiving tanks

The incidence of Listeria species in raw whole milk from farm bulk tanks and from raw milk in storage at a Swedish dairy plant was studied. Listeria monocytogenes was found in 1.0% and Listeria innocua was found in 2.3% of the 294 farm bulk tank (farm tank) milk specimens. One farm tank specimen contained 60 CFU of L. monocytogenes ml(-1). L. monocytogenes was detected in 19.6% and L. innocua was detected in 8.5% of the milk specimens from the silo receiving tanks at the dairy (dairy silos). More dairy silo specimens were positive for both Listeria species during winter than during summer. Restriction enzyme analysis and pulsed-field gel electrophoresis were applied to 65 isolates of L. monocytogenes, resulting in 16 different clonal types. Two clonal types were shared by the farm tank milk and the dairy silo milk. All except one clonal type belonged to serovar 1/2a. In the dairy silo milk five clonal types were found more frequently and for a longer period than the others. No Listeria species were found in any other samples from the plant.     

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.