Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

The microbiological safety of ready-to-eat specialty meats from markets and specialty food shops: A UK wide study with a focus on Salmonella and Listeria monocytogenes

From 2359 specialty meats (continental sausages, cured/fermented, dried meats) sampled from markets and specialty food shops, 98.9% of samples were of satisfactory or acceptable microbiological quality. However, 16 (0.7%) were unsatisfactory as a result of Escherichia coli, Staphylococcus aureus or Listeria spp. contamination (≥10² CFU/g), and nine (0.4%) were unacceptable due to presence of Salmonella spp. or Listeria monocytogenes (>10² CFU/g). Meats with unacceptable levels of L. monocytogenes were within shelf life (range: 8–143 days remaining). Nine different subtypes of L. monocytogenes were detected with sero/AFLP type 1/2c VII predominating (37%), although this subtype was not overrepresented in any particular meat type (P > 0.05). Ninety-six percent of continental sausages and cured/fermented products were stored at <8 °C at premises, including seven of the nine unacceptable samples. These nine meats were all pre-packed prior to supply to retail premises (OR = 0.1 P = 0.003) indicating that contamination with bacterial pathogens occurred earlier in the production chain. Most samples (72.7%, 8/11) with unsatisfactory levels of E. coli were sliced on request, suggesting cross-contamination at point of sale. This study highlights the importance of ensuring that products do not become contaminated before final packaging, that storage conditions are controlled, and that durability dates are an accurate indication of the shelf life of the product so as to minimise the potential for L. monocytogenes to be present at levels hazardous to health at the point of sale.     

Quantifying Listeria monocytogenes prevalence and concentration in minced pork meat and estimating performance of three culture media from presence/absence microbiological testing using a deterministic and stochastic approach

Listeria monocytogenes poses a serious threat to public health, and the majority of cases of human listeriosis are associated with contaminated food. Reliable microbiological testing is needed for effective pathogen control by food industry and competent authorities. The aims of this work were to estimate the prevalence and concentration of L. monocytogenes in minced pork meat by the application of a Bayesian modeling approach, and also to determine the performance of three culture media commonly used for detecting L. monocytogenes in foods from a deterministic and stochastic perspective. Samples (n = 100) collected from local markets were tested for L. monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media according to ISO 11290-1:1996 and 11290-2:1998 methods. Presence of the pathogen was confirmed by conducting biochemical and molecular tests. Independent experiments (n = 10) for model validation purposes were performed. Performance attributes were calculated from the presence–absence microbiological test results by combining the results obtained from the culture media and confirmative tests. Dirichlet distribution, the multivariate expression of a Beta distribution, was used to analyze the performance data from a stochastic perspective. No L. monocytogenes was enumerated by direct-plating (<10 CFU/g), though the pathogen was detected in 22% of the samples. L. monocytogenes concentration was estimated at 14–17 CFU/kg. Validation showed good agreement between observed and predicted prevalence (error = −2.17%). The results showed that all media were best at ruling in L. monocytogenes presence than ruling it out. Sensitivity and specificity varied depending on the culture-dependent method. None of the culture media was perfect in detecting L. monocytogenes in minced pork meat alone. The use of at least two culture media in parallel enhanced the efficiency of L. monocytogenes detection. Bayesian modeling may reduce the time needed to draw conclusions regarding L. monocytogenes presence and the uncertainty of the results obtained. Furthermore, the problem of observing zero counts may be overcome by applying Bayesian analysis, making the determination of a test performance feasible.     

Prevalence and characterization of antimicrobial resistance of foodborne Listeria monocytogenes isolates in Hebei province of Northern China, 2005-2007

A total of 2177 food samples collected from nine cities in northern China during 2005 to 2007 were screened for the presence of Listeria monocytogenes. All L. monocytogenes isolates were subjected to serotyping, antimicrobial susceptibility, pulsed-field gel electrophoresis (PFGE), as well as PCR screening to identify genes responsible for tetracycline resistance [tet(L), tet(M), tet(K), tet(S) and tet(B)], transposon Tn916, and class 1 integron. Contamination with L. monocytogenes was detected in 4.13% (90/2177) of the total samples representing various food products. The pathogen was mainly isolated from frozen food made of wheat flour or rice products (26/252, 10.32%) and raw meat products (46/733, 6.28%). Besides, 3.31% (10/302) of cooked meat, 1.17% (4/343) of seafood, 0.98% (2/204) of non-fermented bean products and 0.62% (2/323) of vegetables samples were contaminated by this bacterium. The L. monocytogenes isolates belonged to five serotypes (1/2a, 1/2b, 1/2c, 4b, and 3a), with serotype 1/2a being dominant (48.88%). Antimicrobial resistance was most frequently observed for ciprofloxacin (17.8%), tetracycline (15.6%) and streptomycin (12.2%). Overall, resistance was observed against 14 out of 18 antimicrobials tested while multiple resistances occurred among 18.9% (17/90) isolates. Interestingly, two isolates were resistant to more than five antimicrobials. Among 14 tetracycline-resistant isolates, 13 carried tet(M) gene including nine possessing Tn916, and one harbored tet(S) gene. PFGE analysis revealed genetic heterogeneity among individual serotypes as well as scattered occurrence of some genotypes without any clear-cut correlation to source or food type. The widespread distribution of epidemiologically important serotypes (1/2a, 1/2b and 4b) of L. monocytogenes, and their resistance to commonly used antibiotics indicate a potential public health risk. Our data also indicate that L. monocytogenes could act as a reservoir of mobile tet genes along the food chain.     

Molecular subtyping and virulence gene analysis of Listeria monocytogenes isolates from food

A total of 67 Listeria monocytogenes isolates from 698 raw meat samples were characterized for molecular serogroup identification and antimicrobial susceptibility. Approximately one third (32.8%) of the isolates belonged to molecular serogroup 1/2a, 3a, followed by 1/2c, 3c (26.9%), 1/2b, 3b, 7 (22.4%), 4b, 4d, 4e (16.4%) and 4a, 4c (1.5%). Most of the L. monocytogenes isolates were susceptible to 14 antimicrobials tested but several were resistant to tetracycline, ciprofloxacin and nitrofurantoin. An additional 30 L. monocytogenes isolates from chicken and produce in our collection were also included to determine the presence of significant virulence markers. All 97 isolates carried inlC and inlJ except for a lineage III isolate 110-1. Most Listeriolysin S (LLS)-carrying isolates (11/12) belonged to lineage I, whereas the remaining one isolate belonged to lineage III. Five 4b, 4d, 4e isolates including two from turkey and three from produce belonged to Epidemic Clone I (ECI). Four molecular serogroup associated mutation types that lead to premature stop codons (PMSCs) in inlA were identified. PFGE and inlA sequence analysis results were concordant, and different virulence potential within 1/2a, 3a and 4b, 4d, 4e isolates were observed. The study revealed that a subset of isolates from meat and produce belonged to ECI, harbored inlC, inlJ and LLS, and produced full length InlA, suggesting that they be capable of causing human illness.     

Prevalence and biofilm-forming ability of Listeria monocytogenes in New Zealand mussel (Perna canaliculus) processing plants

Greenshell™ mussels are New Zealand’s largest seafood export species. Some export markets require compliance with ‘zero’ tolerance legislation for Listeria monocytogenes in 25 g of product. Even though individually quick frozen (IQF) mussel products are labeled ‘to be cooked’, and are not classified as ready-to-eat, some markets still require them to comply with the strict policy. Three mussel processing plants were assessed for the pattern of L. monocytogenes contamination on raw material, environment, food contact surfaces, and in the final product. Cultures (n = 101) obtained from an industrial Listeria monitoring program from August 2007 to June 2009 were characterized by serotyping and pulsed field gel electrophoresis. Using the crystal violet method, isolates were assessed for their ability to form biofilms. This work confirmed the presence of L. monocytogenes in raw and processed product, and the importance of cross-contamination from external and internal environments. Processing plants had L. monocytogenes pulsotypes that were detected more than once over 6 months. No correlation was found between biofilm-forming ability and persistent isolates. Two pulsotypes (including a persistent one), were previously isolated in human cases of listeriosis in New Zealand, but none of the pulsotypes matched those involved in international outbreaks.     

Antimicrobial resistance in Campylobacter coli isolated from pigs in two provinces of China

The aim of this study was to determine the prevalence, antimicrobial resistance and molecular epidemiology of Campylobacter coli isolated from swine in China. A total of 190 C. coli isolates obtained from two slaughter houses and ten conventional pig farms in Shandong (SD, n = 95) and Ningxia (NX, n = 95) provinces were tested for their susceptibility to 14 antimicrobials. A high prevalence (> 95%) of ciprofloxacin and tetracycline-resistant strains was observed in both SD and NX. The erythromycin and clindamycin resistance rates of C. coli from NX (ERY: 54.7% CLI: 43.2%) were higher than those from SD (ERY: 37.9%, CLI: 35.8%). A significant difference (P < 0.05) was observed in erythromycin resistance rate, but not (P > 0.05) in clindamycin resistance rate. while the resistance rates of ampicillin and kanamycin in NX (AMP: 34.7%, KAN: 43.2%) were significantly lower (P < 0.05) than those in SD (AMP: 51.6%, KAN: 71.6%). None of the tested isolates were resistant to phenicols. The majority of the isolates from both provinces (SD: 80% and NX: 73.7%) showed multi-drug resistance profiles. The point mutations of A2075G in the 23S rRNA and C257T in the gyrA gene were detected in 98% (87/89) of macrolide resistant isolates and all ciprofloxacin resistant isolates, respectively. In addition, all tetracycline-resistant isolates harbored the tet(O) gene. The high prevalence of antimicrobial resistance in C. coli strains derived from pigs in China was observed and was likely due to the extensive use of various antimicrobials. Prudent use of antimicrobial agents on farms should be further emphasized to control the dissemination of antimicrobial resistant C. coli.     

Review--Persistence of Listeria monocytogenes in food industry equipment and premises

To understand why Listeria monocytogenes may persist in food industry equipment and premises, notably at low temperature, scientific studies have so far focused on adhesion potential, biofilm forming ability, resistance to desiccation, acid and heat, tolerance to increased sublethal concentration of disinfectants or resistance to lethal concentrations. Evidence from studies in processing plants shows that the factors associated with the presence of L. monocytogenes are those that favor growth. Interestingly, most conditions promoting bacterial growth were shown, in laboratory assays, to decrease adhesion of L. monocytogenes cells. Good growth conditions can be found in so-called harborage sites, i.e. shelters due to unhygienic design of equipment and premises or unhygienic or damaged materials. These sites are hard to eliminate. A conceptual model of persistence/no persistence based on the relative weight of growth vs. outcome of cleaning and disinfection is suggested. It shows that a minimum initial bacterial load is necessary for bacteria to persist in a harborage site and that when a low initial bacterial charge is applied, early cleaning and disinfection is the only way to avoid persistence. We conclude by proposing that there are no strains of L. monocytogenes with unique properties that lead to persistence, but harborage sites in food industry premises and equipment where L. monocytogenes can persist.     

Fate of Listeria monocytogenes during freezing,thawing and home storage of frankfurters

Little information is available regarding the fate of Listeria monocytogenes during freezing, thawing and home storage of frankfurters even though recent surveys show that consumers regularly store unopened packages in home freezers. This study examined the effects of antimicrobials, refrigerated storage, freezing, thawing method, and post-thawing storage (7 °C) on L. monocytogenes on frankfurters. Inoculated (2.1 log CFU/cm²) frankfurters formulated without (control) or with antimicrobials (1.5% potassium lactate plus 0.1% sodium diacetate) were vacuum-packaged, stored at 4 °C for 6 or 30 d and then frozen (−15 °C) for 10, 30, or 50 d. Packages were thawed under refrigeration (7 °C, 24 h), on a countertop (23 ± 2 °C, 8 h), or in a microwave oven (2450 MHz, 1100 watts, 220 s followed by 120 s holding), and then stored aerobically (7 °C) for 14 d. Bacterial populations were enumerated on PALCAM agar and tryptic soy agar plus 0.6% yeast extract. Antimicrobials completely inhibited (p < 0.05) growth of L. monocytogenes at 4 °C for 30 d under vacuum-packaged conditions, and during post-thawing aerobic storage at 7 °C for 14 d. Different intervals between inoculation and freezing (6 or 30 d) resulted in different pathogen levels on control frankfurters (2.1 or 3.9 log CFU/cm², respectively), while freezing reduced counts by <1.0 log CFU/cm². Thawing treatments had little effect on L. monocytogenes populations (<0.5 log CFU/cm²), and post-thawing fate of L. monocytogenes was not influenced by freezing or by thawing method. Pathogen counts on control samples increased by 1.5 log CFU/cm² at d-7 of aerobic storage, and reached 5.6 log CFU/cm² at d-14. As indicated by these results, consumers should freeze frankfurters immediately after purchase, and discard frankfurters formulated without antimicrobials within 3 d of thawing and/or opening.     

Role of general stress-response alternative sigma factors σ(S) (RpoS) and σ(B) (SigB) in bacterial heat resistance as a function of treatment medium pH

This investigation aimed to determine the role of general stress-response alternative sigma factors σ^S (RpoS) and σ^B (SigB) in heat resistance and the occurrence of sublethal injuries in cell envelopes of stationary-phase Escherichia coli BJ4 and Listeria monocytogenes EGD-e cells, respectively, as a function of treatment medium pH. Given that microbial death followed first-order inactivation kinetics (R² > 0.95) the traditional D_T and z values were used to describe the heat inactivation kinetics.Influence of rpoS deletion was constant at every treatment temperature and pH, making a ΔrpoS deletion mutant strain approximately 5.5 times more heat sensitive than its parental strain for every studied condition. Furthermore, the influence of the pH of the treatment medium on the reduction of the heat resistance of E. coli was also constant and independent of the treatment temperature (average z value = 4.9 °C) in both parental and mutant strains.L. monocytogenes EGD-e z values obtained at pH 7.0 and 5.5 were not significantly different (p > 0.05) in either parental or the ?sigB deletion mutant strains (average z value = 4.8 °C). Nevertheless, at pH 4.0 the z value was higher (z = 8.4 °C), indicating that heat resistance of both L. monocytogenes strains was less dependent on temperature at pH 4.0. At both pH 5.5 and 7.0 the influence of sigB deletion was constant and independent of the treatment temperature, decreasing L. monocytogenes heat resistance approximately 2.5 times. In contrast, the absence of sigB did not decrease the heat resistance of L. monocytogenes at pH 4.0.The role of RpoS in protecting cell envelopes was more important in E. coli (4 times) than SigB in L. monocytogenes (1.5 times). Moreover, the role of σ^S in increasing heat resistance seems more relevant in enhancing the intrinsic resilience of the cytoplasmic membrane, and to a lesser extent, outer membrane resilience.Knowledge of environmental conditions related to the activation of alternative sigma factors σ^S and σ^B and their effects on heat resistance would help us to avoid and/or identify situations that increase bacterial stress resistance. Therefore, more efficient food preservation processes might be designed.     

Efficacy of bacteriocin-containing cell-free culture supernatants from lactic acid bacteria to control Listeria monocytogenes in food

Consumer demands have led to an increased interest in the use of natural antimicrobials for food protection. With the objective of developing novel products for enhancing the microbial safety of food, we have tested cell-free culture supernatants (CFS's) of eight antagonistic bacterial strains for their efficacy to inhibit Listeria monocytogenes in different food matrices. The antagonistic strains represented different members of the order Lactobacillales as well as one isolate of Staphylococcus sciuri and all showed strong inhibition of L. monocytogenes on agar plates. Cell-free supernatants were obtained after growing the bacteria in a yeast extract-glucose broth. In six of the CFS's, different class IIa bacteriocins, namely leucocin A, leucocin B, mundticin L, pediocin PA-1, sakacin A, and sakacin X, were identified as the major anti-listerial compounds. For the other two strains, the active substances could not be ascertained conclusively. The minimal effective concentration (MEC) of the individual CFS's to achieve a 2.3 log₁₀ reduction of L. monocytogenes was determined in culture broth, whole milk, and ground beef at 4 °C. While all bacteriocin-containing CFS's were effective in broth at concentrations from 52 to 205 AU/ml, significant higher concentrations were needed when applied in food. Best results were obtained using CFS's containing pediocin PA-1, that displayed only three- and ten-times higher MEC's in milk (307 AU/ml) and ground meat (1024 AU/g) compared to broth, respectively. A twenty-fold increase in the MEC (2048 AU/ml) was observed for a mundticin L-containing fermentate, and a CFS containing leucocin A and B was inactivated more than fifty-fold (> 1280 AU/ml) in both food matrices. Remarkably, the sakacin A and sakacin X containing CFS's displayed very selective inactivation rates, in which sakacin A was only effective in meat (512 AU/g), while sakacin X was only effective in milk (2048 AU/ml). In all cases, inhibition of L. monocytogenes was only transient and surviving or resistant bacteria started growing after prolonged storage. These results highlight the importance of careful testing the effectiveness of bacteriocins in the food systems for which they are intended to be applied against the selected target and non-target bacteria. Furthermore, the outgrowth of surviving or resistant bacterial populations points out that the tested bacteriocins are not suited to assure full inhibition of L. monocytogenes in a food product, if not applied in combination with additional preservative measures.     

Growth characteristics of Listeria monocytogenes as affected by a native microflora in cooked ham under refrigerated and temperature abuse conditions

This study examined the growth characteristics of Listeria monocytogenes as affected by a native microflora in cooked ham at refrigerated and abuse temperatures. A five-strain mixture of L. monocytogenes and a native microflora, consisting of Brochothrix spp., isolated from cooked meat were inoculated alone (monocultured) or co-inoculated (co-cultured) onto cooked ham slices. The growth characteristics, lag phase duration (LPD, h), growth rate (GR, log₁₀ cfu/h), and maximum population density (MPD, log₁₀ cfu/g), of L. monocytogenes and the native microflora in vacuum-packed ham slices stored at 4, 6, 8, 10, and 12 °C for up to 5 weeks were determined. At 4-12 °C, the LPDs of co-cultured L. monocytogenes were not significantly different from those of monocultured L. monocytogenes in ham, indicating the LPDs of L. monocytogenes at 4-12 °C were not influenced by the presence of the native microflora. At 4-8 °C, the GRs of co-cultured L. monocytogenes (0.0114-0.0130 log₁₀ cfu/h) were statistically but marginally lower than those of monocultured L. monocytogenes (0.0132-0.0145 log₁₀ cfu/h), indicating the GRs of L. monocytogenes at 4-8 °C were reduced by the presence of the native microflora. The GRs of L. monocytogenes were reduced by 8-7% with the presence of the native microflora at 4-8 °C, whereas there was less influence of the native microflora on the GRs of L. monocytogenes at 10 and 12 °C. The MPDs of L. monocytogenes at 4-8 °C were also reduced by the presence of the native microflora. Data from this study provide additional information regarding the growth suppression of L. monocytogenes by the native microflora for assessing the survival and growth of L. monocytogenes in ready-to-eat meat products.     

Efficacy of freezing, frozen storage and edible antimicrobial coatings used in combination for control of Listeria monocytogenes on roasted turkey stored at chiller temperatures

The presence and growth of Listeria monocytogenes on ready-to-eat (RTE) turkey is an important food safety issue. The antilisterial efficacy of four polysaccharide-based edible coatings (starch, chitosan, alginate and pectin) incorporating sodium lactate (SL) and sodium diacetate (SD) as well as commercial preparations Opti.Form PD4, NovaGARD™ CB1, Protect-M and Guardian™ NR100 were compared against L. monocytogenes on roasted turkey. Pectin coating treatments incorporating SL/SD, Opti.Form PD4 with or without Protect-M, and NovaGARD™ CB1 displayed higher antimicrobial efficacy against.L. monocytogenes than the other antimicrobials and coating materials. In the second phase of the study, it was investigated whether frozen storage could enhance the antilisterial effectiveness of pectin coating treatments on chilled roasted turkey. Inoculated roasted turkey samples coated with pectin-based treatments were frozen for up to 4 weeks and subsequently stored at 4 °C for 8 weeks. Frozen storage significantly enhanced the antilisterial activity of various coating treatments; with selected treatments reducing the L. monocytogenes populations by as much as 1.1 log CFU/cm² during the subsequent 8-week chilled storage. This study demonstrates that pectin-based antimicrobial edible coatings hold promise in enhancing the safety of RTE poultry products and frozen storage has the potential to enhance their effectiveness.     

Reduction of Listeria monocytogenes on frankfurters treated with lactic acid solutions of various temperatures

United States regulations require ready-to-eat meat and poultry processors to control Listeria monocytogenes using interventions which may include antimicrobials that reduce post-processing contamination by at least 1 log-cycle; if the treatment achieves ≥2 log reductions, the plant is subject to less frequent microbial testing. Lactic acid (LA) may be useful as a post-lethality intervention and its antimicrobial properties may increase with temperature of application. The aim of this study was to evaluate the effect of LA solution concentration and temperature on L. monocytogenes counts of inoculated frankfurters and to identify parameters (concentration, temperature, and time) that achieve 1 and 2 log-unit immediate reductions. Frankfurters were surface-inoculated with a 10-strain mixture of L. monocytogenes (4.4 ± 0.1 log CFU/cm²) and then immersed in distilled water or LA solutions (0–3%) of 4, 25, 40, or 55 °C for 0–120 s. A regression equation for L. monocytogenes reduction included significant (P < 0.05) effects by the terms of concentration, time, temperature, and the interaction of concentration and temperature; other tested parameters (other interactions, quadratic and cubic terms), within the experimental range examined, did not affect (P ≥ 0.05) the extent of reduction. Results indicated that the effectiveness of LA against L. monocytogenes, in addition to concentration, increased with solution temperature (in the range of 0.6–2.8 log CFU/cm²). The developed equation may allow processors to vary conditions of treatment with LA to achieve a 1 or 2 log-unit reduction of the pathogen and comply with United States regulations.     

Design of challenge testing experiments to assess the variability of Listeria monocytogenes growth in foods

The assessment of the evolution of micro-organisms naturally contaminating food must take into account the variability of biological factors, food characteristics and storage conditions. A research project involving eight French laboratories was conducted to quantify the variability of growth parameters of Listeria monocytogenes obtained by challenge testing in five food products. The residual variability corresponded to a coefficient of variation (CV) of approximately 20% for the growth rate (μ_max) and 130% for the parameter K = μ_max × lag. The between-batch and between-manufacturer variability of μ_max was very dependent on the food tested and mean CV of approximately 20 and 35% were observed for these two sources of variability, respectively. The initial physiological state variability led to a CV of 100% for the parameter K. It appeared that repeating a limited number of three challenge tests with three different batches (or manufacturers) and with different initial physiological states seems often necessary and adequate to accurately assess the variability of the behavior of L. monocytogenes in a specific food produced by a given manufacturer (or for a more general food designation).     

Survey of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products by commercial real-time PCR kits

Real-time PCR (RTiPCR) assays including enrichment stage were evaluated for the rapid detection of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products using molecular beacon probes available as commercial kits (WARNEX Genevision, Canada & AES Chemunex detection system, France). The accuracy of the assays was evaluated analyzing 1032 naturally contaminated food samples in combination to the conventional cultural methods. Presence/absence testing of the above pathogens was performed in 25 g samples of each product. In case of L. monocytogenes of 39 positive RTiPCR samples, 37 were confirmed by the cultural method (based on McNemar's test the difference between the two methods is insignificant). The highest incidence of L. monocytogenes in food products was found in desserts and the second highest in frozen pastries. None of the samples were cultural positive but negative in the RTiPCR test. One among the 343 investigated samples was positive for Salmonella spp. by RTiPCR and the cultural method. Out of 333 samples analyzed for E. coli O157:H7 no positive sample was detected. RTiPCR-based methods proved to be powerful tools for fast, sensitive and accurate pathogen detection in raw food ingredients and ready-to-eat products.     

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

We developed a novel filtration-based method that can eliminate dead or severally damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p < 0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R² > 0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.     

Growth kinetics of Listeria monocytogenes and spoilage microorganisms in fresh-cut cantaloupe

The main objective of this study was to investigate the growth kinetics of Listeria monocytogenes and background microorganisms in fresh-cut cantaloupe. Fresh-cut cantaloupe samples, inoculated with three main serotypes (1/2a, 1/2b, and 4b) of L. monocytogenes, were incubated at different temperatures, ranging from 4 to 43 °C, to develop kinetic growth models. During storage studies, the population of both background microorganisms and L. monocytogenes began to increase almost immediately, with little or no lag phase for most growth curves. All growth curves, except for two growth curves of L. monocytogenes 1/2a at 4 °C, developed to full curves (containing exponential and stationary phases), and can be described by a 3-parameter logistic model. There was no significant difference (P = 0.28) in the growth behaviors and the specific growth rates of three different serotypes of L. monocytogenes inoculated to fresh-cut cantaloupe. The effect of temperature on the growth of L. monocytogenes and spoilage microorganisms was evaluated using three secondary models. For L. monocytogenes, the minimum and maximum growth temperatures were estimated by both the Ratkowsky square-root and Cardinal parameter models, and the optimum temperature and the optimum specific growth rate by the Cardinal parameter model. An Arrhenius-type model provided more accurate estimation of the specific growth rate of L. monocytogenes at temperatures <4 °C. The kinetic models developed in this study can be used by regulatory agencies and food processors for conducting risk assessment of L. monocytogenes in fresh-cut cantaloupe, and for estimating the shelf-life of fresh-cut products.     

Characterization and in vitro bioavailability of β-carotene: Effects of microencapsulation method and food matrix

The objectives of this study were to determine the effect of microencapsulation method on physical properties and in vitro release and bioavailability of three types of β-carotene: a spray-dried powder of β-carotene and maltodextrin, commercially available water-dispersible β-carotene powder, and chitosan-coated β-carotene alginate. In vitro digestion trials were conducted with and without food matrices (yogurt, pudding) to elucidate the effect of food matrix on in vitro release and bioavailability. Microencapsulation method significantly affected (p < 0.05) water activity, moisture content, and particle size. The maltodextrin powder had the lowest moisture content (3.5%) and the smallest volume mean diameter (10.5 μm), whereas the chitosan-alginate beads had the lowest water activity (0.195). The maltodextrin powder had the highest surface β-carotene content (39.5%), while the commercial water-dispersible powder had the highest β-carotene content (10%). The microencapsulation method significantly influenced (p < 0.05) release and incorporation into micelles, regardless of food matrix. Water-dispersible β-carotene achieved the highest release (93.3%) and the highest incorporation into micelles (36.4%) in the absence of a food matrix, and the highest release (34.8%) and the highest micelle content (17.0%) with pudding. Food matrix significantly decreased release and micelle incorporation (p < 0.05), with yogurt decreasing release and micelle incorporation more than pudding.     

Multiple-locus variable number of tandem repeat analysis (MLVA) of Listeria monocytogenes directly in food samples

Listeria monocytogenes is the etiologic agent of listeriosis responsible for severe and fatal infections in humans. Listeria contamination occurs quite often in a wide range of foods due to its ubiquitous nature. Isolates need to be characterized to a strain level for accurate diagnosis of Listeria infection, epidemiological studies, investigation of outbreaks and effective prevention and control of food-borne listeriosis. The purpose of this research was to evaluate the multiple-locus variable number of tandem repeat analysis (MLVA) for sub-typing L. monocytogenes isolates in pure cultures and in food matrices. Two multiplex PCR assays were formulated to amplify six specific loci using fluorescently-labeled primers; and the amplicons were analyzed by capillary electrophoresis. The MLVA method resulted in 34 unique DNA fingerprint patterns from 46 L. monocytogenes isolates of 10 serotypes which had 29 or 30 PFGE patterns with a single restriction enzyme and 34 AFLP patterns. The MLVA patterns of the 46 isolates remained unchanged in the presence of pre-enriched food matrices including sausage, ham, chicken, milk and lettuce. The MLVA method successfully typed L. monocytogenes strains spiked in cheese, roast beef, egg salad and vegetable samples after 48 h enrichment at the initial inoculation levels of 1-5 CFU per 25 g of food or higher. The limits of detection (typing) of the MLVA method were 10³-10⁴ CFU/mL of pre-enriched food broth when evaluated using post-spiked sausage, ham, chicken, milk and lettuce samples. The MLVA method was simple, highly discriminatory, and easy to perform with portable (numerical) results. To our knowledge, this is the first report that describes the application of the MLVA method directly to food samples and demonstrates the possibility to obtain rapid and accurate subtyping results before an isolate is obtained.