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.     

Development and validation of a rapid real-time PCR based method for the specific detection of Salmonella on fresh meat

In this study, a combined enrichment/real-time PCR method for the rapid detection of Salmonella on fresh meat carcasses, was designed, developed and validated in-house following requirements outlined in ISO 16140:2003. The method included an 18 h non-selective enrichment in buffered peptone water (BPW) and a 6 h selective enrichment in Rappaport Vasilliadis Soya (RVS) broth, based on the traditional culture method, ISO 6579:2002. The real-time PCR assay included an internal amplification control (IAC), was 100% specific and was sensitive to one cell equivalent. The alternative method was validated against the traditional culture method and relative accuracy of 94.9%, sensitivity of 94.7% and specificity of 100% were determined using 150 fresh meat carcass swabs. This alternative method had a detection limit of 1–10 CFU/100 cm² for fresh meat carcass swabs and was performed in 26 h. Following further inter-laboratory studies, this alternative method could be suitable for implementation in testing laboratories for the analysis of carcass swabs.     

Kinetics of resuscitation and growth of L. monocytogenes as a tool to select appropriate enrichment conditions as a prior step to rapid detection methods

Rapid methods still rely on a prior (shortened) enrichment step before application. Quantitative information is a prerequisite for understanding the resuscitation kinetics of the growth during the enrichment step. In this study various basal and newly introduced selective enrichment broths were evaluated. First, growth parameters (λ, μ_max) of both healthy and sub-lethally injured cells were determined. Next, a selection of enrichment broths was compared for their capacity to support detection within 24 h of low numbers of Listeria monocytogenes in artificially and naturally contaminated food samples. Detection was performed either by phage protein-based capture (Listeria Capture kit, Profos, Regensburg, Germany) combined with plating on chromogenic medium or by fluorescence in situ hybridization (FISH) using the VIT-Listeria kit (Vermicon, Munich, Germany). Kinetics of resuscitation and growth of L. monocytogenes in various enrichment broths showed that for detection of low numbers of sub-lethally injured L. monocytogenes cells at least an overnight enrichment was needed. A selective enrichment broth was needed to enable proliferation of L. monocytogenes within the indigenous bacterial flora present in foods. However, combination of an appropriate enrichment condition with advanced detection techniques may enable a 24 h detection of L. monocytogenes.     

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.     

A predictive model for heat inactivation of Listeria monocytogenes biofilm on buna-N rubber

The purpose of this study was to develop a predictive model for the heat inactivation of Listeria monocytogenes in monoculture (strains Scott A and 3990) and with competing bacteria (Pseudomonas sp. and Pantoea agglomerans) formed on buna-N rubber with and without the presence of food-derived soil. Biofilms were produced on rubber disks in dilute Tryptic Soy broth (dTSB) with incubation for 48 h at 25 °C. Duplicate biofilm samples were heat treated for 1, 3, 5, and 15 min at 70, 72, 75, 77 and 80 °C and tested for survivors using enrichment media. The experiment was repeated six times. A predictive model was developed and plots were generated showing the percent probability of L. monocytogenes inactivation in biofilms after heat treatment. For example, to achieve a 95% probability level of complete inactivation required heat treatment of 76 °C for 6 min. The predicted model was validated using a five-strain cocktail of L. monocytogenes. The validated prediction model indicates that with proper maintenance of the time/temperature controls L. monocytogenes in biofilms on rubber surfaces will be inactivated. This model can be used as a tool in the selection of hot water sanitation processes for rubber surfaces.     

Use of Carnobacterium maltaromaticum cultures and hydroalcoholic extract of Lippia sidoides Cham. against Listeria monocytogenes in fish model systems

Minimally processed refrigerated ready-to-eat fishes may offer health risk of severe infection to susceptible individuals due to contamination by the psychrotolerant bacterium L. monocytogenes. In this work, inhibition of L. monocytogenes by a plant extract and lactic acid bacteria (LAB) was studied in model fish systems kept at 5 °C for 35 days. For that, fillets of tropical fish “surubim” (Pseudoplatystoma sp.) and hydroalcoholic extract of the plant Lippia sidoides Cham. (“alecrim pimenta”) were used. Fish peptone broth (FPB), “surubim” broth and “surubim” homogenate were inoculated with combinations of L. monocytogenes and bacteriocin-producing Carnobacterium maltaromaticum (C2 and A9b⁺) and non bacteriocin-producing C. maltaromaticum (A9b^-), in the presence or absence of extract of “alecrim pimenta” (EAP). In all model systems, monocultures of L. monocytogenes and carnobacteria reached final populations ≥ 10⁸ CFU/ml after 35 days, except for L. monocytogenes in “surubim” homogenate (10⁴ CFU/ml). In FPB, EAP alone and combined with cultures of LAB inhibited L. monocytogenes but carnobacteria without EAP were only weakly antilisterial. In “surubim” broth, EAP alone did not prevent L. monocytogenes growth but cultures of carnobacteria combined or not with EAP inhibited L. monocytogenes, with more pronounced effect being observed for C. maltaromaticum C2, which produced bacteriocin. In “surubim” homogenate, EAP alone and combined with cultures of C. maltaromaticum A9b⁻ and A9b⁺ were strongly inhibitory to L. monocytogenes, while C. maltaromaticum C2 with EAP caused transient inhibition of L. monocytogenes. No significant inhibition of L. monocytogenes was observed for carnobacteria in “surubim” homogenate without EAP. In conclusion, it was observed that the use of EAP and cultures of carnobacteria have potential to inhibit L. monocytogenes in fish systems and the applications should be carefully studied, considering the influence of food matrix.     

A multiplex magnetic capture hybridisation and multiplex Real-Time PCR protocol for pathogen detection in seafood

Seafood could become a source of bacterial pathogens by exposure to contaminated water or through processing practices, thus representing a public health hazard. Conventional culture-based analytical methods take several days to be completed, while the molecular rapid identification of bacterial pathogens is crucial for effective disease control. The developed application consist of a multiplex magnetic capture hybridisation (mMCH) assay for the simultaneous isolation of Salmonella spp. and Listeria monocytogenes DNA from seafood, using paramagnetic amino-modified nanoparticles with capture oligonucleotides, and a triplex Real-Time PCR with an Internal Amplification Control (IAC), in accordance with ISO 22174. The detection probability was 100% with 10 genome equivalents of each target species co-amplified in the same reaction. The complete molecular procedure was tested on raw and smoked salmon fillets artificially contaminated with known amounts of one or both target bacteria (1–10³ cfu/g), directly or after culture enrichment, and compared for equivalence with the standard methods. Results revealed a complete agreement between the two approaches, with a sensitivity of 1 cfu/g, in enriched samples, and higher sensitivity (10²–10³ cfu/g) of the molecular method in samples examined before culture enrichment. The proposed procedure was also able to identify a natural contamination by L. monocytogenes in smoked salmon with a considerable shortening of time.     

Development of multiplex real-time PCR with Internal amplification control for simultaneous detection of Salmonella and Cronobacter in powdered infant formula

Contamination of powdered infant formula (PIF) by the bacteria Cronobacter spp. and Salmonella enterica was deemed a matter of great concern by the World Health Organization and the Food and Agriculture Organization of the United Nations in 2004. Therefore, we developed a rapid and sensitive multiplex real-time PCR assay for the simultaneous detection of Cronobacter and Salmonella in PIF. In addition, an internal amplification control (IAC) was also included for exclusion of false negative results in this study. The quantitative detection range for pure cultures in this optimized multiplex real-time PCR assay was 10³ to 10⁸ CFU/ml for both Salmonella and Cronobacter. When our established multiplex real-time PCR system was applied to artificially contaminated PIF, the detection limit was 10³ CFU/ml for Salmonella and Cronobacter without enrichment. The commercial PIF was then inoculated with Salmonella and Cronobacter at 10, 1 and 0.1 CFU per gram of formula and the single enrichment broth samples were analyzed by multiplex real-time PCR after enrichment for 9, 12, and 24 h. At 12 h post-enrichment, we could detect Salmonella and Cronobacter at initial inoculation levels of approximately 0.1 CFU/g in PIF. Additionally, stable fluorescent IAC signals could be assessed between 29 and 34 cycles of PCR amplification. Results from this study showed that the multiplex real-time PCR assay is an effective method for the rapid and simultaneous detection and quantification of Cronobacter and Salmonella in PIF.     

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.     

The inhibitory effect of natural microflora of food on growth of Listeria monocytogenes in enrichment broths

The aims of this study were to (i) compare the inhibitory effects of the natural microflora of different foods on the growth of Listeria monocytogenes during enrichment in selective and non-selective broths; (ii) to isolate and identify components of the microflora of the most inhibitory food; and (iii) to determine which of these components was most inhibitory to growth of L. monocytogenes in co-culture studies. Growth of an antibiotic-resistant marker strain of L. monocytogenes was examined during enrichment of a range of different foods in Tryptone Soya Broth (TSB), Half Fraser Broth (HFB) and Oxoid Novel Enrichment (ONE) Broth. Inhibition of L. monocytogenes was greatest in the presence of minced beef, salami and soft cheese and least with prepared fresh salad and chicken pâté. For any particular food the numbers of L. monocytogenes present after 24 h enrichment in different broths increased in the order: TSB, HFB and ONE Broth. Numbers of L. monocytogenes recovered after enrichment in TSB were inversely related to the initial aerobic plate count (APC) in the food but with only a moderate coefficient of determination (R²) of 0.51 implying that microbial numbers and the composition of the microflora both influenced the degree of inhibition of L. monocytogenes. In HFB and ONE Broth the relationship between APC and final L. monocytogenes counts was weaker. The microflora of TSB after 24 h enrichment of minced beef consisted of lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas spp., Enterobacteriaceae, and enterococci. In co-culture studies of L. monocytogenes with different components of the microflora in TSB, the lactic acid bacteria were the most inhibitory followed by the Enterobacteriaceae. The least inhibitory organisms were Pseudomonas sp., enterococci and B. thermosphacta. In HFB and ONE Broth the growth of Gram-negative organisms was inhibited but lactic acid bacteria still reached high numbers after 24 h. A more detailed study of the growth of low numbers of L. monocytogenes during enrichment of minced beef in TSB revealed that growth of L. monocytogenes ceased at a cell concentration of about 10² cfu/ml when lactic acid bacteria entered stationary phase. However in ONE Broth growth of lactic acid bacteria was slower than in TSB with a longer lag time allowing L. monocytogenes to achieve much higher numbers before lactic acid bacteria reached stationary phase. This work has identified the relative inhibitory effects of different components of a natural food microflora and shown that the ability of low numbers of L. monocytogenes to achieve high cell concentrations is highly dependent on the extent to which enrichment media are able to inhibit or delay growth of the more effective competitors.     

Multiplex Real-time PCR for the Simultaneous Detection of Salmonella spp. and Listeria monocytogenes in Food Samples

In this study, we have developed a rapid method for the simultaneous detection of Listeria monocytogenes and Salmonella spp. in foods, combining culture enrichment and a multiplex real-time polymerase chain reaction (PCR). The assay used two pre-existing primer-probe sets, labelled with different reporter dyes to enable the direct distinction of the original contaminating agent. Amplification efficiency and inclusivity/exclusivity of the combined assay was successfully assessed. The overall process included the culture enrichment based on the ISO standard, consisting of 24 h incubation in appropriate media (Half Fraser Broth for Listeria and buffered peptone water (BPW) for Salmonella), followed by a single DNA extraction of mixed enrichment aliquots, and real-time PCR detection of the hly and bipA genes of L. monocytogenes and Salmonella spp., respectively. An internal amplification control, co-amplified during the PCR run, was included in the assay to verify the results. The tool was evaluated with a variety of artificially inoculated samples of fresh products and ready to eat and cooked dishes, allowing the identification of the target pathogens down to 5 CFU/25 g of food sample. Moreover, the analysis saved a considerable amount of time compared to the ISO standard, being performed in less than 2 working days. Specificity, sensitivity and accuracy were satisfactorily tested by comparison to the standard methods ISO 11290-2:1998 and ISO 6579:2002, suggesting that the tool has a great potential as a reliable alternative for food safety assurance providing rapid detection of both pathogens in food samples.     

The effect of pH and nitrite concentration on the antimicrobial impact of celery juice concentrate compared with conventional sodium nitrite on Listeria monocytogenes

The objectives of this study were to evaluate the impact of pH and nitrite from celery juice concentrate (CJ) on the growth of Listeria monocytogenes in broth and on ham slices, and to evaluate the impact of pH and nitrite from CJ on quality attributes of the ham. The pH of both broth and ham were increased by the addition of CJ. The CJ was less effective than conventional nitrite at 100 mg/kg nitrite in broth, but in ham, the CJ treatments at both 100 and 200 mg/kg resulted in growth of L. monocytogenes (p > 0.05) similar to that of the conventional nitrite at the same concentrations. Reducing the pH of CJ before addition to the ham had greater impact on L. monocytogenes growth at 200 mg/kg nitrite than at 100 mg/kg. Celery juice concentrate may increase meat product pH which could have implications for the antimicrobial impact of nitrite in some products.     

Determination of the effect of plant essential oils obtained by supercritical fluid extraction on the growth and viability of Listeria monocytogenes in broth and food systems using flow cytometry

The antimicrobial properties of oregano, rosemary and laurel extracts obtained by supercritical fluid extraction were investigated by examining their influence on the growth and viability of Listeria monocytogenes in laboratory medium and broccoli juice at 30 and 8 °C. Important decreases in the L. monocytogenes population were shown in presence of all the extracts obtained from rosemary and one oregano extract. The counts were reduced below the level of detection after 4 h of exposure at 30 °C in laboratory medium. A bactericidal effect was observed also when L. monocytogenes was exposed to rosemary at 30 °C and 8 °C in broccoli juice. Significant reductions in growth rate and an increase in lag phase of L. monocytogenes were observed in presence of some of the laurel and oregano extracts at both temperatures.Flow cytometry was used as a rapid method to determine the antibacterial effect of supercritical extracts and the physiological state of L. monocytogenes. Bacterial viability performed by dual staining of L. monocytogenes with SYTO 9 and propidium iodide revealed three different cell populations, specifically, living, dead and compromised cells. Live cell percentage decreased with the time of exposure, whereas the percentage of compromised cells remained constant and the dead cells increased in the same period.     

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.     

Listeria monocytogenes survival of UV-C radiation is enhanced by presence of sodium chloride, organic food material and by bacterial biofilm formation

The bactericidal effect on food processing surfaces of ceiling-mounted UV-C light (wavelength 254 nm) was determined in a fish smoke house after the routine cleaning and disinfection procedure. The total aerobic counts were reduced during UV-C light exposure (48 h) and the number of Listeria monocytogenes positive samples went from 30 (of 68) before exposure to 8 (of 68). We therefore in a laboratory model determined the L. monocytogenes reduction kinetics by UV-C light with the purpose of evaluating the influence of food production environmental variables, such as presence of NaCl, organic material and the time L. monocytogenes was allowed to adhere to steel before exposure. L. monocytogenes grown and attached in tryptone soy broth (TSB) with glucose were rapidly killed (after 2 min) by UV-C light. However, bacteria grown and adhered in TSB with glucose and 5% NaCl were more resistant and numbers declined with 4-5 log units during exposure of 8-10 min. Bacteria grown in juice prepared from cold-smoked salmon were protected and numbers were reduced with 2-3 log when UV-C light was used immediately after attachment whereas numbers did not change at all if bacteria had been allowed to form a biofilm for 7 days before exposure. It is not known if this enhanced survival is due to physiological changes in the attached bacterial cells, a physical protection of the cells in the food matrix or a combination. In conclusion, we demonstrate that UV-C light is a useful extra bacteriocidal step and that it, as all disinfecting procedures, is hampered by the presence of organic material.     

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.     

Bactericidal activity of lauric arginate in milk and Queso Fresco cheese against Listeria monocytogenes cold growth

Lauric arginate (LAE) at concentrations of 200 ppm and 800 ppm was evaluated for its effectiveness in reducing cold growth of Listeria monocytogenes in whole milk, skim milk, and Queso Fresco cheese (QFC) at 4°C for 15 to 28 d. Use of 200 ppm of LAE reduced 4 log cfu/mL of L. monocytogenes to a nondetectable level within 30 min at 4°C in tryptic soy broth. In contrast, when 4 log cfu/mL of L. monocytogenes was inoculated in whole milk or skim milk, the reduction of L. monocytogenes was approximately 1 log cfu/mL after 24 h with 200 ppm of LAE. When 800 ppm of LAE was added to whole or skim milk, the initial 4 log cfu/mL of L. monocytogenes was nondetectable following 24 h, and no growth of L. monocytogenes was observed for 15 d at 4°C. With surface treatment of 200 or 800 ppm of LAE on vacuum-packaged QFC, the reductions of L. monocytogenes within 24 h at 4°C were 1.2 and 3.0 log cfu/g, respectively. In addition, the overall growth of L. monocytogenes in QFC was decreased by 0.3 to 2.6 and by 2.3 to 5.0 log cfu/g with 200 and 800 ppm of LAE, respectively, compared with untreated controls over 28 d at 4°C. Sensory tests revealed that consumers could not determine a difference between QFC samples that were treated with 0 and 200 ppm of LAE, the FDA-approved level of LAE use in foods. In addition, no differences existed between treatments with respect to flavor, texture, and overall acceptability of the QFC. Lauric arginate shows promise for potential use in QFC because it exerts initial bactericidal activity against L. monocytogenes at 4°C without affecting sensory quality.     

The effect of sodium reduction with and without potassium chloride on the survival of Listeria monocytogenes in Cheddar cheese

Sodium chloride (NaCl) in cheese contributes to flavor and texture directly and by its effect on microbial and enzymatic activity. The salt-to-moisture ratio (S/M) is used to gauge if conditions for producing good-quality cheese have been met. Reductions in salt that deviate from the ideal S/M range could result in changing culture acidification profiles during cheese making. Lactococcus lactis ssp. lactis or Lc. lactis ssp. cremoris are both used as cultures in Cheddar cheese manufacture, but Lc. lactis ssp. lactis has a higher salt and pH tolerance than Lc. lactis ssp. cremoris. Both salt and pH are used to control growth and survival of Listeria monocytogenes and salts such as KCl are commonly used to replace the effects of NaCl in food when NaCl is reduced. The objectives of this project were to determine the effects of sodium reduction, KCl use, and the subspecies of Lc. lactis used on L. monocytogenes survival in stirred-curd Cheddar cheese. Cheese was manufactured with either Lc. lactis ssp. lactis or Lc. lactis ssp. cremoris. At the salting step, curd was divided and salted with a concentration targeted to produce a final cheese with 600 mg of sodium/100 g (control), 25% reduced sodium (450 mg of sodium/100 g; both with and without KCl), and low sodium (53% sodium reduction or 280 mg of sodium/100 g; both with and without KCl). Potassium chloride was added on a molar equivalent to the NaCl it replaced to maintain an equivalent S/M. Cheese was inoculated with a 5-strain cocktail of L. monocytogenes at different times during aging to simulate postprocessing contamination, and counts were monitored over 27 or 50 d, depending on incubation temperature (12 or 5°C, respectively). In cheese inoculated with 4 log₁₀ cfu of L. monocytogenes/g 2 wk after manufacture, viable counts declined by more than 3 log₁₀ cfu/g in all treatments over 60 d. When inoculated with 5 log₁₀ cfu/g at 3 mo of cheese age, L. monocytogenes counts in Cheddar cheese were also reduced during storage, but by less than 1.5 log₁₀ cfu/g after 50 d. However, cheese with a 50% reduction in sodium without KCl had higher counts than full-sodium cheese at the end of 50 d of incubation at 4°C when inoculated at 3 mo. When inoculated at 8 mo postmanufacture, this trend was only observed in 50% reduced sodium with KCl, for cheese manufactured with both cultures. This enhanced survival for 50% reduced-sodium cheese was not seen when a higher incubation temperature (12°C) was used when cheese was inoculated at 3 mo of age and monitored for 27 d (no difference in treatments was observed at this incubation temperature). In the event of postprocessing contamination during later stages of ripening, L. monocytogenes was capable of survival in Cheddar cheese regardless of which culture was used, whether or not sodium had been reduced by as much as 50% from standard concentrations, or if KCl had been added to maintain the effective S/M of full-sodium Cheddar cheese.     

Assessing in-house monitoring efficiency by tracing contamination rates in cheese lots recalled during an outbreak of listeriosis in Austria

A cluster of 34 cases of listeriosis was traced to consumption of quargel cheese, a sour milk specialty, in Austria, Germany and Czech Republic between 2009 and 2010. After recall from the retail market all soft cheese batches (n = 18) were sent for investigation and ISO 11290 based microbiological analysis revealed all red smear-ripened batches (16/18) to be positive for Listeria monocytogenes whereas mold ripened cheeses were negative. The 16 positive batches were grouped into three categories: those having exceeded shelf-life (G1), those around shelf-life (± 4 days, G2) and those within shelf-life (G3). Tracing the contamination levels as measured after recall (CL_R) to the theoretical contamination level after processing (CL₀) was considered to provide an estimate as to whether the in-house monitoring system would have been capable of unraveling the contamination scenario. Growth simulations starting from various hypothetical initial contamination levels of cheese at the plant and considering the potential variability in growth of L. monocytogenes due to model parameters and storage conditions suggested that a very low initial contamination level (e.g., < 1 CFU/g or 5 CFU/100 g) could justify the levels of L. monocytogenes enumerated in recalled samples of G1 and G2 lots. This in turn, may have resulted in low detection probability using ISO 11290:1996. In lots of G3 group, however, high initial contamination levels or temperature abuse at retail are inferred, based on simulated outputs.     

Real-time polymerase chain reaction for the quantitative detection of tetA and tetB bacterial tetracycline resistance genes in food

A new, rapid, sensitive and specific method was developed to directly detect and quantify tetA and tetB in food. Both tet genes are two of the most frequently present tetracycline resistance genes in Gram-negative bacteria. A set of primers and Taqman probes was designed for each gene. The standard curves were performed using Escherichia coli BM13 (C600 RifR)/RP4 and E. coli NCTC 50365, which carry tetA and tetB, respectively. Meat and fish samples inoculated with these reference strains were used as a matrix to construct the standard curves for the analysis of 20 samples of chicken meat and 10 samples of hake (Merlucius merlucius). The limits of detection in pure culture were 5 cfu/mL (0.7 log cfu/mL) in the case of tetA, 50 cfu/mL (1.7 log cfu/mL) for tetB and 5 × 10² cfu/g (2.7 log cfu/g) for both genes in food samples. The results obtained by real-time quantitative polymerase chain reaction (qPCR) were compared to counts of tetracycline-resistant bacteria obtained by plating extracts of poultry and hake samples in culture media supplemented with 16 mg/L of tetracycline. Counts of tetracycline-resistant bacteria obtained by qPCR showed a positive correlation, especially interesting when compared with microbiological counts of tetracycline-resistant Enterobacteriaceae in poultry meat (r = 0.5509) and with tetracycline-resistant mesophilic aerobic bacteria in hake samples (r = 0.7146). The obtained results demonstrate that this method could be a useful tool for the direct quantification of the amount of bacterial strains that carry tetA and/or tetB genes in food samples.