Multi-criteria framework as an innovative tradeoff approach to determine the shelf-life of high pressure-treated poultry.

A multi-criteria framework combining safety, hygiene and sensorial quality was developed to investigate the possibility of extending the shelf-life and/or removing lactate by applying High Hydrostatic Pressure (HHP) in a ready-to-cook (RTC) poultry product. For this purpose, Salmonella and Listeria monocytogenes were considered as safety indicators and Escherichia coli as hygienic indicator. Predictive modeling was used to determine the influence of HHP and lactate concentration on microbial growth and survival of these indicators. To that end, probabilistic assessment exposure models developed in a previous study (Lerasle, M., Guillou, S., Simonin, H., Anthoine, V., Chéret, R., Federighi, M., Membré, J.M. 2014. Assessment of Salmonella and L. monocytogenes level in ready-to-cook poultry meat: Effect of various high pressure treatments and potassium lactate concentrations. International Journal of Food Microbiology 186, 74-83) were used for L. monocytogenes and Salmonella. Besides, for E. coli, an exposure assessment model was built by modeling data from challenge-test experiments. Finally, sensory tests and color measurements were performed to evaluate the effect of HHP on the organoleptic quality of an RTC product. Quantitative rules of decision based on safety, hygienic and organoleptic criteria were set. Hygienic and safety criteria were associated with probability to exceed maximum contamination levels of L. monocytogenes, Salmonella and E. coli at the end of the shelf-life whereas organoleptic criteria corresponded to absence of statistical difference between pressurized and unpressurized products. A tradeoff between safety and hygienic risk, color and taste, was then applied to define process and formulation enabling shelf-life extension. In the resulting operating window, one condition was experimentally assayed on naturally contaminated RTC products to validate the multi-criteria approach. As a conclusion, the framework was validated; it was possible to extend the shelf-life of an RTC poultry product containing 1.8% (w/w) lactate by one week, despite slight color alteration. This approach could be profitably implemented by food processors as a decision support tool for shelf-life determination.

Assessment of Salmonella and Listeria monocytogenes level in ready-to-cook poultry meat: effect of various high pressure treatments and potassium lactate concentrations.

The objective of this study was to develop a probabilistic model in order to determine the contamination level of Salmonella and Listeria monocytogenes in ready-to-cook poultry meat, after a high pressure (HP) treatment. The model included four steps: i) Reception of raw meat materials, mincing and mixing meat, ii) Partitioning and packaging into 200-g modified atmosphere packs, iii) High pressure treatment of the meat, and iv) Storage in chilled conditions until the end of the shelf-life. The model excluded the cooking step and consumption at consumer's home as cooking practices and heating times are highly variable. The initial contamination level of Salmonella and L. monocytogenes was determined using data collected in meat primary processing plants. The effect of HP treatment and potassium lactate on microbial reduction was assessed in minced meat, using a full factorial design with three high pressure treatments (200, 350 and 500 MPa), three holding times (2, 8 and 14 min) and two potassium lactate concentrations (0 or 1.8% w/w). The inactivation curves fitted with a Weibull model highlighted that the inactivation rate was significantly dependent on the HP treatment. From the literature, it was established that Salmonella was not able to grow in the presence of lactate, under modified atmosphere and chilled conditions whereas the growth of L. monocytogenes was determined using an existing model validated in poultry (available in Seafood Spoilage and Safety Predictor software, V. 3.1). Once implemented in the Excel add-in @Risk, the model was run using Monte Carlo simulation. The probability distribution of contamination levels was determined for various scenarios. For an average scenario such as an HP treatment of 350 MPa for 8 min, of 200 g minced meat containing 1.8% lactate (pH 6.1; aw 0.96), conditioned under 50% CO2, the prevalence rate of Salmonella and L. monocytogenes, after a 20-day storage at 6 °C was estimated to be 4.1% and 7.1%, respectively. The contamination level was low considering that the product is going to be cooked by the consumer afterwards: the 99th percentile of the distribution was equal to -2.3log cfu/g for Salmonella and 0.5log cfu/g for L. monocytogenes. More generally, the model developed here from raw material reception up to the end of the shelf-life enables to recommend combinations of HP treatment and lactate formulation to guarantee an acceptable microbial concentration before cooking.

Characterization of isolates of Listeria monocytogenes from sludge using pulsed-field gel electrophoresis and virulence assays.

AIMS: To study the diversity and virulence of Listeria monocytogenes isolated from sludge. METHODS AND RESULTS: A total of 60 isolates of L. monocytogenes from sludge were characterized by serotyping, PFGE typing and using in vitro and in vivo virulence assays. The PFGE patterns were compared with those of food and human isolates to determine whether specific group clones are associated with environmental samples. The 60 isolates gave 44 different combined ApaI/AscI PFGE patterns. The PFGE patterns of most isolates were similar or very similar to those of epidemic isolates. The majority (93%) of isolates were found to be virulent by plaque-forming assay and by mouse virulence assay. CONCLUSIONS: Our findings suggest that L. monocytogenes strains found in non-sanitized sludge are virulent and represent a potential health hazard. Although no case of listeriosis related to sludge spread onto agricultural land has been reported, particular attention to this pathogen is needed. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study dealing with the characterization of L. monocytogenes isolates from non-sanitized sludge samples by molecular typing methods and in vitro and in vivo virulence assays. Our findings provide relevant information for evaluating the health risks associated with spreading sludge onto agricultural land.

Comparison between the biofilm initiation of Campylobacter jejuni and Campylobacter coli strains to an inert surface using BioFilm Ring Test.

AIMS: The adhesion to an inert surface (the first step of biofilm formation) of the two main pathogenic Campylobacter species, Campylobacter jejuni and Campylobacter coli, isolated from diverse origins, was compared. METHODS AND RESULTS: Adhesion assays were conducted in 96-well, polystyrene microtiter plates using the BioFilm Ring Test method. This new technique, based on magnetic bead entrapment, was shown to be suitable for analysing the adhesion of Campylobacter sp. strains by comparing the adhesion of four C. jejuni strains as revealed by the BioFilm Ring Test and immunodetection. Among the 46 strains tested, C. jejuni and C. coli displayed different adhesion capabilities ranging from no adhesion to strong adhesion. However, no strain of C. coli was strongly adherent, and statistically, C. coli adhered less to an inert surface than C. jejuni. In addition, strains isolated from animals or carcasses were less adherent than those isolated from food-processing and clinical cases. CONCLUSIONS: These observations suggest that the food environment and the human body could have selected strains with greater adhesion. SIGNIFICANCE AND IMPACT OF THE STUDY: The adhesion capability of strains could partly explain the cross-contamination or re-contamination of food products by Campylobacter. This property could provide a mode of survival for Campylobacter in the food chain.

Impacts of treatment parameters on the inactivation of Campylobacter jejuni by high pressure: a statistical study of main effects and interactions.

AIM: The influence of environmental (temperature and pH) and biological (strain) parameters on the inactivation of Campylobacter jejuni by high hydrostatic pressure (HHP) was investigated. METHODS AND RESULTS: Two clinical strains harvested in stationary phase were pressurized at 20 degrees C and 37 degrees C within a range of 50-400 MPa, in a phosphate (pH 7.0) or a citrate phosphate buffer (pH 5.6), for 10 min. Treatment efficiencies were determined by logarithmic comparisons of culturable cells on blood agar before and after treatment. Results were statistically compared using an anova of culturable cells after treatment to evaluate the effect of all factors. At least a 7-log reduction in cell numbers was observed for both strains. The pH and the strains had no effect on HHP treatment at 20 degrees C while at 37 degrees C, both pH and strain influenced significantly the HHP treatment on C. jejuni. CONCLUSIONS: The pressure efficacy on C. jejuni eradication was affected by both environmental and biological factors. SIGNIFICANCE AND IMPACT OF THE STUDY: Depending on the treatment conditions, C. jejuni sensitivity to HHP can significantly vary. The determination of the inactivation treatment by HPP has to be normalized considering the interaction of environmental and biological factors.

Determination of rpoA as the most suitable internal control to study stress response in C. jejuni by RT-qPCR and application to oxidative stress.

Campylobacter jejuni represents one of the major causes of bacterial enteritis caused by food in humans. There are still mechanisms to be deciphered to better understand better its physiology and pathogenesis. Study of gene expression levels by RT-qPCR could be used, but to be accurate and reproducible, a good internal control has to be chosen. The aim of this study was to identify a highly stable housekeeping gene in Campylobacter jejuni that could constitute a good internal control to study gene expression variations between different growth phases or stress conditions. Expression levels of six different housekeeping genes (gyrA, ilvC, rpoA, slyD, thiC and rrs) were measured by RT-qPCR under different conditions (exponential phase, stationary phase, cold shock, cold shock+oxidative stress, oxidative stress). The rpoA gene was chosen as the best internal control. In a previous study, 9 proteins were identified as involved in oxidative stress response, among which 3 virulence factors. Expression levels of genes coding for these proteins was evaluated by RT-qPCR using rpoA as an internal control. The results obtained were concordant with what had been observed at the proteomic level, validating the methods used and confirming the hypothesis of a potential link between oxidative stress and virulence factors expression.

Effects of high pressure, subzero temperature, and pH on survival of Listeria monocytogenes in buffer and smoked salmon.

High pressure processing is a novel food preservation technology, applied for over 15 years in the food industry to inactivate spoilage and pathogenic microorganisms. Many studies have shown the differential resistance of bacterial cells to high pressure. Listeria monocytogenes is a bacterium able to grow at refrigerated temperature and to survive for a long time in minimally processed foods such as raw smoked fish. The freezing process does not cause significant decline of L. monocytogenes. The phase diagram of water under pressure permits a pressure treatment under subzero temperature, without the disadvantages of freezing for food quality. The aim of this study was to estimate if combined effects of pressure and subzero temperature could increase the destruction of L. monocytogenes in buffer and in smoked salmon. We investigated effects of high pressure processing (100, 150, and 200 MPa) combined with subzero temperatures (-10, -14, and -18 degrees C) and pH (7.0 and 4.5). Results showed that the most effective high-pressure treatment to inactivate L. monocytogenes was 200 MPa, -18 degrees C, and pH 4.5. The relevance of pressure holding time and the synergistic effect of pressure coupled with the subzero temperature to inactivate bacteria are highlighted. Modifications of physical properties (color and texture) were a lightening of color and an increase of toughness, which might be accepted by consumers, since safety is increased.

Survival of Campylobacter jejuni strains from different origins under oxidative stress conditions: effect of temperature.

Campylobacter jejuni is a microaerophilic pathogen but is able to survive oxidative stress conditions during its transmission to the human host. Strains of different origins (reference, poultry, or human clinical) were tested for survival under oxidative stress conditions. C. jejuni strains were grown in Mueller Hinton broth to obtain late exponential-phase cultures. Then they were exposed to 2 different stresses: (1) cultures were either plated on Columbia agar plates and exposed to atmospheric oxygen or (2) paraquat (a chemical oxidizing agent) was added to liquid cultures to reach a 500-microM concentration. Both of these experimental conditions were realized at 3 different temperatures: 4 degrees C, 25 degrees C, and 42 degrees C. Results obtained with paraquat and atmospheric oxygen were similar. Surprisingly, C. jejuni was found to be very sensitive to oxidative stress at 42 degrees C, which is its optimal growth temperature, whereas it was more resistant at 4 degrees C. A strain effect was observed, but no relationship was found between the origin of the strains and level of resistance. High temperature (42 degrees C) combined with oxidative stress allowed a rapid decrease in the C. jejuni population, whereas low temperature considerably decreased the effect of oxidative stress.

Survival of Campylobacter jejuni in mineral bottled water according to difference in mineral content: application of the Weibull model.

The aim of the study was to examine the hypothesis proposed by Evans et al. [2003. Hazards of healthy living: bottled water and salad vegetables as risk factors for Campylobacter infection. Emerg. Infect. Dis. 9(10), 1219-1225] that mineral bottled water accidentally contaminated by Campylobacter jejuni would represent a risk factor for Campylobacter infection. Culturability of C. jejuni cells inoculated in low- and high-mineral bottled water during storage at 4 degrees C in the dark was performed by surface plating and modelled using the Weibull model. The loss of C. jejuni culturability observed in all conditions tested was shown to be dependent on strain, preculture condition and water composition. Following inoculation of C. jejuni, the rapid loss of culturability was not correlated to complete cell death as the passage into embryonated eggs enabled recovery of cells from the viable but non-culturable state. In conclusion, the sanitary risk associated with contaminated bottled water cannot be excluded although it is presumably low. Culture conditions, strain and water type must be taken into account in the evaluation of the risk factors as they influence significantly Campylobacter survival in water.

Pulsed-light system as a novel food decontamination technology: a review.

In response to consumer preferences for high quality foods that are as close as possible to fresh products, athermal technologies are being developed to obtain products with high levels of organoleptic and nutritional quality but free of any health risks. Pulsed light is a novel technology that rapidly inactivates pathogenic and food spoilage microorganisms. It appears to constitute a good alternative or a complement to conventional thermal or chemical decontamination processes. This food preservation method involves the use of intense, short-duration pulses of broad-spectrum light. The germicidal effect appears to be due to both photochemical and photothermal effects. Several high intensity flashes of broad spectrum light pulsed per second can inactivate microbes rapidly and effectively. However, the efficacy of pulsed light may be limited by its low degree of penetration, as microorganisms are only inactivated on the surface of foods or in transparent media such as water. Examples of applications to foods are presented, including microbial inactivation and effects on food matrices.

Viable but non-culturable Listeria monocytogenes on parsley leaves and absence of recovery to a culturable state.

AIMS: To investigate the presence of viable but non-culturable Listeria monocytogenes during survival on parsley leaves under low relative humidity (RH) and to evaluate the ability of L. monocytogenes to recover from VBNC to culturable state under satured humidity. METHODS AND RESULTS: Under low RH (47-69%) on parsley leaves, the initial number of L. monocytogenes populations counted on non selective media (10(9) L. monocytogenes per leaf on TSA) was reduced by 6 log10 scales in 15 days, whereas number of viable L. monocytogenes counted under the microscope was reduced by 3-4 log10 scales, indicating the presence of VBNC cells. This was demonstrated on three L. monocytogenes strains (EGDe, Bug 1995 and LmP60). Changing from low to 100% RH permitted an increase of the culturable counts of L. monocytogenes and this growth was observed only when residual culturable cells were present. Moreover, VBNC L. monocytogenes inoculated on parsley leaves did not become culturable after incubation under 100% RH. CONCLUSIONS: Dry conditions induced VBNC L. monocytogenes on parsley leaves but these VBNC were likely unable to recover culturability after transfer to satured humidity. SIGNIFICANCE AND IMPACT OF STUDY: Enumeration on culture media presumably under-estimates the number of viable L. monocytogenes on fresh produce after exposure to low RH.

Modelling of Campylobacter survival in frozen chicken meat.

AIMS: To model the survival kinetics of Campylobacter jejuni on frozen chicken meat. METHODS AND RESULTS: Three different types of chicken meat surface (skin, skinned muscle and cut muscle) were inoculated with stationary phase cells of C. jejuni (8 log(10) CFU cm(-2)) and frozen for 5 weeks at -20 degrees C. Bacterial numbers were determined weekly using two different methods of enumeration to quantify uninjured and injured cells. Analysis of variance of the results showed that the type of chicken surface and the method used to enumerate surviving cells were the most significant sources of variations in the numbers recovered (P < 0.0001), much more than the freezing time. To identify an appropriate model for the description of effects of freezing on survival over time, several models were fitted to the count data. Decay was found to be nonlinear. In general, survival was least on skin, better on skinned muscle and best on cut muscle. After 2 weeks, additional inactivation by freezing appeared to be negligible. CONCLUSION: Because of the variability of survival it was not possible to fit and select a general model useful for all the different surfaces types. SIGNIFICANCE AND IMPACT OF THE STUDY: The injured state of the cells leads to variability and the underestimation of bacterial survival. This is an essential factor for the assessment of Campylobacter-associated risk.

Behaviour of Campylobacter jejuni in experimentally contaminated bottled natural mineral water.

AIMS: The main objective of the present study was to estimate the survival of microaerophilic Campylobacter jejuni in filtered natural mineral water at 4 degrees C and 25 degrees C. The influence of the presence of biodegradable organic matter was tested, assuming that the bacterial contamination of a bottled natural mineral water could be associated with contamination by organic matter. METHODS AND RESULTS: Washed Campylobacter cultures were inoculated in natural mineral water and sterile natural mineral water, and incubated in the dark at 4 degrees C and 25 degrees C. The effect of temperature, the biodegradable organic matter added, incubation atmosphere and autochthonous microflora were tested on the cultivability of Camp. jejuni. CONCLUSIONS: The survival of Camp. jejuni in natural mineral water was better at 4 degrees C than at 25 degrees C, and the presence of organic matter led to a deceleration in the loss of cultivability and to the multiplication of Camp. jejuni in natural mineral water. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the fact that, in the event of dual contamination of a bottled natural mineral water (Campylobacter and biodegradable organic matter), the pathogen could survive (and even grow) for a relatively long time, especially at low temperature and in spite of the presence of oxygen.

Recovery methods for detection and quantification of Campylobacter depend on meat matrices and bacteriological or PCR tools.

Campylobacter is one of the main causes of human foodborne bacterial disease associated with meat consumption in developed countries. Therefore, the most effective approach for recovery and detection of Campylobacter from meat should be determined. Two hundred ninety pork skin and chine samples were inoculated with Campylobacter jejuni NCTC 11168 and two strains of Campylobacter coli. Campylobacter cells were then recovered from suspensions and enumerated by direct plating. Campylobacter recovery was evaluated by comparing results for two methods of sample collection (swabbing and mechanical pummeling) and three recovery fluids (peptone water, 5% glucose serum, and demineralized water). End-point multiplex PCR was performed to evaluate the compatibility of the recovery fluids with direct PCR detection techniques. Mean recovery ratios differed significantly between pork skin and chine samples. Ratios were higher for mechanical pummeling (0.53 for pork skin and 0.49 for chine) than for swabbing (0.31 and 0.13, respectively). For pork skin, ratios obtained with peptone water (0.50) and with glucose serum (0.55) were higher than those obtained with demineralized water (0.16). Significant differences were not observed for chine samples. Direct multiplex PCR detection of Campylobacter was possible with pork skin samples. The tools for Campylobacter recovery must be appropriate for the meat matrix to be evaluated. In this study, less than 66% of inoculated Campylobacter was recovered from meat. This underestimation must be taken into account for quantitative risk analysis of Campylobacter infection.

Inactivation of Salmonella Typhimurium and Listeria monocytogenes using high-pressure treatments: destruction or sublethal stress?

AIMS: To investigate potential resuscitation of Listeria monocytogenes and Salmonella Typhimurium after high hydrostatic pressure treatments. METHODS AND RESULTS: Pressure treatments were applied at room temperature for 10 min on bacterial suspensions in buffers at pH 7 and 5.6. Total bacterial inactivation (8 log(10) CFU ml(-1) of bacterial reduction) obtained by conventional plating was achieved regarding both micro-organisms. Treatments at 400 MPa in pH 5.6 and 600 MPa in pH 7 for L. monocytogenes and at 350 MPa in pH 5.6 and 400 MPa in pH 7 for S. Typhimurium were required respectively. A 'direct viable count' method detected some viable cells in the apparently totally inactivated population. Resuscitation was observed for the two micro-organisms during storage (at 4 and 20 degrees C) after almost all treatments. In the S. Typhimurium population, 600 MPa, 10 min, was considered as the treatment achieving total destruction because no resuscitation was observed under these storage conditions. CONCLUSIONS: We suggest a delay before performing counts in treated samples in order to avoid the under-evaluation of surviving cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The resuscitation of pathogen bacteria after physical treatments like high hydrostatic pressure has to be considered from the food safety point of view. Further studies should be performed in food products to study this resuscitation phenomenon.

Influence of kinetic parameters of high pressure processing on bacterial inactivation in a buffer system.

High pressure processing is recently applied in the food industry to inactivate spoilage and pathogenic microorganisms. Bacterial cells exhibit various barosensibility, and the role of pressurization, depressurization and constant pressure stage remain unknown. We investigated the effect of high pressure processing on Salmonella typhimurium and Listeria monocytogenes cells at 400 and 500 MPa respectively in buffer pH 7 at 20 degrees C. We applied various pressurization/depressurization kinetic rates (1, 5 and 10 MPa/s for pressurization and 250, 20 and 5 MPa/s for depressurization), and various pulse series or pressure holding times. Results show that high pressure pulses reduced linearly the number of bacterial cells according to the product of pressure and time: we defined this product as a Barometric Power (BP). Reduction of both microorganisms increased when holding time increased from 5 to 20 min, and better results were obtained when the rate of pressurization and depressurization were increased.

Decrease of enteric micro-organisms from rural sewage sludge during their composting in straw mixture.

AIMS: To study the decrease of enteric micro-organisms including viable nematode eggs, enteroviruses, faecal indicators (Escherichia coli and enterococci) and pathogenic bacteria (Listeria monocytogenes, Salmonella sp. and Clostridium perfringens) of a rural sewage sludge when it is composted for 7 months in mixture with straw. METHODS AND RESULTS: Numbers of the test organisms and the physico-chemical parameters were measured on a monthly basis on the mixture, on the compost after being turned, and on the pile in three positions representing the part by which air is incoming, the bottom of the pile and the part through which air is outgoing. The lowest temperature in the pile was observed at the bottom, where it did not exceed 50 degrees C against 66 degrees C in the two other areas. There were no significant differences between the three areas in terms of micro-organism survival. Infectious enteroviruses were inactivated rapidly and were not found after the first turning whereas some genomes were detected until after the third turning. Escherichia coli and enterococci presented a similar survival rate and their number decreased by 4 log(10) whereas Salmonella decayed at a greater rate than L. monocytogenes. The numbers of C. perfringens decreased gradually to reach a final concentration in the mature compost of about 10(2) CFU g(-1) dry matter (d.m.), which was similar to that of the faecal indicators. CONCLUSIONS: The hygienic effect of sludge composting in mixture with straw results in a significant reduction of enteric micro-organisms, the concentration of the faecal indicators in the final product being < 64 most probable number g(-1) d.m. The concentrations of Salmonella, enteroviruses and viable nematode eggs in the final product were not detectable which is in accordance with the French legislation. SIGNIFICANCE AND IMPACT OF THE STUDY: The results which pointed out the different behaviour of the test micro-organisms reflect the difficulty to propose a relevant indicator of hygienization. Otherwise, they show that composting is an efficient means for hygienization of sludge of rural wastewater treatment, where the straw is available close to their place of production.

Physiological damages of Listeria monocytogenes treated by high hydrostatic pressure.

High hydrostatic pressure is a new food preservation technology known for its capacity to inactivate spoilage and pathogenic microorganisms. This study investigated the damages inflicted on Listeria monocytogenes cells treated by high pressure for 10 min at 400 MPa in pH 5.6 citrate buffer. Under these conditions, no cell growth occurred after 48 h on plate count agar. Scanning electron microscopy (SEM) revealed that cellular morphology was not really affected. Measuring propidium iodide (PI) staining followed by flow cytometry demonstrated that membrane integrity was damaged in a small part of the population, although the membrane potential evaluated by oxonol fluorescence or measured by analytical methods was reduced from - 86 to - 5 mV. These results for the first time showed that such combined methods as fluorescent dyes monitored by flow cytometry and physiological activity measurements provide valuable indications on cellular viability.

Morphological and physiological characterization of Listeria monocytogenes subjected to high hydrostatic pressure.

High hydrostatic pressure is a new food preservation technology known for its capacity to inactivate spoilage and pathogenic microorganisms. That inactivation is usually assessed by the number of colonies growing on solid media after treatment. Under normal conditions the method does not permit recovery of damaged cells and may underestimate the number of cells that will remain viable and grow after a few days in high-pressure-processed foodstuffs. This study investigated the damage inflicted on Listeria monocytogenes cells treated by high pressure for 10 min at 400 MPa in pH 5.6 citrate buffer. Under these conditions, no cell growth occurred after 48 h on plate count agar. Scanning electron microscopy, light scattering by flow cytometry, and cell volume measurements were compared to evaluate the morphological changes in cells after pressurization. All these methods revealed that cellular morphology was not really affected. Esterase activity, as assessed either by enzymatic activity assays or by carboxy fluorescein diacetate fluorescence monitored by flow cytometry, was dramatically lowered, but not totally obliterated, under the effects of treatment. The measurement of propidium iodide uptake followed by flow cytometry demonstrated that membrane integrity was preserved in a small part of the population, although the membrane potential measured by analytical methods or evaluated by oxonol uptake was reduced from -86 to -5 mV. These results showed that such combined methods as fluorescent dyes monitored by flow cytometry and physiological activity measurements provide valuable indications of cellular viability.

Development of a direct viable count procedure for the investigation of VBNC state in Listeria monocytogenes.

A viable but non-culturable (VBNC) bacterial state was originally detected in studies in environmental microbiology. In particular, this state has been demonstrated for a number of human pathogens (Escherichia coli, Salmonella enteritidis, Vibrio cholerae, Legionella pneumophila and Campylobacter jejuni). The presence of VBNC cells poses a major public health problem since they cannot be detected by traditional culturing methods and the cells remain potentially pathogenic under favourable conditions. But, as far as we know, the VBNC state has not been yet described in Listeria monocytogenes. In most studies, this has been assessed by the Kogure procedure based on cellular elongation in the presence of DNA gyrase inhibitors. The antibiotic used was nalidixic acid in order to prevent DNA replication, only efficient in Gram-negative bacteria studies. In this study, we describe a new DVC procedure to detect and count viable of L. monocytogenes suspended in filtered, sterilized distilled water. We used different concentrations of ciprofloxacin, efficient both in Gram-negative and Gram-positive bacteria. Bacteria cells were removed and resuspended in BHI broth, with yeast extract and ciprofloxacin. The mixture was incubated at different incubation times at 37 degrees C. After different incubation times, cells were filtered through an isopore polycarbonate black membrane filter and covered with a DAPI solution or orange acridine. The filters were prepared and examined by epifluorescence microscopy. Elongated cells were counted as viable cells, whereas normal size was regarded as nonactive ones. This method allows determination of ciprofloxacin concentration and incubation time optimal to detect maximum viable cells percentage in L. monocytogenes.