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In Europe, people are increasingly interested in edible insects to meet the growing demand for food diversity and protein supply, and bring expected nutritional and environmental benefits. Among all insect-based foods, insect powder is one of the most promising choices, which presents added advantages in the ability of mixing with versatile ingredients, long shelf-life and higher consumer acceptance (non-recognisable form). As a novel protein source, insects require the well-designed manufacturing process to produce high quality insect powder, while guaranteeing food safety of the final product. This review provides a brief overview of the current consumption patterns and general processing steps of insect powders. With regard to inactivating potential microbial hazards, traditional thermal treatment (e.g. boiling, blanching,) is still the predominant method in insect sector, which shows a desirable microbial reduction but may affects nutritional properties and organoleptic attributes. The use of food preservatives and fermentation have also been incorporated as means for eliminating pathogenic bacteria in some insect products. Recent years, a set of advanced or innovative technologies have been developed for inactivation of microorganisms in various foods while maintaining food quality, such as infrared heating, instant control pressure drop technology, high hydrostatic pressure, and cold plasma. However, except for the initial exploration of very few techniques in the laboratory, most novel technologies have not been attached much importance in insect sector. Eventually, this review highlights the lack of data on the impacts of different processing methods (both conventional and innovative, individual or in combination) for edible insects, encompassing the control of microbial activities, nutritional value, and sensory qualities.

Campylobacteriosis has been recognized as the major bacterial food-borne infection worldwide. Campylobacter, especially C. jejuni, contaminate mainly poultry meat. Although more sensitive than other food-borne pathogens to many stresses, C. jejuni can survive food processing and go on to reach its final reservoir (the human gut). Genomic analyses of this organism indicate a lack of genes described in other gram-negative bacteria to overcome stresses. The high-pressure recovery response of C. jejuni 81-176 was analyzed from two-dimensional electrophoretic profiles of the cytoplasmic proteome. The main cellular mechanisms controlling the down- and upregulated proteins are discussed.

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.

Aims: The main objective of the present study was to estimate the survival of microaerophilic Campylobacter jejuni in filtered natural mineral water at 4°C and 25°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°C and 25°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°C than at 25°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.

International audience; The aim of this study was to assess the efficiency of the embryonated egg model to recover Viable But Non Culturable (VBNC) cells of Listeria monocytogenes. L. monocytogenes cells were incubated in filtered sterilised distilled water. The VBNC state was obtained after a 25 to 47 days incubation period (concentration of culturable cells less than 1 cfu/mL). Fifteen days after the VBNC state was reached, non culturability was checked in various media. One milliliter of each VBNC suspension that contained 10$^{4}$ metabolically active cells (i.e. Direct Viable Count + cells) was inoculated into the vitellus fluid of embryonated and non-embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs (18/32), but not in the non-embryonated eggs (1/32). The recovery rate was higher after culture of the vitellus fluid plus embryo (18/32) than after culture of the vitellus fluid alone (6/32). The results indicate that the embryo likely plays a prominent part in the recovery process. The virulence of recovered cells was assessed by the ability to form plaques in HT-29 cell monolayers and by the ability to colonise mouse spleens. Although the cells were classified as avirulent when in the VBNC state, the virulence was recovered after resuscitation.

The aim of the present study was to determine the effect of the different steps of the cold-smoking process and vacuum storage on the culturability and viability of Listeria monocytogenes strain Scott A inoculated in sterile salmon samples. Additionally, the virulence of L. monocytogenes cells was assessed by intravenous inoculation of immunocompetent mice. Salmon (Salmo salar) portions were inoculated with L. monocytogenes at a level of 6 log CFU/g and were then dry salted (5.9%), smoked (0.74 mg phenol per 100 g), partially frozen (-7 degrees C), vacuum packed, and stored for 10 days at 4 degrees C followed by 18 days at 8 degrees C. Salting represented the only step of the process with a weak but significant listericidal effect (0.6 log reduction). Although the other processing steps had no immediate reduction effect on L. monocytogenes, the combination of steps significantly lowered by 1.6 log CFU/g the number of L. monocytogenes. The culturable count remained less than 7 log CFU/g until the end of the storage period, whereas in unprocessed samples (control) the culturable counts reached values up to 9 log CFU/g. To mimic a postprocess contamination, salmon portions were also inoculated with L. monocytogenes after being cold-smoke processed. A reduction of the culturable count during the 2 first weeks of storage was observed, but then growth occurred and identical values observed for preprocess contamination were reached at the end of the storage. A viable but nonculturable state transition of strain Scott A was not observed, and the cold-smoking process did not affect the virulence of bacteria isolated at the beginning and end of the storage.

Investigations of bacterial survival in natural environments have indicated that some or- ganisms lose culturability on appropriate media under certain conditions and yet still exhibit signs of metabolic activity and thus viability. This reproducible loss of culturability in many bacterial species led to the description of a "Viable But Non Culturable" (VBNC) state. The purpose of this article is to determine environmental and physico-chemical factors which induce the VBNC state in a food-borne pathogen that has become a public concern: Listeria monocytogenes. The factors, i.e. inoculum size, natural sunlight, temperature (4 o Co r 20 o C), NaCl concentration (0% or 7%) and pH (5 or 6) were studied on 4 strains (LO28, ATCC 19115, Scott A, CNL 895807). The culturability of the starved cell suspension was determined in each condition tested by the spread plate count, and the cell activity was determined by the Direct Viable Count technique and CTC-DAPI double staining. A strain effect was found in different test conditions. For the LO 28 and ATCC 19115 strains, the VBNC state was very transient in certain conditions. For the other strains tested (Scott A, CNL 895807), the VBNC state was maintained throughout the observation period. In the dark, the incubation temperature was the main factor in the production of VBNC forms in L. monocytogenes. However, natural sunlight rapidly produced the VBNC state in L. monocytogenes cells in microcosm water. We conclude that because of its ubiquity and the factors studied which are met in the food industry, the presence of VBNC L. monocytogenes cells could pose a major public health problem since they cannot be de- tected by traditional culturing methods. Further investigations are needed to establish virulence before and after resuscitation of VBNC L. monocytogenes cells. VBNC / environmental factors / stress / Listeria monocytogenes

Viable but Non-culturable (VBNC) state in bacteria was detected originally in environmental microbiology studies. 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 moreover the cells remain potentially pathogenic under favourable conditions. But the VBNC state has not been yet described in Listeria monocytogenes . Production of VBNC L. monocytogenes cells was studied using four strains resuspended in microcosm water. Various strains of L. monocytogenes were resuspended in filtered sterilized water adjusted to pH 6·0, incubated at 20°C and 4°C with or without NaCl supplementation (0%, 7%), with gentle shaking at 100 rpm. The culturability of starved cells suspensions was determined by spread plate count (PCA). The cells activity was measured by a Direct Viable Count technique and by CTC-DAPI double staining. Two strains of four (CNL 895807, Scott A) exhibited a VBNC state. They lost their culturability and maintained a cellular activity, even after 10 weeks of starvation in microcosm water.

High hydrostatic pressure treatment is regarded as a possible alternative process for food preservation. One of the primary considerations for industrial applications is the ability of this technique to destroy pathogenic microorganisms. The inactivation of microorganism populations after high pressure treatment is well described, but their residual pathogenicity is less documented. This study compared the virulence of Listeria monocytogenes scott A before and after high pressure treatments. The pressurized samples were composed of 1 ml of Listeria monocytogenes culture diluted into 9 ml of buffer. Two buffers are used: phosphate buffer (pH 7) and citrate buffer (pH 5.6). The virulence of cells is estimated by their association and invasion capacities to Hela cells. These characteristics of pathogenity were compared between untreated and pressurized cells (400 MPa, 10min, in citrate buffer) or (600 MPa, 10min, in phosphate buffer). These results showed that, after treatment in citrate buffer, the...

High hydrostatic pressure is regarded as possible alternative method for food preservation. One of the primary considerations for industrial applications is the ability of this method to eradicate pathogenic micro-organisms. This study subjected L. monocytogenes suspensions, in a phosphate buffer (pH 7·0) or in a citrate phosphate buffer (pH 5·6), to high hydrostatic pressure treatments relative to the following variables: pressure (200–600 MPa), treatment time (3, 10 and 20 min), temperature (4, 20 and 40°C) and the pH of the suspension medium (5·6 and 7·0). An optimal design of 40 runs was obtained using the Fedorov algorithm, and responses were studied by analysis of variance in terms of cell survival on plate count agar. Efficiency was determined by log10comparisons of the numbers of live cells before and after treatment. A statistically significant relationship was found between the four variables considered (pressure, pH, treatment time and temperature), their interactions (treatment time vs pressure, pH vs treatment time, pH vs pressure, pressure vs temperature, treatment time vs temperature) and the inactivation of L. monocytogenes. R -squared statistical analysis indicated that the linear model used accounted for more than 98·5% of the variability in the inactivation ofL. 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.

For many years, environmental microbiologists working on water samples, have reported differences between bacterial counts performed by culture and by microscopy. These observations have led to the demonstration of the viable but non-culturable (VNC) state in bacteria. Some hygienist specialists underlined the risk presented by pathogenic bacteria in the VNC state. The VNC state in bacteria has been studied by a number of authors, but the relation between VNC state and bacterial stress response has not been established yet, while the VNC state is generally described in responses to adverse conditions. Campylobacter jejuni enter the VNC state in response to starvation. In our study, we searched for a protein synthesis in the first hours of the cell starvation exposure. Three Campylobacter jejuni strains were suspended in filtered, sterilized, distilled water, and incubated at 4 degrees C with gentle shaking (100 rpm). After 1, 2, 3, 4 and 5 h of starvation, C. jejuni cells were removed and subjected to a heat shock (55 degrees C, 3 min) and to a conductimetric assay. Results obtained showed that a protein synthesis occurred in the onset of the starvation period, and that these improved the nutrient assimilation and enhanced the heat resistance in starved cells.

The existence of a viable but nonculturable (VBNC) state has been described for Campylobacter jejuni as it had been for a number pathogenic bacteria. Three C. jejuni human isolates were suspended in surface water and subsequently entered the VBNC state. After starvation for 30 days, VBNC cells were inoculated in the yolk sacs of embryonated eggs. Culturable cells were detected in a large proportion of the embryonated eggs inoculated with VBNC C. jejuni cells. Recovered cells kept their adhesion properties.

The infection rate of 60 pigs (10 pigs from each of six farms) by Helicobacter species was studied by two techniques. Histological examination of the cardiac area of the stomach yielded a 58% positive result versus an 80% positive result by PCR with genus-specific primers. Eighty percent of the 16S rRNA gene was amplified, classified in four groups by PCR-restriction fragment length polymorphism, and sequenced. Isolates from all farms except one (farm C) were identified as Helicobacter heilmannii type 1, while those from farm C were identified as H. heilmannii type 2. Attempts to culture this organism in vitro failed. Helicobacter pylori was not found in these animals.

As in many bacteria, including such pathogens as Salmonella enteritidis , enterotoxic Escherichia coli, Vibrio vulnificus, Vibrio cholerae , the occurrence of a viable but non-culturable (VBNC) state has been described for Campylobacter jejuni . This bacteria has been recognized as a leading foodborne pathogen. In this study, VBNC cells from three C. jejuni human isolates were suspended in microcosm water at 4°C and entered the VBNC state. The metabolic activity of these VBNC cells was monitored by CTC reduction. Once in VBNC state, the strains were inoculated per os into newborn mice and 1-day-old chicks so as to evaluate and compare these animal models of VBNC cell recovery. The three strains used in this experiment were revived using the murine model, whereas only two strains were revived with the chick model. The results show that the murine model permits better recovery than the 1-day chick model. All three strains revived exhibited an associative index very similar to that measured in the culturable state. This study indicates that the VBNC state should be considered as playing a role in the epidemiology of Campylobacter infection.

Fifteen hundred food samples were analysed for thermotolerantCampylobacteraccording to the NF ISO 10272 standard during a two-year investigation period using Preston and Park & Sanders methods and three isolation media (Karmali, Butzler and Skirrow agar). The samples, consisting of 660 oysters(Crassostrea gigas), 400 ready-to-eat vegetables, 300 poultry products and 140 raw milk cheeses, were positive 5, 2, 12 and 0 times respectively. All isolates were identified asCampylobacter jejunisubsp.jejuniexcept oneC. coli.Biotype and serotype determination revealed a large diversity among the strains, although biotypes I (59%) and II (18%) were predominant. Serotyping (Lior scheme) showed that 85% of the strains were among the 10 most common Campylobacter serogroups isolated in human campylobacteriosis. Park & Sanders enrichment and isolation on Karmali agar appeared to be the most efficient combination for detecting thermotolerant Campylobacter in the food samples tested.

Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (10 7 cells ml −1 ) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride–4′,6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 μl mg of protein −1 for the culturable form and 10.96 μl mg of protein −1 after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water.

Production of viable but non-culturable (VNC)Campylobacter jejunicells was studied using 36 strains resuspended in microcosm water. Cells in stationary phase were resuspended at 4°C, with gentle stirring at 100 r min−1, in filtered surface water adjusted to pH 6.050. The culturability of starved cell suspensions was determined by spread plate counts, and the activity of cells by the direct viable count technique and CTC–DAPI double staining. Although mostC. jejunistrains died within a few days of suspension in microcosm water, three strains (Bf, 79 and 85) demonstrated a complete decrease of culturability within 15 days of suspension. A constant activity level was found in these three strains, even after 30 days of starvation in microcosm water; and an almost constant concomitant level of adenosine triphosphate was measured in VNC cell suspensions. Scanning electron microscopy ofC. jejuniVNC cells showed that the spiral-shaped cells of strain 85 cultures became coccoid in ageing microcosm-water cell suspensions, whereas most VNC cells of strains Bf and 79 remained spiral-shaped after 30 days of starvation.

A numerical method was developed to simulate the performance of vertical-cavity surface-emitting lasers. A two-dimensional code takes into account the spatial variation of both the carrier distribution and the electromagnetic field inside a cylindrical structure (dependence on the third spatial coordinate, the azimuthal angle, is trivial). The eigenmodes of the electromagnetic field inside the structure are identified. It is explained that these modes are self-oscillations of the combined system of the electromagnetic field and the dielectric and depend on the injection current. The spontaneous-emission spectrum is reproduced at threshold and slightly below it and above it. Due to simplifications in the analysis the modes obtained are restricted to those propagating in directions forming a small angle with the normal to the distributed Bragg reflector mirrors. An estimate is made for the fraction of spontaneous emission into the lasing mode, with the value of 2% in line with experimental determinations.

We present a theoretical model of a loss-compensated symmetric Y-junction acting as an optical beam splitter. We consider silica (SiO/sub 2/) channel waveguides which are assumed to be highly doped with Er/sup 3+/. The model was developed using the beam propagation method (BPM) and a fast-Fourier-transform (FFT)-based algorithm. The analysis showed that considerable gain levels, about 4.2 dB/cm at each port of the Y-junction, can be achieved for erbium concentration 2.5/spl times/10/sup 20/ ions/cm/sup 3/, signal power 1 /spl mu/W and pump power 250 mW.

Effects of high concentration and clustering on the performance of erbium-doped silica waveguide amplifiers are numerically investigated. The model, based on propagation-rate equations of a laser system containing isolated ions and ion-pairs, is numerically solved by combining Runge-Kutta and full-vectorial finite-element methods; it allows the description of both uniform and pair-induced upconversion mechanisms. Numerical results show that cooperative upconversion processes seriously deteriorate amplifier performance at high erbium concentration. Moreover, we show that only the detrimental effect due to uniform upconversion can be reduced by a careful design of waveguide geometry and refractive index profile; performance degradation due to clustering may only be overcome by reducing the fraction of ions in pair using suitable codopants. The proposed model is validated with published experimental results. >

We report the fabrication by PECVD of silicon-rich erbium-doped silica films that exhibit both 1535 nm fluorescence and visible photoluminescence. Fluorescence spectra are presented along with absorption spectra that display a strong band edge in the blue, which we ascribe to the presence of Si microclusters. We are unable to observe characteristic Er3+ absorption bands and propose that excitation of the rare earth is via an energy transfer process from Si microclusters.

We report numerical analysis of planar and channel silica waveguide amplifiers with high erbium concentration. The numerical approach is based on the combined use of a full-vectorial finite-element method and the Runge-Kutta algorithm. It allows one to investigate real structures, accounting for both the active ions interactions by cross-relaxation and upconversion and the effects of waveguide geometry and refractive index profile. We show that, depending on input pump power and waveguide structure, ion-ion interactions affect the amplifier performance for high erbium concentrations. A three-dimensional waveguide designed for single-mode operation ensures higher pumping efficiency and lower noise characteristics than a two-dimensional one and seems to be the best candidate to overcome this drawback. >

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.

International audience; 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.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Infection generally occurs after ingestion of contaminated poultry products, usually conserved at low temperatures. The mechanisms promoting survival of C. jejuni in the cold remain poorly understood despite several investigations. The present study provides insight into the survival mechanism by establishing the involvement of polynucleotide phosphorylase (PNPase), a 3′-5′ exoribonuclease with multiple biological functions in cold survival. The role of PNPase was demonstrated genetically using strains with altered pnp genes (which encode PNPase) created in C. jejuni F38011 and C. jejuni 81-76 backgrounds. Survival assays carried out at low temperatures (4 and 10°C) revealed a difference of 3 log CFU/ml between the wild-type and the pnp deletion (Δ pnp ) strains. This did not result from a general requirement for PNPase because survival rates of the strains were similar at higher growth temperatures (37 or 42°C). trans -Complementation with plasmid pNH04 carrying the pnp gene under the control of its natural promoter restored the cold survival phenotype to the pnp deletion strains (at 4 and 10°C) but not to the same level as the wild type. In this study we demonstrate the role of PNPase in low-temperature survival of C. jejuni and therefore attribute a novel biological function to PNPase directly related to human health.

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°C and 37°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°C while at 37°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.

The GV strain (serotype O103:H2:K-), originally isolated from a diarrheic rabbit, is an enteropathogenic Escherichia coli strain that produces diarrhea without synthesizing the classical enterotoxins and that is not invasive. This strain is characterized by a 117-kb plasmid (pREC-1). Histological study of the gut by scanning electron microscopy and transmission electron microscopy was performed on the GV strain, on a derivative strain cured of pREC-1, and on transconjugants obtained by transfer of pREC-1 to nonpathogenic strains E. coli K-12 and 6100, not belonging to the O103 serogroup. The GV strain adhered to the epithelial cells of the ileum and large intestine, whereas the cured GV strain did not. Transfer of plasmid pREC-1 to E. coli K-12 or 6100 allowed the bacteria to attach to the intestinal mucosa in the same manner as that of the wild-type GV strain. Thus, pREC-1 seems to play an important role in attachment to and colonization of the intestinal tract of rabbits by E. coli serogroup O103. Scanning electron microscopy showed numerous bacteria attached together and closely associated with intestinal villi. Transmission electron microscopy revealed effacing lesions characteristic of enteropathogenic E. coli strains: effacing of microvilli and cuplike projections (pedestal formations) associated with an acute inflammatory and hemorrhagic response. In contrast with the results reported for rabbit pathogenic O15 strains, it appeared that the Peyer's patches were not involved in the early stages of infection with the O103 GV strain. This strain may represent a model for the study of the virulence and pathogenic effects of enteropathogenic and enterohemorrhagic E. coli strains.

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.

Campylobacter jejuni represents one of the leading causes of bacterial enteritis throughout the world. Poultry is an important source of C. jejuni. Despite hygiene measures taken in the production chain, C. jejuni is frequently isolated from poultry meat. C. jejuni is a microaerophilic pathogen, affected by oxidative stress. Freeze-thaw treatment induces cell death by several mechanisms, including oxidative stress. In this article, we investigate the role of oxidative stress in C. jejuni sensitivity during and after a freeze-thaw treatment. This treatment results in dead and sublethally injured cells. The latter population might have an increased sensitivity to oxidative stress. To test this, cells were stored for another 24 h at 4 degrees C under aerobic conditions and compared to cells that were not treated. C. jejuni survival was measured in different media (water, BHI broth, chicken juice, and chicken fillets) to test the environment protective effect. Different strains were tested, including sodB (encoding the superoxide dismutase) and cj1371 (encoding a periplasmic protein) mutants. Cell death was particularly important in water but similar in BHI, chicken juice, and chicken fillets. The sodB mutant was more sensitive to freeze-thaw treatment, suggesting that the killing mechanism involves production of superoxide anions. On the contrary, the cj1371 mutant was more sensitive to storage at 4 degrees C, suggesting that it does not play a role in the detoxification of reactive oxygen species. Storage at 4 degrees C after freeze-thaw treatment increases cell death of oxidative stress-sensitive populations. Sensitization to oxidative stress, freeze-thaw treatment, and further storage at 4 degrees C could be a way to reduce C. jejuni populations on carcasses.

RA2007ACL08-GAR; International audience; Campylobacter jejuni is a microaerophilic pathogen representing one of the major causes of bacterial enteritis in humans. The oxidative stress response after exposure to paraquat, a strong oxidising agent, was analysed by two-dimensional protein electrophoresis and Maldi-ToF mass spectrometry. Oxidative stress and redox-related proteins were overexpressed: FldA flavodoxin and a pyruvate-flavodoxin oxidoreductase encoded by cj1476c. No increase in SodB expression was observed. An additional quantitative RT-PCR analysis showed an increase in katA but not in sodB expression. However, the sodB mutant was very sensitive to paraquat, its basal expression level being essential for oxidative stress resistance. Proteins related to iron homeostasis (Cft and a non-haem iron protein encoded by cj0012c) and general stress response (FusA and MreB) were found overexpressed. Interestingly, a two-component regulator encoded by cj0355c was differentially expressed in the presence of paraquat and could play a role in induction of the C. jejuni oxidative stress response. Virulence factors (CadF, FlaA and a VacJ homolog encoded by cj1371) were also found overexpressed under oxidative stress conditions and a cj1371 mutant showed increased sensitivity to paraquat, suggesting that the Cj1371 periplasmic protein could play a role in C. jejuni oxidative stress resistance.

Pulsed light is a new method intended for the decontamination of food surfaces using short, high frequency pulses of an intense broad spectrum. The effects of broad spectrum pulsed light on the survival of Listeria monocytogenes Scott A, Listeria monocytogenes CNL, Pseudomonas fluorescens MF37 and Photobacterium phosphoreum SF680 populations on agar and in a liquid medium were investigated during this study. The sterilisation system generated 1.5 J cm(-2) per pulse with eight lamps for 300 mu s. In the case of surface-seeded cells, a 7.8, 8.14 and > 7.14 log reduction was obtained for L. monocytogenes, Ps. fluorescens MF37 and Ph. phosphoreum, respectively, after a single pulse of treatment. Inactivation levels were lower for depth-plated cells: indeed, 10 pulses of treatment achieved 1.6, 2.03 and 4.78 log reductions for L. monocylogenes ScottA, L. monocytogenes CN-L and Ps. fluorescens MF37, respectively. After 5 pulses, Ph. phosphoreum exhibited a 4.6 log reduction. Similarly, bacterial cells in suspension treated with 3 pulses were reduced by 0.52, 0.8, 2.07 and 2.05 for L. monocytogenes ScottA, L. monocytogenes CNL, Ps. fluorescens MF37 and Ph. phosphoreum, respectively. No resistance to pulsed light was observed during our experiments.

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 log10 CFU cm−2) and frozen for 5 weeks at −20°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

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 (109 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.

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.

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 log10 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°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.

International audience; Cells of four reference strains (Scott A, LO 28, CNL 895807 and ATCC 19115) and of five recent food isolates (A00M011, A00M018, A00M087, A00M092 and A00M123) of Listeria monocytogenes were grown until late exponential phase in Brain Heart Broth at two different temperatures (37 degrees C and 4 degrees C). Our results show that significant differences exist between the cellular lipid fatty acid profile of reference and recent food isolates. Like the reference strains, and in keeping with previous reports on the cellular lipid fatty acid profile of L. monocytogenes, the recent food isolates were characterised by the presence of ai15:0, i15:0 and ai17:0. In addition, the fatty acid ai13:0 was observed in all of the recent food isolates grown at 4 degrees C, whereas only two reference strains, Scott A and LO 28, showed ai13:0 in their cellular lipid fatty acid profile at 4 degrees C. When grown at 4 degrees C, the recent food isolates showed a mean aiC15/aiC17 ratio of 66, while reference strains were characterised by significantly lower ratios, ranging between 4.3 (ATCC 19115) and 28.9 (Scott A). These results showed that all of the recent food isolates, Scott A and LO28 strains use chain length and anteiso-branching (ai15:0) as their major response to cold temperature adaptation. However, the cold adaptation response of reference strains CNL 895807 and ATCC 19115 appears to be different.

International audience; The virulence of Viable But Non-Culturable (VBNC) cells of 4 strains of Listeria monocytogenes was investigated in both a human adenocarcinoma cell line (HT-29) and a mouse model. LO 28, ATCC 19115 and CNL 895807 strains of Listeria monocytogenes became VBNC when incubated in microcosm water at 20 °C and Scott A strain at 4 °C. No culturable bacteria were detected in the VBNC state, although 104 active cells/mL were found by the Direct Viable Count (DVC) and CTC-DAPI double staining methods. A comparison of virulence in both human adenocarcinoma cell line HT-29 and the mouse model showed that culturable controls were more virulent than VBNC cells, which appeared to be avirulent regardless of the virulence methods applied. Pathogenicity was tested in each model and was lost concomitantly with culturability, whereas some cells were still metabolically active (determined by CTC and DVC). Moreover, amplification of a 388 bp fragment with Immunocapture-PCR revealed the presence of Listeria monocytogenes DNA in all mixed spleen samples after intravenous injection of VBNC cells. These results demonstrate that VBNC cells were present in the mouse spleens. The results of the study suggest that Listeria monocytogenes strains might remain in the aquatic environment for prolonged periods in the VBNC state but these cells were not pathogenic in the conditions tested. These findings demonstrate the value of VBNC studies and show the need to investigate the role of VBNC cells in environmental transmission of Listeria monocytogenes. Further studies are needed in order to investigate the virulence of VBNC cells of Listeria monocytogenes after recovery of a culturable state.

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 °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.

A numerical study of the transient behavior of single-mode Er/sup 3+//Yb/sup 3+/ codoped fiber lasers is presented. The model, based on propagation-rate equations, includes both uniform up-conversion from the /sup 4/I/sub 15/2/ and /sup 4/I/sub 9/2/ erbium levels and the pair-induced energy transfer process from ytterbium to erbium. We show that neglecting the r dependence of the operating fiber laser parameters allows accurate simulation results at minimum numerical effort. Using this numerical model, we characterize the laser relaxation oscillations and the effect of ytterbium on the dynamic behavior and stability with respect to fluctuations of the pump power. Our results confirm that the presence of Yb/sup 3+/ enhances the efficiency of the laser and show that its stability is also significantly improved.

We theoretically analyze the performance of longhaul multichannel optical communication systems based on an integrated WDM power level compensator. By placing an array of Er/sup 3+/-Yb/sup 3+/ co-doped silica waveguides in parallel between a demultiplexer/multiplexer pair and preceeding it by an EDFA, we demonstrate the possibility of transmitting eight unequally spaced WDM channels over long distances, without electronic regenerators and using span lengths as high as 50 km. This technique allows substantial reduction of both four-wave mixing crosstalk and gain peaking.

Campylobacter are a leading cause of human diarrhea. The usual source of infection is contaminated food, particularly poultry but pork has also been described. The veterinary use of antimicrobial drugs has been suggested to be largely responsible for resistance in human isolates of this zoonotic pathogen. A study was carried out to investigate the occurrence and antimicrobial resistance of Campylobacter isolated from French fattening pigs. From March 1998 to June 1999, stomach samples were collected at slaughter from 240 fattening pigs originating from 24 different farms. Half of the pigs were found to be positive for Campylobacter but considerable variation was observed between farms. Isolates all belong to the Campylobacter coli species. Susceptibilities of the strains were determined for five antimicrobial drugs using agar dilution. Resistance to tetracycline and erythromycin was high (79 and 55%, respectively). For nalidixic acid, enrofloxacin and ampicillin, resistance was observed in 34, 15 and 20% of the isolates, respectively. More than one-third of the strains was resistant to at least three antimicrobial drugs. A Thr86Ile modification in GyrA was observed in the enrofloxacin-resistant strains studied. The multiresistant strains analyzed expressed the multidrug transporter CmeB at a high level. Results indicated a high prevalence of C. coli in the stomach of the French pigs examined. In addition, a high proportion of the strains was resistant to antimicrobial drugs, particularly to tetracycline and erythromycin, or were multiresistant.

Continuous wave laser operations of silica-based Er/sup 3+/-Yb/sup 3+/ co-doped waveguides have been numerically analyzed by means of a finite-element method. The theoretical model, based on propagation-rate equations, describes uniform upconversion by a dipole-dipole interaction between Er/sup 3+/ ions, and includes a pair-induced energy transfer process from Yb/sup 3+/-Er/sup 3+/ Numerical results show that single-frequency operation with slope efficiency higher than 50% and threshold pump powers of few mW can be achieved in short and heavily doped waveguides equipped with input dielectric mirrors and output distributed Bragg reflectors. >

High-concentration Er/sup 3+//Yb/sup 3+/ co-doped silica waveguide amplifiers are numerically analyzed. With optimized rare-earth concentrations the effect of Er/sup 3+//Er/sup 3+/ ion-pairs can be neglected and each Er/sup 3+/ ion can be assumed to be paired only to the surrounding Yb/sup 3+/ ions. The rate-equations model includes uniform upconversion mechanisms from /sup 4/I/sub 13/2/ and /sup 4/I/sub 11/2/ erbium levels and an Yb/sup 3+/ to Er/sup 3+/ pair-induced energy transfer process. Numerical results demonstrate the possibility of fabricating short- and high-gain integrated optical amplifiers; it is shown that net gain as high as 3 dB/cm can be obtained. >

Assessment of yeast viability by plate counts, ATP determination and FUN-1 viability staining was performed to study sublethal injury of Saccharomyces cerevisiae cells submitted to low-amperage electrolysis. Lethal effects of electrolysis were confirmed by all methods, demonstrated by the decrease in viable counts observed during electrolysis. FUN-1 viability staining and ATP determination appeared to demonstrate higher survivors than plate counts. To study possible recovery of certain yeast cells damaged by electrolysis thus rendering them nonculturable, yeast suspensions were stored in phosphate buffer at 4 and 20 degrees C. Increase in viable counts and ATP content of treated yeast cells was observed during storage at 20 degrees C, whereas viable counts of treated and control yeast cells were shown to decrease during storage at 4 degrees C. The increase in the number of viable cells appeared to be the result of repair of damaged cells rather than regrowth of few cells remaining culturable. The lethal efficacy of electrolysis might be overestimated by plate counts. Further experiments must be done to evaluate the lethal efficacy of electrolysis on microorganisms in real conditions encountered in food products.

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.

The fabrication of compact active optical waveguides requires high concentrations of rare-earth ions. In highly doped materials the optical gain is affected by cross-relaxation and up-conversion processes. By solving the rate equations for the levels involved in radiative transitions, the optical gain is obtained as a function of the dopant concentration in the case of Er-doped silica pumped at 980 nm. The performance of channel waveguides from the same material is analyzed; in particular, the dependence on Er concentration of threshold pump power and amplifier gain is discussed. >

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.