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Biomedical subjects

M Federighi

Publications and source records attributed to M Federighi.

17 recordsLinked to original sources
PubMed2006

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.

Colony Count, Microbial↗
PubMed2005

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.

Colony Count, Microbial↗
PubMed2005

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.

Animals↗
PubMed2002

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.

Flow Cytometry↗
PubMed2001

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.

Colony Count, Microbial↗
PubMed2000

Effects of high hydrostatic pressure on membrane proteins of Salmonella typhimurium.

Salmonella typhimurium is a leading cause of foodborne diseases. Today high hydrostatic pressure treatments are considered as alternative methods of preservation. To select optimal conditions of treatment, we have to characterize the cell targets of pressure. In this study the action of pressure on the bacterial membrane proteins is analysed. The total membrane extract is obtained by lysis of cells separated by equilibrium density gradient centrifugation. Protein content is analysed by electrophoresis SDS-PAGE and visualised by silver stain. Electrophoretic profiles reveal the presence of three major outer membrane proteins and 12 minor proteins in control bacteria outer membranes. Outer membrane protein content is drastically modified after treatments. In some cases, except for the major proteins OmpA and LamB, other outer membrane proteins seem to totally disappear. LamB is more resistant to hyperbaric exposure when the pH of the media is acidic. This behaviour could be explained by a different conformation adopted by the LamB protein depending on the extracellular pH. This work allows us to define membrane proteins as a target of high hydrostatic pressure treatments. Knowledge of the behaviour of these bacterial membrane proteins subjected to pressure under different conditions (pH, temperature, a(w)...) could allow an increase in the efficiency of treatments.

Bacterial Proteins↗
PubMed2000

Demonstration of a protein synthesis in starved Campylobacter jejuni cells.

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.

Bacterial Proteins↗
PubMed2000

Physiological effects of high hydrostatic pressure treatments on Listeria monocytogenes and Salmonella typhimurium.

The effect of a high hydrostatic pressure treatment on the Gram-positive Listeria monocytogenes strain Scott A and the Gram-negative Salmonella typhimurium strain Mutton (ATCC13 311) has been determined in stationary phase cell suspensions. Pressure treatments were done at room temperature for 10 min in sodium citrate (pH 5.6) and sodium phosphate (pH 7.0) suspension buffers. Increasing pressure treatments resulted in an exponential decrease of cell counts. Salmonella typhimurium suspended at low pH was more sensitive to pressure treatments. Progressive morphological changes were evident with the pressure increase. Cell lysis only appeared with the highest pressure treatments. Cell volume was not affected by pressure treatment. A progressive decrease of deltapH (pHin - pHout), intracellular potassium and ATP contents was demonstrated with the pressure increase. A parallel lowering of membrane potentials was measured.

Adenosine Triphosphate↗
PubMed2000

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.

Acridine Orange↗
PubMed1999

Helicobacter species colonizing pig stomach: molecular characterization and determination of prevalence.

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.

Animals↗
PubMed1999

Recovery in embryonated eggs of viable but nonculturable Campylobacter jejuni cells and maintenance of ability to adhere to HeLa cells after resuscitation.

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.

Animals↗
PubMed1999

Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells.

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 microliter mg of protein-1 for the culturable form and 10.96 microliter 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.

Adenine Nucleotides↗
PubMed1991

Scanning and transmission electron microscopic study of adherence of Escherichia coli O103 enteropathogenic and/or enterohemorrhagic strain GV in enteric infection in rabbits.

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.

Animals↗
PubMed1991

R plasmid in Escherichia coli O103 coding for colonization of the rabbit intestinal tract.

One rabbit pathogenic Escherichia coli strain, belonging to serogroup O103, harbors a self-transferable 117-kb plasmid (pREC-1) encoding resistance to several antibiotics. The role of this R plasmid in the colonization of the digestive tract in specific-pathogen-free (E. coli O103-free) rabbits was studied. Five-week-old rabbits were inoculated with the wild-type strain, with its variant cured of the plasmid, with an E. coli K-12 strain, or with an untypeable E. coli strain from a healthy rabbit. No symptoms and no mortality were observed in animals inoculated with strains without the plasmid pREC-1, but 87.5% of the rabbits infected by the wild strain died, generally with bloody diarrhea, between days 5 and 15 postinfection. The weight gain of animals was strongly reduced. Transfer of the plasmid to the cured strain or to nonvirulent strains led these strains to induce the same pathology but with a lower mortality. Colonization of the gut by the O103 strain and symptoms of bloody diarrhea are thus related to the presence of the pREC-1 plasmid. The GV strain, which does not produce classical heat-labile enterotoxin or heat-stable enterotoxin and is not invasive, could be considered an enteropathogenic E. coli-like strain. The presence of a conjugative plasmid such as pREC-1 encoding both antibiotic resistance and virulence determinants in O103 E. coli from rabbits could represent a prominent epidemiological hazard under selective pressure by antibiotic therapy.

Animals↗
PubMedDate not supplied

Double staining (CTC-DAPI) for detection and enumeration of viable but non-culturable Campylobacter jejuni cells.

Direct microscopic enumeration of viable Campylobacter jejuni cells (ie, respiring bacteria) were performed in both culturable and non-culturable states. Five different C jejuni strains were used, including a reference strain, ATCC 33291. Cells from all five strains were incubated alone with 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), a redox dye. It was reduced by an electron transport chain to an insoluble red fluorescent CTC formazan salt, which accumulated intracellularly. The presence of these red CTC crystals in the bacteria cells was indicative of cellular respiratory activity. Counterstaining with 4'-6 diamino-2 phenylindole (DAPI), which fluoresces in blue, made a suitable contrast and allowed simultaneous enumeration of total and viable bacteria on a single filter. Four hours of incubation with 5 mM CTC under a microaerobic atmosphere was found to be the optimal condition yielding the maximum number of respiring cells (both culturable and non-culturable). When used in combination with standard culture techniques, double staining makes it possible to monitor the viable but non-culturable cells of jejuni obtained by starvation more easily than with the direct viable count procedure.

Animals↗
PubMedDate not supplied

High hydrostatic pressure inactivation of Salmonella typhimurium: effects of pressure, duration, pH and temperature studied by analysis of variance.

High hydrostatic pressure treatments are regarded as possible alternative methods for food preservation. One of the primary considerations for industrial applications is the ability of these methods to eradicate pathogenic microorganisms. This study subjected S. typhimurium suspensions, first in a phosphate buffer (pH 7.0) and then in a citrate phosphate buffer (pH 5.6), to high hydrostatic pressure treatments relative to the following variables: pressure (200-400 MPa), duration (3, 10 and 20 min), temperature (4, 20 and 40 degrees 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 Log10 comparisons of the numbers of live cells before and after treatment. A statistically significant relationship was found between the four variables considered (pressure, pH, duration and temperature), their interactions (duration x pressure, pH x temperature, pH x pressure) and the inactivation of S. typhimurium. R-squared statistical analysis indicated that the linear model used accounted for more than 98% of the variability in the inactivation of S. typhimurium.

Algorithms↗