Influence of the natural microbial flora on the acid tolerance response of Listeria monocytogenes in a model system of fresh meat decontamination fluids

Depending on its composition and metabolic activity, the natural flora that may be established in a meat plant environment can affect the survival, growth, and acid tolerance response (ATR) of bacterial pathogens present in the same niche. To investigate this hypothesis, changes in populations and ATR of inoculated (10(5) CFU/ml) Listeria monocytogenes were evaluated at 35 degrees C in water (10 or 85 degrees C) or acidic (2% lactic or acetic acid) washings of beef with or without prior filter sterilization. The model experiments were performed at 35 degrees C rather than lower (8.0 log CFU/ml) by day 1. The pH of inoculated water washings decreased or increased depending on absence or presence of natural flora, respectively. These microbial and pH changes modulated the ATR of L. monocytogenes at 35 degrees C. In filter-sterilized water washings, inoculated L. monocytogenes increased its ATR by at least 1.0 log CFU/ml from days 1 to 8, while in unfiltered water washings the pathogen was acid tolerant at day 1 (0.3 to 1.4 log CFU/ml reduction) and became acid sensitive (3.0 to >5.0 log CFU/ml reduction) at day 8. These results suggest that the predominant gram-negative flora of an aerobic fresh meat plant environment may sensitize bacterial pathogens to acid.     

Efficacy of Electrolyzed Oxidizing Water for Inactivating Escherichia coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes

The efficacy of electrolyzed oxidizing water for inactivating Escherichia coli O157:H7, Salmonella enteritidis, and Listeria monocytogenes was evaluated. A five-strain mixture of E. coli O157:H7, S. enteritidis, or L. monocytogenes of approximately 10⁸ CFU/ml was inoculated in 9 ml of electrolyzed oxidizing water (treatment) or 9 ml of sterile, deionized water (control) and incubated at 4 or 23°C for 0, 5, 10, and 15 min; at 35°C for 0, 2, 4, and 6 min; or at 45°C for 0, 1, 3, and 5 min. The surviving population of each pathogen at each sampling time was determined on tryptic soy agar. At 4 or 23°C, an exposure time of 5 min reduced the populations of all three pathogens in the treatment samples by approximately 7 log CFU/ml, with complete inactivation by 10 min of exposure. A reduction of ≥7 log CFU/ml in the levels of the three pathogens occurred in the treatment samples incubated for 1 min at 45°C or for 2 min at 35°C. The bacterial counts of all three pathogens in control samples remained the same throughout the incubation at all four temperatures. Results indicate that electrolyzed oxidizing water may be a useful disinfectant, but appropriate applications need to be validated.     

Survival or Growth of Escherichia coli O157:H7 in a Model System of Fresh Meat Decontamination Runoff Waste Fluids and Its Resistance to Subsequent Lactic Acid Stress

A potential may exist for survival of and resistance development by Escherichia coli O157:H7 in environmental niches of meat plants applying carcass decontamination interventions. This study evaluated (i) survival or growth of acid-adapted and nonadapted E. coli O157:H7 strain ATCC 43895 in acetic acid (pH 3.6 ± 0.1) or in water (pH 7.2 ± 0.2) fresh beef decontamination runoff fluids (washings) stored at 4, 10, 15, or 25°C and (ii) resistance of cells recovered from the washings after 2 or 7 days of storage to a subsequent lactic acid (pH 3.5) stress. Corresponding cultures in sterile saline or in heat-sterilized water washings were used as controls. In acetic acid washings, acid-adapted cultures survived better than nonadapted cultures, with survival being greatest at 4°C and lowest at 25°C. The pathogen survived without growth in water washings at 4 and 10°C, while it grew by 0.8 to 2.7 log cycles at 15 and 25°C, and more in the absence of natural flora. E. coli O157:H7 cells habituated without growth in water washings at 4 or 10°C were the most sensitive to pH 3.5, while cells grown in water washings at 15 or 25°C were relatively the most resistant, irrespective of previous acid adaptation. Resistance to pH 3.5 of E. coli O157:H7 cells habituated in acetic acid washings for 7 days increased in the order 15°C > 10°C > 4°C, while at 25°C cells died off. These results indicate that growth inhibition by storage at low temperatures may be more important than competition by natural flora in inducing acid sensitization of E. coli O157:H7 in fresh meat environments. At ambient temperatures in meat plants, E. coli O157:H7 may grow to restore acid resistance, unless acid interventions are applied to inhibit growth and minimize survival of the pathogen. Acid-habituated E. coli O157:H7 at 10 to 15°C may maintain a higher acid resistance than when acid habituated at 4°C. These responses should be evaluated with fresh meat and may be useful for the optimization of decontamination programs and postdecontamination conditions of meat handling.     

Control of Listeria monocytogenes in a Biofilm by Competitive-Exclusion Microorganisms

Biofilms from drains in food processing facilities with a recent history of no detectable Listeria monocytogenes in floor drains were cultured for microorganisms producing antilisterial metabolites. A total of 413 microbial isolates were obtained from 12 drain biofilm samples and were assayed at 15 and 37°C for activities that were bactericidal or inhibitory to L. monocytogenes, by two agar plate assays. Twenty-one of 257 bacterial isolates and 3 of 156 yeast isolates had antilisterial activity. All 24 isolates which produced metabolites inhibitory to L. monocytogenes were assayed for antilisterial activity in coinoculated broth cultures containing tryptic soy broth with yeast extract (TSB-YE). A five-strain mixture of 10³ CFU of L. monocytogenes/ml and 10⁵ CFU of the candidate competitive-exclusion microorganism/ml was combined in TSB-YE and incubated at 37°C for 24 h, 15°C for 14 days, 8°C for 21 days, and 4°C for 28 days. Substantial inhibition of L. monocytogenes growth (4 to 5 log CFU/ml) was observed for nine bacterial isolates at 37°C, two at 15 and 8°C, and three at 4°C. The inhibitory isolates were identified as Enterococcus durans (six isolates), Lactococcus lactis subsp. lactis (two isolates), and Lactobacillus plantarum (one isolate). The anti-L. monocytogenes activity of these isolates was evaluated in biofilms of L. monocytogenes on stainless steel coupons at 37, 15, 8, and 4°C. Results revealed that two isolates (E. durans strain 152 and L. lactis subsp. lactis strain C-1-92) were highly inhibitory to L. monocytogenes (growth inhibition of >5 log₁₀ CFU of L. monocytogenes/cm²). These two bacterial isolates appear to be excellent competitive-exclusion candidates to control L. monocytogenes in biofilms at environmental temperatures of 4 to 37°C.     

Effect of Immersion Solutions Containing Enterocin AS-48 on Listeria monocytogenes in Vegetable Foods

The effect of immersion solutions containing enterocin AS-48 alone or in combination with chemical preservatives on survival and proliferation of Listeria monocytogenes CECT 4032 inoculated on fresh alfalfa sprouts, soybean sprouts, and green asparagus was tested. Immersion treatments (5 min at room temperature) with AS-48 solutions (25 μg/ml) reduced listeria counts of artificially contaminated alfalfa and soybean sprouts by approximately 2.0 to 2.4 log CFU/g compared to a control immersion treatment in distilled water. The same bacteriocin immersion treatment applied on green asparagus had a very limited effect. During storage of vegetable samples treated with immersion solutions of 12.5 and 25 μg of AS-48/ml, viable listeria counts were reduced below detection limits at days 1 to 7 for alfalfa and soybean sprouts at 6 and 15°C, as well as green asparagus at 15°C. Only a limited inhibition of listeria proliferation was detected during storage of bacteriocin-treated alfalfa sprouts and green asparagus at 22°C. Treatment with solutions containing AS-48 plus lactic acid, sodium lactate, sodium nitrite, sodium nitrate, trisodium phosphate, trisodium trimetaphosphate, sodium thiosulphate, n-propyl p-hydroxybenzoate, p-hydoxybenzoic acid methyl ester, hexadecylpyridinium chloride, peracetic acid, or sodium hypochlorite reduced viable counts of listeria below detection limits (by approximately 2.6 to 2.7 log CFU/g) upon application of the immersion treatment and/or further storage for 24 h, depending of the chemical preservative concentration. Significant increases of antimicrobial activity were also detected for AS-48 plus potassium permanganate and in some combinations with acetic acid, citric acid, sodium propionate, and potassium sorbate.     

Use of Ultrasounds to Reduce the Count of Campylobacter coli in Water

The present study aimed to evaluate the effectiveness of low-frequency ultrasounds applied to eliminate Campylobacter spp. from water. The strains used in this research were isolated from water contaminated with sewage. Campylobacter coli alone was detected in the samples and used for further research. The reference strain C. coli ATCC 33559 was simultaneously tested. The isolate was exposed to ultrasounds at frequencies of 37 kHz and 80 kHz in a continuous operation device with ultrapure deionized water. After 5 min of sonication, the count of C. coli decreased by 5.78% (37 kHz) and 6.27% (80 kHz), whereas the temperature increased by 3°C (37 kHz), and 6°C (80 kHz). After 30 min of sonication, the death rates of bacterial cells were 40.15% (37 kHz) and 55.10% (80 kHz), whereas the temperature reached the maximum values of 36°C (37 kHz), and 39°C (80 kHz). Sonication at the frequency of 80 kHz reduced the bacterial count from 6.86 log CFU/ml to 3.08 log CFU/ml, whereas the frequency of 37 kHz reduced the bacterial count from 6.75 log CFU/ml to 4.04 log CFU/ml. Despite significant differences (p < 0.05) in the number of C. coli cells, the cell death rate remained at the same level. Open in a separate window     

Pulsed Electric Field Alters Molecular Chaperone Expression and Sensitizes Listeria monocytogenes to Heat

Pulsed electric field (PEF)-resistant and PEF-sensitive Listeria monocytogenes strains were sublethally treated with electric pulses at 15 kV/cm for 29 μs and held at 25°C for 5 to 30 min prior to protein extraction. The levels of the molecular chaperones GroEL, GroES, and DnaJ were determined by immunoblotting. After 10 to 20 min after sublethal PEF treatment, a transient decrease in molecular chaperone expression was observed in the PEF-sensitive strain (Scott A). The levels of GroEL and DnaJ increased back to the basal expression level within 30 min. A substantial decrease in GroES expression persisted for at least 30 min after PEF treatment. Chaperone expression was suppressed after PEF treatment to a smaller extent in the PEF-resistant (OSY-8578) than in the PEF-sensitive strain, and no clear expression pattern was identified in OSY-8578. Inactivation of Scott A and OSY-8578 in phosphate buffer was compared when lethal PEF (27.5 kV/cm, 144 μs) and heat (55°C, 10 min) were applied in sequence. When PEF and heat treatments were applied separately, the populations of L. monocytogenes Scott A and OSY-8578 decreased 0.5 to 0.6 log CFU/ml. Cells treated first with PEF and incubated at 25°C for 10 min showed substantial sensitivity to subsequent heat treatment; the decrease in counts for Scott A and OSY-8578 was 6.1 and 2.8 log CFU/ml, respectively. The sequence and time lapse between the two treatments were crucial for achieving high inactivation rates. It is concluded that PEF sensitized L. monocytogenes to heat and that maximum heat sensitization occurred when chaperone expression was at a minimum level.     

Antimicrobial Effects of Mustard Flour and Acetic Acid against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Serovar Typhimurium

This study was designed to investigate the individual and combined effects of mustard flour and acetic acid in the inactivation of food-borne pathogenic bacteria stored at 5 and 22°C. Samples were prepared to achieve various concentrations by the addition of acetic acid (0, 0.5, or 1%) along with mustard flour (0, 10, or 20%) and 2% sodium chloride (fixed amount). Acid-adapted three-strain mixtures of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium strains (10⁶ to 10⁷ CFU/ml) were inoculated separately into prepared mustard samples stored at 5 and 22°C, and samples were assayed periodically. The order of bacterial resistance, assessed by the time required for the nominated populations to be reduced to undetectable levels against prepared mustards at 5°C, was S. enterica serovar Typhimurium (1 day) < E. coli O157:H7 (3 days) < L. monocytogenes (9 days). The food-borne pathogens tested were reduced much more rapidly at 22°C than at 5°C. There was no synergistic effect with regard to the killing of the pathogens tested with the addition of 0.5% acetic acid to the mustard flour (10 or 20%). Mustard in combination with 0.5% acetic acid had less bactericidal activity against the pathogens tested than did mustard alone. The reduction of E. coli O157:H7 and L. monocytogenes among the combined treatments on the same storage day was generally differentiated as follows: control < mustard in combination with 0.5% acetic acid < mustard alone < mustard in combination with 1% acetic acid < acetic acid alone. Our study indicates that acidic products may limit microbial growth or survival and that the addition of small amounts of acetic acid (0.5%) to mustard can retard the reduction of E. coli O157:H7 and L. monocytogenes. These antagonistic effects may be changed if mustard is used alone or in combination with >1% acetic acid.     

Selection and Identification of a Listeria monocytogenes Target Strain for Pulsed Electric Field Process Optimization

Nine Listeria monocytogenes strains were treated individually with a continuous pulsed electric field (PEF) apparatus, and their sensitivities to the treatment were compared at 25 kV/cm. When cell suspensions of these strains in 0.1% NaCl (pH 7.0) were treated at 23°C for 144 μs, inactivation ranged from 0.7 to 3.7 log₁₀ CFU/ml. Inactivation by 72-μs PEF treatments at 37°C ranged from 0.3 to 2.5 log₁₀ CFU/ml. L. monocytogenes OSY-8578 was substantially more resistant than other strains when cells were PEF treated in 0.1% NaCl, whereas Scott A was one of the most sensitive strains. The superiority of OSY-8578's resistance to that of Scott A was confirmed in 50% diluted acid whey (pH 4.2). Changes in sensitivity to PEF during phases of growth were minimal in OSY-8578 and substantial in Scott A. Use of L. monocytogenes OSY-8578, therefore, is recommended in studies to optimize PEF processes that target L. monocytogenes. The nine L. monocytogenes strains were genotyped with pulsed-field gel electrophoresis (PFGE) and arbitrarily primed PCR (AP-PCR) techniques. These strains were better differentiated with PFGE than with AP-PCR. The target strain (OSY-8578) was characterized by both molecular typing techniques, but resistance to PEF, in general, was not associated with a particular genotype group.     

Detection of Low Levels of Listeria monocytogenes Cells by Using a Fiber-Optic Immunosensor

Biosensor technology has a great potential to meet the need for sensitive and nearly real-time microbial detection from foods. An antibody-based fiber-optic biosensor to detect low levels of Listeria monocytogenes cells following an enrichment step was developed. The principle of the sensor is a sandwich immunoassay where a rabbit polyclonal antibody was first immobilized on polystyrene fiber waveguides through a biotin-streptavidin reaction to capture Listeria cells on the fiber. Capture of cells on the fibers was confirmed by scanning electron microscopy. A cyanine 5-labeled murine monoclonal antibody, C11E9, was used to generate a specific fluorescent signal, which was acquired by launching a 635-nm laser light from an Analyte 2000 and collected by a photodetector at 670 to 710 nm. This immunosensor was specific for L. monocytogenes and showed a significantly higher signal strength than for other Listeria species or other microorganisms, including Escherichia coli, Enterococcus faecalis, Salmonella enterica, Lactobacillus plantarum, Carnobacterium gallinarum, Hafnia alvei, Corynebacterium glutamicum, Enterobacter aerogenes, Pseudomonas aeruginosa, and Serratia marcescens, in pure or in mixed-culture setup. Fiber-optic results could be obtained within 2.5 h of sampling. The sensitivity threshold was about 4.3 × 10³ CFU/ml for a pure culture of L. monocytogenes grown at 37°C. When L. monocytogenes was mixed with lactic acid bacteria or grown at 10°C with 3.5% NaCl, the detection threshold was 4.1 × 10⁴ or 2.8 × 10⁷ CFU/ml, respectively. In less than 24 h, this method could detect L. monocytogenes in hot dog or bologna naturally contaminated or artificially inoculated with 10 to 1,000 CFU/g after enrichment in buffered Listeria enrichment broth.     

Attachment of and Biofilm Formation by Enterobacter sakazakii on Stainless Steel and Enteral Feeding Tubes

Enterobacter sakazakii has been reported to form biofilms, but environmental conditions affecting attachment to and biofilm formation on abiotic surfaces have not been described. We did a study to determine the effects of temperature and nutrient availability on attachment and biofilm formation by E. sakazakii on stainless steel and enteral feeding tubes. Five strains grown to stationary phase in tryptic soy broth (TSB), infant formula broth (IFB), or lettuce juice broth (LJB) at 12 and 25°C were examined for the extent to which they attach to these materials. Higher populations attached at 25°C than at 12°C. Stainless steel coupons and enteral feeding tubes were immersed for 24 h at 4°C in phosphate-buffered saline suspensions (7 log CFU/ml) to facilitate the attachment of 5.33 to 5.51 and 5.03 to 5.12 log CFU/cm², respectively, before they were immersed in TSB, IFB, or LJB, followed by incubation at 12 or 25°C for up to 10 days. Biofilms were not produced at 12°C. The number of cells of test strains increased by 1.42 to 1.67 log CFU/cm² and 1.16 to 1.31 log CFU/cm² in biofilms formed on stainless steel and feeding tubes, respectively, immersed in IFB at 25°C; biofilms were not formed on TSB and LJB at 25°C, indicating that nutrient availability plays a major role in processes leading to biofilm formation on the surfaces of these inert materials. These observations emphasize the importance of temperature control in reconstituted infant formula preparation and storage areas in preventing attachment and biofilm formation by E. sakazakii.     

Inactivation of Enterohemorrhagic Escherichia coli in Rumen Content- or Feces-Contaminated Drinking Water for Cattle

Cattle drinking water is a source of on-farm Escherichia coli O157:H7 transmission. The antimicrobial activities of disinfectants to control E. coli O157:H7 in on-farm drinking water are frequently neutralized by the presence of rumen content and manure that generally contaminate the drinking water. Different chemical treatments, including lactic acid, acidic calcium sulfate, chlorine, chlorine dioxide, hydrogen peroxide, caprylic acid, ozone, butyric acid, sodium benzoate, and competing E. coli, were tested individually or in combination for inactivation of E. coli O157:H7 in the presence of rumen content. Chlorine (5 ppm), ozone (22 to 24 ppm at 5°C), and competing E. coli treatment of water had minimal effects (<1 log CFU/ml reduction) on killing E. coli O157:H7 in the presence of rumen content at water-to-rumen content ratios of 50:1 (vol/wt) and lower. Four chemical-treatment combinations, including (i) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.05% caprylic acid (treatment A); (ii) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.1% sodium benzoate (treatment B); (iii) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 0.5% butyric acid (treatment C); and (iv) 0.1% lactic acid, 0.9% acidic calcium sulfate, and 100 ppm chlorine dioxide (treatment D); were highly effective (>3 log CFU/ml reduction) at 21°C in killing E. coli O157:H7, O26:H11, and O111:NM in water heavily contaminated with rumen content (10:1 water/rumen content ratio [vol/wt]) or feces (20:1 water/feces ratio [vol/wt]). Among them, treatments A, B, and C killed >5 log CFU E. coli O157:H7, O26:H11, and O111:NM/ml within 30 min in water containing rumen content or feces, whereas treatment D inactivated approximately 3 to 4 log CFU/ml under the same conditions. Cattle given water containing treatment A or C or untreated water (control) ad libitum for two 7-day periods drank 15.2, 13.8, and 30.3 liters/day, respectively, and cattle given water containing 0.1% lactic acid plus 0.9% acidic calcium sulfate (pH 2.1) drank 18.6 liters/day. The amounts of water consumed for all water treatments were significantly different from that for the control, but there were no significant differences among the water treatments. Such treatments may best be applied periodically to drinking water troughs and then flushed, rather than being added continuously, to avoid reduced water consumption by cattle.     

Survival and Heat Resistance of Listeria monocytogenes after Exposure to Alkali and Chlorine

A strain of Listeria monocytogenes isolated from a drain in a food-processing plant was demonstrated, by determination of D values, to be more resistant to the lethal effect of heat at 56 or 59°C following incubation for 45 min in tryptose phosphate broth (TPB) at pH 12.0 than to that of incubation for the same time in TPB at pH 7.3. Cells survived for at least 6 days when they were suspended in TPB at pHs 9.0, 10.0, and 11.0 and stored at 4 or 21°C. Cells of L. monocytogenes incubated at 37°C for 45 min and then stored for 48 or 144 h in TPB at pH 10.0 were more resistant to heat treatment at 56°C than were cells stored in TPB at pH 7.3. The alkaline-stress response in L. monocytogenes may induce resistance to otherwise lethal thermal-processing conditions. Treatment of cells in 0.05 M potassium phosphate buffer (pH 7.00 ± 0.05) containing 2.0 or 2.4 mg of free chlorine per liter reduced populations by as much as 1.3 log₁₀ CFU/ml, while treatment with 6.0 mg of free chlorine per liter reduced populations by as much as 4.02 log₁₀ CFU/ml. Remaining subpopulations of chlorine-treated cells exhibited some injury, and cells treated with chlorine for 10 min were more sensitive to heating at 56°C than cells treated for 5 min. Contamination of foods by L. monocytogenes cells that have survived exposure to processing environments ineffectively cleaned or sanitized with alkaline detergents or disinfectants may have more severe implications than previously recognized. Alkaline-pH-induced cross-protection of L. monocytogenes against heat has the potential to enhance survival in minimally processed as well as in heat-and-serve foods and in foods on holding tables, in food service facilities, and in the home. Cells surviving exposure to chlorine, in contrast, are more sensitive to heat; thus, the effectiveness of thermal processing in achieving desired log₁₀-unit reductions is not compromised in these cells.     

Ability of Shiga Toxin-Producing Escherichia coli and Salmonella spp. To Survive in a Desiccation Model System and in Dry Foods

In order to determine desiccation tolerances of bacterial strains, the survival of 58 diarrheagenic strains (18 salmonellae, 35 Shiga toxin-producing Escherichia coli [STEC], and 5 shigellae) and of 15 nonpathogenic E. coli strains was determined after drying at 35°C for 24 h in paper disks. At an inoculum level of 10⁷ CFU/disk, most of the salmonellae (14/18) and the STEC strains (31/35) survived with a population of 10³ to 10⁴ CFU/disk, whereas all of the shigellae (5/5) and the majority of the nonpathogenic E. coli strains (9/15) did not survive (the population was decreased to less than the detection limit of 10² CFU/disk). After 22 to 24 months of subsequent storage at 4°C, all of the selected salmonellae (4/4) and most of the selected STEC strains (12/15) survived, keeping the original populations (10³ to 10⁴ CFU/disk). In contrast to the case for storage at 4°C, all of 15 selected strains (5 strains each of Salmonella spp., STEC O157, and STEC O26) died after 35 to 70 days of storage at 25°C and 35°C. The survival rates of all of these 15 strains in paper disks after the 24 h of drying were substantially increased (10 to 79 times) by the presence of sucrose (12% to 36%). All of these 15 desiccated strains in paper disks survived after exposure to 70°C for 5 h. The populations of these 15 strains inoculated in dried foods containing sucrose and/or fat (e.g., chocolate) were 100 times higher than those in the dried paper disks after drying for 24 h at 25°C.     

Occurrence of Shewanella algae in Danish Coastal Water and Effects of Water Temperature and Culture Conditions on Its Survival

The marine bacterium Shewanella algae, which was identified as the cause of human cases of bacteremia and ear infections in Denmark in the summers of 1994 and 1995, was detected in seawater only during the months (July, August, September, and October) when the water temperature was above 13°C. The bacterium is a typical mesophilic organism, and model experiments were conducted to elucidate the fate of the organism under cold and nutrient-limited conditions. The culturable count of S. algae decreased rapidly from 10⁷ CFU/ml to 10¹ CFU/ml in approximately 1 month when cells grown at 20 to 37°C were exposed to cold (2°C) seawater. In contrast, the culturable count of cells exposed to warmer seawater (10 to 25°C) remained constant. Allowing the bacterium a transition period in seawater at 20°C before exposure to the 2°C seawater resulted in 100% survival over a period of 1 to 2 months. The cold protection offered by this transition (starvation) probably explains the ability of the organism to persist in Danish seawater despite very low (0 to 1°C) winter water temperatures. The culturable counts of samples kept at 2°C increased to 10⁵ to 10⁷ CFU/ml at room temperature. Most probable number analysis showed this result to be due to regrowth rather than resuscitation. It was hypothesized that S. algae would survive cold exposure better if in the biofilm state; however, culturable counts from S. algae biofilms decreased as rapidly as did counts of planktonic cells.     

Effects of Combined Shear and Thermal Forces on Destruction of Microbacterium lacticum

A twin-screw extruder and a rotational rheometer were used to generate shear forces in concentrated gelatin inoculated with a heat-resistant isolate of a vegetative bacterial species, Microbacterium lacticum. Shear forces in the extruder were mainly controlled by varying the water feed rate. The water content of the extrudates changed between 19 and 45% (wet weight basis). Higher shear forces generated at low water contents and the calculated die wall shear stress correlated strongly with bacterial destruction. No surviving microorganisms could be detected at the highest wall shear stress of 409 kPa, giving log reduction of 5.3 (minimum detection level, 2 × 10⁴ CFU/sample). The mean residence time of the microorganism in the extruder was 49 to 58 s, and the maximum temperature measured in the end of the die was 73°C. The D_75°C of the microorganism in gelatin at 65% water content was 20 min. It is concluded that the physical forces generated in the reverse screw element and the extruder die rather than heat played a major part in cell destruction. In a rotational rheometer, after shearing of a mix of microorganisms with gelatin at 65% (wt/wt) moisture content for 4 min at a shear stress of 2.8 kPa and a temperature of 75°C, the number of surviving microorganisms in the sheared sample was 5.2 × 10⁶ CFU/g of sample compared with 1.4 × 10⁸ CFU/g of sample in the nonsheared control. The relative effectiveness of physical forces in the killing of bacteria and destruction of starch granules is discussed.     

Modeling the Growth/No-Growth Boundaries of Postprocessing Listeria monocytogenes Contamination on Frankfurters and Bologna Treated with Lactic Acid

This study developed models to predict lactic acid concentration, dipping time, and storage temperature combinations determining growth/no-growth interfaces of Listeria monocytogenes at desired probabilities on bologna and frankfurters. L. monocytogenes was inoculated on bologna and frankfurters, and 75 combinations of lactic acid concentrations, dipping times, and storage temperatures were tested. Samples were stored in vacuum packages for up to 60 days, and bacterial populations were enumerated on tryptic soy agar plus 0.6% yeast extract and Palcam agar on day zero and at the end point of storage. The combinations that allowed L. monocytogenes increases of ≥1 log CFU/cm² were assigned the value of 1 (growth), and the combinations that had increases of <l log CFU/cm² were given the value of 0 (no growth). These binary growth response data were fitted to logistic regression to develop a model predicting probabilities of growth. Validation with existing data and various indices showed acceptable model performance. Thus, the models developed in this study may be useful in determining probabilities of growth and in selecting lactic acid concentrations and dipping times to control L. monocytogenes growth on bologna and frankfurters, while the procedures followed may also be used to develop models for other products, conditions, or pathogens.     

Inhibition of Listeria monocytogenes in Cooked Ham by Virulent Bacteriophages and Protective Cultures

Protective cultures can be used successfully as an additional hurdle together with phages to reduce growth of Listeria monocytogenes on sliced cooked ham. Addition of phages resulted in a rapid 10-fold reduction of L. monocytogenes. After 14 to 28 days of storage, a 100-fold reduction was observed in samples with phages and protective culture compared to results for samples with phages alone.Listeriosis in Europe has an average incidence between 2 and 10 reported cases per million population per year (7). Listeria monocytogenes is found in raw and ready-to-eat (RTE) products, poultry, seafood, and dairy products. A review of the incidence and transmission of L. monocytogenes in RTE products has been published by Lianou and Sofos (11). The USDA has implemented a “zero-tolerance” policy for L. monocytogenes in RTE products (2). In the European Union, the limit for common RTE foods is 100 CFU/g (1). Recently, Codex Alimentarius adopted new standards for L. monocytogenes in RTE foods, with a limit of 100 CFU/g in foods where L. monocytogenes cannot grow and absence in foods where the bacterium can grow. However, an alternative approach is accepted. Competent authorities may choose to establish and implement other validated limits (http://www.codexalimentarius.net/web/archives.jsp?lang=en, Alinorm 09/32/REP and Alinorm 09/32/13). L. monocytogenes in cooked products is connected with cross-contamination after heat treatment (11, 12). Bacteriophages have been successfully applied to a number of food products to reduce the level of contaminating L. monocytogenes (6, 8-10, 13, 14). The effect of phages varies with the type of product and is strongly dose dependent (6, 8). Active phages can be recovered from foods after long storage, but the phage particles appear to become immobilized soon after addition to nonliquid foods and therefore, due to limited diffusion, cannot infect bacteria (8). Bacteria surviving phage treatment can later grow in the product. Additional hurdles should therefore be present to inhibit later outgrowth of L. monocytogenes.We have previously employed Lactobacillus sakei TH1 as a protective culture against L. monocytogenes (4, 5). Here we examine the combined use of phages and protective culture to reduce outgrowth of L. monocytogenes on cooked ham.Rifampin (rifampicin)-resistant mutants of L. monocytogenes 2230/92 serotype 1, implicated in a listeriosis outbreak in Norway (12), and L. monocytogenes 167 serotype 4b were grown overnight in brain heart infusion (Difco Laboratories, Detroit, MI) at 37°C without shaking and stored at 4°C for 24 h (3-5). Cells were diluted in 0.9% NaCl and plated on brain heart infusion agar with 200 μg/ml rifampin. L. sakei TH1 was grown at 30°C in MRS (de Man, Rogosa, Sharpe) broth (CM 359; Oxoid, Hampshire, England) (pH 6.2) and plated on MRS agar (5). Listex P100 phages, 2 × 10¹¹ PFU/ml, were from EBI Food Safety (Wageningen, The Netherlands).Ten-gram slices of hams with 2.3% NaCl and 0.01% disodium diphosphate (pH 6.2; a_w > 0.97), made at Nofima''s pilot plant (Aas, Norway), were inoculated with a cold-adapted 1:1 mixture of L. monocytogenes 2230/92 and 167. Bacteria were spread in 100 μl 0.9% NaCl over the 80-cm² surface area of each slice to 10³ CFU/cm² using a bent glass rod. After 1 h at 20°C, phages (5 × 10⁷ PFU/cm² in a total volume of 100 μl) were spread over the same surface. After one additional hour, 10³ CFU/cm² L. sakei TH1 in 100 μl 0.9% NaCl was added where appropriate. The slices were vacuum packed and stored at 10°C. Growth was measured before and after spiking and at 0, 3, 7, 14, and 28 days after homogenizing the slices in 100 ml 0.9% NaCl in a Stomacher homogenizer. No lactic acid bacteria were detected in uninoculated samples. Experiments were performed with three parallel samples. L. monocytogenes alone grew from 10⁴ CFU/g at the onset of the experiment to 10⁷ CFU/g the first 7 days, reached 2 × 10⁸ CFU/g after 14 days, and remained unchanged thereafter (Fig. ​(Fig.1).1). In samples with both L. monocytogenes and phages, a rapid 1-log reduction in L. monocytogenes was observed. Surviving L. monocytogenes, however, grew as well as that in the phage-free controls, reaching >10⁷ CFU/g after 14 days. In samples where both P100 phages and L. sakei TH1 were added, the same initial reduction of L. monocytogenes was observed, but the later outgrowth was reduced by the fast-growing lactic acid bacteria and the L. monocytogenes levels were 2 logs lower than those with P100 phages alone after 28 days of incubation. The phages did not influence the growth and survival of L. sakei TH1. During the 28 days of storage, the pH changed from 6.20 to 6.05 in samples with L. monocytogenes and to 6.00 in samples with both L. monocytogenes and L. sakei TH1. The results were reproduced in a separate repetition of the experiment at 10°C (not shown).Open in a separate windowFIG. 1.Inhibition of L. monocytogenes in cooked ham with bacteriophages and protective culture at 10°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫), L. monocytogenes and 5 × 10⁷ PFU/cm² P100 phages (▪), or L. monocytogenes, 5 × 10⁷ PFU/cm² P100 phages, and 10³ CFU/cm² (approximately 10⁴ CFU/g) protective-culture L. sakei TH1 (▴) and stored at 10°C. Growth of L. sakei TH1 is shown by the broken line.The effect of the protective culture was dose dependent when 10⁴ CFU/g and 10⁶ CFU/g of L. sakei TH1 were added to slices of ham (Fig. ​(Fig.2).2). L. monocytogenes alone grew to 2 × 10⁸ CFU/g after 14 days. When L. sakei TH1 was added at a low concentration (10⁴ CFU/g), L. monocytogenes grew to approximately 4 × 10⁶ CFU/g, while when L. sakei TH1 was added at a high concentration, L. monocytogenes levels were 1 to 2 logs lower. The pHs in the low- and high-inoculum hams were reduced from the initial 6.20 to 6.16 and 6.02, respectively, at day 28. For hams stored at 4°C, slow growth of L. monocytogenes occurred between days 14 and 28 from 10⁴ to 10⁵ CFU/g (P = 0.003) (Fig. ​(Fig.3).3). With phages and L. sakei TH1 added, a rapid 1-log reduction of L. monocytogenes was observed due to the phage attack, and no growth was observed during the 28-day storage period. The L. sakei TH1 strain showed a longer lag phase at this low temperature but nevertheless reached 10⁷ CFU/g at day 14 and thereby inhibited any growth of L. monocytogenes.Open in a separate windowFIG. 2.Inhibition of L. monocytogenes in cooked ham inoculated with large or small amounts of protective culture at 10°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫), L. monocytogenes and 10⁶ CFU/g (10⁵ CFU/cm²) L. sakei TH1 (▴), or L. monocytogenes and 10⁴ CFU/g (10³ CFU/cm²) L. sakei TH1 (▪) and stored at 10°C. Growth of L. sakei TH1 is shown by broken lines. The L. monocytogenes control is the same control as in Fig. ​Fig.11.Open in a separate windowFIG. 3.Inhibition of L. monocytogenes in cooked ham with bacteriophages and protective culture at 4°C. Sliced ham was inoculated with 10³ CFU/cm² (corresponding to approximately 10⁴ CFU/g) L. monocytogenes (⧫) or L. monocytogenes, 5 × 10⁷ PFU/cm² P100 phages, and 10⁴ CFU/g (10³ CFU/cm²) protective-culture, L. sakei TH1 (▴) and stored at 4°C. Growth of L. sakei TH1 is shown by the broken line.Since L. sakei TH1 grows well at low temperatures, prevents growth of L. monocytogenes, and has no negative influence on the organoleptic properties of ham (4, 5), it can successfully be employed as an additional hurdle together with phages.We here chose to perform the storage experiments under “worst-case” conditions. Generally, the contamination levels of L. monocytogenes are lower than in our setup, in the range of 10 to 100 CFU/g (see reference 11 and references therein). Since L. sakei TH1 grows well at low temperatures (Fig. ​(Fig.3),3), its selective advantage will be greater at 4°C than at abuse temperatures. From the above, it is evident that it is possible to optimize L. monocytogenes inhibition by increasing both the phage titer and the starting amount of protective culture. An enhanced effect may also be experienced by modifying phage application, e.g., by using larger liquid volumes (6, 8).Emergence of resistant L. monocytogenes may be a potential problem when treating foods with phages. No emergence of resistance has been detected after phage treatment (6, 8). Such strategies as use of phage mixtures, phage rotation schemes, and treatment of products immediately prior to packaging may reduce eventual resistance problems (8). Some L. monocytogenes strains are naturally phage resistant (6). In these cases, a protective culture still constitutes a powerful hurdle.In conclusion, we have shown here that by applying phages and protective culture as two independent hurdles, it is possible to both reduce the number of L. monocytogenes bacteria on a product and inhibit outgrowth of eventual remaining surviving cells. This is a general method that can potentially be applied to different foods where there is a potential risk for growth of L. monocytogenes, provided a suitable protective culture is available.     

Inactivation of Mycobacterium paratuberculosis by Pulsed Electric Fields

The influence of treatment temperature and pulsed electric fields (PEF) on the viability of Mycobacterium paratuberculosis cells suspended in 0.1% (wt/vol) peptone water and in sterilized cow's milk was assessed by direct viable counts and by transmission electron microscopy (TEM). PEF treatment at 50°C (2,500 pulses at 30 kV/cm) reduced the level of viable M. paratuberculosis cells by approximately 5.3 and 5.9 log₁₀ CFU/ml in 0.1% peptone water and in cow's milk, respectively, while PEF treatment of M. paratuberculosis at lower temperatures resulted in less lethality. Heating alone at 50°C for 25 min or at 72°C for 25 s (extended high-temperature, short-time pasteurization) resulted in reductions of M. paratuberculosis of approximately 0.01 and 2.4 log₁₀ CFU/ml, respectively. TEM studies revealed that exposure to PEF treatment resulted in substantial damage at the cellular level to M. paratuberculosis.     

Variation in Resistance to Hydrostatic Pressure among Strains of Food-Borne Pathogens

Among food-borne pathogens, some strains could be resistant to hydrostatic pressure treatment. This information is necessary to establish processing parameters to ensure safety of pressure-pasteurized foods (N. Kalchayanand, A. Sikes, C. P. Dunne, and B. Ray, J. Food Prot. 61:425–431, 1998). We studied variation in pressure resistance among strains of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Salmonella species at two temperatures of pressurization. Early-stationary-phase cells in 1% peptone solution were pressurized at 345 MPa either for 5 min at 25°C or for 5, 10, or 15 min at 50°C. The viability loss (in log cycles) following pressurization at 25°C ranged from 0.9 to 3.5 among nine L. monocytogenes strains, 0.7 to 7.8 among seven S. aureus strains, 2.8 to 5.6 among six E. coli O157:H7 strains, and 5.5 to 8.3 among six Salmonella strains. The results show that at 25°C some strains of each species are more resistant to pressure than the others. However, when one resistant and one sensitive strain from each species were pressurized at 345 MPa and 50°C, the population of all except the resistant S. aureus strain was reduced by more than 8 log cycles within 5 min. Viability loss of the resistant S. aureus strain was 6.3 log cycles even after 15 min of pressurization. This shows that strains of food-borne pathogens differ in resistance to hydrostatic pressure (345 MPa) at 25°C, but this difference is greatly reduced at 50°C. Pressurization at 50°C, in place of 25°C, will ensure greater safety of foods.