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.     

Use of Hydrostatic Pressure for Inactivation of Microbial Contaminants in Cheese

The objective of this study was to determine the effect of high pressure (HP) on the inactivation of microbial contaminants in Cheddar cheese (Escherichia coli K-12, Staphylococcus aureus ATCC 6538, and Penicillium roqueforti IMI 297987). Initially, cheese slurries inoculated with E. coli, S. aureus, and P. roqueforti were used as a convenient means to define the effects of a range of pressures and temperatures on the viability of these microorganisms. Cheese slurries were subjected to pressures of 50 to 800 MPa for 20 min at temperatures of 10, 20, and 30°C. At 400 MPa, the viability of P. roqueforti in cheese slurry decreased by >2-log-unit cycles at 10°C and by 6-log-unit cycles at temperatures of 20 and 30°C. S. aureus and E. coli were not detected after HP treatments in cheese slurry of >600 MPa at 20°C and >400 MPa at 30°C, respectively. In addition to cell death, the presence of sublethally injured cells in HP-treated slurries was demonstrated by differential plating using nonselective agar incorporating salt or glucose. Kinetic experiments of HP inactivation demonstrated that increasing the pressure from 300 to 400 MPa resulted in a higher degree of inactivation than increasing the pressurization time from 0 to 60 min, indicating a greater antimicrobial impact of pressure. Selected conditions were subsequently tested on Cheddar cheese by adding the isolates to cheese milk and pressure treating the resultant cheeses at 100 to 500 MPa for 20 min at 20°C. The relative sensitivities of the isolates to HP in Cheddar cheese were similar to those observed in the cheese slurry, i.e., P. roqueforti was more sensitive than E. coli, which was more sensitive than S. aureus. The organisms were more sensitive to pressure in cheese than slurry, especially with E. coli. On comparison of the sensitivities of the microorganisms in a pH 5.3 phosphate buffer, cheese slurry, and Cheddar cheese, greatest sensitivity to HP was shown in the pH 5.3 phosphate buffer by S. aureus and P. roqueforti while greatest sensitivity to HP by E. coli was exhibited in Cheddar cheese. Therefore, the medium in which the microorganisms are treated is an important determinant of the level of inactivation observed.     

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 (10⁵ CFU/ml) Listeria monocytogenes were evaluated at 35°C in water (10 or 85°C) or acidic (2% lactic or acetic acid) washings of beef with or without prior filter sterilization. The model experiments were performed at 35°C rather than lower (≤15°C) temperatures to maximize the response of inoculated L. monocytogenes in the washings with or without competitive flora. Acid solution washings were free (<1.0 log CFU/ml) of natural flora before inoculation (day 0), and no microbial growth occurred during storage (35°C, 8 days). Inoculated L. monocytogenes died off (negative enrichment) in acid washings within 24 h. In nonacid (water) washings, the pathogen increased (approximately 1.0 to 2.0 log CFU/ml), irrespective of natural flora, which, when present, predominated (>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°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.     

Use of a Genetically Enhanced, Pediocin-Producing Starter Culture, Lactococcus lactis subsp. lactis MM217, To Control Listeria monocytogenes in Cheddar Cheese

Cheddar cheese was prepared with Lactococcus lactis subsp. lactis MM217, a starter culture which contains pMC117 coding for pediocin PA-1. About 75 liters of pasteurized milk (containing ca. 3.6% fat) was inoculated with strain MM217 (ca. 10⁶ CFU per ml) and a mixture of three Listeria monocytogenes strains (ca. 10³ CFU per ml). The viability of the pathogen and the activity of pediocin in the cheese were monitored at appropriate intervals throughout the manufacturing process and during ripening at 8°C for 6 months. In control cheese made with the isogenic, non-pediocin-producing starter culture L. lactis subsp. lactis MM210, the counts of the pathogen increased to about 10⁷ CFU per g after 2 weeks of ripening and then gradually decreased to about 10³ CFU per g after 6 months. In the experimental cheese made with strain MM217, the counts of L. monocytogenes decreased to 10² CFU per g within 1 week of ripening and then decreased to about 10 CFU per g within 3 months. The average titer of pediocin in the experimental cheese decreased from approximately 64,000 arbitrary units (AU) per g after 1 day to 2,000 AU per g after 6 months. No pediocin activity (<200 AU per g) was detected in the control cheese. Also, the presence of pMC117 in strain MM217 did not alter the cheese-making quality of the starter culture, as the rates of acid production, the pH values, and the levels of moisture, NaCl, and fat of the control cheese and the experimental cheese were similar. Our data revealed that pediocin-producing starter cultures have significant potential for protecting natural cheese against L. monocytogenes.     

Influence of Apple Cultivars on Inactivation of Different Strains of Escherichia coli O157:H7 in Apple Cider by UV Irradiation

This study examined the effect of different apple cultivars upon the UV inactivation of Escherichia coli O157:H7 strains within unfiltered apple cider. Apple cider was prepared from eight different apple cultivars, inoculated with approximately 10⁶ to 10⁷ CFU of three strains of E. coli O157:H7 per ml (933, ATCC 43889, and ATCC 43895), and exposed to 14 mJ of UV irradiation per cm². Bacterial populations for treated and untreated samples were then enumerated by using nonselective media. E. coli O157:H7 ATCC 43889 showed the most sensitivity to this disinfection process with an average 6.63-log reduction compared to an average log reduction of 5.93 for both strains 933 and ATCC 43895. The highest log reduction seen, 7.19, occurred for strain ATCC 43889 in Rome cider. The same cider produced the lowest log reductions: 5.33 and 5.25 for strains 933 and ATCC 43895, respectively. Among the apple cultivars, an average log reduction range of 5.78 (Red Delicious) to 6.74 (Empire) was observed, with two statistically significant (α ≤ 0.05) log reduction groups represented. Within the paired cultivar-strain analysis, five of eight ciders showed statistically significant (α ≤ 0.05) differences in at least two of the E. coli strains used. Comparison of log reductions among the E. coli strains to the cider parameters of °Brix, pH, and malic acid content failed to show any statistically significant relationship (R² ≥ 0.95). However, the results of this study indicate that regardless of the apple cultivar used, a minimum 5-log reduction is achieved for all of the strains of E. coli O157:H7 tested.     

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.     

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.     

Virulent Bacteriophage for Efficient Biocontrol of Listeria monocytogenes in Ready-To-Eat Foods

Food-borne Listeria monocytogenes is a serious threat to human health, and new strategies to combat this opportunistic pathogen in foods are needed. Bacteriophages are natural enemies of bacteria and are suitable candidates for the environmentally friendly biocontrol of these pathogens. In a comprehensive set of experiments, we have evaluated the virulent, broad-host-range phages A511 and P100 for control of L. monocytogenes strains Scott A (serovar 4b) and WSLC 1001 (serovar 1/2a) in different ready-to-eat (RTE) foods known to frequently carry the pathogen. Food samples were spiked with bacteria (1 × 10³ CFU/g), phage added thereafter (3 × 10⁶ to 3 × 10⁸ PFU/g), and samples stored at 6°C for 6 days. In liquid foods, such as chocolate milk and mozzarella cheese brine, bacterial counts rapidly dropped below the level of direct detection. On solid foods (hot dogs, sliced turkey meat, smoked salmon, seafood, sliced cabbage, and lettuce leaves), phages could reduce bacterial counts by up to 5 log units. Variation of the experimental conditions (extended storage over 13 days or storage at 20°C) yielded similar results. In general, the application of more phage particles (3 × 10⁸ PFU/g) was more effective than lower doses. The added phages retained most of their infectivity during storage in foods of animal origin, whereas plant material caused inactivation by more than 1 log₁₀. In conclusion, our data demonstrate that virulent broad-host-range phages, such as A511 and P100, can be very effective for specific biocontrol of L. monocytogenes in contamination-sensitive RTE foods.     

Chemical and Sensory Changes Associated with Microbial Flora of Mediterranean Boque (Boops boops) Stored Aerobically at 0, 3, 7, and 10°C

The development of a microbial population and changes in the physicochemical and sensorial characteristics of Mediterranean boque (Boops boops), called gopa in Greece, stored aerobically at 0, 3, 7, and 10°C were studied. Pseudomonads and Shewanella putrefaciens were the dominant bacteria at the end of the storage period, regardless of the temperature tested. Enterobacteria and Brochothrix thermosphacta also grew, but their population density was always 2 to 3 log₁₀ CFU g⁻¹ less than that of pseudomonads. The concentration of potential indicators of spoilage, glucose and lactic acid, decreased while that of the α-amino groups increased during storage. The concentrations of these carbon sources also decreased on sterile fish blocks inoculated with strains isolated from fish microbial flora. The organic acid profile of sterile fish blocks inoculated with the above-mentioned bacteria and that of naturally spoiled fish differed significantly. An excellent correlation (r = −0.96) between log₁₀ counts of S. putrefaciens or Pseudomonas bacteria with freshness was observed in this study.     

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.     

Size-Dependent Antimicrobial Effects of Novel Palladium Nanoparticles

Investigating the interactions between nanoscale materials and microorganisms is crucial to provide a comprehensive, proactive understanding of nanomaterial toxicity and explore the potential for novel applications. It is well known that nanomaterial behavior is governed by the size and composition of the particles, though the effects of small differences in size toward biological cells have not been well investigated. Palladium nanoparticles (Pd NPs) have gained significant interest as catalysts for important carbon-carbon and carbon-heteroatom reactions and are increasingly used in the chemical industry, however, few other applications of Pd NPs have been investigated. In the present study, we examined the antimicrobial capacity of Pd NPs, which provides both an indication of their usefulness as target antimicrobial compounds, as well as their potency as potential environmental pollutants. We synthesized Pd NPs of three different well-constrained sizes, 2.0±0.1 nm, 2.5±0.2 nm and 3.1±0.2 nm. We examined the inhibitory effects of the Pd NPs and Pd²⁺ ions toward gram negative Escherichia coli (E. coli) and gram positive Staphylococcus aureus (S. aureus) bacterial cultures throughout a 24 hour period. Inhibitory growth effects of six concentrations of Pd NPs and Pd²⁺ ions (2.5×10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸, and 10⁻⁹ M) were examined. Our results indicate that Pd NPs are generally much more inhibitory toward S. aureus than toward E. coli, though all sizes are toxic at ≥10⁻⁵ M to both organisms. We observed a significant difference in size-dependence of antimicrobial activity, which differed based on the microorganism tested. Our work shows that Pd NPs are highly antimicrobial, and that fine-scale (<1 nm) differences in size can alter antimicrobial activity.     

Potential To Reduce Escherichia coli Shedding in Cattle Feces by Using Sainfoin (Onobrychis viciifolia) Forage, Tested In Vitro and In Vivo

There is a growing concern about the presence of pathogens in cattle manure and its implications on human and environmental health. The phytochemical-rich forage sainfoin (Onobrychis viciifolia) and purified phenolics (trans-cinnamic acid, p-coumaric acid, and ferulic acid) were evaluated for their ability to reduce the viability of pathogenic Escherichia coli strains, including E. coli O157:H7. MICs were determined using purified phenolics and acetone extracts of sainfoin and alfalfa (Medicago sativa), a non-tannin-containing legume. Ground sainfoin or pure phenolics were mixed with fresh cattle feces and inoculated with a ciprofloxacin-resistant strain of E. coli, O157:H7, to assess its viability at −20°C, 5°C, or 37°C over 14 days. Forty steers were fed either a sainfoin (hay or silage) or alfalfa (hay or silage) diet over a 9-week period. In the in vitro study, the MICs for coumaric (1.2 mg/ml) and cinnamic (1.4 mg/ml) acids were 10- to 20-fold lower than the MICs for sainfoin and alfalfa extracts. In the inoculated feces, the −20°C treatment had death rates which were at least twice as high as those of the 5°C treatment, irrespective of the additive used. Sainfoin was less effective than coumaric acid in reducing E. coli O157:H7 Cip^r in the inoculated feces. During the animal trial, fecal E. coli numbers declined marginally in the presence of sainfoin (silage and hay) and alfalfa silage but not in the presence of hay, indicating the presence of other phenolics in alfalfa. In conclusion, phenolic-containing forages can be used as a means of minimally reducing E. coli shedding in cattle without affecting animal production.     

A click chemistry approach to pleuromutilin derivatives,evaluation of anti-MRSA activity and elucidation of binding mode by surface plasmon resonance and molecular docking

Novel series of pleuromutilin analogs containing substituted 1,2,3-triazole moieties were designed, synthesised and assessed for their in vitro antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA). Initially, the in vitro antibacterial activities of these derivatives against 4 strains of S. aureus (MRSA ATCC 43300, ATCC 29213, AD3, and 144) were tested by the broth dilution method. Most of the synthesised pleuromutilin analogs displayed potent activities. Among them, compounds 50, 62, and 64 (MIC = 0.5∼1 µg/mL) showed the most effective antibacterial activity and their anti-MRSA activity were further studied by the time-killing kinetics approach. Binding mode investigations by surface plasmon resonance (SPR) with 50S ribosome revealed that the selected compounds all showed obvious affinity for 50S ribosome (K_D = 2.32 × 10⁻⁸∼5.10 × 10⁻⁵ M). Subsequently, the binding of compounds 50 and 64 to the 50S ribosome was further investigated by molecular modelling. Compound 50 had a superior docking mode with 50S ribosome, and the binding free energy of compound 50 was calculated to be −12.0 kcal/mol.     

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.     

Salmonella and Escherichia coli O157:H7 Survival in Soil and Translocation into Leeks (Allium porrum) as Influenced by an Arbuscular Mycorrhizal Fungus (Glomus intraradices)

A study was conducted to determine the influence of arbuscular mycorrhizal (AM) fungi on Salmonella and enterohemorrhagic Escherichia coli O157:H7 (EHEC) in autoclaved soil and translocation into leek plants. Six-week-old leek plants (with [Myc+] or without [Myc−] AM fungi) were inoculated with composite suspensions of Salmonella or EHEC at ca. 8.2 log CFU/plant into soil. Soil, root, and shoot samples were analyzed for pathogens on days 1, 8, 15, and 22 postinoculation. Initial populations (day 1) were ca. 3.1 and 2.1 log CFU/root, ca. 2.0 and 1.5 log CFU/shoot, and ca. 5.5 and 5.1 CFU/g of soil for Salmonella and EHEC, respectively. Enrichments indicated that at days 8 and 22, only 31% of root samples were positive for EHEC, versus 73% positive for Salmonella. The mean Salmonella level in soil was 3.4 log CFU/g at day 22, while EHEC populations dropped to ≤0.75 log CFU/g by day 15. Overall, Salmonella survived in a greater number of shoot, root, and soil samples, compared with the survival of EHEC. EHEC was not present in Myc− shoots after day 8 (0/16 samples positive); however, EHEC persisted in higher numbers (P = 0.05) in Myc+ shoots (4/16 positive) at days 15 and 22. Salmonella, likewise, survived in statistically higher numbers of Myc+ shoot samples (8/8) at day 8, compared with survival in Myc− shoots (i.e., only 4/8). These results suggest that AM fungi may potentially enhance the survival of E. coli O157:H7 and Salmonella in the stems of growing leek plants.     

Selective Enrichment with a Resuscitation Step for Isolation of Freeze-Injured Escherichia coli O157:H7 from Foods

We studied injury of Escherichia coli O157:H7 cells in 11 food items during freeze storage and methods of isolating freeze-injured E. coli O157:H7 cells from foods. Food samples inoculated with E. coli O157:H7 were stored for 16 weeks at −20°C in a freezer. Noninjured and injured cells were counted by using tryptic soy agar and sorbitol MacConkey agar supplemented with cefixime and potassium tellurite. Large populations of E. coli O157:H7 cells were injured in salted cabbage, grated radish, seaweed, and tomato samples. In an experiment to detect E. coli O157:H7 in food samples artificially contaminated with freeze-injured E. coli O157:H7 cells, the organism was recovered most efficiently after the samples were incubated in modified E. coli broth without bile salts at 25°C for 2 h and then selectively enriched at 42°C for 18 h by adding bile salts and novobiocin. Our enrichment method was further evaluated by isolating E. coli O157:H7 from frozen foods inoculated with the organism prior to freezing. Two hours of resuscitation at 25°C in nonselective broth improved recovery of E. coli O157:H7 from frozen grated radishes and strawberries, demonstrating that the resuscitation step is very effective for isolating E. coli O157:H7 from frozen foods contaminated with injured E. coli O157:H7 cells.     

Differential Listeria monocytogenes Strain Survival and Growth in Katiki,a Traditional Greek Soft Cheese,at Different Storage Temperatures

Katiki Domokou is a traditional Greek cheese, which has received the Protected Designation of Origin recognition since 1994. Its microfloras have not been studied although its structure and composition may enable (or even favor) the survival and growth of several pathogens, including Listeria monocytogenes. The persistence of L. monocytogenes during storage at different temperatures has been the subject of many studies since temperature abuse of food products is often encountered. In the present study, five strains of L. monocytogenes were aseptically inoculated individually and as a cocktail in Katiki Domokou cheese, which was then stored at 5, 10, 15, and 20°C. Pulsed-field gel electrophoresis was used to monitor strain evolution or persistence during storage at different temperatures in the case of the cocktail inoculum. The results suggested that strain survival of L. monocytogenes was temperature dependent since different strains predominated at different temperatures. Such information is of great importance in risk assessment studies, which typically consider only the presence or absence of the pathogen.Listeria monocytogenes is a ubiquitous food-borne pathogen associated with outbreaks of listeriosis from consumption of various food commodities, especially dairy products, seafood, and meat (2, 26). The pathogen is of great health concern for the food industry because it is characterized by high mortality rates, amounting to 20 to 30% (14). Due to the severity of illness, especially for pregnant women, neonates, the elderly, and immunodeficient people, the level of the pathogen in food should remain low to ensure safe food products.The new regulation of the European Union (EU) for microbiological criteria for L. monocytogenes in foods has set maximum levels of 100 CFU g⁻¹ at the time of consumption for soft cheeses (8). In fact, the new EC 2073/2005 regulation in annex I lists the microbiological criteria for foodstuffs, which are classified into food safety criteria and process hygiene criteria. According to the new EU regulation, food safety criteria are those which “define the acceptability of a product or a batch of foodstuff applicable to products placed on the market” (8).Legislative amendments regarding the presence of L. monocytogenes in ready-to-eat (RTE) foods are of great importance. Indeed, for the first time RTE foods are legislatively distinguished according to the target population for which they are intended, i.e., whether they are intended for consumption (i) by infants, (ii) by people with special medical conditions (immunocompromised), or (iii) by other target human subpopulations. In the most recent amendment the RTE foods other than those intended for infants or for those with special medical needs are further subdivided into foods that are able to support the growth of L. monocytogenes and those that are not. Products with pH ≤ 5.0 and water activity of ≤0.94 and products with a shelf life of less than 5 days are automatically considered to belong to the category of RTE foods that are unable to support the growth of L. monocytogenes (8). The regulation also states that “other categories of products can also belong to this category, subject to scientific justification.” Last but not least, the food safety criteria for L. monocytogenes are adjusted according to the bacteria''s temporal stage in the food chain. Thus, for RTE foods that are able to support the growth of L. monocytogenes, the new regulation demands the absence of the pathogen (in 25 g) “before the food has left the immediate control of the food business operator, who has produced it” but allows up to 100 CFU g⁻¹ in “products placed on the market during their shelf life.” The 100-CFU g⁻¹ limit also applies throughout the shelf life of marketed RTE foods unable to support L. monocytogenes growth (8).The pH and the water activity of Katiki Domokou (Katiki), a spreadable RTE traditional Greek cheese, are within the limits mentioned in the regulation. This product, a white cheese with a creamy structure, was traditionally produced from goat milk or from a mixture of goat and sheep milk. It has been recognized as a Protected Designation of Origin product since 1994 (www.greekcheese.gr), and its consumption has readily increased in the last few years. The milk is initially pasteurized and cooled at 27 to 28°C. Coagulation is then conducted with or without the addition of rennet, and the mixture is left to stand at 20 to 22°C. The curd is pulped and placed in cloth sacks for draining, with high final moisture (ca. 75%) and low salt content (ca. 1%) and pH (4.3 to 4.5) while it is stored at 4 to 5°C.The quantitative estimation of kinetic parameters related to growth, survival, and death of L. monocytogenes has been described previously (2, 14, 20). The kinetic parameters of L. monocytogenes during storage at different temperatures have been the subject of many studies since temperature abuse of food products is often encountered (25, 28). However, strain characteristics or viability have not been taken into account (or have not been considered) as yet (20). This may explain the variability of findings in regard to different storage conditions (7, 17). Pulsed-field gel electrophoresis (PFGE) is a powerful subtyping tool, a gold standard for epidemiology, which provides repeatable results. It has the ability to generate profiles of a wide range of microorganisms and to discriminate strains with high fidelity (11, 19). PFGE has been used in several studies to type strains of epidemiological interest as well as to trace contaminants in the food chain (12, 13, 18).The purpose of the present study was to assess the survival of five strains of L. monocytogenes inoculated either individually or as a cocktail in Katiki cheese. The cheese was stored at 5, 10, 15, and 20°C over a period of 1 month. PFGE was used to monitor the strain(s) that might survive and/or grow at different temperatures in a complex ecosystem like Katiki. The strains used in the study to form the inoculum consisted of two type strains of serotype 4b and three isolates belonging to our laboratory collection that were isolated from soft cheese and the conveyor belt of RTE foods. The strains were chosen on the basis of their source of isolation since this could be crucial to the interpretation of the data. The population was monitored throughout storage with respect to its quantitative as well as its qualitative evolution.     

Comparison of Different Approaches To Quantify Staphylococcus aureus Cells by Real-Time Quantitative PCR and Application of This Technique for Examination of Cheese

Two different real-time quantitative PCR (RTQ-PCR) approaches were applied for PCR-based quantification of Staphylococcus aureus cells by targeting the thermonuclease (nuc) gene. Purified DNA extracts from pure cultures of S. aureus were quantified in a LightCycler system using SYBR Green I. Quantification proved to be less sensitive (60 nuc gene copies/μl) than using a fluorigenic TaqMan probe (6 nuc gene copies/μl). Comparison of the LightCycler system and the well-established ABI Prism 7700 SDS with TaqMan probes revealed no statistically significant differences with respect to sensitivity and reproducibility. Application of the RTQ-PCR assay to quantify S. aureus cells in artificially contaminated cheeses of different types achieved sensitivities from 1.5 × 10² to 6.4 × 10² copies of the nuc gene/2 g, depending on the cheese matrix. The coefficients of correlation between log CFU and nuc gene copy numbers ranged from 0.979 to 0.998, thus enabling calculation of the number of CFU of S. aureus in cheese by performing RTQ-PCR.     

Analysis of Escherichia coli O157:H7 Survival in Ovine or Bovine Manure and Manure Slurry

Farm animal manure or manure slurry may disseminate, transmit, or propagate Escherichia coli O157:H7. In this study, the survival and growth of E. coli O157:H7 in ovine or bovine feces under various experimental and environmental conditions were determined. A manure pile collected from experimentally inoculated sheep was incubated outside under fluctuating environmental conditions. E. coli O157:H7 survived in the manure for 21 months, and the concentrations of bacteria recovered ranged from <10² to 10⁶ CFU/g at different times over the course of the experiment. The DNA fingerprints of E. coli O157:H7 isolated at month 1 and month 12 were identical or very similar. A second E. coli O157:H7-positive ovine manure pile, which was periodically aerated by mixing, remained culture positive for 4 months. An E. coli O157:H7-positive bovine manure pile was culture positive for 47 days. In the laboratory, E. coli O157:H7 was inoculated into feces, untreated slurry, or treated slurry and incubated at −20, 4, 23, 37, 45, and 70°C. E. coli O157:H7 survived best in manure incubated without aeration at temperatures below 23°C, but it usually survived for shorter periods of time than it survived in manure held in the environment. The bacterium survived at least 100 days in bovine manure frozen at −20°C or in ovine manure incubated at 4 or 10°C for 100 days, but under all other conditions the length of time that it survived ranged from 24 h to 40 days. In addition, we found that the Shiga toxin type 1 and 2 genes in E. coli O157:H7 had little or no influence on bacterial survival in manure or manure slurry. The long-term survival of E. coli O157:H7 in manure emphasizes the need for appropriate farm waste management to curtail environmental spread of this bacterium. This study also highlights the difficulties in extrapolating laboratory data to on-farm conditions.     

Evaluation of Aerated Steam Treatment of Alfalfa and Mung Bean Seeds To Eliminate High Levels of Escherichia coli O157:H7 and O178:H12, Salmonella enterica,and Listeria monocytogenes

Sprouts contaminated with human pathogens are able to cause food-borne diseases due to the favorable growth conditions for bacteria during germination and because of minimal processing steps prior to consumption. We have investigated the potential of hot humid air, i.e., aerated steam, to treat alfalfa and mung bean seeds which have been artificially contaminated with Escherichia coli O157:H7, Salmonella enterica subsp. enterica serovar Weltevreden, and Listeria monocytogenes Scott A. In addition, a recently collected E. coli O178:H12 isolate, characterized by a reduced heat sensitivity, was exposed to the treatment described. Populations of E. coli O157:H7 and S. enterica on alfalfa and mung bean seeds could be completely eliminated by a 300-s treatment with steam at 70 ± 1°C as revealed by enrichment studies. L. monocytogenes and E. coli O178:H12 could not be completely eliminated from artificially inoculated seeds. However, bacterial populations were reduced by more than 5 log CFU/g on alfalfa and by more than 4 log CFU/g on mung bean seeds. The germination rate of mung beans was not affected by the 300-s treatment compared to the germination rate of untreated seeds whereas that of alfalfa seeds was significantly lower by 11.9%. This chemical-free method is an effective alternative to the 20,000-ppm hypochlorite treatment presently recommended by the U.S. Food and Drug Administration (FDA).