DETECTION OF THE VIABLE BUT NONCULTURABLE STATE IN ESCHERICHIA COLI O157:H7

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is recognized as a frequent cause of gastroenteritis ranging from mild to severe bloody diarrhea. The Shiga-like toxin produced by EHEC can result in hemolytic uremic syndrome, now the major cause of acute kidney failure in children in the United States. Inadequately cooked beef is most commonly implicated in the transmission of EHEC, although only a small fraction of cattle appear to harbor the organism. In several studies EHEC positive herds were detected only in the summer months correlating with the occurrence of human infections. Numerous E. coli strains have been shown to enter a viable but nonculturable (VBNC) state as a result of environmental stresses, including low temperature. Using traditional plating methods and the BacLight Molecular Probe, we monitored EHEC strains incubated in river water (RW) and artificial sea water (ASW) at temperatures of 5C and 25C for their entry into the VBNC state. EHEC strains remained culturable for over 40 days in both ASW and RW incubated at 25C. In ASW, these levels were higher than a non-EHEC control. At 5C, the number of culturable EHEC cells dropped gradually in both RW and ASW. Using the BacLight Molecular Probe, we were able to demonstrate that these cells, though not culturable, were viable indicating entry into the VBNC state. Our study suggests that temperature and not salinity is the primary signal for entry into this state .     

EFFECT OF SOLUBLE POLYLACTIC ACID DURING REFRIGERATED STORAGE OF GROUND MEATS INOCULATED WITH ESCHERICHIA COLI O157:H71

Ground beef, ground pork, and commercial breakfast sausage were inoculated (6.5 log₁₀ CFU/mL) with a five strain mixture of Escherichia coli 0157:H7 and treated either with sterile water, or 1% or 2% solutions of soluble polylactic acid (SPLA) in sterile water and stored at 4C for 1, 24, 72 and 168 h. After 168 h, 2% SPLA was significantly (p0.05) more effective than both 1% SPLA and sterile water in reducing E. coli 0157:H7 and resulted in overall reductions of 1.68, 1.70, and 1.32 log₁₀ CFU/mL for beef, pork, and pork sausage, respectively, when compared to control samples. The meat samples treated with 1% and 2% SPLA maintained significantly (p 0.05) lower pH values throughout refrigerated storage of 168 h with the higher concentration sustaining pH values from 3.83 to 3.92. Although the inhibitory effect of this acid increased with storage time, E. coli 0157:H7 survived these acidic conditions, with water activity levels ranging from 0.972 to 0.991.     

PREDICTIVE MODEL FOR THE COMBINED EFFECT OF TEMPERATURE,pH, SODIUM CHLORIDE,AND SODIUM PYROPHOSPHATE ON THE HEAT RESISTANCE OF ESCHERICHIA COLI O157:H71

The effects and interactions of heating temperature (5–62.5C), pH (4 – 8), NaCl (0 – 6%, w/v), and sodium pyrophosphate (0 – 0.3%, w/v) on the heat resistance of a four strain mixture of Escherichia coli O157:H7 in beef gravy were examined. Thermal death times were determined using a submerged coil heating apparatus. The recovery medium was plate count agar supplemented with 1% sodium pyruvate. Decimal reduction times (D-values) were calculated by fitting a survival model to the data with a curve fitting program. The D-values were analyzed by second order response surface regression for temperature, pH, NaCl and sodium pyrophosphate levels. The four variables interacted to affect the inactivation of the pathogen. Thermal resistance of E. coli O157:H7 can be lowered by combining these intrinsic factors. A mathematical model describing the combined effect of temperature, pH, NaCl and sodium pyrophosphate levels on the thermal inactivation of E. coli O157:H7 was developed. The model can predict D-values for any combinations of temperature, pH, NaCl and sodium pyrophosphate that are within the range of those tested.     

CONDITIONS AFFECTING AUTOINDUCER‐2‐BASED QUORUM‐SENSING OF ESCHERICHIA COLI O157:H7 ON FRESH BEEF

ABSTRACT

This study evaluated whether inoculated (none, 1, 5 log colony‐forming units [cfu]/cm²) Escherichia coli O157:H7 would result in detection of autoinducer (AI)‐2‐like activity on beef. Inoculated fresh beef, containing low (LNB) or high (HNB) initial levels of natural flora, was analyzed for bacterial populations and AI‐2‐like activity during aerobic or vacuum‐packaged storage (4, 10, 25C). As expected, no growth of E. coli O157:H7 was detected at 4C, while at 10C, growth was detected only on LNB samples stored aerobically; AI‐2‐like activity was minimal (P ≥ 0.05) at both temperatures. E. coli O157:H7 showed more growth in LNB than HNB, and in aerobically than vacuum‐packaged samples inoculated with 1 log cfu/cm² of the pathogen during storage at 25C. AI‐2‐like activity was generally higher in LNB than HNB samples stored aerobically at 25C, while no significant AI‐2‐like activity was detected in samples stored in vacuum packages. The results indicated that E. coli O157:H7 may exhibit AI‐2‐like activity on aerobically stored beef in the presence of lower initial levels of natural flora, and at temperatures allowing prolific growth of the pathogen. Thus, AI‐2‐based quorum‐sensing of E. coli O157:H7 may not be of importance in beef stored at low temperatures.

PRACTICAL APPLICATIONS

This study presents evidence that Escherichia coli O157:H7 showed autoinducer (AI)‐2 activity and involved in quorum‐sensing on fresh beefcontaining low initial levels of natural flora during aerobic storage at abusive storage temperatures. Thus, AI‐2‐based quorum‐sensing of E. coli O157:H7 may not be important in beef stored at recommended low temperatures.     

INFLUENCE OF STARVATION, TEMPERATURE, AND pH ON CULTURABILITY OF ESCHERICHIA COLI O157:H7

In farm and food processing environments Escherichia coli O157:H7 can survive for days to months. In this study, we investigated the influence of starvation, pH (pH 4 or 7), temperature (4, 10 and 22C), and exposure to chlorine (50, 100, 150, 200 ppm) on culturability of E. coli O157:H7. The culturable cell population in PBS decreased to an undetectable level at 4C and 10C, but not at 22C during the experimental period. Cells suspended in PBS adjusted to pH 4 lost culturability more rapidly than at pH 7. Culturable cells were not detectable after exposure to chlorine, however, viable cell populations in the range of 1 – 2 log remained stable for 5 days. During loss of culturability two distinct morphological cell populations emerged; typical rod shaped and coccoid shaped cells. In this study, independent of exposure to chlorine, conditions of low temperature and low pH had the greatest influence on entry of E. coli O157:H7 into a VNC state.     

BEHAVIOR OF LISTERIA MONOCYTOGENES AND ESCHERICHIA COLI O157:H7 IN FRESH-SLICED CACTUS-PEAR FRUIT

Storage experiments were conducted to follow the behavior of Listeria monocytogenes and Escherichia coli O157:H7, deliberately inoculated on fresh‐cut cactus‐pear fruits before packaging under modified and control atmosphere and stored at four different temperatures (4, 8, 12 and 20C). L. monocytogenes was able to proliferate during storage at different temperature both in control and modified atmosphere. By comparing the sanitary‐risk values with those of shelf life, it is possible to conclude that the storage of cactus‐pear samples at temperatures greater than 4C, both in control and in modified atmospheres, could lead to a significant health‐time risk, and that this is strictly affected by temperature. E. coli O157:H7 was able to proliferate only in the sample stored at 4 and 8C in both package atmospheres. On the contrary, this species was completely suppressed at the higher temperatures. In our study, E. coli O157:H7 appeared to be much less suited for survival on the surface of the fruit than L. monocytogenes.     

INACTIVATION OF ESCHERICHIA COLI O157:H7 BY THE COMBINATION OF ORGANIC ACIDS AND PULSED ELECTRIC FIELD

Inactivation of Escherichia coli O157:H7 by pulsed electric field, benzoic or sorbic acid, alone or in combination was investigated. When the cells of E. coli O157:H7 were suspended in 10% glycerol and treated with a single highvoltage (12.5 kv/cm) electric pulse at 25C, the count decreased by 1.1–1.6 log₁₀ CFU/mL. Presence of benzoic and sorbic acid (1000 ppm) in the suspending medium, at pH 3.4 without electric treatment, decreased the count by 1.9–2.5 and 0.6–1.1 log, respectively. A synergistic killing effect between the high electric pulse and organic acid was observed at pH 3.4, but not at pH 6.4. When the cell suspension of E. coli O157:H7 was treated with five electric pulses in the presence of benzoic or sorbic acid at pH 3.4, the count decreased by 5.6 and 4.2 logs, respectively. Inactivation of the pathogen by combinations of electric pulse and organic acid was enhanced by an increase in temperature, field strength, and number of electric pulses, Inactivation was greater when the cells were suspended in ionic suspending media (0.1% NaCl or 5mM phosphate buffer) than in nonionic media (10% glycerol or 1% sucrose) .     

MODELING TEMPERATURE, pH, NaCl, NITRITE AND LACTATE ON THE SURVIVAL OF ESCHERICHIA COLI O157:H7 IN BROTH

A model was developed to estimate the survival times for a four-strain cocktail of Escherichia coli O157:H7 in BHI broths with pH values from 3.5 to 7.0, NaCl from 0.5 to 15%, Na lactate from 0.0 to 2.0% and NaNO₂ from 0 to 75 ppm. Broths were stored between 4 and 37C for up to 6 months. Samples were removed and enumerated at appropriate intervals. A primary level model with a shoulder period (no decline) and a subsequent log-linear decline was fitted to the survival data and the times for 3, 4, 5, and 6 log₁₀-units of decline determined. A secondary level second-order regression equation with the environmental conditions and declines being the independent variables was fitted to the survival times. Confidence limits about the estimated mean were also estimated. In foods with equivalent conditions that do not permit E. coli O157:H7 growth, this model provides an initial estimate of the survival times.     

CONCURRENT DETECTION OF ESCHERICHIA COLI O157:H7 AND SALMONELLA FROM A SINGLE ENRICHMENT IN 24 H

The objective of this study was to determine if a single assay protocol could result in the concurrent detection of Escherichia coli O157:H7 and Salmonella from a single sample grown in a single enrichment in 24 h. Twenty-five and 375 g of ground beef nonfat dry milk, and dry pet food samples were seeded with low (10 cfu/sample) and high (100 cfu/sample) levels ofE. coli O157:H7 and Salmonella cultures and incubated at 35 and 41C for 18 h for nonselective preenrichment. Incubated samples were analyzed by immunomagnetic separation (IMS) following a 6 h incubation for selective enrichment at 37C using M-broth and enzyme linked immumosorbent assay (ELISA). Depending on the food samples and the inoculation level, the minimum concurrent detection level of E. coli O157:H7 and Salmonella was <1 cfu/g in the samples at the competitor flora level of 10⁵ cfu/g or less in ground beef samples, but in other cases of higher competitor loads and low target inoculations E. coli O157:H7 could not be detected in the presence of the Salmonella.     

GROWTH OF ESCHERICHIA COLI O157:H7 IN THE PRESENCE OF PSEUDOMONAS FLUORESCENS IN SKIMMED MILK AT 7 OR 25C

Escherichia coli O157:H7 STCC 4076 , E. coli O157:H7 STCC 4267 and E. coli STCC 515 were cultured alone or in combination with Pseudomonas fluorescens STCC 378 at 7C or 25C in 10% reconstituted skimmed milk. Culture pH and bacterial population densities were monitored over 40 days. Both E. coli O157:H7 strains grew well after 40 days of incubation at 7C with final pH values between 4.86–4.53. At 25C, both E. coli O157:H7 strains grew during 20 days with final pH values of 4.00–4.14. The pH of the different cultures of this study decreased more at 25C than at 7C. The results suggest that P. fluorescens may inhibit the growth of the other bacteria present in milk at 7C, but this inhibition is weak. In contrast, the growth of E. coli O157:H7 strains in the presence of P. fluorescens appears to be slightly enhanced during most of the incubation period at 25C.     

RAPID DETECTION OF OUTBREAK ESCHERICHIA COLI O157 AND SALMONELLA ON ALFALFA SPROUTS BY IMMUNOMAGNETIC CAPTURE AND TIME‐RESOLVED FLUORESCENCE1

Commercially available alfalfa seeds were inoculated with low levels (～ 4 CFU/g) of pathogenic bacteria and sprouted at 25C. At 48 h, the spent irrigation water and sprouts were separately transferred to brain heart infusion (BHI) broth and enriched for 4 h at 37C and 160 rpm. Specific immunomagnetic beads (IMB) were then applied to capture the E. coli O157 or Salmonella in the enriched media. Separation and concentration of captured pathogens were achieved using magnetic particle concentrators (MPC). IMB captured E. coli O157:H7 and Salmonella spp. then formed sandwiched complexes with europium (Eu) labeled anti‐E. coli O157 antibodies and samarium (Sm) labeled anti‐Salmonella antibodies, respectively. After washing the complexes, the lanthanide labels were extracted out from the complexes by specific chelators to form strongly fluorescent Eu‐ and Sm‐chelates. The specific time‐resolved fluorescence (TRF) associated with Eu or Sm was measured to estimate the extent of capture of the E. coli O157 and Salmonella, respectively. The results indicated that the approach could detect E. coli O157 and many Salmonella spp. from spent irrigation water or sprouts grown from contaminated seeds. Nontargeted bacteria, e.g., native microflora present on the untreated seeds and inoculated Aeromonas and Citrobacter, exhibited no crossreactivity and counts were not significantly different from background fluorescence of the IMB alone. Since pathogen detection was achieved within 6 h, the assay could detect contamination levels as low as 4 CFU/g of seeds and it showed no cross‐reactivity with nonpathogenic microflora present on the sprouts, the developed methodology could be used as a rapid, sensitive and specific screening process for E. coli O157 and Salmonella spp. in sprouts and their irrigation water.     

MODELING THE INACTIVATION KINETICS OF ESCHERICHIA COLI O157:H7 DURING THE STORAGE UNDER REFRIGERATION OF APPLE JUICE TREATED BY PULSED ELECTRIC FIELDS

The aim was to describe the inactivation kinetics of Escherichia coli O157:H7 suspended in apple juice after pulsed electric fields (PEF) and a subsequent storage under refrigeration. Escherichia coli O157:H7 showed a great PEF resistance in apple juice, when survivors were evaluated immediately after PEF. However, PEF-treated cells exhibited a great sensitivity to a subsequent holding in apple juice for 3 days. For instance, although a PEF treatment of 80 pulses at 35.0 kV/cm inactivated less than 0.5 log₁₀ cell cycles, the maintenance of the samples up to 3 days at 4C caused an inactivation of 5.0 log₁₀ cycles. An equation based on the Weibullian-like distribution accurately described the kinetics of cell inactivation.

PRACTICAL APPLICATIONS

The storage time influences the pulsed electric fields (PEF) inactivation of Escherichia coli O157:H7 cells suspended in apple juice. The potential of Weibullian-like distributions to describe survival curves with deviations in their linearity has allowed us to obtain an equation that accurately describes the complete PEF survival profile of E. coli in apple juice, when survivors were evaluated immediately after PEF and also after a subsequent storage under refrigeration. These results underline the possibility of applying PEF to pasteurize acidic foods by taking into account the postprocessing effect of the acidity of the product.     

INACTIVATION OF ESCHERICHIA COLI O157:H7 DURING THE STORAGE UNDER REFRIGERATION OF APPLE JUICE TREATED BY PULSED ELECTRIC FIELDS

The sensitivity of pulsed electric fields (PEF)‐treated E. coli O157:H7 cells to subsequent holding in apple juice has been evaluated. Escherichia coli O157:H7 cells in apple juice were resistant to PEF. A PEF treatment of 400 µs at any electrical field strength was not sufficient to inactivate one log₁₀cycle of cells. However, PEF injured a large proportion of E. coli O157:H7 cells that became sensitive to a subsequent storage under refrigeration in apple juice. The total lethal effect of the combined process depended on the electrical field strength and storage time. The combination of a PEF treatment at 25 kV/cm for 400 µs and a subsequent storage of the apple juice under refrigeration for 48 h allowed five log₁₀cycles of inactivation to be achieved. The combination of PEF and maintenance under refrigeration has been demonstrated to be an effective pasteurization method, by sufficiently reducing the presence of E. coli O157:H7 in apple juice in order to meet U.S. FDA recommendations.     

INACTIVATION OF ESCHERICHIA COLI O157:H7 ON INOCULATED ALFALFA SEEDS WITH OZONATED WATER UNDER PRESSURE1

Alfalfa seeds inoculated with Escherichia coli O157:H7 (～10⁵ CFU/g) were subjected to low hydrostatic pressure. Seeds immersed in ozonated water at 4C were held at 8 and 12‐psi ozone pressure for 2, 4, 8, 16, 32, and 64 min. Alternatively, seeds were continuously sparged with ozone for up to 64 min and then held at 12 psi for 5 min. Controls consisted of sparging and pressurization with air. Thirty‐two minute treatments of continuous ozone sparging followed by pressurization of seeds at 12 psi for 5 min were repeated with the addition of four surfactants (Tween 20, Tween 80, SPAN 20, and SPAN 80) in the treatment water. Enumeration of E. coli O157:H7 on treated, untreated, and control seeds was done on tryptic soy agar supplemented with 50 μg/mL of nalidixic acid. The reduction in population of E. coli O157:H7 on seeds treated with the 8 and 12 psi hydrostatic pressure in ozonated water ranged from 0.74 ?1.56 log₁₀ CFU/g and 0.72 – 1.62 log₁₀ CFU/g, respectively. Control treatments carried out with air pressurization of seeds resulted in maximum population reductions of 1.55 log₁₀ and 1.83 log₁₀ CFU/g for 8 and 12 psi, respectively. For seeds treated with continuous ozone sparging (2 – 64 min) followed by pressurization at 12 psi for 5 min, the highest reduction was 2.03 log₁₀ CFU/g. Reductions were, however, not significantly different (P > 0.05) from control treatment (with air) which reduced the populations by 0.57 – 2.19 log₁₀ CFU/g. The presence of surfactants during continuous sparging of water followed by pressurization at 12 psi was not beneficial. None of the treatments adversely affected the germination of the seeds.     

FACTORS INVOLVED IN RECENT OUTBREAKS OF ESCHERICHIA COLI O157:H7 IN SCOTLAND AND RECOMMENDATIONS FOR ITS CONTROL

This paper describes an outbreak of E. coli O157:H7 that occurred in central Scotland. Edipemiological and microbiological evidence has indicated that the outbreak was comprised of several separate but related incidents, relating to the lunch (attended by around 100 people) held in Wishaw Parish Church Hall, a birthday party held in a public house on November 23, 1996 and retail sales in Lanarkshire and Forth Valley. All isolates of E. coli O157:H7 from individuals in the outbreak belonged to phage type 2 and possessed the verocytotoxin gene VT2. Two hundred sixty-two of these isolates have been subjected to pulsed-field gel electrophoresis (PFGE): all had indistinguishable profiles. There were 496 cases in total. There have been 20 deaths (all adults) associated with the outbreak, the highest number of deaths associated with an outbreak of E. coli O157:H7 infection in the world. Of these, 8 people had attended the luncheon served at Wishaw Old Parish Church on November 27, 1996. The age range of those who died was 69 to 93 years. The outbreak was investigated by the Pennington Group who concluded that the circumstances of the central Scotland outbreak, and the inevitable uncertainties that may surround outbreaks generally, provide strong justification for the precautionary, preventative measures proposed and the recommendations made.     

QUANTITATIVE DETECTION OF ESCHERICHIA COLI O157:H7 IN GROUND BEEF BY IMMUNOMAGNETIC SEPARATION AND POLYMERASE CHAIN REACTION

A quantitative assay for viable Escherichia coli O157:H7 in ground beef based on immunomagnetic separation (IMS) and the polymerase chain reaction (PCR) was developed. Bacterial cells inoculated into ground beef, were captured by immunomagnetic beads (IMB) after a 6 h non-selective enrichment. The percent capture of the target cells was consistent for the inoculation levels of 0.7 to 70 colony-forming-units (CFU)/g. Captured bacteria were lysed with PCR buffer containing 0.2 mg/mL proteinase K at 65°C for 30 min. DNA sequences of Shiga-like toxin 1 and 2 (Stx 1 and 2) were amplified independently. Log-linear relationships were observed between CFU of E. coli O157:H7 inoculated into ground beef and the integrated fluorescent image of the PCR products with Stx 1 and 2 primers after enrichment. The quantitative range was between 0.7 to 70 CFU/g.