Elimination of Escherichia coli O157:H7 from Fermented Dry Sausages at an Organoleptically Acceptable Level of Microencapsulated Allyl Isothiocyanate

Four sausage batters (17.59% beef, 60.67% pork, and 17.59% pork fat) were inoculated with two commercial starter culture organisms (>7 log₁₀ CFU/g Pediococcus pentosaceus and 6 log₁₀ CFU/g Staphylococcus carnosus) and a five-strain cocktail of nonpathogenic variants of Escherichia coli O157:H7 to yield 6 to 7 log₁₀ CFU/g. Microencapsulated allyl isothiocyanate (AIT) was added to three batters at 500, 750, or 1,000 ppm to determine its antimicrobial effects. For sensory analysis, separate batches with starter cultures and 0, 500, or 750 ppm microencapsulated AIT were produced. Sausages were fermented at ≤26°C and 88% relative humidity (RH) for 72 h. Subsequently sausages were dried at 75% RH and 13°C for at least 25 days. The water activity (a_w), pH, and levels of starter cultures, E. coli O157:H7, and total bacteria were monitored during fermentation and drying. All sausages showed changes in the initial pH from 5.57 to 4.89 and in a_w from 0.96 to 0.89 by the end of fermentation and drying, respectively. Starter culture numbers were reduced during sausage maturation, but there was no effect of AIT on meat pH reduction. E. coli O157:H7 was reduced by 6.5 log₁₀ CFU/g in sausages containing 750 and 1,000 ppm AIT after 21 and 16 days of processing, respectively. E. coli O157:H7 numbers were reduced by 4.75 log₁₀ CFU/g after 28 days of processing in treatments with 500 ppm AIT, and the organism was not recovered from this treatment beyond 40 days. During sensory evaluation, sausages containing 500 ppm AIT were considered acceptable although slightly spicy by panelists.     

Fate of Escherichia coli O157:H7 as affected by pH or sodium chloride and in fermented, dry sausage.

The influence of pH adjusted with lactic acid or HCl or sodium chloride concentration on survival or growth of Escherichia coli O157:H7 in Trypticase soy broth (TSB) was determined. Studies also determined the fate of E. coli O157:H7 during the production and storage of fermented, dry sausage. The organism grew in TSB containing less than or equal to 6.5% NaCl or at a pH of 4.5 to 9.0, adjusted with HCl. When TSB was acidified with lactic acid, the organism grew at pH 4.6 but not at pH 4.5. A commercial sausage batter inoculated with 4.8 x 10(4) E. coli O157:H7 per g was fermented to pH 4.8 and dried until the moisture/protein ratio was less than or equal to 1.9:1. The sausage chubs were then vacuum packaged and stored at 4 degrees C for 2 months. The organism survived but did not grow during fermentation, drying, or subsequent storage at 4 degrees C and decreased by about 2 log10 CFU/g by the end of storage. These studies reveal the importance of using beef containing low populations or no E. coli O157:H7 in sausage batter, because when initially present at 10(4) CFU/g, this organism can survive fermentation, drying, and storage of fermented sausage regardless of whether an added starter culture was used.     

Combined effect of high-pressure treatments and bacteriocin-producing lactic acid bacteria on inactivation of Escherichia coli O157:H7 in raw-milk cheese

The effect of high-pressure (HP) treatments combined with bacteriocins of lactic acid bacteria (LAB) produced in situ on the survival of Escherichia coli O157:H7 in cheese was investigated. Cheeses were manufactured from raw milk inoculated with E. coli O157:H7 at approximately 10(5) CFU/ml. Seven different bacteriocin-producing LAB were added at approximately 10(6) CFU/ml as adjuncts to the starter. Cheeses were pressurized on day 2 or 50 at 300 MPa for 10 min or 500 MPa for 5 min, at 10 degrees C in both cases. After 60 days, E. coli O157:H7 counts in cheeses manufactured without bacteriocin-producing LAB and not pressurized were 5.1 log CFU/g. A higher inactivation of E. coli O157:H7 was achieved in cheeses without bacteriocin-producing LAB when 300 MPa was applied on day 50 (3.8-log-unit reduction) than if applied on day 2 (1.3-log-unit reduction). Application of 500 MPa eliminated E. coli O157:H7 in 60-day-old cheeses. Cheeses made with bacteriocin-producing LAB and not pressurized showed a slight reduction of the pathogen. Pressurization at 300 MPa on day 2 and addition of lacticin 481-, nisin A-, bacteriocin TAB 57-, or enterocin AS-48-producing LAB were synergistic and reduced E. coli O157:H7 counts to levels below 2 log units in 60-day-old cheeses. Pressurization at 300 MPa on day 50 and addition of nisin A-, bacteriocin TAB 57-, enterocin I-, or enterocin AS-48-producing LAB completely inactivated E. coli O157:H7 in 60-day-old cheeses. The application of reduced pressures combined with bacteriocin-producing LAB is a feasible procedure to improve cheese safety.     

Survival of Escherichia coli O157:H7 and Salmonella typhimurium inoculated on chicken by aqueous chlorine dioxide treatment

Inactivation of Escherichia coli O157:H7 and Salmonella typhimurium was evaluated on inoculated chicken by aqueous chlorine dioxide (ClO2) treatment. Chicken samples were inoculated with 6-7 log CFU/g of Escherichia coli O157:H7 and Salmonella typhimurium, respectively. The chicken samples were then treated with 0, 50, and 100 ppm of ClO2 solution and stored at 4 +/- 1 degrees C. Aqueous ClO2 treatment decreased the populations of the pathogenic bacteria on the chicken breast and drumstick. In particular, 100 ppm ClO2 treatment on the chicken breast and drumstick reduced Escherichia coli O157:H7 and Salmonella typhimurium by 1.00-1.27 and 1.37-1.44 log CFU/g, respectively. Aqueous ClO2 treatment on the growth of the bacteria was continuously in effect during storage, resulting in the decrease of the populations of Escherichia coli O157:H7 and Salmonella typhimurium. These results suggest that aqueous ClO2 treatment should be useful in improving the microbial safety of chicken during storage.     

Reduction of Escherichia coli O157:H7 populations in cattle by addition of colicin E7-producing E. coli to feed

A cattle trial using artificially inoculated calves was conducted to determine the effect of the addition of colicinogenic Escherichia coli strains capable of producing colicin E7 (a 61-kDa DNase) to feed on the fecal shedding of serotype O157:H7. The experiment was divided into three periods. In period 1, which lasted 24 days, six calves were used as controls, and eight calves received 10(7) CFU of E. coli (a mixture of eight colicinogenic E. coli strains) per g of feed. Both groups were orally inoculated with nalidixic acid-resistant E. coli O157:H7 strains 7 days after the treatment started. In periods 2 and 3, the treatment and control groups were switched, and the colicinogenic E. coli dose was increased 10-fold. During period 3, which lasted as long as period 1, both groups were reinoculated with E. coli O157:H7. The numbers of E. coli O157:H7 were consistently greater in the control groups during the three periods, but comparisons within each time period determined a statistically significant (P < 0.05) difference only at day 21 of period 1. However, when the daily average counts were compared between the period 1 control group and the period 3 treatment group that included the same six animals, an overall reduction of 1.1 log(10) CFU/g was observed, with a maximum decrease of 1.8 log(10) CFU/g at day 21 (overall statistical significance, P = 0.001). Serotype O157:H7 was detected in 44% of the treatment group's intestinal tissue samples and in 64% of those from the control group (P < 0.04). These results indicated that the daily addition of 10(8) CFU of colicin E7-producing E. coli per gram of feed could reduce the fecal shedding of serotype O157:H7.     

Death of enterohemorrhagic Escherichia coli O157:H7 in real mayonnaise and reduced-calorie mayonnaise dressing as influenced by initial population and storage temperature.

This study was undertaken to determine the survivability of low-density populations (10(0) and 10(2) CFU/g) of enterohemorrhagic Escherichia coli O157:H7 inoculated into real mayonnaise and reduced-calorie mayonnaise dressing and stored at 20 and 30 degrees C, temperatures within the range used for normal commercial mayonnaise distribution and storage. Inactivation patterns at 5 degrees C and inactivation of high-inoculum populations (10(6) CFU/g) were also determined. The pathogen did not grow in either mayonnaise formulation, regardless of the inoculum level or storage temperature. Increases in storage temperature from 5 to 20 degrees C and from 20 to 30 degrees C resulted in dramatic increases in the rate of inactivation. Populations of E. coli O157:H7 in the reduced-calorie and real formulations inoculated with a population of 0.23 to 0.29 log10 CFU/g and held at 30 degrees C were reduced to undetectable levels within 1 and 2 days, respectively; viable cells were not detected after 1 day at 20 degrees C. In mayonnaise containing an initial population of 2.23 log10 CFU/g, viable cells were not detected after 4 days at 30 degrees C or 7 days at 20 degrees C; tolerance was greater in real mayonnaise than in reduced-calorie mayonnaise dressing stored at 5 degrees C. The tolerance of E. coli O157:H7 inoculated at the highest population density (6.23 log 10 CFU/g) was less in reduced-calorie mayonnaise dressing than in real mayonnaise at all storage temperatures. In reduced-calorie mayonnaise dressing and real mayonnaise initially containing 2.23 log10 CFU/g, levels were undetectable after 28 and 58 days at 5 degrees C, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combined Effect of High-Pressure Treatments and Bacteriocin-Producing Lactic Acid Bacteria on Inactivation of Escherichia coli O157:H7 in Raw-Milk Cheese

The effect of high-pressure (HP) treatments combined with bacteriocins of lactic acid bacteria (LAB) produced in situ on the survival of Escherichia coli O157:H7 in cheese was investigated. Cheeses were manufactured from raw milk inoculated with E. coli O157:H7 at approximately 10⁵ CFU/ml. Seven different bacteriocin-producing LAB were added at approximately 10⁶ CFU/ml as adjuncts to the starter. Cheeses were pressurized on day 2 or 50 at 300 MPa for 10 min or 500 MPa for 5 min, at 10°C in both cases. After 60 days, E. coli O157:H7 counts in cheeses manufactured without bacteriocin-producing LAB and not pressurized were 5.1 log CFU/g. A higher inactivation of E. coli O157:H7 was achieved in cheeses without bacteriocin-producing LAB when 300 MPa was applied on day 50 (3.8-log-unit reduction) than if applied on day 2 (1.3-log-unit reduction). Application of 500 MPa eliminated E. coli O157:H7 in 60-day-old cheeses. Cheeses made with bacteriocin-producing LAB and not pressurized showed a slight reduction of the pathogen. Pressurization at 300 MPa on day 2 and addition of lacticin 481-, nisin A-, bacteriocin TAB 57-, or enterocin AS-48-producing LAB were synergistic and reduced E. coli O157:H7 counts to levels below 2 log units in 60-day-old cheeses. Pressurization at 300 MPa on day 50 and addition of nisin A-, bacteriocin TAB 57-, enterocin I-, or enterocin AS-48-producing LAB completely inactivated E. coli O157:H7 in 60-day-old cheeses. The application of reduced pressures combined with bacteriocin-producing LAB is a feasible procedure to improve cheese safety.     

Genomic analysis of Pediococcus starter cultures used to control Listeria monocytogenes in turkey summer sausage.

The pulsed-field technique of clamped homogeneous electric field electrophoresis was employed to characterize and size genomic DNA of three pediocin-producing (Ped+) and two non-pediocin-producing (Ped-) strains of Pediococcus acidilactici. Comparison of genomic fingerprints obtained by digestion with the low-frequency-cleavage endonuclease AscI revealed identical restriction profiles for four of the five strains analyzed. Summation of results for 10 individually sized AscI fragments estimated the genome length to be 1,861 kb for the four strains (H, PAC1.0, PO2, and JBL1350) with identical fingerprints. Genomic analysis of the pediocin-sensitive, plasmid-free strain P. acidilactici LB42 with the unique fingerprint revealed nine AscI fragments and a genome length of about 2,133 kb. Ped- (JBL1350) and Ped+ (JBL1095) starter cultures (one each) were used to separately prepare turkey summer sausage coinoculated with a four-strain Listeria monocytogenes mixture (ca. 10(5) CFU/g). The starter cultures produced equivalent amounts of acid during fermentation, but counts of L. monocytogenes were reduced to a greater extent in the presence of the Ped+ starter culture (3.4 log10 unit decrease) than in the presence of the Ped- starter culture (0.9 log10 unit decrease). Although no listeriae were recovered from sausages following the cook/shower, appreciable pediocin activity was recovered from sausages prepared with the Ped+ strain for at least 60 days during storage at 4 degrees C. The results of this study revealed genomic similarities among pediococcal starter cultures and established that pediocins produced during fermentation provide an additional measure of safety against listerial proliferation in turkey summer sausage.     

Genomic analysis of Pediococcus starter cultures used to control Listeria monocytogenes in turkey summer sausage.

The pulsed-field technique of clamped homogeneous electric field electrophoresis was employed to characterize and size genomic DNA of three pediocin-producing (Ped+) and two non-pediocin-producing (Ped-) strains of Pediococcus acidilactici. Comparison of genomic fingerprints obtained by digestion with the low-frequency-cleavage endonuclease AscI revealed identical restriction profiles for four of the five strains analyzed. Summation of results for 10 individually sized AscI fragments estimated the genome length to be 1,861 kb for the four strains (H, PAC1.0, PO2, and JBL1350) with identical fingerprints. Genomic analysis of the pediocin-sensitive, plasmid-free strain P. acidilactici LB42 with the unique fingerprint revealed nine AscI fragments and a genome length of about 2,133 kb. Ped- (JBL1350) and Ped+ (JBL1095) starter cultures (one each) were used to separately prepare turkey summer sausage coinoculated with a four-strain Listeria monocytogenes mixture (ca. 10(5) CFU/g). The starter cultures produced equivalent amounts of acid during fermentation, but counts of L. monocytogenes were reduced to a greater extent in the presence of the Ped+ starter culture (3.4 log10 unit decrease) than in the presence of the Ped- starter culture (0.9 log10 unit decrease). Although no listeriae were recovered from sausages following the cook/shower, appreciable pediocin activity was recovered from sausages prepared with the Ped+ strain for at least 60 days during storage at 4 degrees C. The results of this study revealed genomic similarities among pediococcal starter cultures and established that pediocins produced during fermentation provide an additional measure of safety against listerial proliferation in turkey summer sausage.     

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 degrees 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 degrees 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.     

Shedding of Escherichia coli O157:H7 in dairy cattle housed in a confined environment following waterborne inoculation

A study of Escherichia coli O157:H7 transmission and shedding was conducted with bull calves housed in individual pens within a confined environment. For comparative purposes, the numbers and duration of E. coli O157:H7 shedding in naturally infected calves were monitored after a single purchased calf (calf 156) tested positive prior to inoculation. During the next 8 days, the calves in adjacent pens and a pen directly across a walkway from calf 156 began to shed this serotype O157:H7 strain. Five of the eight calves in this room shed this O157:H7 strain at some time during the following 8 weeks. The numbers of E. coli O157:H7 isolates shed in these calves varied from 60 to 10(5) CFU/g of feces, and the duration of shedding ranged from 17 to >31 days. The genomic DNAs from isolates recovered from these calves were indistinguishable when compared by using XbaI digestion and pulsed-field gel electrophoresis. Inoculation of calves with 1 liter of water containing ca. 10(3) to 10(4) CFU of E. coli O157:H7/ml resulted in shedding in 10 of 12 calves (trial 1, 4 of 4 calves; trial 2, 6 of 8 calves). The inoculated calves shed the inoculation strain (FRIK 1275) as early as 24 h after administration. The duration of shedding varied from 18 to >43 days at levels from 10(2) to 10(6) CFU/g of feces. The numbers of doses necessary to initiate shedding varied among calves, and two calves in trial 2 never shed FRIK 1275 after four doses (ca. 10(6) CFU per dose). Results from this study confirm previous reports of animal-to-animal and waterborne dissemination of E. coli O157:H7 and highlight the need for an effective water treatment to reduce the spread of this pathogen in cattle.     

Detection and recovery rates achieved using direct plate and enrichment/immunomagnetic separation methods for Escherichia coli O157:H7 in minced beef and on bovine hide

AIMS: To assess the detection and recovery rates achieved with commonly used cultural methods for the enumeration and recovery of Escherichia coli O157:H7 from minced beef and bovine hide. METHODS AND RESULTS: Minced beef and bovine hide were inoculated with varying concentrations (log(10) 1.58-2.58 CFU g(-1) and log(10) 2.42-4.49 CFU 100 cm(2) respectively) of E. coli O157:H7 and recovered using a direct plate method or an enrichment/immunomagnetic separation (IMS) method and then plated onto SMAC or SMAC-CT in both cases. The direct plate method detected the pathogen consistently from minced beef samples with an average recovery of 69.2-91.2%. From faecal material on the bovine hide the recovery of the pathogen ranged from 1.80 to 64.5% with fresh faeces depending on the inocula while from dried faeces on hide the results ranged from no recovery at all to 25.1%. Enrichment/IMS recovered E. coli O157:H7 at all inocula levels tested in minced beef while the pathogen was only detected consistently at an average inocula level of log(10) 2.73 CFU 100 cm(2) from fresh faeces and log(10) 4.49 CFU 100 cm(2) from dried faeces on bovine hide. CONCLUSIONS: The direct count enumeration method for E. coli O157:H7 underestimated the numbers of pathogens present. The enrichment/IMS procedure consistently detected the pathogen from minced beef but did not always detect E. coli O157:H7 from faeces on bovine hide. SIGNIFICANCE AND IMPACT OF THE STUDY: Overall this study highlights that any microbial data, used in either predictive microbiology or risk assessment, must take account of the sensitivity and associated performance of the methods employed, in order to make an accurate reflection of the true microbiology of the examined sample.     

Postprocessing in vitro digestion challenge to evaluate survival of Escherichia coli O157:H7 in fermented dry sausages

Fermented dry sausages, inoculated with Escherichia coli O157:H7 during batter preparation, were submitted to an in vitro digestion challenge to evaluate the extent to which passage through the human gastrointestinal tract could inactivate the pathogenic cells, previously stressed by the manufacturing process. The numbers of surviving E. coli O157:H7 cells remained constant after a 1-min exposure of the finely chopped sausage to synthetic saliva or during the following 120-min exposure to synthetic gastric juice at an initial pH of 2.0. However, significant (P < or = 0.05) growth of the pathogen (1.03 to 2.16 log10 CFU/g) was observed in a subsequent 250-min exposure to a synthetic pancreatic juice at pH 8.0. In a different set of experiments, fractions from the gastric suspension were transferred into the synthetic pancreatic juice at 30-min intervals to mimic the dynamics of gastric emptying. Concurrently, the pH of the remaining gastric fluid was reduced to 3.0, 2.5, and 2.0 to simulate the gradual reacidification of the stomach contents after the initial buffering effect resulting from meal ingestion. Under these new conditions, pathogen growth during pancreatic challenge was observed for the first few fractions released from the stomach (90 min of exposure [pH 2.5]), but growth was no longer possible in the fractions submitted to the most severe gastric challenge (120 min of exposure [pH < 2.2]).     

Fate of pGFP-bearing Escherichia coli O157:H7 in ground beef at 2 and 10 degrees C and effects of lactate, diacetate, and citrate

Although beef has been implicated in the largest outbreaks of Escherichia coli O157:H7 infection in the United States, studies on the fate of this pathogen have been limited. Problems in such studies are associated with detection of the pathogen at levels considerably lower than the levels of the competing microorganisms. In the present study, a green fluorescent protein-expressing E. coli O157:H7 strain was used, and the stable marker allowed us to monitor the behavior of the pathogen in ground beef stored aerobically from freshness to spoilage at 2 and 10 degrees C. In addition, the effects of sodium salts of lactate (SL) (0.9 and 1.8%), diacetate (SDA) (0.1 and 0.2%), and buffered citrate (SC) (1 and 2%) and combinations of SL and SDA were evaluated. SC had negligible antimicrobial activity, and SL delayed microbial growth, while SDA and SL plus SDA were most inhibitory to the total-aerobe population in the meat. At 2 degrees C, the initial numbers of E. coli O157:H7 (3 and 5 log(10) CFU/g) decreased by approximately 1 log(10) CFU/g when spoilage was manifest (>7 log(10) CFU of total aerobes/g), irrespective of the treatment. There was no decline in the numbers of the pathogen during storage at 10 degrees C. Our results showed that the pathogen was resistant to the salts tested and confirmed that refrigerated meat contaminated with the pathogen remains hazardous.     

Survival of Escherichia coli O157:H7 in feces from corn- and barley-fed steers

The survival of Escherichia coli O157:H7 in feces from steers fed corn (CO) or barley (BA) was evaluated at -10, +4 and +22 degrees C. Fecal pats were inoculated with a four-strain mixture of nalidixic-acid resistant E. coli O157:H7 at two levels: 10(3) CFU g(-1) (low, L) and 105 CFU g(-1) (high, H). At -10 degrees C, duration of survival of E. coli O157:H7 was reduced (p < 0.05) in CO-L (35 days) compared to BA-L (49 days), likely due to the effects of fecal volatile fatty acids in combination with a fecal pH of <6.5. At 4 degrees C, E. coli O157:H7 was detected in BA-H, CO-H, CO-L and BA-L for 77, 77, 56 and 63 days, respectively, with no difference (p > 0.05) observed in the duration of survival or rate of decline of E. coli O157:H7 among treatments. Survival of E. coli O157:H7 was twice as likely (p < 0.05) at 22 degrees C than at 4 degrees C and -10 degrees C. While pH and dry matter content increased, and volatile fatty acid concentrations decreased over 84 days at all three temperatures, these changes were most pronounced at 22 degrees C. Survival of E. coli O157:H7 for extended periods of time in feces from both corn- and barley-fed animals was demonstrated, thus fecal material may serve as a vector for the transmission of the organism. The greater survival of E. coli O157:H7 at 22 degrees C suggests that temperature may play a role in the seasonality of transmission and prevalence of this bacterium in feedlot cattle. The reported greater prevalence of E. coli O157:H7 in cattle fed barley as compared to those fed corn does not appear to be related to elevated risk of transmission arising from differential survival of the bacterium in feces.     

Reduction of Escherichia coli O157:H7 by stimulated Pediococcus acidilactici

This study was conducted to determine if stimulating the growth of meat starter culture (Pediococcus acidilactici) in a laboratory medium (Brain Heart Infusion broth +2.3% NaCl + 1.5% sucrose; LBHI) and during meat fermentation would control Escherichia coli O157:H7. In LBHI medium without P. acidilactici, the numbers of E. coli O157:H7 increased from 4.00 to 8.34 log10 cfu ml-1, whereas in the presence of P. acidilactici (approximately 6.0 log10 cfu ml-1) in LBHI, LBHIM (LBHI + 0.005% MnSO4), LBHIO (LBHI + 0.3 unit ml-1 Oxyrase), and LBHIMO (LBHI + M + O), the numbers of E. coli O157:H7 increased from 4.00 to 8.05, 7.50, 7.99, and 6.50 log10 cfu ml-1, respectively, after incubation at 40 degrees C for 15 h. During salami fermentation, the numbers of E. coli O157:H7 changed from 7.00 to 6.40 and 5.10 log10 cfu g-1 without and with P. acidilactici (approximately 7.0 log10 cfu g-1), respectively. Stimulated P. acidilactici by M, O, and MO further reduced the number of E. coli O157:H7 from 7.00 to 4.00, 4.80, and 3.65 log10 cfu g-1, respectively. The combination of MO was a better growth stimulator for P. acidilactici, which controlled E. coli O157:H7 in both systems (P < 0.05).     

Destruction of Salmonella typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes in chicken manure by drying and/or gassing with ammonia

Escherichia coli O157:H7 and Listeria monocytogenes were able to grow for a period of 2 days in fresh chicken manure at 20 degrees C with a resulting 1-2 log units increase in CFU; Salmonella typhimurium remained stable. Prolongation of the storage time to 6 days resulted in a 1-2 log decreases of S. typhimurium compared to the initial count and a 3-4 log decrease of E. coli O157:H7; the number of L. monocytogenes did not decrease below the initial. These changes were accompanied by an increase in pH and accumulation of ammonia in the manure. The destruction of the three microorganisms was greatly increased by drying the manure to a moisture content of 10% followed by exposure to ammonia gas in an amount of 1% of the manure wet weight; S. typhimurium and E. coli O157:H7 were reduced by 8 log units, L. monocytogenes by 4.     

Detection and quantification of Escherichia coli O157:H7 in environmental samples by real-time PCR

AIMS: To apply the real-time Polymerase chain reaction (PCR) method to detect and quantify Escherichia coli O157:H7 in soil, manure, faeces and dairy waste washwater. METHODS AND RESULTS: Soil samples were spiked with E. coli O157:H7 and subjected to a single enrichment step prior to multiplex PCR. Other environmental samples suspected of harbouring E.coli O157:H7 were also analysed. The sensitivity of the primers was confirmed with DNA from E.coli O157:H7 strain 3081 spiked into soil by multiplex PCR assay. A linear relationship was measured between the fluorescence threshold cycle (C T ) value and colony counts (CFU ml(-1)) in spiked soil and other environmental samples. The detection limit for E.coli O157:H7 in the real-time PCR assay was 3.5 x 10(3) CFU ml(-1) in pure culture and 2.6 x 10(4) CFU g(-1) in the environmental samples. Use of a 16-h enrichment step for spiked samples enabled detection of <10 CFU g(-1) soil. E. coli colony counts as determined by the real-time PCR assay, were in the range of 2.0 x 10(2) to 6.0 x 10(5) CFU PCR (-1) in manure, faeces and waste washwater. CONCLUSIONS: The real-time PCR-based assay enabled sensitive and rapid quantification of E. coli O157:H7 in soil and other environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to quantitatively determine cell counts of E.coli O157:H7 in large numbers of environmental samples, represents considerable advancement in the area of pathogen quantification for risk assessment and transport studies.     

Experimental Escherichia coli O157:H7 carriage in calves.

Nine weaned calves (6 to 8 weeks of age) were given 10(10) CFU of a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 by oral-gastric intubation. After an initial brief period of pyrexia in three calves and transient mild diarrhea in five calves, calves were clinically normal throughout the 13- to 27-day study. The population of E. coli O157:H7 in the faces decreased dramatically in all calves during the first 2 weeks after inoculation. Thereafter, small populations of E. coli O157:H7 persisted in all calves, where they were detected intermittently in the feces and rumen contents. While withholding food increased fecal shedding of E. coli O157:H7 by 1 to 2 log10/g in three of four calves previously shedding small populations of E. coli O157:H7, the effect of fasting on fecal shedding of E. coli O157:H7 was variable in calves shedding larger populations. At necropsy, E. coli O157:H7 was not isolated from sites outside the alimentary tract. E. coli O157:H7 was isolated from the forestomach or colon of all calves at necropsy. Greater numbers of E. coli O157:H7 were present in the gastrointestinal contents than in the corresponding mucosal sections, and there was no histologic or immunohistochemical evidence of E. coli O157:H7 adhering to the mucosa. In conclusion, under these experimental conditions, E. coli O157:H7 is not pathogenic in weaned calves, and while it does not appear to colonize mucosal surfaces for extended periods, E. coli O157:H7 persists in the contents of the rumen and colon as a source for fecal shedding.     

Multiplex fluorogenic real-time PCR for detection and quantification of Escherichia coli O157:H7 in dairy wastewater wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C(T)) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C(T) and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 x 10(-5) pg of E. coli O157:H7 DNA ml(-1) equivalent to approximately 6.4 x 10(3) CFU of E. coli O157:H7 ml(-1) based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were >/=3.5 x 10(4) CFU g(-1). E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g(-1) with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.