Transmission of Escherichia coli O157:H7 in cattle is influenced by the level of environmental contamination.

A pen infection-transmission experiment was conducted to elucidate the role of pathogen strain and environmental contamination in transmission of Escherichia coli O157:H7 (ECO157) in cattle. Five steers were inoculated with a three-strain mixture of ECO157 and joined with five susceptible steers in each of two experimental replicates. Faecal and environmental samples were monitored for ECO157 presence over 30 days. One ECO157 strain did not spread. Transmission rates for the other two strains were estimated using a generalized linear model developed based on a modified 'Susceptible-Infectious-Susceptible' mathematical model. Transmission rates estimated for the two strains (0·11 and 0·14) were similar. However, the rates significantly (P = 0·0006) increased 1·5 times for every 1-unit increase in the level of environmental contamination measured as log10 c.f.u. Depending on the level of environmental contamination, the estimated basic reproduction numbers varied from <1 to 8. The findings indicate the importance of on-farm measures to reduce environmental contamination for ECO157 control in cattle that should be validated under field conditions.

Growth and survival parameter estimates and relation to RpoS levels in serotype O157:H7 and non-O157 Shiga toxin-producing Escherichia coli.

AIMS: This study analysed the growth and survival of 18 strains of the six serotypes of non-O157 Shiga toxin-producing Escherichia coli (STEC) (O26, O45, O103, O111, O121 and O145) most frequently implicated in human illness and compared them with Escherichia coli O157:H7 strain ATCC43895. METHODS AND RESULTS: The data from growth in Luria-Bertani broth (LB)-HCl (pH 4.0, 4.5, 4.8), LB-lactate (pH 4.5 and 4.8) and LB-NaCl (5%, 7%) were fitted to modified Gompertz equations to enable quantitative comparisons across strains and media conditions. Serogroup O45 strains had growth rates that were equal to or significantly greater than the O157:H7 control strain in all growth conditions tested. The growth rate was independent from the maximum growth achieved, but three strains (103A, 121A and 45B) had significantly faster growth and greater maximum cell densities in LB-NaCl 5% (strain 103A), LB-HCl pH 4·0 (strain 121A) and LB-NaCl 7% (strain 45B). Survival in LB-HCl pH 3.0 of four strains (103C, 111B, 26B and 26C) was significantly greater and five strains (26A, 45A, 111A, 121A and 145A) were significantly reduced in comparison with the O157:H7 control strain. None of the STEC strains had greater survival in LB-NaCl 12% than the O157:H7 control strain. A significant association was found between the exponential phase, but not stationary phase, RpoS level and survival of STEC. CONCLUSIONS: Some STEC strains had growth or survival properties that exceeded those of the O157:H7 control strain, but none of the non-O157 STEC had both significantly greater growth and survival properties. STEC survival was associated with the exponential-phase RpoS level. SIGNIFICANCE AND IMPACT OF THE STUDY: Results from this study define the variability in growth and survival of STEC strains that will be useful defining food product formulations and process interventions to control STEC. The presence of exponential phase σ(s) expands the significance of this alternative sigma factor.

Effectiveness of environmental decontamination as an infection control measure.

The effectiveness of environmental decontamination (ED) as a measure in the control of infectious diseases is controversial. This work quantifies the effectiveness of ED by analysing the transmission of pathogens from the environment to susceptible hosts in a Susceptible-Infected-Susceptible model. Analysis of the model shows that ED can render a population disease-free only when the duration of infection (D) is within a certain range. As host-to-host transmission rate is increased, D falls outside this range and the higher levels of ED have a diminishing return in reducing the number of infected hosts at endemic equilibrium. To avoid this, ED can be combined with other control measures, such as treating infected individuals to push the duration of infection into the specified range. We propose decision criteria and minimum ED efforts required for control policies to be effective.

Isolation of Escherichia coli O157:H7 from the gall bladder of inoculated and naturally-infected cattle.

To determine if Escherichia coli O157:H7 is capable of residing in the gall bladder of cattle, inoculation studies were conducted with O157:H7 strain 86-24 in weaned Holstein calves. Strain 86-24 was isolated from the gall bladders of five calves 36 days after inoculation. Two other calves contained the inoculation strain in the distal colon but the organism was absent in their gall bladders. A second trial in which the calves were euthanized 15 days after inoculation found strain 86-24 in six of seven inoculated calves but only in colon and/or rumen samples. In a third trial that inoculated eight calves with a four-strain cocktail of O157:H7 strains, the gall bladders from all eight animals were positive 9 days after inoculation. The colon and rumen samples from these calves were also positive. E. coli O157:H7 isolates recovered from bile samples and subtyped by pulsed field gel electrophoresis found that three of the four inoculation strains were present in one or more of the calves. Thus, residence in the gall bladder is not restricted to a single strain. Additional evidence of the ability to localize in the gall bladder of cattle was provided by testing the bile from 150 gall bladders (five collection dates, 30 samples each) obtained at an abbatoir and the isolation of E. coli O157:H7 from four samples (2.7%). This study establishes that E. coli O157:H7 can reside transiently or permanently at a low level in the gall bladder of cattle.

Characterization of Escherichia coli O157:H7 from downer and healthy dairy cattle in the upper Midwest region of the United States.

While cattle in general have been identified as a reservoir of Escherichia coli O157:H7, there are limited data regarding the prevalence and clonality of this pathogen in downer dairy cattle and the potential impact to human health that may occur following consumption of meat derived from downer dairy cattle. In the present study, conducted at two slaughter facilities in Wisconsin between May and October of 2001, we established a higher prevalence of E. coli O157:H7 in fecal and/or tissue samples obtained aseptically from intact colons of downer dairy cattle (10 of 203, 4.9%) than in those from healthy dairy cattle (3 of 201, 1.5%). Analyses of 57 isolates, representing these 13 positive samples (one to five isolates per sample), by pulsed-field gel electrophoresis, revealed 13 distinct XbaI restriction endonuclease digestion profiles (REDP). Typically, isolates from different animals displayed distinct REDP and isolates from the same fecal or colon sample displayed indistinguishable REDP. However, in one sample, two different, but highly related, REDP were displayed by the isolates recovered. Antimicrobial susceptibility testing indicated that 10 of the 57 isolates, recovered from 2 (1 downer and 1 healthy animal) of the 13 positive samples, were resistant to at least 1 of 18 antimicrobials tested. However, there was no appreciable difference in the frequency of resistance of isolates recovered from downer and healthy dairy cattle, and not all isolates with the same REDP displayed the same antimicrobial susceptibility profile. Lastly, it was not possible to distinguish between isolates recovered from downer and healthy cattle based on their XbaI REDP or antimicrobial susceptibility. These results indicate that downer cattle had a 3.3-fold-higher prevalence of E. coli O157:H7 than healthy cattle within the time frame and geographic scope of this study.

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.

Contribution of dps to acid stress tolerance and oxidative stress tolerance in Escherichia coli O157:H7.

An Escherichia coli O157:H7 dps::nptI mutant (FRIK 47991) was generated, and its survival was compared to that of the parent in HCl (synthetic gastric fluid, pH 1.8) and hydrogen peroxide (15 mM) challenges. The survival of the mutant in log phase (5-h culture) was significantly impaired (4-log(10)-CFU/ml reduction) compared to that of the parent strain (ca. 1.0-log(10)-CFU/ml reduction) after a standard 3-h acid challenge. Early-stationary-phase cells (12-h culture) of the mutant decreased by ca. 4 log(10) CFU/ml while the parent strain decreased by approximately 2 log(10) CFU/ml. No significant differences in the survival of late-stationary-phase cells (24-h culture) between the parent strain and the mutant were observed, although numbers of the parent strain declined less in the initial 1 h of acid challenge. FRIK 47991 was more sensitive to hydrogen peroxide challenge than was the parent strain, although survival improved in stationary phase. Complementation of the mutant with a functional dps gene restored acid and hydrogen peroxide tolerance to levels equal to or greater than those exhibited by the parent strain. These results demonstrate that decreases in survival were from the absence of Dps or a protein regulated by Dps. The results from this study establish that Dps contributes to acid tolerance in E. coli O157:H7 and confirm the importance of Dps in oxidative stress protection.

Role of rpoS in acid resistance and fecal shedding of Escherichia coli O157:H7.

Acid resistance (AR) is important to survival of Escherichia coli O157:H7 in acidic foods and may play a role during passage through the bovine host. In this study, we examined the role in AR of the rpoS-encoded global stress response regulator sigma(S) and its effect on shedding of E. coli O157:H7 in mice and calves. When assayed for each of the three AR systems identified in E. coli, an rpoS mutant (rpoS::pRR10) of E. coli O157:H7 lacked the glucose-repressed system and possessed reduced levels of both the arginine- and glutamate-dependent AR systems. After administration of the rpoS mutant and the wild-type strain (ATCC 43895) to ICR mice at doses ranging from 10(1) to 10(4) CFU, we found the wild-type strain in feces of mice given lower doses (10(2) versus 10(3) CFU) and at a greater frequency (80% versus 13%) than the mutant strain. The reduction in passage of the rpoS mutant was due to decreased AR, as administration of the mutant in 0.05 M phosphate buffer facilitated passage and increased the frequency of recovery in feces from 27 to 67% at a dose of 10(4) CFU. Enumeration of E. coli O157:H7 in feces from calves inoculated with an equal mixture of the wild-type strain and the rpoS mutant demonstrated shedding of the mutant to be 10- to 100-fold lower than wild-type numbers. This difference in shedding between the wild-type strain and the rpoS mutant was statistically significant (P

Survival of Escherichia coli O157:H7 in ground-beef patties during storage at 2, -2, 15 and then -2 degrees C, and -20 degrees C.

The survival of Escherichia coli O157:H7 and of a nonpathogenic control strain of E. coli was monitored in raw ground beef that was stored at 2 degrees C for 4 weeks, -2 degrees C for 4 weeks, 15 degrees C for 4 h and then -2 degrees C for 4 weeks, and -20 degrees C. Irradiated ground beef was inoculated with one E. coli control strain or with a four-strain cocktail of E. coli O157:H7 (ca. 10(5) CFU/g), formed into patties (30 to 45 g), and stored at the appropriate temperature. The numbers of the E. coli control strain decreased by 1.4 log 10 CFU/g, and pathogen numbers declined 1.9 log 10 CFU/g when patties were stored for 4 weeks at 20 degrees C. When patties were stored at -2 degrees C for 4 weeks, the numbers of the E. coli control strain and the serotype O157:H7 strains decreased 2.8 and 1.5 log 10 CFU/g, respectively. Patties stored at 15 degrees C for 4 h prior to storage at -2 degrees C for 4 weeks resulted in 1.6 and 2.7 log 10-CFU/g reduction in the numbers of E. coli and E. coli O157:H7, respectively. Storage of retail ground beef at 15 degrees C for 4 h (tempering) did not result in increased numbers of colony forming units per gram, as determined with violet red bile, MRS lactobacilli, and plate-count agars. Frozen storage (-20 degrees C) of ground-beef patties that had been inoculated with a single strain of E. coli resulted in approximately a 1 to 2 log 10-CFU/g reduction in the numbers of the control strain and individual serotype O157:H7 strains after 1 year. There was no significant difference between the survival of the control strain and the O157:H7 strains, nor was there a difference between O157:H7 strains. These data demonstrate that tempering of ground-beef patties prior to low-temperature storage accelerated the decline in the numbers of E. coli O157:H7.

Lethality of a heat- and phosphate-catalyzed glucose by-product to Escherichia coli O157:H7 and partial protection conferred by the rpoS regulon.

A by-product of glucose produced during sterilization (121 degrees C, 15 lb/in2, 15 min) at neutral pH and in the presence of phosphate (i.e., phosphate-buffered saline) was bactericidal to Escherichia coli O157:H7 (ATCC 43895). Other six-carbon (fructose and galactose) and five-carbon (arabinose, ribose, and xylose) reducing sugars also produced a toxic by-product under the same conditions. Fructose and the five-carbon sugars yielded the most bactericidal activity. Glucose concentrations of 1% (wt/vol) resulted in a 99.9% decline in the CFU of stationary-phase cells per milliliter in 2 days at 25 degrees C. An rpoS mutant (pRR10::rpoS) of strain 43895 (FRIK 816-3) was significantly (P < 0.001) more sensitive to the glucose-phosphate by-product than the parent strain, as glucose concentrations from 0.05 to 0.25% resulted in a 2- to 3-log10 reduction in CFU per milliliter in 2 days at 25 degrees C. Likewise, log-phase cells of the wild-type strain, 43895, were significantly more sensitive (P < 0.001) to the glucose-phosphate by-product than were stationary-phase cells, which is consistent with the stability of rpoS and the regulation of rpoS-regulated genes. The bactericidal effect of the glucose-phosphate by-product was reduced when strains ATCC 43895 and FRIK 816-3 were incubated at a low temperature (4 degrees C). Also, growth in glucose-free medium (i.e., nutrient broth) did not alleviate the sensitivity to the glucose-phosphate by-product and excludes the possibility of substrate-accelerated death as the cause of the bactericidal effect observed. The glucose-phosphate by-product was also bactericidal to Salmonella typhimurium, Shigella dysenteriae, and a Klebsiella sp. Attempts to identify the glucose-phosphate by-product were unsuccessful. These studies demonstrate the production of a glucose-phosphate by-product bactericidal to E. coli O157:H7 and the protective effects afforded by rpoS-regulated gene products. Additionally, the detection of sublethally injured bacteria may be compromised by the presence of this by-product in recovery media.

Comparative survival of Salmonella typhimurium DT 104, Listeria monocytogenes, and Escherichia coli O157:H7 in preservative-free apple cider and simulated gastric fluid.

This study compared the survival of three-strain mixtures (ca. 10(7) CFU ml(-1) each) of Salmonella typhimurium DT104, Listeria monocytogenes, and Escherichia coli O157:H7 in pasteurized and unpasteurized preservative-free apple cider (pH 3.3-3.5) during storage at 4 and 10 degrees C for up to 21 days. S. typhimurium DT104 populations decreased by <4.5 log10 CFU ml(-1) during 14 days storage at 4 and 10 degrees C in pasteurized cider, and by > or =5.5 log10 CFU ml(-1) during 14 days in unpasteurized cider stored at these temperatures. However, after 7 days at 4 degrees C, the S. typhimurium DT104 populations had decreased by only about 2.5 log10 CFU ml(-1) in both pasteurized and unpasteurized cider. Listeria monocytogenes populations decreased below the plating detection limit (10 CFU ml(-1)) within 2 days under all conditions tested. Survival of E. coli O157:H7 was similar to that of S. typhimurium DT104 in pasteurized cider at both 4 and 10 degrees C over the 21-days storage period, but E. coli O157:H7 survived better (ca. 5.0 log10 CFU ml(-1) decrease) than S. typhimurium DT104 (> 7.0 log10 CFU ml(-1) decrease) after 14 days at 4 degrees C in unpasteurized cider. In related experiments, when incubated in simulated gastric fluid (pH 1.5) at 37 degrees C, S. typhimurium DT104 and L. monocytogenes were eliminated (5.5-6.0 log10 CFU ml(-1) decrease) within 5 and 30 min, respectively, whereas E. coli O157:H7 concentrations decreased only 1.60-2.80 log10 CFU ml(-1) within 2 h.

Viability of Escherichia coli O157:H7 in salami following conditioning of batter, fermentation and drying of sticks, and storage of slices.

The fate of Escherichia coli O157:H7 was monitored in salami during conditioning of batter, fermentation and drying of sticks, and storage of slices. The raw batter (75% pork: 25% beef, wt/wt, fat content about 20%) was inoculated with a pediococcal starter culture (about 10(8) CFU/g) and a five-strain cocktail of E. coli O157:H7 ( > or = 2 x 10(7) CFU/g) and stuffed into 104-mm diameter fibrous casings. After being refrigerated at 4 degrees C or being tempered at 13 degrees C, frozen at -20 degrees C, and thawed at 4 degrees C, or being frozen at -20 degrees C, and thawed at 4 degrees C, the inoculated batter was fermented at 24 degrees C and 90% relative humidity (RH) to pH < or = 4.8, dried at 13 degrees C and 65% RH to a moisture/protein ratio of < or = 1.9:1, and then stored at 4 or 21 degrees C under air or vacuum. For salami sticks sampled immediately after drying, appreciable differences were evident among the various batter-conditioning treatments; pathogen numbers were reduced from original levels by 2.1, 1.6, or 1.1 log10 units when batter was tempered, frozen, and thawed, frozen and thawed, or refrigerated, respectively. Similarly, regardless of storage temperature or atmosphere, within 7 days salami slices cut from sticks prepared from batter that was tempered, frozen, and thawed (2.7- to 4.9-log10-unit reduction) or frozen and thawed (2.3- to 4.8-log10-unit reduction) displayed a greater impact on pathogen numbers than slices cut from sticks prepared from batter that was refrigerated (1.6- to 3.1-log10-unit reduction). The effects of batter conditioning notwithstanding, a greater reduction in levels of E. coli O157:H7 was observed when slices were stored at 21 degrees C compared to otherwise similar slices stored at 4 degrees C. After storage for 60 days the pathogen was only detected by enrichment in slices stored at 21 degrees C, whereas pathogen levels ranged from 1.4 to 4.5 log10 CFU/g in slices stored at 4 degrees C. Differences related to storage atmosphere were first observed after slices were stored for 21 days. Such differences were more readily demonstrable after 60 and 90 days, with pathogen numbers for treatments that were statistically different ranging from 0.6- to 1.5-log10 units higher on slices stored under vacuum than in air. These data emphasize the need to implement multiple barriers to appreciably reduce numbers of E. coli O157:H7 in salami.

Survival of Escherichia coli O157:H7 in full- and reduced-fat pepperoni after manufacture of sticks, storage of slices at 4 degrees C or 21 degrees C under air and vacuum, and baking of slices on frozen pizza at 135, 191 and 246 degrees C.

Pepperoni batter was prepared with fat contents of about 15, 20, and 32% (wt/wt) and inoculated with a pediococcal starter culture and > or = 2.0 x 10(7) CFU/g of a five-strain inoculum of Escherichia coli O157:H7. The batter was fermented at 96 degrees F (ca. 36 degrees C and 85% relative humidity (RH) to pH < or = 4.8 and then dried at 55 degrees F (ca. 13 degrees C) and 65% RH to a moisture/protein ratio of < or = 1.6:1. For storage, slices were packaged under air or vacuum and stored at 39 degrees F (ca. 4 degrees C) and 70 degrees F (ca. 21 degrees C). For baking, frozen slices were placed on retail frozen cheese pizzas that were subsequently baked at 275 degrees F (ca. 135 degrees C), 375 degrees F (ca. 191 degrees C), or 475 degrees F (ca. 246 degrees C) for 0 to 20 min. Appreciable differences related to fat levels were observed after drying; pathogen numbers decreased by 1.04, 1.31 and 1.62 log10 units in sticks prepared from batter at initial fat levels of 15, 20, and 32%, respectively. During storage, the temperature rather than the atmosphere had the greater effect on pathogen numbers, with similar viability observed among the three fat levels tested. At 70 degrees F (ca. 21 degrees C), compared to original levels, pathogen numbers decreased by > or = 5.56 and > or = 4.53 log10 units within 14 days in slices stored under air and vacuum, respectively, whereas at 39 degrees F (ca. 4 degrees C) numbers decreased by < or = 2.43 log10 CFU/g after 60 days of storage under either atmosphere. Baking, as expected, resulted in greater reductions in pathogen numbers as the temperature and/or time of baking increased. However, it was still possible to recover the pathogen by enrichment after baking frozen slices on frozen pizza at 475 degrees F (ca. 246 degrees C) for 10 min or at 375 degrees F (ca. 191 degrees C) for 15 min. The calculated D values for all three temperatures tested increased as the fat content of the batter increased from 15 to 20 to 32%. The present study confirmed that fermentation and drying were sufficient to reduce levels of E. coli O157:H7 in pepperoni sticks by < 2.0 log10 CFU/g. Storage of slices for at least 14 days at ambient temperature under air resulted in a > 5.5-log10-unit total reduction of the pathogen. Baking slices on frozen pizza for at least 15 min at 475 degrees F (ca. 246 degrees C) or 20 min at 375 degrees F (ca. 191 degrees C) was necessary to reduce pathogen numbers to below detection by both direct plating and enrichment.

Longitudinal study of Escherichia coli O157:H7 dissemination on four dairy farms in Wisconsin.

A 14-month longitudinal study was conducted on four dairy farms (C, H, R, and X) in Wisconsin to ascertain the source(s) and dissemination of Escherichia coli O157:H7. A cohort of 15 heifer calves from each farm were sampled weekly by digital rectal retrieval from birth to a minimum of 7 months of age (range, 7 to 13 months). Over the 14 months of the study, the cohort heifers and other randomly selected cattle from farms C and H tested negative. Farm R had two separate periods of E. coli O157:H7 shedding lasting 4 months (November 1995 to February 1996) and 1 month (July to August 1996), while farm X had at least one positive cohort animal for a 5-month period (May to October 1996). Heifers shed O157:H7 strains in feces for 1 to 16 weeks at levels ranging from 2.0 x 10(2) to 8.7 x 10(4) CFU per g. E. coli O157:H7 was also isolated from other noncohort cattle, feed, flies, a pigeon, and water associated with the cohort heifers on farms R and/or X. When present in animal drinking water, E. coli O157:H7 disseminated through the cohort cattle and other cattle that used the water source. E. coli O157:H7 was found in water at < 1 to 23 CFU/ml. Genomic subtyping by pulsed-field gel electrophoresis demonstrated that a single O157:H7 strain comprised a majority of the isolates from cohort and noncohort cattle, water, and other positive samples (i.e., from feed, flies, and a pigeon, etc.) on a farm. The isolates from farm R displayed two predominant XbaI restriction endonuclease digestion profiles (REDP), REDP 3 and REDP 7, during the first and second periods of shedding, respectively. Six additional REDP that were > or = 89% similar to REDP 3 or REDP 7 were identified among the farm R isolates. Additionally, the REDP of an O157:H7 isolate from a heifer on farm R in 1994 was indistinguishable from REDP 3. Farm X had one O157:H7 strain that predominated (96% of positive samples had strains with REDP 9), and the REDP of an isolate from a heifer in 1994 was indistinguishable from REDP 9. These results suggest that E. coli O157:H7 is disseminated from a common source on farms and that strains can persist in a herd for a 2-year period.

Genomic comparisons and Shiga toxin production among Escherichia coli O157:H7 isolates from a day care center outbreak and sporadic cases in southeastern Wisconsin.

Contour-clamped homogeneous electric field pulsed-field gel electrophoresis (CHEF-PFGE) was used to compare Wisconsin isolates of Escherichia coli O157:H7, including 39 isolates from a 1994 day care center outbreak, 28 isolates from 18 individuals from the surrounding geographic area with sporadic cases occurring during the 3 months before the outbreak, and 3 isolates, collected in 1995, from patients with hemolytic-uremic syndrome (HUS) who were from eastern Wisconsin counties other than those inhabited by the day care center and sporadic-case individuals. The technique of CHEF-PFGE using XbaI identified seven highly related restriction endonuclease digestion profiles (REDPs) (93 to 98% similarity) among the 39 day care center isolates and nine XbaI REDPs (63 to 93% similarity) among the 28 isolates from sporadic-case individuals, including REDP 33, which was exhibited by both day care and sporadic-case isolates. PFGE analyses of sequential E. coli O157:H7 isolates from symptomatic day care center attendees revealed that the REDPs of 25 isolates from eight patients were indistinguishable whereas the REDPs of 2 of 6 isolates from two patients differed slightly (93 to 95% similarity). The REDPs of the three isolates from 1995 HUS patients were 78 to 83% similar, with REDP 26 being exhibited by one HUS-associated isolate and an isolate from one day care attendee who did not develop HUS. The genes for both Shiga toxins I and II (stx1 and stx2, respectively) were detected in all but one isolate (sporadic case), and Shiga toxin production by the day care center isolates was not significantly different from that of the other isolates, including the three HUS-associated isolates. Analyses of E. coli O157:H7 isolates from both the day care center outbreak and sporadic cases by CHEF-PFGE permitted us to define the REDP variability of an outbreak and geographic region and demonstrated that the day care center outbreak and a HUS case in 1995 were caused by E. coli O157:H7 strains endemic to eastern Wisconsin.

Survey of retail cheeses, dairy processing environments and raw milk for Escherichia coli O157:H7.

Escherichia coli was isolated from 58% (11/19) of retail soft and semi-soft cheeses tested, but E. coli O157:H7 was not detected. The presence of E. coli in retail cheeses and the lack of baseline data on the prevalence of serotype O157:H7 strains prompted us to survey ingredients and the environment in 15 cheese and dairy plants. Escherichia coli O157:H7 was not detected in any of the 1104 samples tested, including 42 raw milk samples. These results suggest that serotype O157:H7 is not prevalent within dairy product ingredients and processing environments.

Association of multiple-antibiotic-resistance profiles with point and nonpoint sources of Escherichia coli in Apalachicola Bay.

A total of 765 Escherichia coli isolates from point and nonpoint sources were collected from the Apalachicola National Estuarine Research Reserve, and their multiple-antibiotic-resistance (MAR) profiles were determined with 10 antibiotics. E. coli isolates from point sources showed significantly greater resistance (P < 0.05) to antibiotics and higher MAR indices than isolates from nonpoint sources. Specifically, 65 different resistance patterns were observed among point source isolates, compared to 32 among nonpoint source isolates. Examples of this contrast in MAR profiles included percentages of isolates with resistance to chlortetracycline-sulfathiazole of 33.7% and to chlortetracycline-penicillin G-sulfathiazole of 14.5% for point source isolates versus 15.4 and 1.7%, respectively, for nonpoint source isolates. MAR profile homology, based on coefficient similarity, showed that isolates from point sources were markedly more diverse than isolates from nonpoint sources. Seven clusters were observed among point source isolates, with a coefficient value of approximately 1.8. In contrast, only four clusters were observed among nonpoint source isolates. Covariance matrices of data displayed six very distinct foci representing nonpoint source E. coli isolates. Importantly, E. coli isolates obtained directly from human and animal feces also clustered among point and nonpoint sources, respectively. We conclude that E. coli MAR profiles were associated with point and nonpoint sources of pollution within Apalachicola Bay and that this method may be useful in facilitating management of other estuaries.

Genomic analyses of Salmonella enteritidis phage type 4 strains from Austria and phage type 8 strains from the United States.

Forty illness associated phage-type (PT) 4 and PT 8 strains of Salmonella enteritidis were analyzed by the pulsed-field technique of clamped homogeneous electric fields (CHEF) electrophoresis. Using NotI and XbaI, the 40 strains were subdivided by each enzyme into seven restriction endonuclease digestion profiles (REDP). The 35 PT 4 isolates from Austria were subdivided into six NotI and five XbaI REDP, while the five PT 8 isolates from the United States displayed a single NotI and two XbaI REDP. When highly-concentrated, uncleaved genomic DNA was subjected to CHEF electrophoresis, plasmid DNA in the size range of 350 kb relative to a linear DNA standard was discernible in 38 of the 40 strains. Subsequent isolation and restriction analyses of plasmid DNA from one strain (E40) revealed a single plasmid (pE40; ca. 54 kb) with one XbaI and two NotI cleavage sites that was similar in size to the S. enteritidis virulence plasmid pRQ29. Hybridization of the PE40 probe with S. enteritidis genomic DNAs identified a 54 kb fragment within the XbaI REDP and two fragments, 20 and 34 kb, in NotI REDP of plasmid-positive strains. It was not possible to identify plasmid-specific bands in NotI REDP without hybridization due to comigrating chromosomal and plasmid DNA fragments. Regardless of PT, all 40 S. enteritidis strains showed highly related REDP. The similarity between PT 4 and PT 8 strains as further revealed by Dice similarity coefficients was 90% to 95% for NotI REDP and 79% to 93% for XbaI REDP. These results support the hypothesis that the pandemic observed today is the result of the efficient spread of a single clone, or clusters of closely related clones, of S. enteritidis.

Pulsed-field gel electrophoresis and ribotype profiles of clinical and environmental Vibrio vulnificus isolates.

Vibrio vulnificus belongs to the autochthonous bacterial flora of warm estuarine waters. It can cause life-threatening extraintestinal disease in persons who have underlying illness and who consume raw shellfish or contact wounds with estuarine water. Currently, very little is known about genetic diversity within this species. In this report, we describe high-level variation in restriction fragment length polymorphism profiles among 53 clinical and 78 environmental isolates, as determined by pulsed-field gel electrophoresis. In contrast, ribotype profiles showed greater similarity. When combined ribotype profiles of clinical and environmental isolates were analyzed, four predominant clusters were observed. Interestingly, a low number (16%) of clinical isolates were found in cluster C, compared with clusters A, B, and D (range, 50 to 83%). In addition, 83% of all Hawaiian isolates were located in a single cluster, indicating a possible relationship between geography and genotype. We also report that spontaneous translucent colonial morphotypes were distinct by both restriction fragment length polymorphism and biochemical profiles, compared with opaque parent strains.

Prevalence and clonal nature of Escherichia coli O157:H7 on dairy farms in Wisconsin.

A survey was conducted between March and October of 1994 to determine the prevalence and identify the sources of serotype O157:H7 isolates of Escherichia coli in Wisconsin dairy herds. A stratified sample of 400 farms was identified, and 70 farms with weaned calves less than 4 months old were included in the study. During the prevalence study, 5 of the 70 farms (herd prevalence, 7.1 +/- 4.5%) and fecal samples from 10 of 560 calves (animal prevalence, 1.8%) tested positive for serotype O157:H7. In a follow-up study, the five O157:H7-positive farms and seven of the O157:H7-negative farms identified in the prevalence study were visited again. An additional 517 fecal samples from cattle of various ages were tested, and a total of 15 animals from four of the five herds that were previously positive and 4 animals from two of seven herds that were previously negative tested positive for E. coli O157:H7. Observations made during the follow-up study suggested that horizontal transmission was an important means of E. coli O157:H7 dissemination on the farms. A total of 302 environmental samples, were examined, and 2 animal drinking water samples from one previously negative farm and 1 animal drinking water sample from a previously positive farm contained E. coli O157:H7. Analyses by the pulsed-field gel electrophoresis technique of contour-clamped homogeneous electric field electrophoresis revealed that isolates from the same farm displayed identical or very similar XbaI restriction endonuclease digestion profiles (REDP), whereas isolates from different farms typically displayed different REDP. However, more than one REDP was usually observed for a given herd over the 8-month sampling period. Analyses of multiple isolates from an animal revealed that some animals harbored O157:H7 strains that had different REDP, although the REDP of isolates obtained from the same fecal sample were very similar. Collectively, 160 bovine isolates obtained from 29 different animals and three water isolates displayed 20 distinct XbaI REDP. Our data revealed that there are several clonal types of serotype O157:H7 isolates in Wisconsin and indicated that there is probably more than one source of this pathogen on the dairy farms studied. However, animal drinking water was identified as one source of E. coli O157:H7 on one farm.