Spontaneous reactivation of Shiga toxins in Escherichia coli O157:H7 cells caused by transposon excision

IS1203v is an insertion sequence which has been found in inactivated Shiga toxin 2 genes (stx2) of Escherichia coli O157:H7. Using PCR amplification, we detected the wild-type stx2 genes in colonies of E. coli O157:H7 which possessed stx2 genes inactivated by insertion of IS1203v. This suggests that IS1203v is excised from the inactivated stx2 genes in E. coli O157:H7. We isolated the cells possessing the wild-type stx2 genes, and confirmed Stx2 productivities by reversed passive latex agglutination. We also analyzed the frequency of the appearance of the Stx2-producing cells using a quantitative PCR method. As a result, the frequency was 3.00 x 10(-6) with culturing for 24 h at 37 degrees C, and this increased to 8.83 x 10(-5) when E. coli O157:H7 possessing the inactivated stx2 genes was transformed by an expression plasmid harboring the IS1203v transposase. These results showed that some Stx2-nonproducing E. coli O157:H7 strains could be spontaneously changed into Stx2-producing cells.     

Identification of an insertion sequence, IS1203 variant, in a Shiga toxin 2 gene of Escherichia coli O157:H7

An insertion sequence composed of 1310 bp was found in Shiga toxin 2 genes of some isolates of Escherichia coli O157:H7. This insertion sequence showed extremely high homology with IS1203 of E. coli O111:H(-). This IS1203 variant was inserted in the region encoding the amino-terminus of the B subunit with a duplication of 3 bp at the target site, resulting in inactivation of the Shiga toxin 2 gene.     

uspA of Shigella sonnei

One of the strategies that bacteria utilize to combat environmental stress is to synthesize stress-responding proteins. In Escherichia coli, adverse environmental factors, such as starvation, heat, and the presence of acid, oxidants, heavy metals, and antibiotics, trigger the expression of the universal stress protein (USP). The gene of the USP, uspA, in E. coli K-12 and E. coli O157:H7 has been identified and sequenced. In this study, the nucleotide sequence of uspA in a strain of Shigella sonnei implicated in the 1998 parsley-related outbreak of shigellosis was determined. Within an 800-bp region sequenced, there were 17 bp mismatches between the uspA of S. sonnei and that of E. coli K-12. Among the 17 mismatched nucleotides, 8 were within the structure gene of uspA. A total of 12 bp variations were identified between the uspA of S. sonnei and that of E. coli O157:H7, of which 5 bp were internal to the coding region of uspA. However, unlike the mismatches between the uspA of E. coli K-12 and the same gene of E. coli O157:H7 and S. sonnei that resulted in a single amino acid substitution and changed an alanine to an arginine at position 140, the mismatches between S. sonnei and E. coli O157:H7 were silent and did not result in any amino acid substitution.     

Fecal shedding of Escherichia coli O157, Salmonella, and Campylobacter in Swiss cattle at slaughter

Fecal samples from 2,930 slaughtered healthy cattle were examined with the following goals: (i) to monitor the shedding of Escherichia coli O157, Salmonella, and Campylobacter in cattle; and (ii) to further characterize the isolated strains. The percentage of the 2,930 samples that tested positive for E. coli O157 by PCR was 1.6%. Thirty-eight strains from different animals that agglutinated with Wellcolex E. coli O157 were isolated. Of the six sorbitol-negative strains, five tested positive for stx2 genes (two times for stx2c and three times for stx2), and one strain tested positive for stx1 and stx2c genes. All sorbitol-negative strains belonged to the serotypes O157:H7- and O157:H7 and harbored the eae type gamma 1 and ehxA genes. The 32 sorbitol-positive strains tested negative for stx genes and belonged to the serotypes O157:H2, O157:H7, O157:H8, O157:H12, O157:H19, O157:H25, O157:H27, O157:H38, O157:H43, O157:H45, and O157:H-. All O157:H45 strains harbored the eae subtype alpha 1 and therefore seem to be atypical enteropathogenic E. coli strains. Whereas none of 1,000 examined samples was positive for Salmonella, 95 of 935 (10.2%) samples were positive for Campylobacter, and all strains were identified as C. jejuni. Sixteen Campylobacter strains were resistant to tetracycline, five were resistant to nalidixic acid/ciprofloxacin, four were resistant to streptomycin, and one was resistant to nalidixic acid/ciprofloxacin and streptomycin. Fecal shedding of zoonotic pathogens in slaughter animals is strongly correlated with the hazard of carcass contamination. Therefore, the maintenance of slaughter hygiene is of crucial importance.     

Selection of recently isolated colicinogenic Escherichia coli strains inhibitory to Escherichia coli O157:H7

Escherichia coli strains were screened for their ability to inhibit E. coli O157:H7. An initial evaluation of 18 strains carrying previously characterized colicins determined that only colicin E7 inhibited all of the E. coli O157:H7 strains tested. A total of 540 strains that had recently been isolated from humans and nine different animal species (cats, cattle, chickens, deer, dogs, ducks, horses, pigs, and sheep) were tested by a flip-plating technique. Approximately 38% of these strains were found to inhibit noncolicinogenic E. coli K12 strains. The percentage of potentially colicinogenic E. coli per animal species ranged from 14% for horse isolates to 64% for sheep strains. Those isolates that inhibited E. coli K12 were screened against E. coli O157:H7, and 42 strains were found to be capable of inhibiting all 22 pathogenic strains tested. None of these 42 strains produced bacteriophages, and only 24 isolates inhibited serotype O157:H7 in liquid culture. The inhibitory activity of these strains was completely eliminated by treatment with proteinase K. When mixtures of these 24 colicinogenic strains were grown in anaerobic continuous culture, the four-strain E. coli O157:H7 population was reduced at a rate of 0.25 log10 cells per ml per h, which was fivefold faster than the washout rate. Two strains originally isolated from cat feces (F16) and human feces (H30) were identified by repetitive sequences polymerase chain reaction as the predominant isolates in continuous cultures. The results of this work indicate that animal species other than cattle can be sources of anti-O157 colicinogenic strains, and these results also lead to the identification of at least two isolates that could potentially be used in preharvest control strategies.     

Influence of transportation stress and animal temperament on fecal shedding of Escherichia coli O157:H7 in feedlot cattle

To test the influence of transportation stress and temperament on shedding of Escherichia coli O157:H7, cattle (n=150) were classified at various stages of production as Excitable, Intermediate or Calm based on a variety of disposition scores. Presence of E. coli O157:H7 was determined by rectal swabs from live animals and from colons collected postmortem. Percentage of cattle shedding E. coli O157:H7 at arrival at the feedlot was approximately equal among temperament groups. Before shipment to the processing facility, a higher (P=0.03) proportion of cattle from the Calm group shed E. coli O157:H7 compared to the other temperament groups. When pooled across all sampling periods, cattle from the Calm group had a greater percentage test positive for E. coli O157:H7. Neither the acute stressor of transportation nor a more excitable temperament led to increased shedding of E. coli O157:H7 in cattle.     

Polymerase chain reaction assay for detection of Escherichia coli O157: H7 and Escherichia coli O157: H-.

The DNA band patterns generated by polymerase chain reaction (PCR) using the du2 primer and template DNAs from various strains of Escherichia coli and non-E. coli bacteria were compared. Among three to five prominent bands produced, the three bands at about 1.8, 2.7, and 5.0 kb were detected in all of the E. coli O157 strains tested. Some nonpathogenic E. coli and all pathogenic E. coli except E. coli O157 showed bands at 1.8 and 5.0 kb. It seems that the band at 2.7 kb is specific to E. coli O157. Sequence analysis of the 2.7-kb PCR product revealed the presence of a DNA sequence specific to E. coli O157:H- and E. coli O157:H7. Since the DNA sequence from base 15 to base 1,008 of the PCR product seems to be specific to E. coli O157, a PCR assay was carried out with various bacterial genomic DNAs and O157-FHC1 and O157-FHC2 primers that amplified the region between base 23 and base 994 of the 2.7-kb PCR product. A single band at 970 bp was clearly detected in all of the strains of E. coli O157:H- and E. coli O157:H7 tested. However, no band was amplified from template DNAs from other bacteria, including both nonpathogenic and pathogenic E. coli except E. coli O157. All raw meats inoculated with E. coli O157:H7 at 3 x 10(0) to 3.5 x 10(2) CFU/25 g were positive both for our PCR assay after cultivation in mEC-N broth at 42 degrees C for 18 h and for the conventional cultural method.     

Comparative evaluation of different chromogenic/fluorogenic media for detecting Escherichia coli O157:H7 in food.

Escherichia coli O157:H7 is a serious and common human pathogen that can cause diarrhoea, haemorrhagic colitis, and haemolytic uraemic syndrome (HUS). This study evaluated the enrichment, detection and confirmation procedures for the isolation of E. coli O157:H7 from raw ground beef and raw drinking milk. The purpose of this investigation was to compare Rainbow Agar O157 (RB; Biolog, Hayward, USA), Biosynth Culture Medium O157:H7 (BCM O157:H7; Biosynth, Staad, Switzerland) and Fluorocult HC (HC; Merck, Darmstadt, Germany) with the conventional Sorbitol MacConkey Agar (SMAC, Merck) using mEC + n (raw ground beef) and mTSB + n (raw milk) enrichment media. Single-path GLISA test (Gold Labeled Immuno Sorbent Assay; Merck) was used as the confirmation test. Growth of 466 strains of gram-negative rods isolated from food samples and 46 known E. coli strains from type culture and other collections (34 E. coli O157:H7 strains and 12 serotypes other than E. coli O157:H7) was examined on the agar media. The E. coli O157:H7 strains could readily be isolated and recognized uniquely by their typical black/grey colonies on RB and blue/black colonies on BCM O157:H7. Examination of the 46 known strains of E. coli reference strains showed false negative results on BCM O157:H7 (3.0%), RB (8.8%), HC (5.9%) and SMAC (5.9%) agars. On BCM O157:H7 no false negative results were found with the typical E. coli O157:H7 (beta-D-glucuronidase and sorbitol negative strains). One of two atypical E. coli O157:H7 strains (beta-D-glucuronidase positive) showed similar colouration to the typical strains and was mis-identified by each of the three media (RB, BCM O157:H7, and SMAC agar media). None of the 60 food samples tested yielded E. coli O157:H7. Examination of the food samples, showed that RB gave the lowest number of false positives. The percentages were RB (2.1%), BCM O157:H7 (3.3%), HC (6.2%), and SMAC (57.3%).     

Control of Escherichia coli O157:H7 with sodium metasilicate

Three intervention strategies-trisodium phosphate, lactic acid, and sodium metasilicate--were examined for their in vitro antimicrobial activities in water at room temperature against a three-strain cocktail of Escherichia coli O157:H7 and a three-strain cocktail of "generic" E. coli. Both initial inhibition and recovery of injured cells were monitored. When 3.0% (wt/wt) lactic acid, pH 2.4, was inoculated with E. coli O157:H7 (approximately 6 log CFU/ml), viable microorganisms were recovered after a 20-min exposure to the acid. After 20 min in 1.0% (wt/wt) trisodium phosphate, pH 12.0, no viable E. coli O157:H7 microorganisms were detected. Exposure of E. coli O157:H7 to sodium metasilicate (5 to 10 s) at concentrations as low as 0.6%, pH 12.1, resulted in 100% inhibition with no recoverable E. coli O157:H7. No difference in inhibition profiles was detected between the E. coli O157:H7 and generic strains, suggesting that nonpathogenic strains may be used for in-plant sodium metasilicate studies.     

Acid and alcohol tolerance of Escherichia coli O157:H7 in pulque, a typical Mexican beverage

Pulque is a traditional Mexican fermented alcoholic beverage produced from the nectar of maguey agave plants. No data exist on the behavior of Escherichia coli O157:H7 in agave nectar and pulque. An initial trial was done of the behavior of E. coli O157:H7 during fermentation of nectar from a single producer, a nectar mixture from different producers and "seed" pulque. A second trial simulating artisanal pulque production was done by contaminating fresh nectar with a cocktail of three E. coli O157:H7 strains, storing at 16 ° and 22 °C for 14 h, adding seed pulque and fermenting until pulque was formed. A third trial used pulque from the second trial stored at 22 °C as seed to ferment fresh nectar at 22 °C for 48 h (fermentation cycle). This procedure was repeated for an additional two fermentation cycles. During incubation at 16 ° or 22 °C in the first trial, the E. coli O157:H7 strains multiplied in both the single producer nectar and nectar mixture, reaching maximum concentration at 12h. E. coli O157:H7 cell concentration then decreased slowly, although it survived at least 72 h in both fermented nectars. E. coli O157:H7 did not multiply in the seed pulque but did survive at least 72 h. In the second trial, the numbers of E. coli O157:H7 increased approximately 1.5 log CFU/ml at 22 °C and 1.2 log CFU/ml at 16 °C after 14 h. After seed pulque was added, E. coli O157:H7 concentration decreased to approximately 2 log CFU/ml, and then remained constant until pulque was produced. In the third trial, the E. coli O157:H7 cells multiplied and survived during at least three nectar fermentation cycles. The results suggest that E. coli O157:H7 can develop acid and alcohol tolerance in pulque, and constitutes a public health risk for pulque consumers.     

Detection and frequency of VT1, VT2 and eaeA genes in Escherichia coli O157 and O157:H7 strains isolated from cattle, cattle carcasses and abattoir environment in Istanbul

The aim of this study was to detect VT1, VT2 and eaeA genes and to determine the frequency of these genes in Escherichia coli O157 and O157:H7 strains isolated from cattle, cattle carcasses and environmental samples of the 5 abattoirs located in Istanbul, Turkey. For this, the presence of VT1, VT2 and eaeA genes in 26 strains of E. coli O157:H7 and 6 strains of O157 was investigated by multiplex-PCR. The results have shown that eaeA gene was detected in all O157 and O157:H7 strains tested. Both VT2 and eaeA genes were detected in 4 (80%) of 5 strains of E. coli O157 and eaeA alone in 1 strain of O157. In 27 strains of O157:H7, 5 (18.5%) strains were found to be positive for VT1, VT2 and eaeA genes, 19 (70.3%) strains for both VT2 and eaeA and, 3 (11.1%) strains for only eaeA gene. Either VT1 alone or VT2 alone was not detected in any strains tested. eaeA gene alone in 2 strains, VT2-eaeA genes in 9 strains and VT1-VT2-eaeA genes in 2 strains were detected in 13 of E. coli O157:H7 strains isolated from cattle. eaeA alone in 1 strain, VT2-eaeA genes in 5 strains and VT1-VT2-eaeA genes in 2 strains were detected in 8 of E. coli O157:H7 strains isolated from carcasses. VT2-eaeA genes in 5 strains (isolated from hands, apron, knife and floor) and VT1-VT2-eaeA genes in 1 strain (isolated from knife) were also detected in 6 of E. coli O157:H7 strains isolated from environmental samples. This study reveals that most of the strains are found to be toxigenic and it is most likely that strains isolated from carcasses and abattoir environment originated from cattle feces. Therefore, HACCP systems are necessary from farm to table especially in the abattoirs to prevent contamination of meat and abattoir environment with intestinal content.     

Colicin concentrations inhibit growth of Escherichia coli O157:H7 in vitro

Escherichia coli O157:H7 is a virulent foodborne pathogen that causes severe human illness and inhabits the intestinal tract of food animals. Colicins are antimicrobial proteins produced by E. coli strains that inhibit or kill other E. coli. In the present Study, the efficacy of three pore-forming colicins (El, N, and A) were quantified in vitro against E. coli O157:H7 strains 86-24 and 933. Colicins E1 and N reduced the growth of E. coli O157:H7 strains, but the efficacy of each colicin varied among strains. Colicin E1 was more effective against both strains of E. coli O157:H7 than colicins A and N and reduced (P < 0.05) populations of E. coli O157:H7 at concentrations <0.1 microg/ml. These potent antimicrobial proteins may potentially provide an effective and environmentally sound preharvest strategy to reduce E. coli O157:H7 in food animals.     

Determination of 5-log pathogen reduction times for heat-processed, acidified vegetable brines

Recent outbreaks of acid-resistant food pathogens in acid foods, including apple cider and orange juice, have raised concerns about the safety of acidified vegetable products. We determined pasteurization times and temperatures needed to assure a 5-log reduction in the numbers of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella strains in acidified cucumber pickle brines. Cocktails of five strains of each pathogen were (separately) used for heat-inactivation studies between 50 and 60 degrees C in brines that had an equilibrated pH value of 4.1. Salmonella strains were found to be less heat resistant than E. coli O157:H7 or L. monocytogenes strains. The nonlinear killing curves generated during these studies were modeled using a Weibull function. We found no significant difference in the heat-killing data for E. coli O157:H7 and L. monocytogenes (P = 0.9709). The predicted 5-log reduction times for E. coli O157:H7 and L. monocytogenes were found to fit an exponential decay function. These data were used to estimate minimum pasteurization times and temperatures needed to ensure safe processing of acidified pickle products and show that current industry pasteurization practices offer a significant margin of safety.     

Implementation of targeted interventions to control Escherichia coli O157:H7 in a commercial abattoir

The objective of this study was to define locations on the carcass with highest contamination of E. coli O157 throughout the harvest process and implement targeted interventions to reduce or eliminate contamination. To establish a pathogen baseline, samples were collected at the foreshank, hindshank, inside round, neck and midline area and evaluated for E. coli O157:H7 presence. Environmental samples were also collected in the harvest area and the fabrication area of the facility. E. coli O157:H7 prevalence was highest on the foreshank, hindshank and inside rounds in the baseline study and steam vacuums/cones were implemented as an intervention in these specific areas on the harvest floor. At pre-evisceration, foreshank prevalence of E. coli O157:H7 was significantly (P<0.05) reduced from 21.7% to 3.1% after the application of steam interventions. At the final rail, foreshank prevalence in the baseline study was 4.2% while no E. coli O157:H7 was detected post-intervention implementation. E. coli O157:H7 on hindshanks and inside rounds was significantly reduced after intervention implementation from 24.2 to 11.5% and 37.5 to 16.7%, respectively at the final rail. Pathogen contamination of environmental samples collected in fabrication declined from 6.7% to 0.7% after slaughter interventions were implemented. Data indicate the identifying areas of contamination on the carcass and implementing interventions can significantly reduce E. coli O157 on the carcasses and in the fabrication environment.     

Characterization of an E2-type colicin and its application to treat alfalfa seeds to reduce Escherichia coli O157:H7

Several outbreaks of Escherichia coli O157:H7 infections have been associated with contaminated alfalfa seeds. A recently isolated E. coli strain Hu194 was capable of inhibiting 22 strains of E. coli O157:H7 and this inhibition was mediated by the production of a colicin named Hu194. The objectives of this study were to test the efficacy of treating alfalfa seeds with colicin Hu194 against E. coli O157:H7 strains, and to characterize this antimicrobial protein. Significant reductions (approximately 5 log CFU ml-1) in the viable cell counts of strains 43890 and 43895 were observed after 1-day incubation with semi-crude colicin, and after 2 days for strain 3081. Strain 43890 was successfully eliminated (5 log CFU g-1) from inoculated alfalfa seeds after soaking in a colicin suspension at a concentration of 10,000 AU/g. Treatment of alfalfa seeds inoculated with strains 43895 and 3081 required 20-fold higher concentrations of colicin Hu194 to achieve as much as 3 log CFU g-1 reductions. The genes encoding the colicin Hu194 operon were located on a 6 kb plasmid, and the sequence analysis revealed that this colicin was an E-type DNAse. From the sequence data, the estimated molecular masses of colicin Hu194, its immunity protein and lysis protein were 61.3, 10.0 and 4.8 kDa, respectively. Based on DNA and protein sequence comparisons with other E-type colicin, colicin Hu194 belonged to the type E2-colicin cluster. However, cross-immunity tests between E-group colicins suggested that Hu194 colicin was divergent from the previously characterized E2 colicins.     

Survival of enterohemorrhagic Escherichia coli O157:H7 in retail mustard

Escherichia coli O157:H7 survival in acid foods such as unpasteurized apple cider and fermented sausage is well documented. Researchers have determined that E. coli O157:H7 can survive in refrigerated acid foods for weeks. The potential of acid foods to serve as a vector of E. coli O157:H7 foodborne illness prompted this study to determine the fate of this organism in retail mustard containing acetic acid when stored at room and refrigerated temperatures. Various retail brands of dijon, yellow, and deli style mustard, pH ranging from 3.17 to 3.63, were inoculated individually with three test strains of E. coli O157:H7. Samples were inoculated with approximately 1.0 x 10(6) CFU/g, incubated at room (25+/-2.5 degrees C) and refrigerated (5+/-3 degrees C) temperatures, and assayed for surviving test strains at predetermined time intervals. An aliquot was appropriately diluted and plated using sorbitol MacConkey agar (SMAC). When the test strain was not recoverable by direct plating, the sample was assayed by enrichment in modified tryptic soy broth and recovered using SMAC. Growth of E. coli O157:H7 test strains was inhibited in all retail mustard styles. E. coli O157:H7 was not detected in dijon style mustard beyond 3 h at room and 2 days at refrigerated temperatures. Survival in yellow and deli style mustard was not detected beyond 1 h. Overall, test strain survival was greater at refrigerated than room temperature. Retail mustard demonstrated the ability to eliminate effectively any chance contamination by this organism within hours to days, suggesting that these products are not a likely factor in E. coli O157:H7 foodborne illness.     

Forage feeding to reduce preharvest Escherichia coli populations in cattle,a review

Although Escherichia coli are commensal organisms that reside within the host gut, some pathogenic strains of E. coli can cause hemorrhagic colitis in humans. The most notable enterohemorrhagic E. coli (EHEC) strain is O157:H7. Cattle are asymptomatic natural reservoirs of E. coli O157:H7, and it has been reported that as many as 30% of all cattle are carriers of this pathogen, and in some circumstances this can be as high as 80%. Feedlot and high-producing dairy cattle are fed large grain rations in order to increase feed efficiency. When cattle are fed large grain rations, some starch escapes ruminal microbial degradation and passes to the hind-gut where it is fermented. EHEC are capable of fermenting sugars released from starch breakdown in the colon, and populations of E. coli have been shown to be higher in grain fed cattle, and this has been correlated with E. coli O157:H7 shedding in barley fed cattle. When cattle were abruptly switched from a high grain (corn) diet to a forage diet, generic E. coli populations declined 1000-fold within 5 d, and the ability of the fecal generic E. coli population to survive an acid shock similar to the human gastric stomach decreased. Other researchers have shown that a switch from grain to hay caused a smaller decrease in E. coli populations, but did not observe the same effect on gastric shock survivability. In a study that used cattle naturally infected with E. coli O157:H7, fewer cattle shed E. coli O157:H7 when switched from a feedlot ration to a forage-based diet compared with cattle continuously fed a feedlot ration. Results indicate that switching cattle from grain to forage could potentially reduce EHEC populations in cattle prior to slaughter; however the economic impact of this needs to be examined.     

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.     

Role of bacterial association and penetration on destruction of Escherichia coli O157:H7 in beef tissue by high pH

This study was undertaken to determine if association with collagen enables Escherichia coli O157:H7 to resist high-pH treatments and to determine the effects of high pH on the survival of E. coli O157:H7 within different layers of beef tissue. E. coli O157:H7 was inoculated onto purified bovine type I collagen on 12-mm2 circular glass coverslips, plain 12-mm2 circular glass coverslips (control), and 12-mm2 irradiated (cobalt-60) lean beef tissue. The rates of destruction of E. coli O157:H7 inoculated on coverslips in pH 10.5 NaHCO3-NaOH buffer at 35 degrees C were determined at various sampling times. E. coli O157:H7 cells associated with collagen and treated in the same manner were also examined using scanning electron microscopy to determine if association with collagen enabled the organism to resist high-pH treatments. The inoculated tissue was treated in pH 13.0 NaHCO3-NaOH buffer at 25 degrees C, and penetrating cells of E. coli O157:H7 were recovered using a cryostat technique. There was no significant difference (P < 0.05) between the rates of destruction of collagen-associated E. coli O157:H7 and non-collagen-associated E. coli O157:H7 following exposure to high-pH treatments. Scanning electron micrographs showed that collagen-associated E. coli O157:H7 cells appeared physically damaged by exposure to high-pH treatments, and association of E. coli O157:H7 to collagen did not increase the resistance of the organism to destruction by high-pH rinses. No significant differences were seen between 20 ml of NaHCO3-NaOH buffer at pH 13.0 (treatment) and 20 ml of distilled water at pH 7.0 (control) when E. coli O157:H7 cells were recovered in beef tissue at depths of up to 2,000 microm (P < 0.05). The ability of E. coli O157:H7 to penetrate beef tissue may be an important factor in reducing the effectiveness of high-pH treatments in killing this organism on beef tissue. This finding should be considered in the future when designing treatments to decontaminate beef carcasses.     

Influence of acid adaptation on the tolerance of Escherichia coli O157:H7 to some subsequent stresses

Three stains of Escherichia coli O157:H7, including ATCC 43889, ATCC 43895, and 933, were first subjected to acid adaptation at a pH of 5.0 for 4 h. Thermal tolerance at 52 degrees C and survival of the acid-adapted as well as the nonadapted cells of E. coli O157:H7 in the presence of 10% sodium chloride, 0.85% bile salt, or 15.0% ethanol were investigated. Results showed that the effect of acid adaptation on the survival of E. coli O157:H7 varied with the strains and types of subsequent stress. Acid adaptation caused an increase in the thermal tolerance of E. coli O157:H7 ATCC 43889 and ATCC 43895, but no significant difference in the thermal tolerance was noted between acid-adapted and nonadapted cells of E. coli O157:H7 933. Although the magnitude of increase varied with strains of test organisms, acid adaptation generally led to an increase in the tolerance of E. coli O157:H7 to sodium chloride. On the other hand, the susceptibility of acid-adapted cells of the three strains of E. coli O157:H7 tested did not show a significant difference from that of their nonadapted counterparts when stressed with bile salt. The acid-adapted cells of E. coli O157:H7 ATCC 43889 and ATCC 43895 were less tolerant than the nonadapted cells to ethanol, whereas the tolerance of adapted and nonadapted cells of E. coli O157:H7 933 showed no significant differences.