Growth and survival of E. coli O157:H7 during the manufacture and ripening of a smear-ripened cheese produced from raw milk

AIMS: The behaviour of Escherichia coli O157:H7 was studied during the manufacture and ripening of a smear-ripened cheese produced from raw milk. METHODS AND RESULTS: Cheese was manufactured on a laboratory scale using milk (20 l) inoculated with E. coli O157:H7, and enumeration was carried out using CT-SMAC. From an initial level of 1.52 +/- 0.03 log cfu ml-1 in the milk (34 +/- 2 cfu ml-1), the numbers increased to 3.4 +/- 0.05 log cfu g-1 in the cheese at day 1. During ripening, the numbers decreased to <1 cfu g-1 and <10 cfu g-1 in the rind and core, respectively, after 21 days, although viable cells were detected by enrichment after 90 days. The presence of E. coli O157:H7 in the cheese was confirmed by latex agglutination and by multiplex PCR. CONCLUSION: The results indicate that the manufacturing procedure encouraged substantial growth of E. coli O157:H7 to levels that permitted survival during ripening and extended storage. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of low numbers of E. coli O157:H7 in milk, destined for raw milk cheese manufacture, could constitute a threat to the consumer.     

The survival characteristics of a non-toxigenic strain of Escherichia coli O157:H7

The survival characteristics of a non-toxigenic, antibiotic-resistant strain of Escherichia coli O157:H7 in bovine faeces were investigated. Faecal samples were inoculated with 10(8-9) cfu g-1 of the organism and (i) stored in closed plastic containers at 10 degrees C, (ii) stored in closed plastic containers placed outside or (iii) decanted onto the surface of grazing land. Recovery and enumeration on Sorbitol MacConkey Agar (SMAC) and Tryptic Soya Agar (TSA) revealed that the E. coli O157:H7 numbers in both enclosed samples (i and ii) had decreased by 4.5-5.5 log10 cfu g-1 within 99 d. Numbers in samples decanted onto grassland (iii) decreased by 4.0-5.0 log10 cfu g-1 within 50 d but the organism was still detectable in the surrounding soil for up to 99 d. Persistence of E. coli O157:H7 in bovine faeces and contaminated pastures may therefore be an important factor in the initial infection and re-infection of cattle.     

Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef

In this study, enrichment procedures and two recovery methods, a membrane surface adhesion technique and an immunomagnetic separation (IMS), were compared for use in conjunction with a multiplex polymerase chain reaction (PCR) method with a view to describing a fast (24 h) and economical test for detection of Escherichia coli O157:H7 in meat samples. The study showed no significant difference between three different enrichment media (BHI, E. coli (E.C.) broth+novobiocin, modified tryptone soya broth (mTSB)+novobiocin) or two incubation temperatures (37 or 41.5 degrees C) for growth of E. coli O157:H7 in minced beef. Minced beef samples inoculated with E. coli O157:H7 at 40 cfu g(-1) were incubated at 37 degrees C for 16 h in E.C. broth+novobiocin reaching numbers of (log(10)7.82-8.70). E. coli O157:H7 were recovered by attachment to polycarbonate membranes immersed in the enriched cultures for 15 min or by immunomagnetic separation. Subsequent treatment of recovered membranes or IMS beads with lysis buffer and phenol/chloroform/isoamyl alcohol was used to extract the DNA from the extracted E. coli O157:H7 cells. The results show when E. coli O157:H7 was present at high levels in the enriched meat sample (log(10)9.6-7.5 cfu ml(-1); >16-h enrichment), the membrane and IMS techniques recovered similar levels of the pathogen and the microorganism was detectable by PCR using both methods. At lower levels of E. coli O157:H7 (log(10)6.4), only the IMS method could recover the pathogen but at levels below this neither method could recover sufficient numbers of the pathogens to allow detection. The conclusion of the study is that with sufficient enrichment time (16 h) the membrane surface adhesion membrane extraction method used in combination with multiplex PCR has the potential for a rapid and economical detection method.     

Persistence of Escherichia coli O157:H7 in barley silage: effect of a bacterial inoculant

AIMS: The effect of a lactic acid producing bacterial (LAB) inoculant on the elimination of Escherichia coli O157:H7 from barley forage was assessed. METHODS AND RESULTS: Triplicate mini-silos were prepared for four treatments and six sampling times (1, 3, 7, 15, 30 and 42 d post-ensiling). The treatments were (i) 10(5) cfu g(-1) Pediococcus pentosaceus and Propionibacterium jenzenii (P2); (ii) 10(5) cfu g(-1) E. coli O157:H7 strain 3081 and 10(5) cfu g(-1) E. coli Biotype 1 strains 719IE10, 719IE14 and 614ME49 (EC); (iii) P2 + EC; and (iv) the control (sterile distilled water). Triplicate mini-silos were opened at each sampling time for pH, volatile fatty acid (VFA) and lactate determinations and E. coli, E. coli O157:H7 and LAB were enumerated. On d 3 and 7, numbers of E. coli O157:H7 in P2 + EC were significantly lower than in EC (P < 0;05). Escherichia coli O157:H7 was not detected in P2 + EC and EC at 7 and 15 d post-ensiling, respectively. On d 15 through 42, E. coli Biotype 1 was not detected in P2 + EC or EC. Populations of LAB were higher in P2 and P2 + EC than in the control and EC on d 3 and 7 (P < 0.05). After 3 d of ensiling, lactate levels were higher (P < 0.05) and pH was lower (P < 0.05) in P2 and P2 + EC as compared to the control and EC. Bacteriocins of P2 were not found to be inhibitory to E. coli O157:H7 using the agar-spot procedure. Escherichia coli O157:H7 inoculated into the control silage at a level of 10(3) cfu g(-1) and exposed to aerobic conditions at 22 degrees C was not detected after 1 d and remained undetectable for the 28 d exposure period. CONCLUSIONS: Silage inoculant P2 increased lactate levels and decreased pH more rapidly during ensiling, which appeared to hasten the elimination of E. coli O157:H7 from the silage. SIGNIFICANCE AND IMPACT OF THE STUDY: Results emphasize the importance of adequate ensiling since E. coli O157:H7 may be maintained and spread among cattle through feed.     

A chromogenic plating medium for isolating Escherichia coli O157:H7 from beef

There was no significant difference (P > 0.05) in the percentages of Escherichia coli O157:H7 cells recovered on BCM O157:H7 (+) agar (69.7%) and MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid (MSA-BCIG) (76.8%) vs Tryptic soy agar. Three E. coli O157:H7 strains (ATCC 35150, 43890 and 43894) were separately inoculated into raw ground beef at low (mean 0.32 cfu g-1) and high (mean 3.12 cfu g-1) levels. Using the United States Department of Agriculture (USDA) m-EC + novobiocin enrichment broth, BCM O157:H7 (+) medium surpassed MSA-BCIG agar with overall percentage sensitivities for BCM O157:H7 (+) of 92.1 and 94.4 compared with 52.6 and 84.7 for MSA-BCIG at low and high levels, respectively. A comparison of BCM O157:H7 (+) and MSA-BCIG agars using naturally contaminated beef samples was made utilizing presumptively positive enrichment broths previously identified by rapid methods. The E. coli O157:H7 cells in these broths were concentrated with Dynabeads anti-E. coli O157 before inoculating the agars. The respective percentage sensitivity and specificity values were 90.0 and 78.5 for BCM O157:H7 (+) and 70.0 and 46.4 for MSA-BCIG. Thus, under identical pre-plating conditions, BCM O157:H7 (+) medium displayed a greater sensitivity than MSA-BCIG for detecting E. coli O157:H7 in artificially inoculated beef, and both greater sensitivity and specificity upon examining naturally contaminated beef samples.     

Survival of Escherichia coli O157:H7 in bottled natural mineral water

A non-verotoxin-producing isolate of Escherichia coli O157:H7 was inoculated at final concentrations of 10(3) or 10(6) ml-1 into natural non-carbonated mineral water (MW), sterile natural mineral water (SMW) and sterile distilled deionized water (SDDW) and stored at 15 degrees C for 10 weeks. Samples were examined every 7 d for the presence of E. coli O157:H7 using a resuscitative/selective agar procedure. The MW samples were also plated onto a non-selective agar, R2A, to enumerate E. coli O157:H7 and the autochthonous flora. There was a significant difference in the survival of E. coli O157:H7 (10(3) ml-1 inoculum) between the MW and the SDDW at time periods 0, 7, 14 (P < 0.005) 21, 28, 35 (P < 0.001) and 42 d (P < 0.05) and between the MW and the SMW at time periods 7, (P < 0.05) 14, 21 (P < 0.005) 28 (P < 0.01) and 35 d (P < 0.05), with the pathogen surviving longest in the MW samples. In contrast, at 10(6) ml-1, no significant differences in the survival of E. coli O157:H7 were observed between the water types. The presence of E. coli O157:H7 (10(3) ml-1) in the MW samples did not have an antagonistic effect on the recovery of the autochthonous flora. Transmission electron microscopy analysis demonstrated that the E. coli O157:H7 cells lyse during storage, releasing their contents into the surrounding environment. These substances may have been utilized by the autochthonous flora and thereby explain why the numbers of flora recovered from the inoculated MW samples were higher than those recovered from the uninoculated samples.     

Isolation of shiga toxin-producing Escherichia coli (STEC) from foods using EHEC agar

Enterohaemorrhagic Escherichia coli (EHEC) agar was evaluated for its ability to recover one isolate of each of three serotypes (O157:H7, O26 and O113:H21) of shiga toxin-producing E. coli (STEC) from raw mince, pasteurized milk and salami after enrichment. The method detected around one colony-forming unit (cfu) in 25 ml in milk, but was less sensitive with salami, requiring 10-1000 cfu 25 g-1 (depending on serotype) for detection. In raw minced beef any enterohaemolysin-producing colonies were outnumbered by other colonies and only one of 12 enrichments yielded the inoculum serotype. Additional tests were conducted on 15 retail meat products. One 25-g sample of each product was processed as purchased, while another was inoculated with 157-185 cfu of a cocktail of E. coli O157, O113 and O26 cultures. Recovery was easily achieved with cooked meat products and salami. Recovery from raw minced meat was again difficult, but sometimes possible. Testing more suspect colonies than were tested in this study would presumably increase the sensitivity of the method.     

Enumeration of Escherichia coli O157 in cattle faeces using most probable number technique and automated immunomagnetic separation

AIMS: To determine the numbers of Escherichia coli O157 present in the faeces of naturally infected cattle. METHODS AND RESULTS: A combination of the most probable number (MPN) technique and automated immunomagnetic separation (AIMS) was used to enumerate E. coli O157 in cattle faeces from both pasture-fed and grain-fed animals. A total of 22 E. coli O157 positive faecal samples were enumerated for E. coli O157 (10 from pasture-fed and 12 from grain-fed animals). The numbers of E. coli O157 in cattle faeces varied from undetectable (<3 MPN g-1 of faeces) to 2.4 x 104 MPN g-1. There was no significant difference (P = 0.06) between the numbers of E. coli O157 in pasture-fed or grain-fed cattle faeces, although the geometric mean (antilog of the mean of log10 transformed MPN values) was higher in grain-fed (130 MPN g-1) than in pasture-fed (13 MPN g-1). CONCLUSIONS: Although the number of samples tested is small, the results indicate that E. coli O157 make up a small proportion of the total E. coli population present in cattle faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the numbers of E. coli O157 present in cattle will assist in developing more robust quantitative risk assessments and formulating intervention strategies.     

Adsorption of endogenous polyphenols relieves the inhibition by fruit juices and fresh produce of immuno-PCR detection of Escherichia coli O157:H7

The immuno-polymerase chain reaction (PCR) approaches facilitate rapid (8 h) detection of Escherichia coli O157:H7 in contaminated dairy products and ground beef samples with detection sensitivities approaching 1 colony forming unit (cfu) g-1 ml-1. However, no PCR products were obtained when the method was applied to identify E. coli O157:H7 in tainted apple juice. Enzyme-linked immuno-assay (ELISA) results suggested non-specific binding of endogenous polyphenols (ubiquitous in plant products) to antibodies present on the surface of the immunobeads, making the latter unavailable for capturing the target bacteria Treatment of the test sample, prior to IMS, with a synthetic fining agent, polyvinylpyrrolidone, restored the full function and sensitivity of the immuno-PCR. The study demonstrates the suitability of the improved method as a generic strategy for rapid screening of fruit juices and plant produce for E. coli O157:H7.     

Growth of Escherichia coli O157:H7 during sprouting of alfalfa seeds

AIMS: Escherichia coli O157:H7 was monitored daily during sprouting of alfalfa seeds inoculated at high (3.92 log10 cfu g(-1)) and low (1.86 log10 cfu g(-1)) levels to assess the extent of pathogen growth during production. METHODS AND RESULTS: Sprouts and rinse water were tested by direct and membrane filter plating on modified sorbitol MacConkey agar and BCM O157:H7(+) agar; the antibody-direct epifluorescent filter technique; and rapid immunoassays. The pathogen reached maximum populations after one and two days of sprouting seeds inoculated at high and low levels, respectively; in either case, populations of 5-6 log10 cfu g(-1) were reached. Detection limits of two rapid immunoassays, Reveal and VIP, without enrichment were determined to be 5-7 log10 cfu ml(-1). CONCLUSION: These results show the ability of E. coli O157:H7 to grow to high levels during sprouting; however, because these levels may be below detection limits, it is necessary to include enrichment when monitoring sprout production for E. coli O157:H7 by the rapid test kits. SIGNIFICANCE AND IMPACT OF THE STUDY: The data indicate that sprouts may harbor high levels of pathogens. The appropriate use of rapid test methods for pathogen monitoring during sprouting is indicated.     

Detection of Escherichia coli O157:H7 in soil and water using multiplex PCR

AIMS: To evaluate the suitability of a multiplex PCR-based assay for sensitive and rapid detection of Escherichia coli O157:H7 in soil and water. METHODS AND RESULTS: Soil and water samples were spiked with E. coli O157:H7 and subjected to two stages of enrichment prior to multiplex PCR. Detection sensitivities were as high as 1 cfu ml(-1) drinking water and 2 cfu g(-1) soil. Starvation of E. coli O157:H7 for 35 d prior to addition to soil did not affect the ability of the assay to detect initial cell numbers as low as 10 cfu g(-1) soil. Use of an 8-h primary enrichment enabled detection of as few as 6 cfu g(-1) soil, and 10(4) cfu g(-1) soil with a 6-h primary enrichment. When soil was inoculated with 10(5) cfu g(-1), the PCR assay indicated persistence of E. coli O157:H7 during a 35 d incubation. However, when soil was inoculated with lower numbers of pathogen, PCR amplification signals indicated survival to be dependent on cell concentration. CONCLUSIONS: A multiplex PCR-based assay, in combination with an enrichment strategy enabled sensitive and rapid detection of E. coli O157:H7 in soil and water. SIGNIFICANCE AND IMPACT OF THE STUDY: The ability to sensitively detect E.coli O157:H7 in environmental material within one working day represents a considerable advancement over alternative more time-consuming methods for detection of this pathogen.     

Fate of Escherichia coli O157:H7 in unpasteurized milk stored at 8 degrees C

Escherichia coli O157:H7 and verocytotoxins were not found in any of 100 unpasteurized milk samples obtained from the bulk tanks of eight dairy farms located in the Puglia and Basilicata areas. Seven E. coli O157:H7 (EHEC) strains were inoculated separately into raw milk samples and then examined periodically to determine the fate of EHEC as influenced by the storage temperature (8 degrees C) and time. There was essentially no change in the viable population of three EHEC strains for up to 14 d. The remaining four strains showed an increase in population from < 2 log to 3 log cfu ml-1 in a time period of between 9 and 17 d. The results indicate good survival or even multiplication of E. coli O157:H7 in raw milk when stored at 8 degrees C and reaffirm the need for pasteurization and holding the milk at < or = 5 degrees C.     

Survival of Escherichia coli O157:H7 in broth and processed salami as influenced by pH, water activity, and temperature and suitability of media for its recovery.

The survival of unheated and heat-stressed (52 degrees C, 30 min) cells of Escherichia coli O157:H7 inoculated into tryptic soy broth (TSB) adjusted to various pHs (6.0, 5.4, and 4.8) with lactic acid and various water activities (a(w)s) (0.99, 0.95, and 0.90) with NaCl and incubated at 5, 20, 30, and 37 degrees C was studied. The performance of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), and modified eosin methylene blue agar in supporting colony development of incubated cells was determined. Unheated cells of E. coli O157:H7 grew to population densities of 10(8) to 10(9) CFU ml-1 in TSB (pHs 6.0 and 5.4) at an a(w) of 0.99. Regardless of the pH and a(w) of TSB, survival of E. coli O157:H7 was better at 5 degrees C than at 20 or 30 degrees C. At 30 degrees C, inactivation or inhibition of growth was enhanced by reduction of the a(w) and pH. A decrease in the a(w) (0.99 to 0.90) of TSB in which the cells were heated at 52 degrees C for 30 min resulted in a 1.5-log10 reduction in the number of E. coli O157:H7 cells recovered on TSA; pH did not significantly affect the viability of cells. Recovery was significantly reduced on MSMA when cells were heated in TSB with reduced pH or a(w) for an increased length of time. With the exception of TSB (a(w), 0.90) incubated at 37 degrees C, heat-stressed cells survived for 24 h in recovery broth. TSB (a(w), 0.99) at pH 6.0 or 5.4 supported growth of E. coli O157:H7 cells at 20 or 37 degrees C, but higher numbers of heated cells survived at 5 or 20 degrees C than at 37 degrees C. The ability of unheated and heat-stressed E. coli O157:H7 cells to survive or grow as affected by the a(w) of processed salami was investigated. Decreases of about 1 to 2 log10 CFU g-1 occurred soon after inoculation of salami (pHs 4.86 and 4.63 at a(w)s of 0.95 and 0.90, respectively). Regardless of the physiological condition of the cells before inoculation into processed salami at an a(w) of either 0.95 or 0.90, decreases in populations occurred during storage at 5 or 20 degrees C for 32 days. If present at < or = 100 CFU g-1, E. coli O157:H7 would unlikely survive storage at 5 degrees C for 32 days. However, contamination of salami with E. coli O157:H7 at 10(4) to 10(5) CFU g-1 after processing would pose a health risk to consumers for more than 32 days if storage were at 5 degrees C. Regardless of the treatment conditions, performance of the media tested for the recovery of E. coli O157:H7 cells followed the order TSA > modified eosin methylene blue agar > MSMA.     

Exposure to non-acid fresh meat decontamination washing fluids sensitizes Escherichia coli O157:H7 to organic acids

AIMS: To investigate whether Escherichia coli O157:H7 maintains acid tolerance in water meat decontamination washing fluids. METHODS AND RESULTS: A rifampicin-resistant derivative of E. coli O157:H7 strain ATCC 43895 was inoculated (10(5) cfu ml(-1)) in spray-washings from meat sprayed with cold (10 degrees C) or hot (85 degrees C) water, stored at 10 degrees C for up to 14 days, and its acid tolerance was assessed at 2 and 8 days by exposure to broth or new washings adjusted to pH 3.5 or 3.7 with lactic or acetic acid. The pathogen survived in the water washings, but it was outgrown by the natural, Pseudomonas-like flora, and it was sensitized to acid. CONCLUSIONS: The acid tolerance of E. coli O157:H7 decreases following exposure to non-acid, but otherwise stressful, conditions prevailing in water meat washings at 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings suggest that the more intense use of water-based technologies should be included in meat decontamination strategies because they may contribute to enhanced meat safety by inducing acid sensitization in E. coli O157:H7.     

Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milk

AIM: To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E. coli O157) in raw and pasteurized milk. METHODS AND RESULTS: Raw milk, pasteurized milk with 1.5% fat content and pasteurized milk with 3.5% fat content were spiked with E. coli O157 at low levels. The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37 degrees C. Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFosstrade mark). Uninjured E. coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml-1 sample). Injured organisms were detected at levels of about 4 cfu 10 ml-1 sample. Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37 degrees C) and mTSBn (16 h, 37 degrees C), successively. CONCLUSIONS: The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml-1 milk sample. Injured organisms can be detected and isolated at a level almost as low as this. A resuscitation step is not recommended for the detection and isolation of injured and non-injured E. coli O157 from milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E. coli O157 requires a very sensitive method. Both methods described here are useful for such analysis.     

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.     

Detection of Escherichia coli O157:H7 in raw meat by immunomagnetic separation and multiplex PCR

The aim of this research was to elaborate fast and sensitive method ofdetection of E. coli O157:H7 in food samples. Raw ground meat obtained from retail was artificially inoculated with low numbers of E. coli O157:H7. 18 h enrichment culture allowed pathogenic bacteria to multiply to the levels detectable in multiplex PCR. Immunomagnetic separation with magnetic beads coated with an antibody against E. coli O157:H7 were used to concentrate target bacteria and to separate PCR inhibitors. A portion of the bacterial suspension was used in a multiplex PCR to amplify eae (attaching and effacing) gene, stx (shiga toxin) genes and 90 kbp plasmid. The sensitivity of E. coli O157:H7 detection method was shown to be 1 cfu per 25 g of food sample. The total analysis can be completed within 24 h, whilst traditional methods involves enrichment, direct plating and confirmation tests with entire time at least 3 days.     

Effects of acid adaptation, product pH, and heating on survival of Escherichia coli O157:H7 in pepperoni

The thermotolerance of E. coli O157:H7 cells (strain 380-94) heated in pepperoni is reported. Information on the pattern of thermal inactivation of E. coli O157:H7 in pepperoni was applied in the development of heating processes designed to reduce E. coli O157:H7 numbers therein by 5 log(10) units.     

Antimicrobial activity of a 14-residue peptide against Escherichia coli O157:H7

An amphiphilic, cationic peptide composed of eight leucines and six lysines was synthesized by solid phase peptide synthesis (SPPS). The synthetic peptide was bactericidal within 10 min at concentrations as low as 3 microg ml - 1 against mid-exponential Escherichia coli O157:H7 suspended in buffer. Concentrations of 25 microg ml - 1 caused up to 7 log10 cfu ml - 1 reductions. When tested against E. coli O157:H7 grown in TSB, the peptide was bactericidal and bacteriostatic at concentrations of 50 and 25 microg ml - 1, respectively. An inhibitory effect was also observed against stationary phase cells. The synthetic peptide caused the release of u.v.-absorbing materials from the E. coli O157:H7 as well as an increase in its O.D.600 nm. Intracellular K+ and ATP depletion were also observed. These results suggest that the peptide increased the cell membrane permeability but it did not lyse the cells.     

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.