Survival of Escherichia coli O157:H7 in dry sausage fermented by probiotic lactic acid bacteria

Probiotic Lactobacillus rhamnosus GG, L rhamnosus E‐97800, L rhamnosus LC‐705 and commercial Pediococcus pentosaceus were studied for their ability to inhibit the growth of Escherichia coli O157:H7 in dry sausage. The strains were able to produce technologically high‐quality dry sausage. The number of E coli O157:H7 decreased from approximately 5 to approximately 2 log cfu g⁻¹ It was concluded that the above‐mentioned strains and the commercial culture were equally effective in inhibiting E coli O157:H7. © 2000 Society of Chemical Industry     

Survival of Escherichia coli O157:H7 in meat product brines containing antimicrobials

Brine solution injection of beef contaminated with Escherichia coli O157:H7 on its surface may lead to internalization of pathogen cells and/or cross-contamination of the brine, which when recirculated, may serve as a source of new product contamination. This study evaluated survival of E. coli O157:H7 in brines formulated without or with antimicrobials. The brines were formulated in sterile distilled water (simulating the composition of freshly prepared brines) or in a nonsterile 3% meat homogenate (simulating the composition of recirculating brines) at concentrations used to moisture-enhance meat to 110% of initial weight, as follows: sodium chloride (NaCl, 5.5%) + sodium tripolyphosphate (STP, 2.75%), NaCl + sodium pyrophosphate (2.75%), or NaCl + STP combined with potassium lactate (PL, 22%), sodium diacetate (SD, 1.65%), PL + SD, lactic acid (3.3%), acetic acid (3.3%), citric acid (3.3%), nisin (0.0165%) + ethylenediamine tetraacetic acid (EDTA, 200 mM), pediocin (11000 AU/mL) + EDTA, sodium metasilicate (2.2%), cetylpyridinium chloride (CPC, 5.5%), or hops beta acids (0.0055%). The brines were inoculated (3 to 4 log CFU/mL) with rifampicin-resistant E. coli O157:H7 (8-strain composite) and stored at 4 or 15 °C (24 to 48 h). Immediate (0 h) pathogen reductions (P < 0.05) of 1.8 to ≥ 2.4 log CFU/mL were observed in brines containing CPC or sodium metasilicate. Furthermore, brines formulated with lactic acid, acetic acid, citric acid, nisin + EDTA, pediocin + EDTA, CPC, sodium metasilicate, or hops beta acids had reductions (P < 0.05) in pathogen levels during storage; however, the extent of pathogen reduction (0.4 to > 2.4 log CFU/mL) depended on the antimicrobial, brine type, and storage temperature and time. These data should be useful in development or improvement of brine formulations for control of E. coli O157:H7 in moisture-enhanced meat products. PRACTICAL APPLICATION: Results of this study should be useful to the meat industry for developing or modifying brine formulations to reduce the risk of E. coli O157:H7 in moisture-enhanced meat products.     

Use of deodorized yellow mustard powder to control Escherichia coli O157:H7 in dry cured Westphalian ham

Dry cured (uncooked) hams with low water activity and pH ≥5.6 seem a likely food vehicle for Escherichia coli O157:H7. In previous work, isothiocyanates produced from mustard glucosinolates by bacterial myrosinase-like activity converted deodorized mustard into a potent antimicrobial in dry sausage. In this study its value in controlling E. coli O157:H7 survival in Westphalian ham was investigated. Hams were inoculated with a 7.5 log cfu g(-1) cocktail of E. coli O157:H7, 4% or 6% (w/w) deodorized yellow mustard powder was surface applied and monitored 80 d for pathogen survival. In one trial to accelerate formation of isothiocyanate, a Staphylococcus (S.) carnosus meat starter culture was added to hams at 45 d (after salt equilibration). At 21 d, E. coli O157:H7 was reduced by 3 log cfu g(-1) on hams treated with mustard powder compared to only a 1 log cfu g(-1) reduction in the control. By 45 d, mustard powder caused a reduction of >5 log cfu g(-1)E. coli O157:H7, whereas it took 80 d for numbers in control hams to be similarly reduced. Although the commercial process used caused a 5 log cfu g(-1) reduction of E. coli O157:H7 in 80 d, 4% or 6% deodorized mustard accelerated this reduction and the S. carnosus starter culture may have contributed to the maintenance of this effect.     

Ecology of Escherichia coli O157:H7 in commercial dairies in southern Alberta

Shedding of Escherichia coli O157:H7 was monitored monthly over a 1-yr period by collecting pooled fecal pats (FECAL) and manila ropes orally accessed for 4 h (ROPE) from multiple pens of cattle in 5 commercial dairies in southern Alberta, Canada. Using immunomagnetic separation, E. coli O157:H7 was isolated from cows on 4 of the dairies and from 13.5% of FECAL and 1.1% of ROPE samples. Pulsed-field gel electrophoresis of XbaI- and SpeI-digested bacterial DNA of the 65 isolates produced 23 unique restriction endonuclease digestion patterns, although 92% of the isolates belonged to 3 restriction endonuclease digestion pattern clusters sharing a minimum 90% homology. Collection of positive isolates was 15 times more likely from June through September. Across dairies, peak somatic cell count occurred in July, August, September, and November. The likelihood of positive isolates was 2.6 times higher in calves and heifers compared with mature cows. This study indicates that ROPE would be of little value for the detection of E. coli O157:H7 in dairy herds unless oral contact with ROPE could be increased in mature animals. Additionally, mitigation strategies for E. coli O157:H7 should be targeted to the months of July, August, and September and toward immature animals for maximum impact. All farms displayed unique combinations of seasonality of shedding and diversity of E. coli O157:H7 subtypes. The fact that seasonal prevalence of E. coli O157:H7 largely coincided with peak somatic cell count within climatically controlled dairy barns suggests that similar environmental factors may be enhancing fecal shedding E. coli O157:H7 and the incidence of mastitis.     

Individual and combined application of dry heat with high hydrostatic pressure to inactivate Salmonella and Escherichia coli O157:H7 on alfalfa seeds

Alfalfa sprouts are recurrently implicated in outbreaks of food-borne illnesses as a result of contamination with Salmonella or Escherichia coli O157:H7. In the majority of these outbreaks, the seeds themselves have been shown to be the most likely source of contamination. The aims of this study were to comparatively assess the efficacy of dry heat treatments alone or in conjunction with high hydrostatic pressure (HHP) to eliminate a ∼5 log CFU/g load of Salmonella and E. coli O157:H7 on alfalfa seeds. Dry heat treatments at mild temperatures of 55 and 60 °C achieved ≤1.6 and 2.2 log CFU/g reduction in the population of Salmonella spp. after a 10-d treatment, respectively. However, subjecting alfalfa seeds to more aggressive temperatures of 65 °C for 10 days or 70 °C for 24 h eliminated a ∼5 log population of Salmonella and E. coli O157:H7. We subsequently showed that the sequential application of dry heating followed by HHP could substantially reduce the dry heating exposure time while achieving equivalent decontamination results. Dry heating at 55, 60, 65 and 70 °C for 96, 24, 12 and 6 h, respectively followed by a pressure treatment of 600 MPa for 2 min at 35 °C were able to eliminate a ∼5 log CFU/g initial population of both pathogens. Finally, we evaluated the impact of selected treatments on the seed germination percentages and yield ratios and showed that dry heating at 65 °C for 10 days did not bring about any considerable decrease in the germination percentage. However, the sprout yield of treated alfalfa seeds was reduced by 21%. Dry heating at 60 and 65 °C for 24 and 12 h respectively followed by the pressure treatment of 600 MPa for 2 min at 35 °C did not significantly (P > 0.05) affect the germination percentage of alfalfa seeds although a reduction in the sprouting yield was observed.     

Immunosensors for rapid detection of Escherichia coli O157:H7 - perspectives for use in the meat processing industry

This review critically evaluates different types of immunosensors proposed for rapid identification of Escherichia coli O157:H7. The methods are compared with approved USDA-FSIS standard procedures for determination of this pathogen in raw or ready-to-eat meat products. Major advantages and disadvantages for each method are highlighted. Our analysis suggests that application of immunosensors in the meat-processing industry may be limited to identification of uncontaminated samples after conventional selective enrichment in broth. Use for detection appears limited at the present time.     

Characterization of interactions between Escherichia coli O157:H7 with epiphytic bacteria in vitro and on spinach leaf surfaces

This study characterized the types of interactions between Escherichia coli O157:H7 and spinach phylloepiphytic bacteria and identified those that influence persistence of E. coli O157:H7 on edible plants. A total of 1512 phylloepiphytic bacterial isolates were screened for their ability to inhibit or to enhance the growth of E. coli O157:H7 in vitro and on spinach leaf surfaces. Fifteen different genera, the majority belonging to Firmicutes and Enterobacteriaceae, reduced growth rates of E. coli O157:H7 in vitro by either nutrient competition or acid production. Reduced numbers of E. coli O157:H7 were recovered from detached spinach leaves that were co-inoculated with epiphytic isolates belonging to five genera. A 1.8 log reduction in E. coli O157:H7 was achieved when co-inoculated with Erwinina perscinia and 20% cellobiose, a carbon source used by the phylloepiphytes but not E. coli O157:H7. The reduction on leaves was significantly less than reduction measured in vitro. Phylloepiphytic bacteria belonging to eight different genera, increased numbers of E. coli O157:H7 when co-cultured in vitro on spent medium and when co-cultured on detached spinach leaves. The results, showing reduction of E. coli O157:H7 numbers by natural epiphytic bacteria, support the hypothesis that native plant microbiota can be used for bio-control of foodborne pathogens, however, other epiphytes may promote the persistence of enteric pathogens on the phyllosphere.     

Efficacy of a post enrichment acid treatment for isolation of Escherichia coli O157:H7 from alfalfa sprouts

The enrichment, detection and isolation procedure in the current US FDA BAM have been shown effective for Escherichia coli O157:H7 in a wide variety of foods. Recently reported modifications to the enrichment protocol, including post-enrichment immunomagnetic separation (IMS) procedures have improved sensitivity of the method for alfalfa sprouts. However, cultural isolation on selective agar plates still presents a challenge in this food matrix.     

乳酸菌对肠出血性大肠杆菌O157:H7抑制作用的研究

为探讨乳酸菌对肠出血性大肠杆菌O157：H7 ATCC43895(E．Coli O157：H7)的抑制作用，在培养基上进行了研究。将E．Coli O157：H7与干酪乳杆菌干酪亚种、植物乳杆菌、发酵乳杆菌、乳酸乳球菌和瑞士乳杆菌同时接种在培养基中，E．Coli O157：H7的活性不受影响；将E．Coli O157：H7接种到培养了24h的乳酸茵培养液中，E．Coli O157：H7活性显下降。以乳酸调整的低pH值对E．Coli O157：H7有一定的杀灭作用。本研究表明：乳酸菌的代谢产物乳酸对E．Coli O157：H7有杀灭作用。     

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.     

大肠杆菌O157:H7的冷冻损伤及解冻存活

为探讨冷冻后残存的大肠杆菌O157:H7（Escherichia coli O157:H7）在解冻后的存活情况,本研究首先比较4 株E. coli O157:H7冷冻后的死亡和损伤情况,进而采用无营养的磷酸盐缓冲液作为基质研究冷冻后不同解冻方式对E. coli O157:H7存活的影响。结果表明：4 株E. coli O157:H7 －20 ℃冷冻24、48、72 h后均发生了一定程度的死亡和损伤,冷冻时间越长细菌致死和致伤程度越明显,且存在菌株差异,冷冻72?h时菌株CICC21530的损伤率最高,为87.70%。采用混合菌株进行解冻实验,4?株E.?coli?O157:H7磷酸盐缓冲液菌液冷冻后立即置于20、30、37?℃解冻,细菌发生了进一步的死亡,解冻温度越高死亡越明显,3?个温度组在解冻48?h时菌落数均显著低于冷冻72?h时菌落数（P＜0.05）。进一步探讨缓慢解冻方式对菌体存活的影响,菌液冷冻后先置于4?℃一定时间（0、2、6、12?h）,再置于37?℃不同时间（5、10、30?min）观察菌株存活情况,结果表明4?℃缓慢解冻时间越长,越有利于细菌的存活,4?℃、12?h/37?℃、5～30?min解冻方式下改良山梨醇麦康凯琼脂上菌落数仍显著低于胰蛋白胨大豆琼脂上的菌落数（P＜0.05）,表明仍有损伤菌的存在。本实验提示采用缓慢解冻反而有利于残存菌的存活,冷冻食品风险评估时应重视残存菌尤其是损伤菌的检测和控制。     

Survival of Escherichia coli O157:H7, nitrite content and sensory acceptance of low‐salt Chinese paocai fermented by screened lactic acid bacteria

Starter cultures of low‐salt Chinese paocai were screened from 135 lactic acid bacteria (LAB) by evaluating their antimicrobial activity and growth characteristics. Furthermore, the survival of Escherichia coli O157:H7, nitrite contents and sensory acceptability of the starter‐fermented paocai were evaluated. LAB BC92, which was identified as Lactobacillus pentosus, was used as a starter culture to produce paocai. In comparison with naturally fermented paocai, the disappearance time of E. coli O157:H7 in starter‐fermented paocai decreased from 5.29 to 3.82 days, and the maximum nitrite content decreased from 11.73 to 8.64 mg kg^?1, and the nitrite reduction time decreased from 4 to 3 days. In addition, starter fermentations were able to accelerate the flavour formation and shorten the paocai ripening period. The paocai that was fermented naturally and fermented by BC92 all had good sensory acceptance, and no significant difference (P > 0.05) was observed.     

Cloning of genes encoding colicin E2 in Lactococcus lactis subspecies lactis and evaluation of the colicin-producing transformants as inhibitors of Escherichia coli O157:H7 during milk fermentation

Colicin E2 (ColE2) is a proteinaceous bacterial toxin produced by some strains of Escherichia coli and other members of the Enterobacteriaceae that exhibits inhibitory activity against some strains of E. coli O157:H7. A 2.0-kb DNA fragment, containing the ColE2 structural gene ceaB and immunity gene ceiB from E. coli NCTC 50133 (pColE2-P9), was cloned into the lactococcal plasmid vector pNZ2103. The lysis gene, celB, was not cloned. The plasmid, pLR-E2, encoding the cloned genes was transformed into E. coli DH5α and Lactococcus lactis ssp. lactis LM0230 and PN-1 using electroporation. The bacteriocin ColE2 was expressed in transformants of both E. coli and L. lactis ssp. lactis. Secretion of ColE2 into media was verified by spot-on-lawn assays and measurement of ColE2 activity in the growth medium of transformants. The level of ColE2 produced by transformants containing pLR-E2 was similar to that produced by the parental strain, E. coli NCTC 50133 (pColE2-P9). Evaluation of a ColE2-producing transformant of L. lactis ssp. lactis as a starter culture revealed that, although ColE2 was produced by transformants and could be detected in milk during fermentation, the inhibitory activity of ColE2 against E. coli O157:H7 was significantly decreased in milk compared with buffered growth medium.     

Development of PCR assays for detection of Escherichia coli O157:H7 in meat products

A multiplex polymerase chain reaction (PCR) procedure based on fliC_h7 and rfbE genes was developed for the detection of Escherichia coli O157:H7 in raw pork meat and ready-to-eat (RTE) meat products. Two different DNA extraction procedures were evaluated for application on meat products. MasterPure™ DNA Purification kit in combination with immunomagnetic separation was found to be the best method in a meat system. The optimized PCR included an enrichment step in brilliant green bile 2% broth at 37 °C. This method was applied to artificially inoculated meat and RTE meat products with different concentrations of E. coli O157:H7. The results indicate that the PCR assay developed could sensitively and specifically detect E. coli O157:H7 in raw pork meat and RTE meat products in approximately 10 h, including a 6 h enrichment step. Thus, this method could be proposed for screening E. coli O157:H7 in raw pork and RTE meat products.     

Survival of Escherichia coli O157:H7 in cucumber fermentation brines

Abstract: Bacterial pathogens have been reported on fresh cucumbers and other vegetables used for commercial fermentation. The Food and Drug Administration currently has a 5‐log reduction standard for E. coli O157:H7 and other vegetative pathogens in acidified pickle products. For fermented vegetables, which are acid foods, there is little data documenting the conditions needed to kill acid resistant pathogens. To address this knowledge gap, we obtained 10 different cucumber fermentation brines at different stages of fermentation from 5 domestic commercial plants. Cucumber brines were used to represent vegetable fermentations because cabbage and other vegetables may have inhibitory compounds that may affect survival. The 5‐log reduction times for E. coli O157:H7 strains in the commercial brines were found to be positively correlated with brine pH, and ranged from 3 to 24 d for pH values of 3.2 to 4.6, respectively. In a laboratory cucumber juice medium that had been previously fermented with Lactobacillus plantarum or Leuconostoc mesenteroides (pH 3.9), a 5‐log reduction was achieved within 1 to 16 d depending on pH, acid concentration, and temperature. During competitive growth at 30 °C in the presence of L. plantarum or L. mesenteroides in cucumber juice, E. coli O157:H7 cell numbers were reduced to below the level of detection within 2 to 3 d. These data may be used to aid manufacturers of fermented vegetable products determine safe production practices based on fermentation pH and temperature. Practical Application: Disease causing strains of the bacterium E. coli may be present on fresh vegetables. Our investigation determined the time needed to kill E. coli in cucumber fermentation brines and how E. coli strains are killed in competition with naturally present lactic acid bacteria. Our results showed how brine pH and other brine conditions affected the killing of E. coli strains. These data can be used by producers of fermented vegetable products to help assure consumer safety.     

Efficacy of trimming chilled beef during fabrication to control Escherichia coli O157:H7 surrogates on subsequent subprimals

Effectiveness of trimming external carcass surfaces from subprimals during fabrication to reduce Escherichia coli O157:H7 surrogates was evaluated. Carcass sides (n = 10 sides) were inoculated along the hide pattern opening before entering the blast chill cooler with a gelatin slurry containing a bacterial cocktail of three rifampicin-resistant, nonpathogenic E. coli biotype I strains. Following a 48 h chill, sides were fabricated to produce eight subprimals. Microbiological samples were taken from the original carcass fat surface area, initial lean surface area, trimmed fat surface area (where applicable), and trimmed lean surface area (where applicable). Newly exposed lean surfaces had lower (P < 0.05) counts of rifampicin-resistant E. coli than did the external fat surfaces. However, fat and lean surfaces that were not inoculated became contaminated during the fabrication process. Trimming external surfaces reduced levels of pathogens, but under normal fabrication processes, pathogens were still spread to newly exposed surfaces.     

Fate of Escherichia coli O157:H7 in field-inoculated lettuce

Impact of drip and overhead sprinkler irrigation on the persistence of attenuated Escherichia coli O157:H7 in the lettuce phyllosphere was investigated using a split-plot design in four field trials conducted in the Salinas Valley, California, between summer 2007 and fall 2009. Rifampicin-resistant attenuated E. coli O157:H7 ATCC 700728 (BLS1) was inoculated onto the soil beds after seeding with a backpack sprayer or onto 2- or 4-week-old lettuce plant foliage with a spray bottle at a level of 7 log CFU ml⁻¹. When E. coli O157:H7 was inoculated onto 2-week-old plants, the organism was recovered by enrichment in 1 of 120 or 0 of 240 plants at 21 or 28 days post-inoculation, respectively. For the four trials where inoculum was applied to 4-week-old plants, the population size of E. coli O157:H7 declined rapidly and by day 7, counts were near or below the limit of detection (10 cells per plant) for 82% or more of the samples. However, in 3 out 4 field trials E. coli O157:H7 was still detected in lettuce plants by enrichment 4-weeks post-inoculation. Neither drip nor overhead sprinkler irrigation consistently influenced the survival of E. coli O157:H7 on lettuce.     

Survival of Escherichia coli O157:H7 and Salmonella enterica serovars Typhimurium in iceberg lettuce and the antimicrobial effect of rice vinegar against E. coli O157:H7

The microbiological safety of fresh produce is a significant concern of consumers and industry. After applying at an inoculated level (about 10(6) CFUg(-1)) of E. coli O157:H7 and Salmonella enterica serovars Typhimurium on shredded iceberg lettuce and water samples individually, they were stored at 4 degrees C for 14 days and 22 degrees C for 7 days to monitor the growth and survival of pathogens. The results showed that at the end of 4 degrees C storage, populations of two pathogens in lettuce and water decreased approximately 1 log CFUg(-1). However, microbial levels on shredded lettuce increased 3 logs within 3 days at 22 degrees C. Vinegar (acetic acid) had been used to reduce populations of foodborne pathogens in foods; hence, the antimicrobial effect of rice vinegar on the survival of E. coli O157:H7 in inoculated lettuce (10(4) and 10(7) CFUg(-1)) is examined in this study. Results were observed that the treatment of inoculated lettuce (10(7) CFUg(-1)) with commercial vinegar containing 5% acetic acid (pH 3.0) for 5 min would reduce 3 logs population at 25 degrees C. Less than a 1-log decrease in bacterial numbers was recovered during 5 min exposure to 0.5% (pH 3.26) acetic acid.     

Sucrose monolaurate improves the efficacy of sodium hypochlorite against Escherichia coli O157:H7 on spinach

It is well-recognized that chlorine has limited efficacy when applied to inactivate pathogens on fresh produce. One of the many factors limiting efficacy is the high interfacial tension of chlorine-based sanitizers that limits the access of chlorine to the microorganisms. In this work, we investigated the efficacy of sodium hypochlorite (200 ppm, pH 6.0) at 4 and 20 °C against Escherichia coli O157:H7 inoculated on baby spinach leaves as affected by the surfactant sucrose monolaurate (SML) at below (100 ppm), above (250 ppm), and well above (10,000 ppm) the critical micelle concentration (CMC) of ~ 200 ppm at 20 °C. The surfactant-containing chlorine treatments were compared to those with buffer only, surfactant only, and chlorine only. Significantly improved inactivation, as evidenced by survival of E. coli O157:H7 was achieved when 250 or 10,000 ppm SML was added with chlorine. This is attributed to the reduction of interfacial tension between the sanitizing solutions and spinach surface. Treatments at 20 °C resulted in greater mean inactivation than those at 4 °C but the difference was not significant. Comparisons of SML concentrations in treatment solutions before and after sanitization showed that SML decreased more at a lower temperature and when chlorine was present, resulting from adsorption of SML onto spinach matrix. Our work illustrates the importance of using surfactants at concentrations above the CMC to enhance the efficacy of chlorine sanitization.