Antimicrobial Effects of Mustard Flour and Acetic Acid against Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica Serovar Typhimurium

This study was designed to investigate the individual and combined effects of mustard flour and acetic acid in the inactivation of food-borne pathogenic bacteria stored at 5 and 22°C. Samples were prepared to achieve various concentrations by the addition of acetic acid (0, 0.5, or 1%) along with mustard flour (0, 10, or 20%) and 2% sodium chloride (fixed amount). Acid-adapted three-strain mixtures of Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella enterica serovar Typhimurium strains (10⁶ to 10⁷ CFU/ml) were inoculated separately into prepared mustard samples stored at 5 and 22°C, and samples were assayed periodically. The order of bacterial resistance, assessed by the time required for the nominated populations to be reduced to undetectable levels against prepared mustards at 5°C, was S. enterica serovar Typhimurium (1 day) < E. coli O157:H7 (3 days) < L. monocytogenes (9 days). The food-borne pathogens tested were reduced much more rapidly at 22°C than at 5°C. There was no synergistic effect with regard to the killing of the pathogens tested with the addition of 0.5% acetic acid to the mustard flour (10 or 20%). Mustard in combination with 0.5% acetic acid had less bactericidal activity against the pathogens tested than did mustard alone. The reduction of E. coli O157:H7 and L. monocytogenes among the combined treatments on the same storage day was generally differentiated as follows: control < mustard in combination with 0.5% acetic acid < mustard alone < mustard in combination with 1% acetic acid < acetic acid alone. Our study indicates that acidic products may limit microbial growth or survival and that the addition of small amounts of acetic acid (0.5%) to mustard can retard the reduction of E. coli O157:H7 and L. monocytogenes. These antagonistic effects may be changed if mustard is used alone or in combination with >1% acetic acid.     

Combined effect of mild heat and acetic acid treatment for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an asparagus puree

AIMS: This study was conducted to validate combined heat and acid treatments for inactivating Escherichia coli O157:H7, Listeria monocytogenes and Salmonella typhimurium in an acidified brine containing, or pickled, asparagus model food. METHODS AND RESULTS: A mixture of three strains of E. coli O157:H7, L. monocytogenes and S. typhimurium were inoculated onto pickled asparagus samples. Combinations of various concentrations of acetic acid [0%, 0.25%, 0.5%, 0.75%, 1%, 1.5% and 2% (v/v)] and various temperatures (40 degrees C, 50 degrees C, 60 degrees C and 75 degrees C) were investigated. Following treatment, asparagus samples were stored at room temperature and enumerated at 0, 0.5, 1, 2 and 3 days. Heat and acetic acid treatments were synergistic. The inhibitory effects of these combined treatments on the tested foodborne pathogens were also effective during storage. Loss of green colour in the pickled asparagus significantly increased with increasing concentrations of acetic acid. CONCLUSIONS: Using a combination of mild heat and acetic acid treatments can successfully control E. coli O157:H7, L. monocytogenes and S. typhimurium in pickled asparagus, combinations of heat and acid are synergistic and effective treatments can be selected to reduce adverse effect on colour which occur during product storage. SIGNIFICANCE AND IMPACT OF THE STUDY: Mild heating plus acetic acid treatment are synergistic, so combined treatments can be developed, which would lower the temperature and amount of acetic acid required for minimally processed vegetables while maintaining pathogen control.     

Evaluation of the pH-dependent,stationary-phase acid tolerance in Listeria monocytogenes and Salmonella Typhimurium DT104 induced by culturing in media with 1% glucose: a comparative study with Escherichia coli O157:H7

AIMS: To comparatively evaluate the adaptive stationary-phase acid tolerance response (ATR) in food-borne pathogens induced by culturing in glucose-containing media, as affected by strain variability and antibiotic resistance, growth temperature, challenge pH and type of acidulant. METHODS AND RESULTS: Antibiotic resistant or sensitive strains of Listeria monocytogenes, Salmonella including S. Typhimurium DT104, and Escherichia coli O157:H7 were cultured (30 degrees C for 24 h; 10 degrees C for up to 14 days) in trypticase soya broth with yeast extract (TSBYE) with 1% or without glucose to induce or prevent acid adaptation, respectively. Cultures were subsequently exposed to pH 3.5 or 3.7 with lactic or acetic acid at 25 degrees C for 120 min. Acid-adapted cultures were more acid tolerant than nonadapted cultures, particularly those of L. monocytogenes and Salmonella. No consistent, positive or negative, influence of antibiotic resistance on the pH-inducible ATR or acid resistance (AR) was observed. Compared with 30 degrees C cultures, growth and acid adaptation of L. monocytogenes and S. Typhimurium DT104 at 10 degrees C markedly reduced their ATR and AR in stationary phase. E. coli O157:H7 had the greatest AR, relying less on acid adaptation. A 0.2 unit difference in challenge pH (3.5-3.7) caused great variations in survival of acid-adapted and nonadapted cells. CONCLUSIONS: Culturing L. monocytogenes and Salmonella to stationary phase in media with 1% glucose induces a pH-dependent ATR and enhances their survival to organic acids; thus, this method is suitable for producing acid-adapted cultures for use in food challenge studies. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacterial pathogens may become acid-adapted in foods containing glucose or other fermentable carbohydrates. Low storage temperatures may substantially decrease the stationary-phase ATR of L. monocytogenes and S. Typhimurium DT104, but their effect on ATR of E. coli O157:H7 appears to be far less dramatic.     

Thermal tolerance of acid-adapted and unadapted Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in cantaloupe juice and watermelon juice

AIMS: A study was performed to determine D values of acid-adapted and unadapted cells of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in cantaloupe juice and watermelon juice. METHODS AND RESULTS: Salmonella enterica serotype Poona, S. enterica serotype Saphra, two strains of E. coli O157:H7, and two strains of L. monocytogenes were grown in tryptic soy broth (TSB) and TSB supplemented with 1% glucose for 24 h at 37 degrees C. Decimal reduction times (D values) of cells suspended in unpasteurized cantaloupe juice and watermelon juice were determined. Acid-adapted cells of Salmonella and E. coli O157:H7, but not L. monocytogenes, had increased thermal tolerance compared with cells that were not acid-adapted. There was no correlation between soluble solids content of the two types of juice and thermal resistance. CONCLUSIONS: Growth of Salmonella and E. coli O157:H7 in cantaloupe juice, watermelon juice, or other acidic milieu, either in preharvest or postharvest environments, may result in cross protection to heat. The pasteurization conditions necessary to achieve elimination of pathogens from these juices would consequently have to be more severe if cells are habituated to acidic environments. SIGNIFICANCE AND IMPACT OF THE STUDY: Insights from this study provide guidance to developing pasteurization processes to eliminate Salmonella, E. coli O157:H7, and L. monocytogenes in cantaloupe juice and watermelon juice.     

Comparative acid stress response of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella Typhimurium after habituation at different pH conditions

AIMS: The aim of the study was to evaluate the effect of habituation at different pH conditions on the acid resistance of Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella enterica serotype Typhimurium, and to identify potential differences between the adaptive responses of the three pathogens. METHODS: Stationary phase cells of L. monocytogenes, E. coli O157:H7 and S. Typhimurium, grown in glucose-free media, were exposed to pH 3.5 broth directly or after habituation for 90 min at various pH conditions from 4.0 to 6.0. Survivors at pH 3.5 were determined by plating on tryptic soy agar and incubating at 30 degrees C for 48 h. The kinetics (death rate) of the pathogens at pH 3.5 was calculated by fitting the data to an exponential model. RESULTS: Habituation to acidic environments provided protection of the pathogens against lethal acid conditions. This acid protection, however, was found to be pH dependent. For example, for E. coli O157:H7 an increased acid resistance was observed after habituation at a pH range from 4.0 to 5.5, while the maximum acid tolerance was induced at pH 5.0. Furthermore, the effect of low pH habituation was different among pathogens. For L. monocytogenes, E. coli O157:H7 and S. Typhimurium, the pH range within which habituation resulted to increased acid resistance was 5.0-6.0, 4.0-5.5 and 4.0-5.0, respectively, while the maximum acid tolerance was induced after habituation at pH 5.5, 5.0 and 4.5, respectively. SIGNIFICANCE: Acid stress conditions are common within current food processing technologies. The information on adaptive responses of L. monocytogenes, E. coli O157:H7 and S. Typhimurium after habituation to different pH environments provided in the present study, could lead to a more realistic evaluation of food safety concerns and to a better selection of processes in order to avoid adaptation phenomena and to minimize the potential for food safety risks.     

Inactivation of barotolerant strains of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 by ultra high pressure and <Emphasis Type="Italic">tert</Emphasis>-butylhydroquinone combination

Antimicrobial efficacy of ultra-high-pressure (UHP) can be enhanced by application of additional hurdles. The objective of this study was to systematically assess the enhancement in pressure lethality by TBHQ treatment, against barotolerant strains of Escherichia coli O157:H7 and Listeria monocytogenes. Two L. monocytogenes Scott A and the barotolerant OSY-328 strain, and two E. coli O157:H7 strains, EDL-933 and its barotolerant mutant, OSY-ASM, were tested. Cell suspensions containing TBHQ (50 ppm, dissolved in dimethyl sulfoxide) were pressurized at 200 to 500 MPa (23+/-2 degrees C) for 1 min, plated on tryptose agar and enumerated the survivors. The TBHQ-UHP combination resulted in synergistic inactivation of both pathogens, with different degrees of lethality among strains. The pressure lethality threshold, for the combination treatment, was lower for E. coli O157:H7 (> or = 200 MPa) than for L. monocytogenes (> 300 MPa). E. coli O157:H7 strains were extremely sensitive to the TBHQ-UHP treatment, compared to Listeria strains. Interestingly, a control treatment involving DMSO-UHP combination consistently resulted in higher inactivation than that achieved by UHP alone, against all strains tested. However, sensitization of the pathogens to UHP by the additives (TBHQ in DMSO) was prominently greater for UHP than DMSO. Differences in sensitivities to the treatment between these two pathogens may be attributed to discrepancies in cellular structure or physiological functions.     

Effect of environmental stresses on antibody-based detection of Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes

AIMS: To study the reaction patterns of selected antibodies to Escherichia coli O157:H7, Salmonella enterica serotype Enteritidis and Listeria monocytogenes cells exposed to various environmental stresses. METHODS AND RESULTS: Escherichia coli O157:H7, Salmonella Enteritidis and L. monocytogenes cells subjected to different environmental stress of temperatures (4 and 45 degrees C), NaCl (5.5%), oxidative stress (15 mmol(-1) H2O2), acidic pH (5.5) and ethanol (5%) for 3 h (short-term stress) or for 5 days (long-term stress) were analysed by ELISA and Western blotting. The ELISA results indicated that most stresses caused 12-16% reductions in reaction for anti-E. coli O157:H7 and 20-48% reductions for anti-Salmonella polyclonal antibodies during short-term stress, whereas the most stresses exhibited enhanced reaction (44-100% increase) with the anti-L. monocytogenes polyclonal antibody. During long-term stress exposure to combined stress conditions of pH 5.5, 3.5% NaCl at 12 degrees C or at 4 degrees C, antibody reactions to the three pathogens were highly variable with the combined stress at 4 degrees C showing the most reductions (8-40%). Likewise, there were about 18-59% reductions in antibody reactions with pathogens when cultured in hotdog samples with the combined stress conditions. Western blot analyses of crude cell surface antigens from both short- and long-term stressed cells revealed that the changes in antibody reactions observed in ELISA were either because of repression, expression or possible denaturation of antigens on the surface of cells. CONCLUSIONS: Overall, the antibody reactions were significantly reduced in pathogens exposed to both short- and long-term environmental stresses in culture medium or in meat sample because of expression, repression or denaturation of specific antigens in cells. SIGNIFICANCE AND IMPACT OF THE STUDY: In order to ensure the reliable detection of foodborne pathogens using antibody-based methods, the influence of stress on antibody reactions should be thoroughly examined and understood first as the physiological activities in cells are often altered in response to a stress.     

Enhancing the antimicrobial effects of bovine lactoferrin against Escherichia coli O157:H7 by cation chelation, NaCl and temperature

AIM: To evaluate the effect of NaCl, growth medium and temperature on the antimicrobial activity of bovine lactoferrin (LF) against Escherichia coli O157:H7 in the presence of different chelating agents. METHODS AND RESULTS: LF (32 mg ml(-1)) was tested against E. coli O157:H7 strain 3081 in Luria broth (LB) and All Purpose Tween (APT) broth with metal ion chelators sodium bicarbonate (SB), sodium lactate (SL), sodium hexametaphosphate (SHMP), ethylene diamine tetraacetic acid (EDTA) or quercetin at 0.5 and 2.5% NaCl at 10 and 37 degrees C. LF and the chelators were tested against four other E. coli O157:H7 strains in LB at 2.5% NaCl and 10 degrees C. LF alone was bacteriostatic against strains 3081 and LCDC 7283 but other strains grew. Antimicrobial effectiveness of LF was reduced in APT broth but enhanced by SB at 2.5% NaCl and 10 degrees C where 4.0 log(10) CFU ml(-1) inoculated cells were killed. EDTA enhanced antimicrobial action of the LF-SB combination. SL alone was effective against E. coli O157:H7 but a reduction in its activity at 2.5% NaCl and 10 degrees C was reversed by LF. The combinations LF-SHMP and LF-quercetin were more effective at 37 degrees C and NaCl effects varied. CONCLUSIONS: LF plus SB or SL were bactericidal toward the same 3/5 E. coli O157:H7 strains and inhibited growth of the others at 2.5% NaCl and 10 degrees C. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of LF with either SL or SB shows potential for reducing viability of E. coli O157:H7 in food systems containing NaCl at reduced, but growth permissive temperature.     

A comparative heat inactivation study of indigenous microflora in beef with that of Listeria monocytogenes,Salmonella serotypes and Escherichia coli O157:H7

AIMS: Thermal inactivation of a mixture of five strains of Listeria monocytogenes, four strains of Escherichia coli O157:H7 and eight serotypes of Salmonella were compared with that of indigenous microflora in 75% lean ground beef. METHODS AND RESULTS: Inoculated meat was packaged in bags that were completely immersed in a circulating water bath and held at 55, 57.5 and 60 degrees C for predetermined lengths of time. The surviving cell population was enumerated by spiral plating heat-treated samples onto tryptic soya agar supplemented with 0.6% yeast extract and 1% sodium pyruvate. D-values, determined by linear regression, in beef were 77.49, 21.9, and 10.66 min at 55, 57.5, and 60 degrees C, respectively, for indigenous microflora (z = 5.81 degrees C). When either of the three pathogens were heated in beef, their D-values calculated were significantly lower (P < 0.05) than those of indigenous microflora at all temperatures. The slope of the thermal death time curve for L. monocytogenes, E. coli O157:H7 and indigenous microflora were similar. Using a survival model for nonlinear survival curves, the D1-values at all temperatures for L. monocytogenes were significantly higher (P < 0.05) compared with those for Salmonella serotypes, E. coli O157:H7 or indigenous microflora. However, higher recovery of a subpopulation of the indigenous microflora in beef exposed to heating at 55, 57.5 or 60 degrees C resulted in significantly higher (P < 0.05) D2-values at all three temperatures, compared with those of the three pathogens at the same test temperatures. CONCLUSIONS: If the thermal process is designed to ensure destruction of indigenous microbial flora, it should also provide an adequate degree of protection against L. monocytogenes, Salmonella serotypes or E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study will assist the retail food industry in designing acceptance limits on critical control points that ensure safety, without introducing pathogens in a retail food environment, against L. monocytogenes, E. coli O157:H7 and Salmonella in cooked ground beef.     

Potential role of Diploscapter sp. strain LKC25, a bacterivorous nematode from soil, as a vector of food-borne pathogenic bacteria to preharvest fruits and vegetables

Diploscapter, a thermotolerant, free-living soil bacterial-feeding nematode commonly found in compost, sewage, and agricultural soil in the United States, was studied to determine its potential role as a vehicle of Salmonella enterica serotype Poona, enterohemorrhagic Escherichia coli O157:H7, and Listeria monocytogenes in contaminating preharvest fruits and vegetables. The ability of Diploscapter sp. strain LKC25 to survive on agar media, in cow manure, and in composted turkey manure and to be attracted to, ingest, and disperse food-borne pathogens inoculated into soil or a mixture of soil and composted turkey manure was investigated. Diploscapter sp. strain LKC25 survived and reproduced in lawns of S. enterica serotype Poona, E. coli O157:H7, and L. monocytogenes on agar media and in cow manure and composted turkey manure. Attraction of Diploscapter sp. strain LKC25 to colonies of pathogenic bacteria on tryptic soy agar within 10, 20, 30, and 60 min and 24 h was determined. At least 85% of the worms initially placed 0.5 to 1 cm away from bacterial colonies migrated to the colonies within 1 h. Within 24 h, > or =90% of the worms were embedded in colonies. The potential of Diploscapter sp. strain LKC25 to shed pathogenic bacteria after exposure to bacteria inoculated into soil or a mixture of soil and composted turkey manure was investigated. Results indicate that Diploscapter sp. strain LKC25 can shed pathogenic bacteria after exposure to pathogens in these milieus. They also demonstrate its potential to serve as a vector of food-borne pathogenic bacteria in soil, with or without amendment with compost, to the surface of preharvest fruits and vegetables in contact with soil.     

Interactions between food-borne pathogens and protozoa isolated from lettuce and spinach

The survival of Salmonella enterica was recently shown to increase when the bacteria were sequestered in expelled food vacuoles (vesicles) of Tetrahymena. Because fresh produce is increasingly linked to outbreaks of enteric illness, the present investigation aimed to determine the prevalence of protozoa on spinach and lettuce and to examine their interactions with S. enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Glaucoma sp., Colpoda steinii, and Acanthamoeba palestinensis were cultured from store-bought spinach and lettuce and used in our study. A strain of Tetrahymena pyriformis previously isolated from spinach and a soil-borne Tetrahymena sp. were also used. Washed protozoa were allowed to graze on green fluorescent protein- or red fluorescent protein-labeled enteric pathogens. Significant differences in interactions among the various protist-enteric pathogen combinations were observed. Vesicles were produced by Glaucoma with all of the bacterial strains, although L. monocytogenes resulted in the smallest number per ciliate. Vesicle production was observed also during grazing of Tetrahymena on E. coli O157:H7 and S. enterica but not during grazing on L. monocytogenes, in vitro and on leaves. All vesicles contained intact fluorescing bacteria. In contrast, C. steinii and the amoeba did not produce vesicles from any of the enteric pathogens, nor were pathogens trapped within their cysts. Studies of the fate of E. coli O157:H7 in expelled vesicles revealed that by 4 h after addition of spinach extract, the bacteria multiplied and escaped the vesicles. The presence of protozoa on leafy vegetables and their sequestration of enteric bacteria in vesicles indicate that they may play an important role in the ecology of human pathogens on produce.     

Culture conditions influencing phytase production of Mitsuokella jalaludinii,a new bacterial species from the rumen of cattle

AIMS: To determine the effectiveness of combined treatments with chemicals, heat and ultrasound in killing or removing Salmonella and Escherichia coli O157:H7 on alfalfa seeds intended for sprout production. METHODS AND RESULTS: Alfalfa seeds inoculated with Salmonella or E. coli O157:H7 were treated with ultrasound (38.5-40.5 kHz) in solutions containing 1% Ca(OH)(2), 1% Tween 80, 1% Ca(OH)(2) plus 1% Tween 80, 160 microg ml(-1) Tsunami 200 and 0.5% Fit at 23 and 55 degrees C for 2 and 5 min. Highest reductions were in chemical solutions at 55 degrees C, but seed viability was also reduced compared with treatment at 23 degrees C. Inactivation of Salmonella and E. coli O157:H7 was generally enhanced by simultaneous treatments with ultrasound, chemicals and heat. CONCLUSIONS: Ultrasound treatment, in combination with chemicals and heat, had a modest enhancing effect on the effectiveness of chemicals in killing or removing pathogens on alfalfa seeds. Overall, treatment with 1% Ca(OH)(2) was most effective in killing Salmonella and E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of 1% Ca(OH)(2) instead of 20,000 microg ml(-1) chlorine, which is currently recommended as a sanitizer for seeds intended for sprout production in the US, should be considered. Ultrasound treatment of alfalfa seeds containing Salmonella or E. coli O157:H7, in combination with chemical treatment, contributes to achieving greater reductions in populations of these pathogens, thereby reducing the risk of contamination and the presence of pathogens in sprouts produced from these seeds.     

Survival and growth of Escherichia coli O157:H7 in ground, roasted beef as affected by pH, acidulants, and temperature.

A study was undertaken to determine the fate of Escherichia coli O157:H7 in ground, roasted beef as influenced by the combined effects of pH, acidulants, temperature, and time. There was essentially no change in the viable population of E. coli O157:H7 when beef salads (pH 5.40 to 6.07) containing up to 40% mayonnaise were incubated at 5 degrees C for up to 72 h. At 21 and 30 degrees C, significant (P < or = 0.05) increases in populations of the organism occurred in salads containing 16 to 32% mayonnaise (pH 5.94 to 5.55) between 10 and 24 h of incubation. Death was more rapid as the pH of acidified beef slurries incubated at 5 degrees C was decreased from 5.98 to 4.70. E. coli O157:H7 grew in control slurries (pH 5.98) and in slurries containing citric and lactic acids (pHs 5.00 and 5.40) incubated at 21 degrees C for 24 h; decreases occurred in slurries acidified to pHs 4.70, 5.00, and 5.40 with acetic acid or pH 4.70 with citric or lactic acid. At 30 degrees C, populations decreased in slurries acidified to pHs 4.70 and 5.00 with acetic acid. Citric and lactic acids failed to prevent significant increases in populations in slurries at pH 4.70 to 5.40 between 10 and 24 h of incubation. The order of effectiveness of acidulants in inhibiting growth was acetic acid > lactic acid > or = citric acid. The same order was observed for inactivation of E. coli O157:H7 in acidified (pH 5.00) beef slurry heated at 54 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of acid adaptation of Escherichia coli O157:H7 on efficacy of acetic acid spray washes to decontaminate beef carcass tissue

Exposure to low pH and organic acids in the bovine gastrointestinal tract may result in the induced acid resistance of Escherichia coli O157:H7 and other pathogens that may subsequently contaminate beef carcasses. The effect of acid adaptation of E. coli O157:H7 on the ability of acetic acid spray washing to reduce populations of this organism on beef carcass tissue was examined. Stationary-phase acid resistance and the ability to induce acid tolerance were determined for a collection of E. coli O157:H7 strains by testing the survival of acid-adapted and unadapted cells in HCl-acidified tryptic soy broth (pH 2.5). Three E. coli O157:H7 strains that were categorized as acid resistant (ATCC 43895) or acid sensitive (ATCC 43890) or that demonstrated inducible acid tolerance (ATCC 43889) were used in spray wash studies. Prerigor beef carcass surface tissue was inoculated with bovine feces containing either acid-adapted or unadapted E. coli O157:H7. The beef tissue was subjected to spray washing treatments with water or 2% acetic acid or left untreated. For strains ATCC 43895 and 43889, larger populations of acid-adapted cells than of unadapted cells remained on beef tissue following 2% acetic acid treatments and these differences remained throughout 14 days of 4 degrees C storage. For both strains, numbers of acid-adapted cells remaining on tissue following 2% acetic acid treatments were similar to numbers of both acid-adapted and unadapted cells remaining on tissue following water treatments. For strain ATCC 43890, there was no difference between populations of acid-adapted and unadapted cells remaining on beef tissue immediately following 2% acetic acid treatments. These data indicate that adaptation to acidic conditions by E. coli O157:H7 can negatively influence the effectiveness of 2% acetic acid spray washing in reducing the numbers of this organism on carcasses.     

Survival or growth of Escherichia coli O157:H7 in a model system of fresh meat decontamination runoff waste fluids and its resistance to subsequent lactic acid stress

A potential may exist for survival of and resistance development by Escherichia coli O157:H7 in environmental niches of meat plants applying carcass decontamination interventions. This study evaluated (i) survival or growth of acid-adapted and nonadapted E. coli O157:H7 strain ATCC 43895 in acetic acid (pH 3.6 +/- 0.1) or in water (pH 7.2 +/- 0.2) fresh beef decontamination runoff fluids (washings) stored at 4, 10, 15, or 25 degrees C and (ii) resistance of cells recovered from the washings after 2 or 7 days of storage to a subsequent lactic acid (pH 3.5) stress. Corresponding cultures in sterile saline or in heat-sterilized water washings were used as controls. In acetic acid washings, acid-adapted cultures survived better than nonadapted cultures, with survival being greatest at 4 degrees C and lowest at 25 degrees C. The pathogen survived without growth in water washings at 4 and 10 degrees C, while it grew by 0.8 to 2.7 log cycles at 15 and 25 degrees C, and more in the absence of natural flora. E. coli O157:H7 cells habituated without growth in water washings at 4 or 10 degrees C were the most sensitive to pH 3.5, while cells grown in water washings at 15 or 25 degrees C were relatively the most resistant, irrespective of previous acid adaptation. Resistance to pH 3.5 of E. coli O157:H7 cells habituated in acetic acid washings for 7 days increased in the order 15 degrees C > 10 degrees C > 4 degrees C, while at 25 degrees C cells died off. These results indicate that growth inhibition by storage at low temperatures may be more important than competition by natural flora in inducing acid sensitization of E. coli O157:H7 in fresh meat environments. At ambient temperatures in meat plants, E. coli O157:H7 may grow to restore acid resistance, unless acid interventions are applied to inhibit growth and minimize survival of the pathogen. Acid-habituated E. coli O157:H7 at 10 to 15 degrees C may maintain a higher acid resistance than when acid habituated at 4 degrees C. These responses should be evaluated with fresh meat and may be useful for the optimization of decontamination programs and postdecontamination conditions of meat handling.     

Differences in growth of Salmonella enterica and Escherichia coli O157:H7 on alfalfa sprouts

Sprout producers have recently been faced with several Salmonella enterica and Escherichia coli O157:H7 outbreaks. Many of the outbreaks have been traced to sprout seeds contaminated with low levels of human pathogens. Alfalfa seeds were inoculated with S. enterica and E. coli O157:H7 strains isolated from alfalfa seeds or other environmental sources and sprouted to examine growth of these human pathogens in association with sprouting seeds. S. enterica strains grew an average of 3.7 log(10) on sprouting seeds over 2 days, while E. coli O157:H7 strains grew significantly less, an average of 2.3 log(10). The initial S. enterica or E. coli O157:H7 inoculum dose and seed-sprouting temperature significantly affected the levels of both S. enterica and E. coli O157:H7 on the sprouts and in the irrigation water, while the frequency of irrigation water replacement affected only the levels of E. coli O157:H7. Colonization of sprouting alfalfa seeds by S. enterica serovar Newport and E. coli O157:H7 strains transformed with a plasmid encoding the green fluorescent protein was examined with fluorescence microscopy. Salmonella serovar Newport colonized both seed coats and sprout roots as aggregates, while E. coli O157:H7 colonized only sprout roots.     

基于单碱基延伸标签反应的常见食源性致病菌基因芯片检测方法的建立

针对8种常见的食源性致病菌(金黄色葡萄球菌、副溶血弧菌、单核细胞增生李斯特菌、沙门氏菌、阪崎肠杆菌、志贺氏菌、肠出血性大肠杆菌O157:H7和空肠弯曲杆菌),建立了基于单碱基延伸标签反应原理的基因芯片检测方法。筛选和整合8种食源性致病菌基因组中的特异性序列和相应PCR引物,致病菌靶DNA片段被扩增和纯化作为单碱基延伸标签反应的模板,反应产物在DNA芯片上与探针进行杂交反应,然后通过扫描基片的荧光强度进行判断。实验结果表明,可采用基于单碱基延伸标签反应的基因芯片方法同时特异性检测8种食源性致病菌,基因组DNA多重检测灵敏度可达到0.1pg,以鼠伤寒沙门氏菌为单一检测对象的细菌纯培养物灵敏度可达到5×102CFU/mL。本方法可以快速灵敏地检测食源性致病菌,为食源性疾病的诊断和防治提供了一个有效的方法。     

Combined effects of water activity, temperature and chemical treatments on the survival of Salmonella and Escherichia coli O157:H7 on alfalfa seeds

AIMS: The objective of this study was to determine the combined effects of water activity (a(w)), chemical treatment and temperature on Salmonella and Escherichia coli O157:H7 inoculated onto alfalfa seeds. METHODS AND RESULTS: Alfalfa seeds inoculated with Salmonella or E. coli O157:H7 and adjusted to various a(w) values were subjected to simultaneous and separate treatments with chemicals and heat. The rate of death of both pathogens was correlated with increased a(w) (0.15-0.60) and temperature (5-37 degrees C) over a 52-week storage period. Higher seed a(w) enhanced the inactivation of pathogens on seeds heated at 50-70 degrees C for up to 24 h. Treatment of seeds with water, 1% Ca(OH)2, 1% Tween 80, 1% Ca(OH)2 plus 1% Tween 80 or 40 mg l(-1) Tsunami 200 at 23 or 55 degrees C for 2 min significantly (alpha=0.05) reduced populations of Salmonella and E. coli O157:H7. CONCLUSIONS: Overall, at the combinations of temperature and concentrations of chemicals tested, 1% Ca(OH)2 was most effective in killing Salmonella and E. coli O157:H7 without reducing seed viability. SIGNIFICANCE AND IMPACT OF THE STUDY: None of the treatments evaluated in this study, whether applied separately or in combination, eliminated Salmonella or E. coli O157:H7 on alfalfa seeds without sacrificing the viability of the seeds. It remains essential that practices to prevent the contamination of alfalfa seeds be strictly followed in order to minimize the risk of Salmonella and E. coli O157:H7 infections associated with sprouts produced from these seeds.     

The synergistic effect of nisin and lactoferrin on the inhibition of Listeria monocytogenes and Escherichia coli O157:H7

AIMS: The goal of this study was to determine whether nisin and lactoferrin would act synergistically to inhibit the growth of Listeria monocytogenes and Escherichia coli O157:H7. METHODS AND RESULTS: Lactoferrin and nisin separately or in combination were suspended in peptone yeast glucose broth and following inoculation with L. monocytogenes or E. coli O157:H7 growth inhibition of each pathogen was determined. At 1000 microg ml(-1) lactoferrin L. monocytogenes was effectively inhibited. However, E. coli O157:H7 initially was inhibited and then grew to cell density similar to the control. A combination of 500 microg ml(-1) of lactoferrin and 250 IU ml(-1) of nisin effectively inhibited the growth of E. coli O157:H7, whereas, 250 microg ml(-1) of lactoferrin and 10 IU ml(-1) of nisin were inhibitory to L. monocytogenes. CONCLUSIONS: The results suggest that lactoferrin and nisin act synergistically to inhibit the growth of L. monocytogenes and E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: Natural preservatives that are active against gram-positive and gram-negative pathogens are desirable to the food industry and consumers. This study demonstrates that lactoferrin and nisin work synergistically reducing the levels required independently inhibiting growth of two major foodborne pathogens. Previous reported results indicated a low level of antimicrobial activity; however, this work was not performed in low divalent cation concentration media. It has been suggested that nondivalent cation-limiting medium such as trypticase soy broth (TSB), can reduce or completely eliminate the inhibitory activity. Further knowledge of these interactions can increase the understanding of the antimicrobial activity of lactoferrin. This should make the use of these compounds by industry more attractive.     

Factors controlling acid tolerance of Listeria monocytogenes: effects of nisin and other ionophores.

The acid tolerance of a Listeria monocytogenes serotype 4b strain was studied by measuring its ability to survive at an acidic pH at 37 degrees C. The acid tolerance of L. monocytogenes was much lower than those of Escherichia coli O157:H7 and Shigella flexneri strains. This observation suggested a higher infective dose for L. monocytogenes than E. coli O157:H7 and Shigella. The susceptibility of L. monocytogenes to acidic pH was dependent upon growth medium pH and growth phase of the culture. Nisin and some other ionophores reduced the acid tolerance of both stationary-phase and log-phase cultures of L. monocytogenes. These studies indicated that nisin might be a useful candidate for controlling acid tolerance of L. monocytogenes.