Influence of soil microbes on Escherichia coli O157:H7 survival in soil rinse and artificial soil.

AIMS: This research investigated the influence of soil microbiota on Escherichia coli O157:H7 survival in soil rinse and artificial soil. Additionally, the influence of selected soil bacteria on E. coli O157:H7 in soil environments was determined. METHODS AND RESULTS: Escherichia coli O157:H7 counts (log CFU per ml or g-1 ) were determined by spread plating: (i) artificial soil amended with soil rinse (filter-sterilized and unfiltered) at 30°C; (ii) unfiltered soil rinse (50 ml) treated with cycloheximide (200 µg ml-1 ), vancomycin (40 µg ml-1 ), heat (80°C, 15 min) and no treatment (control) for 7 days at 30°C and (iii) filtered soil rinse with selected soil bacterial isolates over 7 days. There was a significant difference (P = 0·027) in E. coli O157:H7 counts after 35 days between artificial soils amended with filtered (4·45 ± 0·29) and non-filtered (1·83 ± 0·33) soil rinse. There were significant differences (P < 0·05) in E. coli O157:H7 counts after 3 days of incubation between soil rinse treatments (heat (7·04 ± 0·03), cycloheximide (6·94 ± 0·05), vancomycin (4·26 ± 0·98) and control (5·00 ± 0·93)). Lastly, a significant difference (P < 0·05) in E. coli O157:H7 counts was observed after 3 days of incubation at 30°C in filtered soil rinse when incubated with Paenibacillus alvei versus other soil bacterial isolates evaluated. CONCLUSIONS: Soil microbiota isolated from Florida sandy soil influenced E. coli O157:H7 survival. Specifically, P. alvei reduced E. coli O157:H7 by over 3 log CFU per ml after 3 days of incubation at 30°C in filtered soil rinse. SIGNIFICANCE AND IMPACT OF THE STUDY: This research identified soil bacterial isolates that may reduce E. coli O157:H7 in the soil environment and be used in future biocontrol applications.

Escherichia coli O157 survival in liquid culture and artificial soil microcosms with variable pH, humic acid and clay content.

AIMS: This research was performed to investigate the influence of clay and humic acid on Escherichia coli O157 survival in model soils. Additionally, the influence of pH and humic acid on E. coli O157 in liquid culture was investigated. METHODS AND RESULTS: Artificial soil microcosms were prepared with sand, kaolinite, bentonite and humic acid. Artificial soil microcosms pH was adjusted (6·0-7·0) with aluminium sulphate before E. coli O157 inoculation. After 56 days of incubation at 30°C, significant differences in E. coli O157 log CFU per gram were observed between 0 and 1000 ppm (P < 0·0001) and 0 and 5000 ppm (P < 0·0001) humic acid in 1·5% clay soils, but not in 7·5 or 15% clay soils. Significant differences (P < 0·05) in E. coli O157 log CFU per ml were observed in liquid culture influenced by humic acid concentrations after 8 h at 37°C. CONCLUSIONS: The developed model soils support E. coli O157 populations over 28 days, and higher clay soils may aid in E. coli O157 survival. SIGNIFICANCE AND IMPACT OF THE STUDY: These results provide insights into physicochemical properties of soil that may influence E. coli O157 in the environment and help explain E. coli O157 survival in various soils and geographical regions.

Sodium metasilicate affects growth of Campylobacter jejuni in fresh, boneless, uncooked chicken breast fillets stored at 4 degrees Celsius for 7 days.

The objectives of this study were to determine the antimicrobial effects of sodium metasilicate (SMS) treatments against Campylobacter jejuni in fresh, boneless, uncooked chicken breast fillets and to ascertain the effects of SMS treatments on pH. The fillets were inoculated with C. jejuni, treated with 0% SMS and no inoculum (negative control), 0% SMS and inoculum (positive control), 1 and 2% SMS solutions, and stored at 4 ± 1°C. All samples were analyzed after 0, 1, 3, 5, and 7 d storage for C. jejuni, psychrotrophic organisms, and pH. Campylobacter jejuni and psychrotrophic counts for samples treated with 1 and 2% SMS solutions were similar (P > 0.05) to the positive control on all storage days. The pH values for 2% SMS marinade treatments were higher (P < 0.05) when compared with the negative and positive controls through 7 d of storage. Based on the findings in this study, a second study was conducted to determine the level of SMS necessary to reduce C. jejuni by at least 1 log cfu/g. The treatments were the same as previously discussed, except SMS was used at levels of 1 and 2% of the weight of the meat instead of percentage of the solution. Chicken fillets treated with 1 and 2% SMS (by weight of meat) resulted in 1.12 to 1.26 and 3.27 to 3.79 log cfu/g reductions in C. jejuni, respectively, when compared with the positive control. Except for d 0, psychrotrophic counts for samples treated with 2% SMS were lower (P < 0.05) than negative and positive controls on all storage days. The pH values were higher (P < 0.05) for all SMS treatments when compared with the negative and positive controls. This study revealed that SMS, when used at elevated levels in excess of the USDA Food Safety and Inspection Service 2% approved level, could function to control Campylobacter jejuni and extend the shelf life of raw poultry by retarding the growth of psychrotrophic bacteria.

Sodium metasilicate affects growth of Salmonella Typhimurium in fresh, boneless, uncooked chicken breast fillets stored at 4°C for 7 days.

The objectives of this study were to determine the antimicrobial effects of sodium metasilicate (SMS) against Salmonella and psychrotrophic organisms in fresh, boneless, uncooked chicken breast fillets and to ascertain the effects of SMS treatments on pH. Chicken breast fillets were inoculated with Salmonella enterica serovar Typhimurium treated with 0% SMS and no inoculum (negative control), 0% SMS and inoculum (positive control), 1% SMS, or 2% SMS solutions and stored at 4 ± 1°C. All samples were analyzed after 0, 1, 3, 5, and 7 d for Salmonella, psychrotrophic organisms, and pH. The fillets that were treated with 1 or 2% SMS had lower (P < 0.05) Salmonella counts as compared with those of the positive control at 3 through 7 d. Reductions in Salmonella Typhimurium were 0.83 to 0.91 log cfu/g and 1.04 to 1.16 log cfu/g for 1 and 2% SMS treatments, respectively. The psychrotrophic counts were similar (P > 0.05) for all treatments. The pH values for samples treated with 1 or 2% SMS were higher (P < 0.05) when compared with those of the negative and positive controls. This study revealed that SMS could restrict the growth of pathogenic Salmonella Typhimurium in fresh poultry.

Salmonella SdiA recognizes N-acyl homoserine lactone signals from Pectobacterium carotovorum in vitro, but not in a bacterial soft rot.

Genomes of Salmonella enterica isolates, including those linked to outbreaks of produce-associated gastroenteritis, contain sdiA, which encodes a receptor of N-acyl homoserine lactones (AHL). AHL are the quorum-sensing signals used by bacteria to coordinately regulate gene expression within -their populations. Because S. enterica does not produce its own AHL, SdiA is hypothesized to function in the interspecies cross-talk with AHL-producing bacteria. Under laboratory conditions, S. enterica responded to AHL from phytobacteria by upregulating expression of srgE. AHL-dependent expression of srgE required a functional sdiA. Essentially, no sdiA-dependent resolution of the srgE recombinase-based (RIVET) reporter was observed inside a soft rot formed on a tomato by an AHL-producing strain of Pectobacterium carotovorum. The results of the control experiments suggest that sdiA is not expressed inside tomato, pepper, green onion, or carrot affected by the soft rot, and the lack of sdiA expression in planta prevents Salmonella spp. from responding to AHL. Despite its inability to detect and respond to AHL during colonization of soft rots, S. enterica reached higher final cell numbers inside a tomato soft rot compared with its growth in intact tomato fruit. The synergistic effect was the strongest under the conditions that are typical for the Florida fall/winter production season.

Phosphate-regulated inactivation of the kinase PHO80-PHO85 by the CDK inhibitor PHO81.

A complex consisting of the cyclin-dependent kinase (CDK) PHO85 and the cyclin PHO80 phosphorylates and is thought to inactivate the transcription factor PHO4 when yeast cells are grown in medium containing high concentrations of phosphate. The CDK inhibitor PHO81 inhibits the kinase activity of the PHO80-PHO85 complex when Saccharomyces cerevisiae cells are grown in medium depleted of phosphate. A region of PHO81 with similarity to the mammalian CDK inhibitor p16INK4 is sufficient for inhibition in vitro. These studies demonstrate that CDK inhibitors are used to regulate kinases involved in processes other than cell cycle control and suggest that the ankyrin repeat motif may be commonly used for interaction with cyclin-CDK complexes.

Survival of Escherichia coli O157:H7 and Salmonella on Bruised and Unbruised Tomatoes from Three Ripeness Stages at Two Temperatures.

Tomatoes are one of the major fresh produce commodities consumed in the United States. Harvesting tomato fruit at a later stage of development can enhance consumer acceptance but can also increase damage due to bruising. Bruising can affect the quality of whole tomatoes by causing an unacceptable appearance and accelerating decay. Bruising may also facilitate bacterial attachment to the fruit surface and support growth of pathogens. This study evaluated the survival and/or proliferation of Escherichia coli O157:H7 and Salmonella on the surface of artificially bruised and unbruised tomatoes at three ripeness stages (breaker, pink, and red) and two storage temperatures (10 and 20°C). A total of 1,440 tomatoes, 720 for each organism, were analyzed. Both E. coli O157:H7 and Salmonella counts declined significantly ( P < 0.05) on the bruised and unbruised tomatoes over the 7-day storage period, by approximately 2.5 and 2.0 log, respectively. E. coli O157:H7 was not detected on pink tomatoes on day 7, whereas Salmonella persisted on the tomato surfaces throughout the 7-day study at all ripeness stages. Bruising had no significant effect ( P > 0.05) on the survival of E. coli O157:H7 (CFU per tomato) compared with the unbruised tomatoes, in most cases. Tomatoes from the red ripeness stage showed a significant effect ( P < 0.05) of bruising on Salmonella survival at both 10 and 20°C. Similar to the colony count results, the frequency (presence or absence) of inoculated tomatoes with detectable levels of inoculated bacteria decreased significantly ( P < 0.05) over time. At the lower temperature, E. coli O157:H7 was recovered from significantly higher ( P < 0.05) numbers of breaker and pink tomatoes, whereas there was no effect of temperature on the overall survival of E. coli O157:H7 on red tomatoes. Results from this study are essential for understanding the effects of bruising on produce safety and for producers and packers to develop mitigation strategies to control pathogenic and spoilage organisms.

Shigella as a foodborne pathogen and current methods for detection in food.

Shigella, the causative agent of shigellosis or "bacillary dysentery," has been increasingly involved in foodborne outbreaks. According to the Centers for Disease Control and Prevention's Emerging Infections Program, Foodborne Diseases Active Surveillance Network (FoodNet), Shigella was the third most reported foodborne bacterial pathogen in 2002. Foods are most commonly contaminated with Shigella by an infected food handler who practices poor personal hygiene. Shigella is acid resistant, salt tolerant, and can survive at infective levels in many types of foods such as fruits and vegetables, low pH foods, prepared foods, and foods held in modified atmosphere or vacuum packaging. Survival is often increased when food is held at refrigerated temperatures. Detection methods for Shigella include conventional culture methods, immunological methods, and molecular microbiological methods. Conventional culture of Shigella in foods is often problematic due to the lack of appropriate selective media. Immunological methods for Shigella have been researched, yet there is only one commercially available test kit. Molecular microbiological methods such as PCR, oligonucleotide microarrays, and rep-PCR have also been developed for the detection and identification of Shigella. This manuscript reviews the general characteristics, prevalence, growth and survival, and methods for detection of Shigella in food.

A genetic study of signaling processes for repression of PHO5 transcription in Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, transcription of a secreted acid phosphatase, PHO5, is repressed in response to high concentrations of extracellular inorganic phosphate. To investigate the signal transduction pathway leading to transcriptional regulation of PHO5, we carried out a genetic selection for mutants that express PHO5 constitutively. We then screened for mutants whose phenotypes are also dependent on the function of PHO81, which encodes an inhibitor of the Pho80p-Pho85p cyclin/cyclin-dependent kinase complex. These mutations are therefore likely to impair upstream functions in the signaling pathway, and they define five complementation groups. Mutations were found in a gene encoding a plasma membrane ATPase (PMA1), in genes required for the in vivo function of the phosphate transport system (PHO84 and PHO86), in a gene involved in the fatty acid synthesis pathway (ACC1), and in a novel, nonessential gene (PHO23). These mutants can be classified into two groups: pho84, pho86, and pma1 are defective in high-affinity phosphate uptake, whereas acc1 and pho23 are not, indicating that the two groups of mutations cause constitutive expression of PHO5 by distinct mechanisms. Our observations suggest that these gene products affect different aspects of the signal transduction pathway for PHO5 repression.

Comparison of conventional culture methods and FTA filtration-nested PCR for the detection of Shigella boydii and Shigella sonnei on tomato surfaces.

Detection of Shigella boydii UI02 and Shigella sonnei UI05 artificially inoculated onto tomatoes was evaluated using enrichment protocols of the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) and the American Public Health Association's Compendium of Methods for the Microbiological Examination of Food (CMMEF), enrichment in Enterobacteriaceae enrichment (EE) broth supplemented with 1.0 microg/ml novobiocin and incubated at 42 degrees C, and FTA filtration-nested PCR. To assess the effect of natural tomato microflora on recovery, conventional culture enrichments were repeated using rifampin-adapted inocula and enrichment medium supplemented with 50 microg/ml rifampin. The lowest detection levels for S. boydii UI02 were > 5.3 x 10(5) (BAM, CMMEF, and EE broth) and 6.2 CFU per tomato (FTA filtration-nested PCR). For S. sonnei UI05, the lowest detection levels were 1.9 x 10(1) (BAM), 1.5 x 10(3) (CMMEF), 1.1 x 10(1) (EE broth), and 7.4 CFU per tomato (FTA filtration-nested PCR). Natural tomato microflora had a large impact on recovery of S. sonnei UI05 and completely inhibited recovery of S. boydii UI02. EE broth was inhibitory to S. boydii UI02. FTA filtration-nested PCR provided superior detection (P < 0.05) compared with the conventional culture enrichment protocols.

Comparison of chromogenic Shigella spp. plating medium with standard media for the recovery of Shigella boydii and Shigella sonnei from tomato surfaces.

Isolation of Shigella spp. from food is difficult because of a lack of appropriate selective media and the presence of low numbers of shigellae relative to competitive microorganisms. Chromogenic Shigella spp. plating medium (CSPM) was evaluated for use with the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) enrichment procedure for isolation of artificially contaminated Shigella boydii UI02 and Shigella sonnei UI05 from tomato surfaces. Tomatoes were inoculated with various concentrations of S. boydii UI02 or S. sonnei UI05 and rinsed using a shake-rub-shake procedure. Tomato rinses were enriched overnight according to the BAM procedure and streaked for isolation on CSPM, Salmonella-Shigella agar (SSA), and MacConkey agar (MAC). To access the isolation of S. boydii UI02 and S. sonnei UI05 without competition from natural tomato microflora, experiments were repeated using rifampin-adapted inocula and enrichments supplemented with 50 microg/ml rifampin. Isolation of S. boydii UI02 and S. sonnei UI05 with or without natural tomato microflora was not significantly different (P > 0.05) on CSPM, MAC, or SSA. Colony color enhancements created by CSPM may ease differentiation of Shigella colonies from those of closely related competitors.

Continuous online processing of fecal- and ingesta-contaminated poultry carcasses using an acidified sodium chlorite antimicrobial intervention.

The objective of this study was to determine the effectiveness of the combined use of an inside-outside-bird-washer for the removal of visible contamination and an online acidified sodium chlorite (ASC) spray system in reducing microbial levels on contaminated poultry carcasses. Specifically, we attempted to determine if this technique (referred to as continuous online processing [COP]) would (i) eliminate the need for offline reprocessing of contaminated carcasses, (ii) meet Zero Fecal Tolerance standards, and (iii) attain significant reductions in titers of some of the commonly found bacterial species. Carcasses were sampled for Ercherichia coli, Salmonella, and Campylobacter at five stations along the processing lines in a series of five commercial plant studies to compare the efficacy of the COP system to that of offline processing. The microbiological quality of fecally contaminated carcasses was found to be significantly better following COP treatment (E. coli, 0.59 log10 CFU/ml; Salmonella, 10.0% incidence) than after standard offline reprocessing (E. coli, 2.37 log10 CFU/ml; Salmonella, 31.6% incidence). Zero Fecal Tolerance requirements were met by all but 2 (0.2%) of the 1.127 carcasses following COP. COP also significantly reduced the titers of Campylobacter; residual titers were 1.14 log10 CFU/ml (49.1% incidence) following COP, compared to 2.89 log10 CFU/ml (73.2% incidence) in carcasses that underwent offline reprocessing. These results support the combined use of an inside-outside-bird-washer for the removal of visible contamination and an online ASC spray system to reduce microbial levels in commercially processed poultry.

Validation of thiosulfate for neutralization of acidified sodium chlorite in microbiological testing.

At low pH, acidified sodium chlorite (ASC) has antimicrobial activity against a variety of foodborne contaminants. To evaluate the antimicrobial efficacy of ASC at specific time points posttreatment, it is necessary to halt the action of the disinfectant by removing residual chlorite or by increasing the pH. In this study, thiosulfate was investigated at varying concentrations for its effect on microbial survival and was investigated at a concentration of 0.1% in the presence of ASC for its effect on the antimicrobial and chemical activity of the test solution. Additionally, sodium thiosulfate was tested in two buffering systems, buffered peptone water (BPW) and Butterfield's phopshate buffer (BPB), for its ability to inactivate ASC chemistry. The results of this study show that, at a concentration of 0.1%, sodium thiosulfate has no deleterious effect on Escherichia coli survival and is effective in halting the antimicrobial action of ASC by eliminating the production of residual chlorite. The BPW alone and BPB in combination with thiosulfate were found to be effective inactivators of ASC chemistry.

Adaptation of Salmonella spp. in juice stored under refrigerated and room temperature enhances acid resistance to simulated gastric fluid.

The objective of this study was to evaluate the acid resistance of Salmonella spp. adapted in juices stored under refrigeration and room temperatures to simulated gastric fluid (SGF, pH 1.5). Five Salmonella serovars, Agona, Gaminara, Michigan, Montevideo, and Poona were used in this study. Apple, orange, and tomato juices inoculated with five serovars were stored at refrigeration (7 degrees C) and room temperature (20 degrees C) for 24 h for adaptation. Acid resistances of serovars adapted in juice were determined in SGF at 37 degrees C. All acid-adapted Salmonella serovars in juices displayed enhanced survival time compared to non-adapted controls. Among serovars, S. Poona adapted in apple at 20 degrees C and orange juices at 7 and 20 degrees C showed >2.0 log cfu/ml survivors, while the other serovars decreased to non-detectable level or <2.0 log cfu/ml for 100 s in SGF. Unlike apple and orange juices, all serovars adapted in tomato juice survived with >2.0 log cfu/ml for 100 s. For D-values, all Salmonella serovars adapted in apple and tomato juice enhanced their acid resistances compared to orange juices. S. Agona adapted in tomato juice at 7 degrees C and S. Poona in apple juice at 20 degrees C had the highest D-values with 82.9 and 82.5s, respectively. Results showed that the adaptation in juice increased acid resistance in SGF and varied by serovar, juice type, and adaptation temperature. Therefore, this study indicates that the introduction of Salmonella spp. to an acidic juice environment during processing can enhance their ability to survive in a human stomach, possibly increasing the risk of a Salmonella outbreak by juice.

Model reduction by extended quasi-steady-state approximation.

We extend the quasi-steady-state approximation (QSSA) with respect to the class of differential systems as well as with respect to the order of approximation. We illustrate the first extension by an example which cannot be treated in the frame of the classical approach. As an application of the second extension we prove that the trimolecular autocatalator can be approximated by a fast bimolecular reaction system. Finally we describe a class of singularly perturbed systems for which a higher order QSSA can easily be obtained.

Marking snails with numbered, colored discs: a technique for identifying individual specimens.

Numbered, colored discs, measuring 2.5 mm in diameter, were evaluated under laboratory conditions as tags for marking and identifying individual snails. Discs were affixed to the shells of specimens of Helisoma duryi (Pulmonata: Planorbidae) with an adhesive supplied with the discs and with waterproof epoxy. All snails survived the initial tagging procedure and showed no observable behavioral responses to the tagging. The supplied adhesive, intended for use with terrestrial insects, was inadequate for securing the tags for prolonged periods under aquatic conditions. Use of waterproof epoxy, however, resulted in 100% tag retention for over 5 months without significant mortality. The permanence of the marks, their coding of individuals, the rapidity and ease with which the tags can be applied without causing behavioral changes or snail mortality suggest that this technique will be useful in studies requiring identification of individual aquatic snails.

[Comparative studies of growth hormone secretion in acromegaly after isolated and combined application of insulin hypoglycemia, LH-RH- and TRH tests (author's transl)]. / Vergleichende Untersuchungen zur Wachstumshormonsekretion bei Akromegalie nach isolierter und kombinierter Anwendung des Insulinhypoglykämie-, LH-RH- und TRH-Tests.

Growth hormone (GH) release was measured in 17 patients with active acromegaly following the administration of insulin, LH-RH and TRH given intravenously either combined or each separately. The simultaneous application of insulin and the hypothalamic releasing hormones resulted in a striking increase of plasma GH in 15 out of 17 patients. Inappropriate stimulation of GH release was found in 9 out of the 17 patients with acromegaly, when TRH was given as the only hormone; conversely this phenomenon due to LH-RH application was observed in 4 cases. In insulin-induced hypoglycemia GH release could be stimulated in 5 patients. After selective, transsphenoidal hypophysectomy, 4 of 13 patients still showed a definite stimulation of GH release after the combined use of test substances. Two of these also exhibited a comparable stimulation of GH after TRH, indicating adenoma cells remaining active after operation. The combined insulin-induced hypoglycemia/LH-RH/TRH-test is therefore advisable for patients with acromegaly, since GH release as well as other hypophyseal partial functions can be tested. The performance of individual tests is essential for evaluating selective stimulation of GH release.

Ozone depuration of Vibrio vulnificus from the southern quahog clam, Mercenaria campechiensis.

Southern quahog clams, Mercenaria campechiensis, were dosed with Vibrio vulnificus and placed in a pilot-scale depuration system using ozonated recirculated artificial seawater. Twenty-four hours of treatment with ozone-treated recirculating artificial seawater reduced the numbers of V. vulnificus in the shellfish meats by an average of 2 log units when compared to natural die-off in control clams. The oxidant levels (up to 3 mg/liter) did not adversely affect shellfish pumping during the depuration process.