Search

The effects of acidic electrolysed water (AEW) as ‘green’ technology on the microbiological and physicochemical properties of fresh-cut red cabbages were studied. Fresh-cut red cabbages and artificially inoculated red cabbages with Salmonella typhimurium DT104 were washed with distilled water (DW) and different available chlorine concentrations (ACC) of AEW for different times. AEW treatments significantly reduced the populations of native aerobic bacteria, molds, and yeasts, and artificially inoculated S. typhimurium DT104 compared with the DW-treated and untreated red cabbage samples. The effectiveness of AEW treatments was greatly enhanced with increasing ACC and treatment times. S. typhimurium DT104 were not detected in the washing water that were collected after the red cabbages treated by AEW. The surface colour, pH, and total phenolic contents did not significantly change when the red cabbages were washed with DW and AEW containing 100 mg/L available chlorine for 3 min. The anthocyanin contents and antioxidant activities of red cabbage were significantly reduced by 18.5 per cent for cyanidin, 22.1 per cent for pelargonidin, and 11.2 per cent for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, however, the impacts on the nutritional benefits of red cabbage were considered as limited and acceptable. The optimal process condition of AEW for washing red cabbage was 100 mg/L ACC for 3 min. In these conditions, most of the native microflora were inactivated, and artificially inoculated S. typhimurium DT104 on the red cabbage were reduced by 40.2 per cent [3.67 log CFU/g (log10 colony-forming units per gram)] and with minimal losses of nutrients and antioxidant activity, as well as no requirement of decontamination treatment on the washing water after AEW treatment.

Disease outbreaks of verotoxin-producing Escherichia coli (VTEC) O157:H7 and non-O157 serotypes associated with leafy green vegetables are becoming a growing concern. A better understanding of the behaviour of VTEC, particularly non-O157 serotypes, on lettuce under stress conditions is necessary for designing more effective control strategies. Hydrogen peroxide can be used as a sanitizer to reduce the microbial load in leafy green vegetables, particularly in fresh produce destined for organic market. In this study, we tested the hypothesis that hydrogen peroxide treatment of contaminated lettuce affects in the same manner transcription of stress-associated and virulence genes in VTEC strains representing O157 and non-O157 serotypes. Six VTEC isolates representing serotypes O26:H11, O103:H2, O104:H4, O111:NM, and O145:NM and O157:H7 were included in this study. The results indicate that 50 mM H2O2 caused a population reduction of 2.4 - 2.8 log10 (compared to non-treated control samples) in all six VTEC strains present on Romaine lettuce. Following the treatment, the transcription of genes related to oxidative stress (oxyR and sodA), general stress (uspA and rpoS), starvation (phoA), acid stress (gadA, gadB and gadW), and virulence (stx1A, stx2A and fliC) were dramatically downregulated in all six VTEC serotypes (P≤0.05) compared to not treated control samples. Therefore, VTEC O157:H7 and non-O157 serotypes on lettuce showed similar survival rates and gene transcription profiles in response to 50 mM H2O2 treatment. Thus, the results derived from this study provide a basic understanding of the influence of H2O2 treatment on the survival and virulence of VTEC O157:H7 and non-O157 serotypes on lettuce.

A better understanding of the functionality of probiotics and dietary fibres with prebiotic activity is required for the development of improved synbiotic preparations. In this study, utilization of β(2–1) fructans, galactooligosaccharides, and plant polysaccharides as prebiotics by lactobacilli, bifidobacteria, and pediococci was investigated. Our results demonstrate that prebiotics with linear chains consisting of galactose units are better utilized by probiotics than are those consisting of glucose and fructose units, and the ability of probiotic bacteria to utilize prebiotics is strain-specific. In addition, rye fructooligosaccharides represent a prebiotic fibre that supports the growth of a wide range of probiotic cultures and as such has a potential to improve the successfulness of probiotic treatments. This study also demonstrates dietary fibre utilization by pediococci and provides data supporting the possible use of pediococci as a probiotic in synbiotic combinations.

BACKGROUND: There is interest in applying bacteriophages to control Salmonella in pig production and pork processing. The following reports on the prevalence of Salmonella infecting bacteriophages within Ontario pig farms and associated with the holding area of a pork slaughterhouse. RESULTS:Salmonella infecting bacteriophages were present in 30 and 28 of the effluent manure samples collected from 36 farms using S. Typhimurium DT104 or S. Heidelberg as host cell respectively. Bacteriophages were recovered in 95–100% of the 48 samples taken from holding pens within a high capacity slaughterhouse over a 12 month period. Bacteriophages isolated from farms exhibited similar host ranges which differed to that of slaughterhouse isolates. Salmonella (n = 21) from the slaughterhouse were susceptible to the endogenous bacteriophages. Despite being susceptible to the resident phages, the Salmonella populations were found to be genetically stable with the same genotypes being recovered over successive visits. Salmonella isolated from the farms were frequently resistant to the endogenous phages. CONCLUSIONS: Bacteriophages are prevalent in the pig slaughterhouse environment although they do not have a significant impact on the genetic structure of Salmonella populations. However, there was evidence that the Salmonella population structure on farms is influenced by the presence of infecting phages. Copyright © 2010 Society of Chemical Industry

Aims: To determine the microflora profiles of pre- and postharvest tomatoes contaminated with Salmonella montevideo or S. typhimurium DT104. Methods and Results: Salmonella montevideo or S. typhimurium was inoculated onto the flowers of tomato plants with the microflora of the subsequent fruit examined using a combination of Source Carbon Utilization and 16S rDNA-PCR profiling. From 16S rDNA profiles it was evident that tomatoes derived from Salmonella inoculated plants harboured a different microbial population compared to nontreated controls. The same result was observed for tomatoes inoculated at postharvest and subsequently stored for 14 days at 15°C. From sequencing analysis it was found that tomatoes derived from Salmonella inoculated plants but testing negative for the enteric pathogen, frequently harboured Enterobacter and Bacillus spp. In contrast, both bacterial types were not found associated with tomatoes testing positive for Salmonella. Conclusions: Salmonella introduced onto tomatoes at pre- or postharvest alters the composition of the microbial community. The presence of Enterobacter and Bacillus spp negatively affects the persistence of Salmonella on preharvest tomatoes. Significance and Impact of the Study: Salmonella appears to modify rather than become integrated into the microbial communities associated with tomatoes. Yet, the presence of antagonistic bacteria appears to reduce the persistence of the enteric pathogen.

Salmonellosis is an important disease in humans and is associated with contaminated food, including pork products. Salmonella infection is invasive in humans, but it usually remains latent within the swine population, creating reservoirs for carcass contamination. Although abattoirs implement stringent procedures during carcass processing, some raw pork products still have Salmonella contamination. To reduce the presence of Salmonella, a dynamic picture of the pork production chain is needed that includes management practices aimed at health and welfare of swine and practices within swine operations that affect the environment and community health. Swine practices indirectly influence the spread of zoonotic enteric pathogens. Pathogens in food animals can escape detection, and critical control points often are missed. Preharvest growth of swine by enhancement of normal gut flora and targeting intestinal pathogens through nonantibiotic approaches might improve food safety and reduce antibiotic residues. In light of the threat posed by multidrug-resistant pathogens, old dogma is being revisited with optimism for potential utility in promoting pre- and postharvest pork safety. This review includes possible approaches that can be implemented in swine operations and postslaughter during pork processing with simultaneous omission of subtherapeutic antibiotics to control Salmonella. We emphasize the vital roles of the veterinarians, pig producers, industry, food research scientists, and government guidelines for the strategic implementation of approaches to Salmonella control across the pork production and processing chains.

Probiotics are defined as live microorganisms that confer a health benefit to the host when administered in adequate amounts. In addition to human health benefits, probiotics can improve various aspects of growth and performance in livestock and poultry, as well as control undesirable microorganisms in food animals. Studies indicate that probiotics can prevent or treat certain conditions, including atopic disease in infants, food allergy, infection after surgery, acute diarrhea, and symptoms associated with irritable bowel syndrome. Understanding the complete mechanism, effectiveness, and potential use of probiotics is limited by the availability and sensitivity of current methods (i.e., culturing techniques). In recent years, real-time polymerase chain reaction (PCR) and microarrays have become prominent and promising methods to examine quantitative changes of specific members of the microbial community and the influence of probiotics on the structure and function of human and animal intestinal ecosystems. Culture-independent studies have established that only a fraction of organisms present in feces are cultivable, therefore, results obtained by cultivation are limited. Conversely, in-depth knowledge of microbial genomes has enabled real-time PCR and microarrays to be more sensitive and has resulted in precise methods for comprehensive analysis of the complex gut microbiota. Additionally, these technologies can assess the influence of intestinal microorganisms on host metabolism, nutrient status, and disease. This paper reviews method technologies and applications of real-time PCR and microarray assays as they relate to the effect and use of probiotics on the intestinal microbiota and gastrointestinal disease.

Aim: To determine the mechanisms by which a stabilized oxychloro (SOC)-based sanitizer, applied to decontaminate seeds destined for sprout production, inactivates Escherichia coli O157:H7 ph1 and Salmonella serotype Meleagridis. Materials and Results: The action of SOC on the metabolism, membrane and DNA integrity of Salmonella and E. coli O157:H7 was studied. In both pathogens, there was an oxidative burst and depletion of intracellular glutathione (GSH) upon initial exposure to 200 ppm SOC. Metabolic activity, measured via bioluminescence, decreased over a 4-h period in E. coli O157:H7 ph1 cells exposed to SOC. Membrane integrity, assessed through viability staining, decreased progressively over 23 h when exposed to SOC. The appearance of auxotrophic mutants suggested that DNA damage had also occurred. Enzymes rich in disulfide bonds (alkaline phosphatase and protease) were sensitive to the chlorite-based sanitizer. Through challenging other microbial types, it was found that Gram positive had higher tolerance to SOC than Gram negatives with the exception of Salmonella. MS2 bacteriophage was highly sensitive; however, Bacillus endospores were not inactivated by SOC. Conclusions: SOC inactivates E. coli O157:H7 and Salmonella through GSH oxidation and disruption of disulfide bonds. Ultimately, membrane damage resulting from prolonged exposure to SOC leads to the loss of cell viability. Significance and Impact of the Study: The results provide a basis for understanding why extended treatment times are required to inactivate bacteria using SOC.

Aims: To evaluate the efficacy of a stabilized oxychloro-based (SOC) sanitizer to decontaminate mung beans artificially or naturally contaminated with Escherichia coli O157:H7 or Salmonella. Methods and Results: Naturally contaminated beans were produced by introducing a five-strain cocktail of E. coli O157:H7 or Salmonella onto the flowers of growing mung bean plants. Escherichia coli O157:H7 was only sporadically recovered from sprout lots (three testing positive from 10 tested) derived from harvested beans. In contrast, Salmonella was recovered from 18 of 20 lots screened. Pathogens present on naturally contaminated seed could be successfully inactivated with SOC applied at 200 ppm for 24 h at 28°C. SOC treatment could also decontaminate artificially inoculated mung bean batches containing different levels of contaminated seed. SOC inactivated E. coli O157:H7, but not Salmonella introduced onto damaged (scarified) beans. Conclusions: SOC sanitizer could inactivate Salmonella or E. coli O157:H7 naturally or artificially introduced onto mung beans. However, the SOC treatment failed to inactivate Salmonella introduced onto damaged mung beans. Significance and Impact of the Study: SOC sanitizer represents an effective method for decontaminating undamaged mung beans.

Understanding the survival mechanisms used by Shiga toxin-producing Escherichia coli (STEC), including O157:H7 and non-O157 serotypes, is important for minimizing contamination of fresh produce and occurrence of foodborne outbreaks. Recent outbreaks linked to leafy green vegetables and sprouted seeds have prompted researchers to focus on investigating decontamination strategies. Several studies showed that hydrogen peroxide (H2O2) treatment has been effective in reducing pathogens on fresh produce. As such, the effect of hydrogen peroxide on stress-associated and virulence gene expression in six STEC isolates was investigated in this study. Logarithmic phase cells of E. coli O157:H7 (EDL933) and non-O157 serotypes, including E. coli O26:H11 (EC20070549), O103:H2 (EC19970811), O104:H4 (NML#11-3088), O111:NM (EC20070546) and O145:NM (EC19970355) were exposed to 2.5mM H2O2 for 40 min and gene expression was evaluated using quantitative real-time PCR. Different patterns of gene expression were observed in E. coli O157:H7 and non-O157 serotypes. Particularly, Shiga toxin gene stx2 was upregulated in O157:H7, but not in O104:H4. Moreover, stx1 was significantly upregulated in STEC O157:H7, but only slightly upregulated Stx1-positive non-O157 serotypes. However genes related to motility (fliC) and intimin gene (eae) were downregulated in most strains. Stress-associated sodA gene encoding manganese superoxide dismutase was significantly upregulated in all serotypes. The dps gene coding for non-specific DNA binding protein was upregulated in O145:NM, O111:NM, O103:H2 and O26:H11. However genes related to cold shock (cspC) and acid resistance (gadW) were significantly downregulated in all strains tested. The results of this study provide a basic understanding of the oxidative stress impact on survival and virulence of non-O157 serotype STEC strains.

The efficacy of a stabilized oxychloro-based food grade sanitizer to decontaminate seeds destined for sprout production has been evaluated. By using mung bean seeds as a model system, it was demonstrated that the sanitizer could be used to inactivate a five-strain cocktail of Escherichia coli O157:H7 or Salmonella introduced onto beans at 10(3) to 10(4) CFU/g. Salmonella was more tolerant to stabilized oxychloro than was E. coli O157:H7, with sanitizer levels of >150 and >50 ppm, respectively, being required to ensure pathogen-free sprouts. The decontamination efficacy was also found to be dependent on treatment time (>8 h optimal) and the seed-to-sanitizer ratio (>1:4 optimal). Stabilized oxychloro treatment did not exhibit phytotoxic effects, as germination and sprout yields were not significantly (P > 0.05) different as compared with untreated controls. Although human pathogens could be effectively eliminated from mung beans, the aerobic plate count of native microflora on sprouts grown from treated seed was not significantly (P > 0.05) different from the controls. The diversity of microbial populations (determined through 16S rRNA denaturing gradient gel electrophoresis analysis) associated with bean sprouts was not significantly affected by the sanitizer treatment. However, it was noted that Klebsiella and Herbasprillum (both common plant endophytes) were absent in sprouts derived from decontaminated seed but were present in control sprouts. When a further range of seed types was evaluated, it was found that alfalfa, cress, flax, and soybean could be decontaminated with the stabilized oxychloro sanitizer. However, the decontamination efficacy with other seed types was less consistent. It appears that the rate of seed germination and putative activity of sanitizer sequestering system(s), in addition to other factors, may limit the efficacy of the decontamination method.

DNA microarrays represent the latest advance in molecular technology. In combination with bioinformatics, they provide unparalleled opportunities for simultaneous detection of thousands of genes or target DNA sequences and offer tremendous potential for studying food-borne microorganisms. This review provides an up-to-date look at the application of DNA microarray technology to detect food-borne pathogenic bacteria, viruses, and parasites. In addition, it covers the advantages of using microarray technology to further characterize microorganisms by providing information for specific identification of isolates, to understand the pathogenesis based on the presence of virulence genes, and to indicate how new pathogenic strains evolved epidemiologically and phylogenetically.Key words: DNA microarrays, food-borne pathogens, detection.

Increasing levels of environmental pollution demand specific and sensitive methods for detection of genotoxic agents in water, food products and environmental samples. Tests for genotoxicity assessment are often based on biosensor strains that respond to DNA damage induced by chemicals. In the present study, fluorescent reporter Escherichia coli strains have been developed, which contain a plasmid-borne transcriptional fusion between the DNA-damage inducible recA promoter and the green fluorescent protein gene (gfp) or a gene encoding a red-shifted, higher intensity GFP variant (mutant 3). GFP-based biosensors allowed the detection of a dose-dependent response to genotoxic agents such as mitomycin C (MMC), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and nalidixic acid (NA). A reporter strain carrying recA'-gfp mutant 3 fusion gave more dramatic and sensitive response than a strain containing the wild-type gfp. These results indicate that recA'-gfp mutant 3-based biosensor is potentially useful for detection of genotoxins.

A 16-kbp DNA region that contains genes involved in the biosynthesis of the capsule of Mannheimia ( Pasteurella ) haemolytica A1 has been characterized. The gene cluster can be divided into three regions like those of the typical group II capsule biosynthetic clusters in gram-negative bacteria. Region 1 contains four genes ( wzt , wzm , wzf , and wza ) which code for an ATP-binding cassette transport apparatus for the secretion of the capsule materials across the membranes. The M. haemolytica A1 wzt and wzm genes were able to complement Escherichia coli kpsT and kpsM mutants, respectively. Further, the ATP binding activity of Wzt was demonstrated by its affinity for ATP-agarose, and the lipoprotein nature of Wza was supported by [ 3 H]palmitate labeling. Region 2 contains six genes; four genes ( orf1 / 2 / 3 / 4 ) code for unique functions for which no homologues have been identified to date. The remaining two genes ( nmaA and nmaB ) code for homologues of UDP– N -acetylglucosamine-2-epimerase and UDP– N -acetylmannosamine dehydrogenase, respectively. These two proteins are highly homologous to the E. coli WecB and WecC proteins (formerly known as RffE and RffD), which are involved in the biosynthesis of enterobacterial common antigen (ECA). Complementation of an E. coli rffE/D mutant with the M. haemolytica A1 nmaA/B genes resulted in the restoration of ECA biosynthesis. Region 3 contains two genes ( wbrA and wbrB ) which are suggested to be involved in the phospholipid modification of capsular materials.

A PCR method for the quantitation of Cryptosporidium parvum oocysts in municipal drinking water samples was investigated. Quantitative PCR uses an internal standard (IS) template with unknown target numbers to compare to standards of known concentrations in a standard curve. The IS template was amplified using the same primers used to amplify a portion of a 358 bp gene fragment that encodes a repetitive oocyst wall protein in C. parvum. Municipal water samples spiked with known numbers of C. parvum oocysts were tested by quantitative PCR using the IS and the Digene SHARP Signal System Assay for PCR product detection. The absorbance readings for target DNA and IS templates versus the number of molecules of the target DNA were plotted to generate standard curves for estimating oocyst numbers. The method allowed the quantitation of oocysts from log 3 to log 5 spiked into municipal water samples.

Cryptosporidium parvum is a protozoan parasite responsible for an increasing number of outbreaks of gastrointestinal illness worldwide. In this report, we describe development of sample preparation protocols for polymerase chain reaction (PCR)-based detection of C. parvum in fecal material and environmental water samples. Two of these methods were found adequate for isolation of Cryptosporidium DNA from filtered water pellet suspensions. The first involved several filtration steps, immunomagnetic separation and freeze-thaw cycles. The second method involved filtration, addition of EnviroAmp lysis reagent, freeze-thaw cycles and precipitation of the DNA with isopropanol. Using nested PCR, we detected 100 oocysts/ml of filtered water pellet suspension, with either of the above sample preparation procedures. Nested PCR increased sensitivity of the assay by two to three orders of magnitude as compared to the primary PCR. The detection limit for seeded fecal samples was 10-fold higher than for filtered environmental water pellet suspension. Nested PCR results showed 62.4 and 91.1% correlation with immunofluorescence assay (IFA) for fecal samples and filtered environmental water pellet suspensions, respectively. This correlation decreased to 47.2% and 44.4%, respectively, when only IFA positive samples were analyzed. However, in fecal samples contaminated with a high number (10(5)/g) of C. parvum oocysts, this correlation was 100%.

International audience; The green fluorescent protein gene (gfp) was introduced into a p-nitrophenol-metabolizing strain of Moraxella sp. by chromosomal integration. The gfp-marked transformants, designated Moraxella sp. strains G21 and G25, exhibited green fluorescence under UV light. Molecular characterization by PCR and Southern hybridization showed the presence of gfp in both transformants. Both transformants and the parent strain degraded 720 μM of p-nitrophenol with nitrite release within 4 h after inoculation in minimal medium supplemented with yeast extract. Transformants degraded up to 1440 μM p-nitrophenol and mineralized about 60% of 720 μM p-nitrophenol, both in broth and in soil, to the same extent as the parent strain. Insertion of gfp did not adversely affect the expression of p-nitrophenol-degrading genes in the transformants. Survival studies indicated that individual green fluorescent colonies of transformants can be detected up to 2 weeks after inoculation in soil. These marked strains could be of value in studies on microbial survival in the environment.

The enteric protozoan, Cryptosporidium parvum, the causative agent for cryptosporidiosis, has been isolated from drinking water, fecal samples from humans and animals, and environmental samples such as sediment and soil. The currently available water sampling methods for detection of this parasite are labor-intensive and the efficiency of oocyst recovery is poor. A recent improved method utilizing membrane filtration and dissolution followed by polymerase chain reaction (PCR) amplification, and confirmatory nested PCR was evaluated for the sensitive and specific detection of C. parvum oocysts. Detection of PCR products by the ELISA-based Digene SHARP Signal™ System Assay was assessed for sensitivity. Seventy-two municipal water samples ranging in volume from 230 to 1,000 l from southwestern Ontario, Canada were spiked with varying concentrations of formalin-killed C. parvum oocysts for use in this study. Oocyst recovery on the filters was determined by the Merifluor immunofluoresence assay for Cryptosporidium/Giardia. Oocyst detection using the PCR assay showed an 84.7% correlation with immunofluoresence assay (IFA) results. During optimization studies, the correlation between PCR and IFA reached 98%. The sensitivity of a primary PCR assay ranged from 1 to 10 oocysts per reaction, which was equivalent to 102 to 103 oocysts per 100 l municipal water. The PCR assay also showed potential for application to untreated water samples and naturally contaminated municipal water from a recent Cryptosporidium outbreak. Further application of nested PCR may improve overall sensitivity and specificity for detecting C. parvum in municipal water samples since combined primary and nested PCR results showed 97.2% correlation with IFA. The Digene SHARP Signal™ System assay offers a sensitive and specific alternative for detection of C. parvum amplification products.

Inflammation is a physiological response to infections and tissue injury; however, abnormal immune responses can give rise to chronic inflammation and contribute to disease progression. Various dietary components, including probiotic lactic acid bacteria and prebiotics, have the potential to modulate intestinal inflammatory responses. One factor in particular, the chemokine interleukin-8 (IL-8, CXCL-8), is one of the major mediators of the inflammatory response. The purpose of this study was to investigate modulation of the inflammatory host response induced by Salmonella enterica serovar Typhimurium DT104 in the presence of selected probiotics and lactic acid bacteria (LAB) isolated from human sources, dairy products, and farm animals. IL-8 gene expression and protein production in HT-29 cells were evaluated by real-time PCR and ELISA, respectively. Pre-incubation of HT-29 cells with Lactobacillus kefir IM002, Bifidobacterium adolescentis FRP 61, Bifidobacterium longum FRP 68 and FRP 69, Bifidobacterium breve FRP 334, and Leuconostoc mesenteroides IM080 significantly inhibited IL-8 secretion induced by Salmonella Typhimurium DT104. Co-culture of selected probiotics and Salmonella Typhimurium DT104 reduced IL-8 production, while potential probiotics and LAB had no effect on IL-8 secretion in HT-29 cells preincubated with Salmonella Typhimurium DT104 prior to adding probiotics. Lactobacillus kefir IM002 supernatant also significantly reduced IL-8 production. In conclusion, our study suggests that probiotic bifidobacteria and LAB modulate cytokine induction and possess anti-inflammatory properties; however, the effectiveness is strain dependent.

Summary Flagellar hooks were purified from Helicobacter pylori and Helicobacter mustelae. The 70 × 16nm H. pylori hook was composed of FIgE subunits of 78kDa, while the 72 × 16nm H. mustelae hook was composed of 87kDa subunits. N-terminal sequence was obtained for the FIgH proteins of both species, and for an internal H. mustelae FlgE peptide. Degenerate oligonucleotide primers allowed amplification of a 1.2 kb fragment from the H. mustelae chromosome, which carried part of the flgE gene. The corresponding H. pylori gene was cloned by immunoscreening of a genomic library constructed in λZAP Express, The translated H. pylori flgE sequence indicated a protein with limited homology with the hook proteins from Salmonella typhimurium and Treponema phagedenis. Mutants of H. pylori and H. mustelae defective in hook production generated by allele replacement were non-motile and devoid of flagellar filaments but produced both flagellin subunits, which were localized in the soluble fraction of the cell. The level of flagellin production was unchanged in the mutants, indicating that the regulation of flagellin expression in Helicobacter differs from that in the Enterobacteriaceae.

There is interest in applying bacteriophages to control Salmonella in pig production and pork processing. The following reports on the prevalence of Salmonella infecting bacteriophages within Ontario pig farms and associated with the holding area of a pork slaughterhouse.Salmonella infecting bacteriophages were present in 30 and 28 of the effluent manure samples collected from 36 farms using S. Typhimurium DT104 or S. Heidelberg as host cell respectively. Bacteriophages were recovered in 95-100% of the 48 samples taken from holding pens within a high capacity slaughterhouse over a 12 month period. Bacteriophages isolated from farms exhibited similar host ranges which differed to that of slaughterhouse isolates. Salmonella (n = 21) from the slaughterhouse were susceptible to the endogenous bacteriophages. Despite being susceptible to the resident phages, the Salmonella populations were found to be genetically stable with the same genotypes being recovered over successive visits. Salmonella isolated from the farms were frequently resistant to the endogenous phages.Bacteriophages are prevalent in the pig slaughterhouse environment although they do not have a significant impact on the genetic structure of Salmonella populations. However, there was evidence that the Salmonella population structure on farms is influenced by the presence of infecting phages.

Publisher Summary Microarray analysis can be used for detection and genotyping as well as for an unbiased genome-scale examination of gene expression patterns and is recognized as a large-scale and high-throughput screening technique. The diverse types of microarrays have applications in clinical, environmental, and food microbiology, microbial ecology, and in human, animal, and plant disease diagnosis. Array technology is able to examine a large portion of an organism's genome (DNA microarray) or proteome (protein array) in a single assay and has become an important technique used to study the influence of probiotics on host health or intestinal microbial interactions. Microarray can be used to detect microorganisms in the gut and investigate quantitative changes in populations of specific members within the microbial community and the influence of probiotics on the structure and function of human intestinal ecosystems. Microarray can also be applied to pathogen detection, characterizing microorganisms based on sequence variation among isolates and identifying the presence or absence of genes.

Summary Forthy-three strains of Streptococcus pneumoniae were tested for their ability to bind radiolabeled laminin, collagen types I, II and IV, fibronectin, and vitronectin. Two basement membrane components, laminin and type IV collagen, interacted with many S. pneumoniae strains. All strains bound laminin and 28 (65%) bound collagen type IV. Approximately 60% of the strains bound vitronectin but only a few strains showed low binding of fibronectin and collagen type I and II.

Foodborne outbreaks attributed to the contamination of fresh produce with Escherichia coli O157:H7 are a growing concern. In particular, leafy-green vegetables, including lettuce and spinach, are susceptible to contamination by irrigation water, manure, and food processing and storage practices. The survival of E. coli O157:H7 and natural microflora on Romaine lettuce stored at 4 degrees C and 15 degrees C over a 9-day period was evaluated by plate counts. A two-step reverse-transcription comparative quantitative real-time PCR assay was employed to evaluate expression of genes coding for the A subunit of Shiga-toxin 1 and 2 (stx1A and stx2A), intimin (eaeA), flagellin (fliC), sigmaS--general stress sigma factor (rpoS) and iron superoxide dismutase (sodB) in E. coli O157:H7. Results indicate that reducing the storage temperature from 15 degrees C to 4 degrees C significantly (P

International audience; The advancement in functional genomics, such as DNA microarrays along with the genome availability of important pathogens as well as of human and livestock species has allowed scientists to study the expression of thousands of genes in a single step. In the past decade, DNA arrays have been employed to study infectious processes of pathogens, in diagnostics, and to study host-pathogen interactions. The generation of enormous data sets by microarray experiments also stimulated the growth of a new generation of analytical software. The information provided by microarray experiments has been useful in generating new hypotheses for future research. The concept of DNA array technology has been utilized in the development of novel diagnostic methods. This review highlights the application of microarrays in the field of veterinary research.

The interaction of a range of Salmonella serovars with pre- and postharvest tomatoes was evaluated. Serovars were selected on the basis of previous association in tomato-linked outbreaks of salmonellosis (Salmonella Javiana, Salmonella Montevideo, and Salmonella Newport) or those typically isolated from animal or clinical infections (Salmonella Dublin, Salmonella Enteritidis, Salmonella Hadar, Salmonella Infantis, Salmonella Typhimurium, and Salmonella Senftenberg). Salmonella serovars introduced onto the flowers of growing plants were recovered on and within the developing tomato fruit. Of all the Salmonella serovars tested, Montevideo appeared to be more adapted to survival within tomatoes and was recovered from 90% of the fruit screened. All of the Salmonella serovars could persist and grow when introduced onto unripened (green) tomato fruit. In general, growth (internal and external) was promoted at the high incubation temperature (25 degrees C) and high relative humidity (95%), although this was serovar dependent. The growth and persistence of Salmonella introduced on and into ripened (red) tomatoes was serovar dependent. Salmonella serovars Enteritidis, Typhimurium, and Dublin were less adapted to grow in or on intact red tomatoes than were serovars Hadar, Montevideo, or Newport. The results illustrated that a diverse range of Salmonella serovars can become established within and/or on preharvest tomatoes. The majority of Salmonella can grow and become established both on and within unripened tomatoes, but growth on ripened fruit was serovar dependent. The results provide a possible explanation why only a narrow range of Salmonella serovars are associated with foodborne illness outbreaks linked to tomatoes.

Probiotics are known to have an inhibitory effect against the growth of various foodborne pathogens, however, the specific role of probiotics in Shiga-toxin-producing Escherichia coli (STEC) virulence gene expression has not been well defined. Shiga toxins are members of a family of highly potent bacterial toxins and are the main virulence marker for STEC. Shiga toxins inhibit protein synthesis in eukaryotic cells and play a role in hemorrhagic colitis and hemolytic uremic syndrome. STEC possesses Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2), both of which have A and B subunits. Although STEC containing both Stx1 and Stx2 has been isolated from patients with hemorrhagic colitis, Stx2 is more frequently associated with human disease complications. Thus, the effect of Lactobacillus, Pediococcus, and Bifidobacterium strains on stx2A expression levels in STEC was investigated. Lactic acid bacteria and bifidobacteria were isolated from farm animals, dairy, and human sources and included L. rhamnosus GG, L. curvatus, L. plantarum, L. jensenii, L. acidophilus, L. casei, L. reuteri, P. acidilactici, P. cerevisiae, P. pentosaceus, B. thermophilum, B. boum, B. suis and B. animalis. E. coli O157:H7 (EDL 933) was coincubated with sub-lethal concentrations of each probiotic strain. Following RNA extraction and cDNA synthesis, relative stx2A mRNA levels were determined according to a comparative critical threshold (Ct) real-time PCR. Data were normalized to the endogenous control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the level of stx2A expression between treated and untreated STEC was compared. Observed for all probiotic strains tested, stx2A was down-regulated, when compared to the control culture. Probiotic production of organic acids, as demonstrated by a decrease in pH, influenced stx2A gene expression.

Lactic acid bacteria (LAB) are often utilized to control food-borne pathogens on produce and on cooked, fermented, or refrigerated meats. Most research to date has focused on the inhibition of Listeria monocytogenes, Escherichia coli O157:H7, Salmonella, Clostridium botulinum, and spoilage microorganisms. LAB are excellent candidates for reducing pathogen levels on foods because they inhibit the growth of these microorganisms through various mechanisms without causing unacceptable sensory changes. This review provides an up-to-date look at research directed at maximizing the use of LAB by selecting the most appropriate strains, by learning how to apply them to foods most effectively, and by gaining an understanding of the mechanism by which they inhibit pathogens.Key words: bacterial competition, lactic acid bacteria, food-borne pathogens, meat products, produce.

A gfp-labeled p-nitrophenol-degrading Moraxella strain G21 was used to study grazing of a Tetrahymena thermophila strain in liquid medium. This allowed visualization of the feeding process. Under an epifluorescent microscope, individual G21 fluorescent cells could be seen in vacuoles within the protozoans. Most of the G21 cells appeared to be lysed by T. thermophila and green fluorescent protein released from the bacteria yielded brightly fluorescent food vacuoles inside the protozoans, Examination of population dynamics of a mixed culture of T. thermophila and Moraxella sp. G21 showed that the protozoan reduced the bacterial density from 7.6 to 5.8 log CFU/ml in 2 days. Encapsulating the bacteria in alginate prevented grazing by the protozoans and the density of G21 cells in the beads increased steadily from about 8.3 to 8.9 log CFU/ml in 15 days regardless of the presence of the protozoans.

In this review, we examine numerous applications of the green fluorescent protein (GFP) marker gene in environmental microbiology research. The GFP and its variants are reviewed and applications in plant-microbe interactions, biofilms, biodegradation, bacterial-protozoan interactions, gene transfer, and biosensors are discussed. Methods for detecting GFP-marked cells are also examined. The GFP is a useful marker in environmental microorganisms, allowing new research that will increase our understanding of microorganisms in the environment.

Xylose reductase catalyzes the NAD(P)H-dependent reduction of xylose to xylitol and is essential for growth on xylose by yeasts. To understand the nature of coenzyme binding to the Pichia stipitis xylose reductase, we investigated the role of the strictly conserved Lys270 in the putative IPKS coenzyme binding motif by site-directed mutagenesis. The Lys270Met variant exhibited lower enzyme activity than the wild-type enzyme. The apparent affinity of the variant for NADPH was decreased 5–16-fold, depending on the substrate used, while the apparent affinity for NADH, measured using glyceraldehyde as the substrate, remained unchanged. This resulted in 4.3-fold higher affinity for NADH over NADPH using glyceraldehyde as the substrate. The variant also showed a 14-fold decrease in Km for xylose, but only small changes were observed in Km values for glyceraldehyde. The wild-type enzyme, but not the Lys270Met variant, was susceptible to modification by the Lys-specific pyridoxal 5′-phosphate. Results of our chemical modification and site-directed mutagenesis study indicated that Lys270 is involved in both NADPH and d-xylose binding in the P. stipitis xylose reductase.

Antisera raised in rabbits to whole cells of Helicobacter pylori recognized as a major antigen a protein with an apparent molecular weight of 20,000. The antigen was purified by differential solubilization with N-octyl-beta-D-glucopyranoside, urea, and sodium dodecyl sulfate followed by molecular sieving. The mass of the protein, Lpp20, was 18,283 Da as determined by mass spectrometry. The lpp20 gene encoding this protein was cloned in Escherichia coli by using the vector lambda EMBL3, and plasmid subclones expressed the full-length protein from the native H. pylori promoter. lpp20 was mapped to the same 358-kb NruI fragment as flaB. DNA sequence analysis showed that the gene was 525 bp long and encoded a 175-amino-acid protein with a molecular weight of 19,094 containing a 21-residue typical lipoprotein signal peptide and consensus prolipoprotein processing site. The mass of the deduced 154-residue mature protein was 16,865 Da. Growth of E. coli cells expressing the cloned H. pylori lpp20 gene in the presence of [3H]palmitic acid resulted in radiolabelled Lpp20 while treatment of the E. coli cells with globomycin caused accumulation of unprocessed Lpp20, consistent with Lpp20 being a lipoprotein. Lpp20 cofractionated with the cytoplasmic membrane fraction, although a proportion of the protein was also found in the outer membrane. A mutant generated by mutant-allele exchange displayed normal viability, showing that Lpp20 belonged to the nonessential class of lipoproteins.

Many strains of Streptococcus pyogenes are capable of binding type IV collagen. In the present study, all 50 S. pyogenes strains isolated from patients with acute glomerulonephritis showed high or moderate affinity for radiolabelled type IV collagen. A majority of strains of other sources, such as reference strains of various M-types and strains isolated from patients with pharyngeal infections also bound type IV collagen; however, a number of weak binders or non-binders were found among those. The collagen type IV binding component(s) on S. pyogenes were susceptible to proteinase K digestion, partially sensitive to trypsin but insensitive to pepsin treatment at pH 5.5. According to tests with three M-positive strains and their M-negative derivatives, the binding was not dependent on M-protein. The binding was saturable with time and inhibited by unlabelled type IV collagen. Partially inhibition was found with type II collagen, gelatin and fibrinogen but not with a number of other serum proteins.