Magdalena Kostrzynska

The effect of probiotics and organic acids on Shiga-toxin 2 gene expression in enterohemorrhagic Escherichia coli O157:H7.

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.

Escherichia coli O157:H7 stress and virulence gene expression on Romaine lettuce using comparative real-time PCR.

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<0.05) reduced the growth of Escherichia coli O157:H7 on Romaine lettuce, however, viable populations remained after the end of both storage periods. At end of the storage period, a 0.430 and 0.180 log decrease in E. coli O157:H7 was observed at 4 degrees C and 15 degrees C, respectively. Under both storage temperatures, total aerobic plate counts increased over the duration of the experiment. An increase in E. coli O157:H7 fold expression was observed with stx2A. Although stx1A exhibited upregulation for all storage conditions, variable gene expression was observed throughout the storage period. In addition, fliC was up-regulated during storage at 15 degrees C, while transcription at 4 degrees C storage changed only slightly. Expression of eaeA was variable at 15 degrees C with a tendency towards down-regulation, however, this gene was slightly up-regulated when stored at 4 degrees C. A slight upregulation of rpoS and sodB was also observed at 4 degrees C. In conclusion, our results suggest E. coli O157:H7 may become more virulent with prolonged storage of Romaine lettuce, particularly when stored at refrigerated temperatures.

Current and future uses of real-time polymerase chain reaction and microarrays in the study of intestinal microbiota, and probiotic use and effectiveness

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.

Utilization of different types of dietary fibres by potential probiotics

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.

Approaches for reducing Salmonella in pork production.

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.

Lactic acid bacteria and bifidobacteria attenuate the proinflammatory response in intestinal epithelial cells induced by Salmonella enterica serovar Typhimurium

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.

The effect of oxidative stress on gene expression of Shiga toxin-producing Escherichia coli (STEC) O157:H7 and non-O157 serotypes

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.

Prevalence of Salmonella infecting bacteriophages associated with Ontario pig farms and the holding area of a high capacity pork processing facility.

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.

Changes in Gene Transcription Induced by Hydrogen Peroxide Treatment of Verotoxin-Producing Escherichia coli O157:H7 and Non-O157 Serotypes on Romaine Lettuce

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 behavior of VTEC, particularly non-O157 serotypes, on lettuce under stress conditions is necessary for designing more effective control strategies. Hydrogen peroxide (H2O2) can be used as a sanitizer to reduce the microbial load in leafy green vegetables, particularly in fresh produce destined for the organic market. In this study, we tested the hypothesis that H2O2 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, 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.

Microarray Analysis of Probiotics Effectiveness

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 organisms 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.