Enteric Microbes Research Articles

DEFB1 gene 5′ untranslated region (UTR) polymorphisms in inflammatory bowel diseases

Inflammatory bowel diseases (IBDs) are multifactorial disorders resulting from an abnormal immune response driven by the presence of normal luminal flora. IBDs are associated with the production of nonspecific mediators of inflammation that initiate inflammatory processes and tissue destruction ()()(1-3). Crohn's disease (CD) and ulcerative colitis (UC) are the two main forms of IBDs. An accepted hypothesis for IBD pathogenesis is that an uncorrected balance between the host defenses and the commensal microbiota in the gut of CD and UC patients might promote the disease by causing bacterial invasion, inflammation and loss of tolerance (4). A key role in the host defense is performed by the intestinal epithelium, which acts as a physical barrier that limits the access of enteric microbes that are able to produce endogenous antimicrobial peptides (AMPs) (5). Human defensins, which are classified as α-defensins (HDs) and β-defensins (HBDs) based on the arrangement of three disulfide bridges (6), are antimicrobial peptides that represent a wide spectrum of activity against pathogens. Human β-defensin-1 (hBD-1), the first described β-defensin, is characterized by antimicrobial, chemotactic and immuno-enhancing activities ()()(7-9). The hBD-1 protein, which is encoded by the DEFB1 gene (8p23.1), is constitutively expressed by epithelial cells of a wide variety of tissues, but its expression can vary between individuals and can be modified during the inflammatory process. A decrease in hBD-1 expression was reported in the mucosa of CD and UC patients ()()(10-12). Impaired production of defensins appears to contribute to the pathogenesis of IBDs (13,14), and a correlation between DEFB1 expression and single-nucleotide polymorphisms (SNPs) present in the regulatory region of the gene has been reported (15,16). Therefore, we analyzed the possible association of 5′ untranslated region (UTR) DEFB1 SNPs, namely c.-52G>A (rs1799946) c.-44C>G (rs1800972) and c.-20G>A (rs11362), with the susceptibility to inflammatory bowel diseases in a group of Italian IBD patients (CD and UC) and healthy control patients.

Standard Colonic Lavage Alters the Natural State of Mucosal-Associated Microbiota in the Human Colon

Background & AimsPast studies of the human intestinal microbiota are potentially confounded by the common practice of using bowel-cleansing preparations. We examined if colonic lavage changes the natural state of enteric mucosal-adherent microbes in healthy human subjects.MethodsTwelve healthy individuals were divided into three groups; experimental group, control group one, and control group two. Subjects in the experimental group underwent an un-prepped flexible sigmoidoscopy with biopsies. Within two weeks, subjects were given a standard polyethylene glycol-based bowel cleansing preparation followed by a second flexible sigmoidoscopy. Subjects in control group one underwent two un-prepped flexible sigmoidoscopies within one week. Subjects in the second control group underwent an un-prepped flexible sigmoidoscopy followed by a second flexible sigmoidoscopy after a 24-hour clear liquid diet within one week. The mucosa-associated microbial communities from the two procedures in each subject were compared using 16S rRNA gene based terminal restriction fragment length polymorphism (T-RFLP), and library cloning and sequencing.ResultsClone library sequencing analysis showed that there were changes in the composition of the mucosa-associated microbiota in subjects after colonic lavage. These changes were not observed in our control groups. Standard bowel preparation altered the diversity of mucosa-associated microbiota. Taxonomic classification did not reveal significant changes at the phylum level, but there were differences observed at the genus level.ConclusionStandard bowel cleansing preparation altered the mucosal-adherent microbiota in all of our subjects, although the degree of change was variable. These findings underscore the importance of considering the confounding effects of bowel preparation when designing experiments exploring the gut microbiota.

Historical Perspectives

Élie Metchnikoff is often considered the “father of innate immunity” because of his identification of phagocytosis and its primary role in fighting infection. Today, Metchnikoff is recognized for his description of lactic acid-producing bacteria in the intestine and how enteric microbes may promote or hinder intestinal health and human longevity. The work of Metchnikoff and Tissier at the Pasteur Institute that established probiotic research and provided early insights into the intestinal microbiome are summarized in this review.

Human microbial ecology: Lactobacilli, probiotics, selective decontamination

Health care-associated infections are closely associated with different medical interventions which interrupt the balance of human microbiota. The occasional predominance of opportunistic pathogens may lead to their translocation into the lymph nodes and bloodstream, causing endogenous (primary or secondary) hospital infections. The question is raised as to if there is a possibility for prevention of the imbalance of GI microbiota during medical interventions in critically ill patients. Prophylactic selective decontamination of the digestive tract (SDD) simultaneously applies three to four different antimicrobials for the suppression of enteric aerobic microbes, which are potentially pathogenic microorganisms. However, there is no convincing evidence that the indigenous beneficial intestinal microbiota are preserved, resulting in reduced mortality of high-risk patients. In this overview, we have evaluated the antimicrobial treatment guidelines of the Infectious Diseases Society of America (IDSA) for intra-abdominal infections in adults and seniors according to their safety for different Lactobacillus spp. The data from our group and in the literature have shown that all tested lactobacilli strains (nearly one hundred) were insusceptible to metronidazole while different species of lactobacilli of the three fermentation groups expressed particular antibiotic susceptibility to vancomycin, cefoxitin, ciprofloxacin and some new tetracyclines. We have relied on microbial ecology data showing that the GI tracts of adults and the elderly are simultaneously colonised at least with several (four to a maximum of 12) Lactobacillus species expressing variable intrinsic insusceptibility to the aforementioned antimicrobials, according to the provided data in table. This finding offers the possibility of preserving the colonisation of the intestine with some beneficial lactobacilli during antimicrobial treatment in critically ill patients with health care-associated infections. Several probiotic Lactobacillus spp. strains are intrinsically resistant to antimicrobials and can be used during antibacterial therapy, however, their application as an additive to antimicrobial treatment in critically ill patients needs to be investigated in well-designed clinical trials.

You Aren't Always What You Eat

The maxim ‘‘you are what you eat’’ goes only so far. The bacteria inhabiting our guts, which outnumber our own cells by perhaps 10 to 1, are commonly thought to reflect our diets. But other factors from geography to host physiology can also affect gut microbes, and sorting out their provenance is critical because they can affect our health for good or ill, from enhancing immune function to increasing the risk of stomach cancer. New research in this issue of PLoS Biology by Howard Ochman and colleagues counters the prevailing view that diet shapes the makeup of gut microbes, revealing that the host animal is a stronger determinant of these bacterial worlds in our digestive tracts. On the face of it, gut microbe composition could be determined by either the host or its environment. Mammals are born with sterile digestive tracts and usually get their first gut microbes from their mothers, but thereafter acquire new ones from their environment. Moreover, there is evidence on both sides of the host versus environment debate. The evolutionary relationships of some gut bacteria are known to match those of their hosts, notably Helicobacter pylori, which is found in about half of people and is linked to stomach ulcers and cancer. However, work on great apes had suggested that diet is the major determinant of gut microbe composition. To sort out whether gut microbe composition depends primarily on the host or its environment, Ochman and colleagues compared fecal samples from five closely related hominid species: Eastern and Western lowland gorillas, bonobos, and three subspecies of chimpanzees from several African countries as well as people from Africa and North America. By using fecal samples from locations separated by such considerable distances, the researchers effectively knocked out any effects of geography and the local environmental on gut microbes. The researchers constructed two phylogenetic trees from the fecal samples: one of the great ape hosts, which was based on mitochondrial DNA, and the other of their gut microbe communities. Gut microbe diversity and abundance were based on small subunit ribosomal RNA genes, which are unique to bacteria and other prokaryotes. In turn, the ‘‘gut microbe’’ tree was based on the relative abundance of the various microbe species in each fecal sample, much as physical measures such as femur lengths from, say, mouse to elephant are used to infer evolutionary relationships. The resulting gut microbe tree mirrored that for hominids; the patterns of relationships among the gut microbe communities were identical to those among the five great ape species. This congruence neatly establishes hosts as the major force behind the makeup of gut microbe communities. Further, the concordance of the two phylogenetic trees suggests that the gut microbe communities were shaped by divergences in host physiology over the course of great ape evolution. The disparity with previous findings is partly because this new work entailed surveying many more fecal bacteria sequences per host, with a median of 28,000 sequences. In contrast, in previous studies only 100 to 200 bacterial sequences were surveyed per host, which is insufficient for gauging the diversity and abundance of species in the complex microbe communities living in our digestive tracts. This work brings us closer to understanding the acquisition and evolution of the gut flora that can affect our health for better or for worse. Even though new microbes are constantly arriving in digestive tracts, their makeup is largely determined by the distinctive physiologies of people and our closest relatives. Gut microbe communities are thus predictable rather than variable, as they would be if determined largely by the host’s external environment. Perhaps this is not altogether surprising, given that we hosts essentially are the environment for the worlds of enteric microbes within us. In this case at least, what we are trumps what we eat.

Regional Mucosa-Associated Microbiota Determine Physiological Expression of TLR2 and TLR4 in Murine Colon

Many colonic mucosal genes that are highly regulated by microbial signals are differentially expressed along the rostral-caudal axis. This would suggest that differences in regional microbiota exist, particularly mucosa-associated microbes that are less likely to be transient. We therefore explored this possibility by examining the bacterial populations associated with the normal proximal and distal colonic mucosa in context of host Toll-like receptors (TLR) expression in C57BL/6J mice housed in specific pathogen-free (SPF) and germ-free (GF) environments. 16S rRNA gene-based terminal restriction fragment length polymorphism (T-RFLP) and clone library analysis revealed significant differences in the community structure and diversity of the mucosa-associated microbiota located in the distal colon compared to proximal colon and stool, the latter two clustering closely. Differential expression of colonic TLR2 and TLR4 along the proximal-distal axis was also found in SPF mice, but not in GF mice, suggesting that enteric microbes are essential in maintaining the regional expression of these TLRs. TLR2 is more highly expressed in proximal colon and decreases in a gradient to distal while TLR4 expression is highest in distal colon and a gradient of decreased expression to proximal colon is observed. After transfaunation in GF mice, both regional colonization of mucosa-associated microbes and expression of TLRs in the mouse colon were reestablished. In addition, exposure of the distal colon to cecal (proximal) microbiota induced TLR2 expression. These results demonstrate that regional colonic mucosa-associated microbiota determine the region-specific expression of TLR2 and TLR4. Conversely, region-specific host assembly rules are essential in determining the structure and function of mucosa-associated microbial populations. We believe this type of host-microbial mutualism is pivotal to the maintenance of intestinal and immune homeostasis.

Isolation and Identification of Pathogenic Bacteria and Fungi from Some Sudanese Banknote Currency

Sudanese Banknotes in different values were critically evaluated to show the level of contamination with pathogenic microbes using standard techniques.The fungal genera Trichophyton sp, Microsporum sp, Epidermophyton sp, Taenia sp, Aspergillus sp and Saccharomyces sp were isolated and identified.The genera of bacteria that isolated were Escherichia coli, Citrobacter sp, Klebsiella. sp, Proteus sp, Bacillus sp, Corynebacterium sp and staphylococcus sp. The potential of Sudanese currency notes to act as environmental vehicles for transmission of pathogenic fungi & bacteria was assessed. The lower values of banknotes were highly contaminated and there is a negative correlation between banknotes value and microbial contamination. The results suggest that currency notes may be contaminated, especially with bacteria and enteric microbes and may serve as a source of contamination or infection . Personal hygiene and electronic or ceredit banking to reduce the risk of infection is recommended.

PTU-048 Correlating urinary metabolic profiles with serological and genetic determinants of inflammatory bowel disease

Introduction Subgroups of inflammatory bowel disease (IBD) patients have specific serological and genetic associations, reflecting abnormal immunogenetic responses to enteric microbes. We have recently shown significant differences between the urinary metabolic profiles of Crohn9s disease (CD) patients, ulcerative colitis (UC) patients and healthy control individuals.1 The differentiating metabolites included several strongly influenced by gut microbial metabolism. Methods It was hypothesised that the urinary metabolic profiles of CD patients would differ depending on possession of 1) anti-Saccharomyces cerevisiae antibodies (ASCA) and antibodies to an E coli outer membrane porin (anti-Omp C), and 2) CD-associated CARD15/NOD2 polymorphisms. It was hypothesised that profiles of UC patients would differ depending on their possession of perinuclear anti-neutrophil cytoplasmic antibodies (pANCA). Urinary metabolic profiles were acquired from 74 CD and 50 UC patients, with nuclear magnetic resonance spectroscopy. Sera were assayed for ASCA IgA and IgG, anti-Omp C (CD) and pANCA (UC) status; CD patients were genotyped for CD-associated CARD15/NOD2 polymorphisms. Multivariate analysis was carried out to investigate differences between the CD cohort depending on ASCA, anti-Omp C and CARD15/NOD2 status, and the UC cohort depending on pANCA status. This involved partial least squares discriminant analysis with orthogonal signal correction (OSC-PLS-DA), with leave-one-out cross-validation of the multivariate models created. Results OSC-PLS-DA modelling discriminated the profiles of CD patients possessing ASCA (58% of the cohort) from those who did not, with a statistically significant Q 2 value of 0.31 (sensitivity 88%, specificity 60%). The major contributory metabolites to this model included the gut microbe-related metabolites hippurate, lower in the group seropositive for ASCA, and trimethylamine-N-oxide (TMAO), higher in this group. No significant distinctions were found between CD urinary profiles analysed according to anti-Omp C or CARD15/NOD2 status, nor UC profiles when stratified by pANCA status. Conclusion The urinary metabolic profiles of CD patients with ASCA differ significantly from those without. The involvement of hippurate and TMAO in this multivariate model suggests that gut microbial metabolism differs in CD patients possessing ASCA.

New Triterpenoids and Other Constituents from a Special Microbial-Fermented Tea—Fuzhuan Brick Tea

Fuzhuan brick tea, a special microbial-fermented tea prepared from the leaves of Camellia sinensis var. sinensis, is a traditional beverage used in China throughout history. Phytochemical investigation of this material led to the identification of three new triterpenoids, 3beta,6alpha,13beta-trihydroxyolean-7-one (1), 3beta-acetoxy-6alpha,13beta-dihydroxyolean-7-one (2), and 3beta-O-(8-hydroxyoctanoyl)-12-oleanene (3), together with 11 known compounds, friedelin (4), beta-amyrone (5), beta-amyrin (6), alpha-spinasterone (7), alpha-spinasterol (8), 22,23-dihydro-alpha-spinasterone (9), 22,23-dihydro-alpha-spinasterol (10), alpha-phytol (11), alpha-tocopherol (12), alpha-tocoquinone (13), and caffeine (14). The structures of 1-13 were determined by spectroscopic and chemical methods. Compounds 1 and 2 are the first two examples of triterpenoids possessing a 6-hydroxy-7-one function. All of the compounds, except 6, 8, 10, 11, and 14, were isolated from tea and Camellia spp. for the first time. The antibacterial activities of 1 were assessed against some enteric pathogenic microbes. Compound 1 showed no cytotoxic activity against A-549, Bel-7402, and HCT-8 cell lines.

The Single IgG IL-1–Related Receptor Controls TLR Responses in Differentiated Human Intestinal Epithelial Cells

Intestinal epithelial cells (IECs) are constantly exposed to enteric microbes. Although IECs express TLRs that recognize bacterial products, the activation of these TLRs is strictly controlled through poorly understood mechanisms, producing a state of hyporesponsiveness and preventing unwanted inflammation. The single IgG IL-1-related receptor (Sigirr) is a negative regulator of TLRs that is expressed by IECs and was recently shown to inhibit experimental colitis. However, the importance of Sigirr in IEC hyporesponsiveness and its distribution within the human colon is unknown. In this study, we investigated the role of Sigirr in regulating epithelial-specific TLR responses and characterized its expression in colonic biopsy specimens. Transformed and nontransformed human IECs were cultured as monolayers. Transient gene silencing and stable overexpression of Sigirr was performed to assess innate IEC responses. Sigirr expression in human colonic biopsy specimens was examined by immunohistochemistry. Bacterial infection of IECs and exposure to flagellin transiently decreased Sigirr protein expression, concurrent with secretion of the neutrophil chemokine IL-8. Sigirr gene silencing augmented chemokine responses to bacterial flagellin, Pam3Cys, and the cytokine IL-1beta. Conversely, stable overexpression of Sigirr diminished NF-kappaB-mediated IL-8 responses to TLR ligands. We also found that Sigirr expression increased as IECs differentiated in culture. This observation was confirmed in biopsy sections, in which Sigirr expression within colonic crypts was prominent in IECs at the apex and diminished at the base. Our findings show that Sigirr broadly regulates innate responses in differentiated human IECs; therefore, it may modulate epithelial involvement in infectious and inflammatory bowel diseases.

Policy interventions needed to manage bacterial build-up in municipal effluent irrigated agroforestry plantations

Mortality of total enteric bacteria, Escherichia coli, other coliforms and Salmonella spp. was monitored under municipal effluent irrigated agroforestry plantations via periodic field and in vitro studies. The results of the field study showed no significant differences among agroforestry systems in any of the monitored bacterial populations. Microbial populations were higher in summer (January 2003) than in other seasons. The in vitro study, two pot experiments during October 2003 (pre-summer) and July 2004 (winter), revealed significant effects of moisture on Most Probable Number (MPN) of the total bacteria and E. coli. The moisture levels did not affect the Salmonella spp. and other coliform bacteria, suggesting that certain microbes may be found at low irrigation levels and vice-versa. Enteric microbes were recorded until the 27th day after irrigation in October 2003 and for less than 15 days in July 2004. Thus inclusion of more pathogens in environmental guidelines and stringent exercise of the 'exclusion' in practice are suggested.

Gut barrier disruption by an enteric bacterial pathogen accelerates insulitis in NOD mice

Increased exposure to enteric microbes as a result of intestinal barrier disruption is thought to contribute to the development of several intestinal inflammatory diseases; however, it less clear whether such exposure modulates the development of extra-intestinal inflammatory and autoimmune diseases. The goal of this study was to examine the potential role of pathogenic enteric microbes and intestinal barrier dysfunction in the pathogenesis of type 1 diabetes. Using NOD mice, we assessed: (1) intrinsic barrier function in mice at different ages by measuring serum levels of FITC-labelled dextran; and (2) the impact on insulitis development of infection by strains of an enteric bacterial pathogen (Citrobacter rodentium) either capable (wild-type) or incapable (lacking Escherichia coli secreted protein F virulence factor owing to deletion of the gene [DeltaespF]) of causing intestinal epithelial barrier disruption. Here we demonstrate that prediabetic (12-week-old) NOD mice display increased intestinal permeability compared with non-obese diabetes-resistant and C57BL/6 mice. We also found that young (4-week-old) NOD mice infected with wild-type C. rodentium exhibited accelerated development of insulitis in concert with infection-induced barrier disruption. In contrast, insulitis development was not altered in NOD mice infected with the non-barrier-disrupting DeltaespF strain. Moreover, C. rodentium-infected NOD mice demonstrated increased activation and proliferation of pancreatic-draining lymph node T cells, including diabetogenic CD8(+) T cells, compared with uninfected NOD mice. This is the first demonstration that a loss of intestinal barrier integrity caused by an enteric bacterial pathogen results in the activation of diabetogenic CD8(+) T cells and modulates insulitis.

Characterization of TEM-, SHV- and AmpC-type β-lactamases from cephalosporin-resistant Enterobacteriaceae isolated from swine

Extended-spectrum β-lactamase (ESBL) and AmpC-producing Enterobacteriaceae are an increasing problem in human medicine and an emerging problem in the veterinary field. Our study, therefore, focused on assessing the prevalence of β-lactamases isolated from swine. Sixty-six Salmonella enterica serovar Typhimurium ( S. Typhimurium), 33 Salmonella enterica serovar Enteritidis ( S. Enteritidis), 26 Klebsiella pneumonia ( K. pneumoniae) and 130 Escherichia coli ( E. coli) pig isolates collected from 1999–2006 were screened for β-lactam resistance by the disk diffusion test (DDT) and micro-broth dilution. Among the isolates, five E. coli and five K. pneumoniae exhibited reduced susceptibility to the cephalosporins tested. PCR, plasmid profiling and Southern blot hybridization showed the presence of multiple β-lactamases in these isolates of animal origin. Hybridization patterns of the DHA-1 specific probe indicated that dissemination of DHA-1 related β-lactamases could be attributed to plasmids of one common size among the enteric microbes of animal origin. To the best of our knowledge, this study reports the first identification of SHV-28 and DHA-1 from microbes of animal origin.

DISSOLVED AIR FLOTATION (DAF) FOR PRIMARY AND TERTIARY TREATMENT OF MUNICIPAL WASTEWATERS

ABSTRACT Tertiary treatment of municipal wastewater by dissolved air flotation was studied on a pilot‐scale. The effects of coagulant dose, flocculation pattern, dispersion water recycle ratio and hydraulic surface load on process performance were evaluated. The treatment of primary effluents by dissolved air flotation was investigated to assess the suitability of this process for the treatment of heavily polluted effluents and wastewater treatment plant by‐passes. The tertiary dissolved air flotation process typically achieved 90–99 % reductions in the numbers of enteric microbes (total coliforms, enterococci and F‐RNA coliphages). The average reductions of total phosphorus and chemical oxygen demand were 55–81 % and 28–39 %, respectively. Increasing the polyaluminium chloride coagulant dose from 2 to 10 mgAl3+ l−1 and the dispersion water recycle ratio from 11 to 22 % improved the efficiency of the process. Changes in the flocculation conditions (range of G‐values 10–55 s−1; retention time 4–8 min) and hydraulic surface load (5 or 10 m h−1) did not clearly affect the process efficiency. The dissolved air flotation process decreased the numbers of enteric microbes and reduced total phosphorus, chemical oxygen demand and suspended solids from the primary treated wastewaters on average by 98–99.8%, 90 %, 47% and 77 %, respectively. The dissolved air flotation process was demonstrated to be a suitable method for efficient tertiary treatment of wastewaters, as well as for the elimination of peak pollution loads or by‐pass wastewaters during the treatment plant overloading situations.

Toll like receptor-5: protecting the gut from enteric microbes

The intestine is normally colonized by a large and diverse commensal microbiota and is occasionally exposed to a variety of potential pathogens. In recent years, there has been substantial progress made in identifying molecular mechanisms that normally serve to protect the intestine from such enteric bacteria and which may go awry in chronic idiopathic inflammatory diseases of the gut. One specific molecular interaction that appears to play a key role in governing bacterial-intestinal interactions is that of the bacterial protein flagellin with toll-like receptor 5. This article reviews studies performed in vitro, in mice, and in humans that indicate an important role for the flagellin-TLR5 interaction in regulating both the innate and adaptive immune responses in the intestine.

Deletion of TLR5 results in spontaneous colitis in mice

Activation of TLRs by bacterial products results in rapid activation of genes encoding products designed to protect the host from perturbing microbes. In the intestine, which is colonized by a large and diverse population of commensal bacteria, TLR signaling may not function in a simple on/off mode. Here, we show that the flagellin receptor TLR5 has an essential and nonredundant role in protecting the gut from enteric microbes. Mice lacking TLR5 (TLR5KO mice) developed spontaneous colitis, as assessed by well-defined clinical, serologic, and histopathologic indicators of this disorder. Compared with WT littermates, TLR5KO mice that had not yet developed robust colitis exhibited decreased intestinal expression of TLR5-regulated host defense genes despite having an increased bacterial burden in the colon. In contrast, such TLR5KO mice displayed markedly increased colonic expression of hematopoietic-derived proinflammatory cytokines, suggesting that elevated levels of bacterial products may result in activation of other TLRs that drive colitis in TLR5KO mice. In accordance, deletion of TLR4 rescued the colitis of TLR5KO mice in that mice lacking both TLR4 and TLR5 also had elevated bacterial loads in the colon but lacked immunological, histopathological, and clinical evidence of colitis. That an engineered innate immune deficiency ultimately results in spontaneous intestinal inflammation supports the notion that an innate immune deficiency might underlie some instances of inflammatory bowel disease.

Multistate Evaluation of an Ultrafiltration-Based Procedure for Simultaneous Recovery of Enteric Microbes in 100-Liter Tap Water Samples

Multistate Evaluation of an Ultrafiltration-Based Procedure for Simultaneous Recovery of Enteric Microbes in 100-Liter Tap Water Samples

Mechanisms of Disease: protease functions in intestinal mucosal pathobiology

Of all our organ systems, the gastrointestinal tract contains the highest levels of endogenous and exogenous proteases (also known as proteinases and peptidases); however, our understanding of their functions and interactions within the gastrointestinal tract is restricted largely to nutrient digestion. The gut epithelium is a sensor of the luminal environment, not only controlling digestive, absorptive and secretory functions, but also relaying information to the mucosal immune, vascular and nervous systems. These functions involve a complex array of cell types that elaborate growth factors, cytokines and extracellular matrix (ECM) proteins, the activity and availability of which are regulated by proteases. Proteolytic activity must be tightly regulated in the face of diverse environmental challenges, because unrestrained or excessive proteolysis leads to pathological gastrointestinal conditions. Moreover, enteric microbes and parasites can hijack proteolytic pathways through 'pathogen host mimicry'. Understanding how the protease balance is maintained and regulated in the intestinal epithelial cell microenvironment and how proteases contribute to physiological and pathological outcomes will undoubtedly contribute to the identification of new potential therapeutic targets for gastrointestinal diseases.

Multistate Evaluation of an Ultrafiltration-Based Procedure for Simultaneous Recovery of Enteric Microbes in 100-Liter Tap Water Samples

Ultrafiltration (UF) is increasingly being recognized as a potentially effective procedure for concentrating and recovering microbes from large volumes of water and treated wastewater. Because of their very small pore sizes, UF membranes are capable of simultaneously concentrating viruses, bacteria, and parasites based on size exclusion. In this study, a UF-based water sampling procedure was used to simultaneously recover representatives of these three microbial classes seeded into 100-liter samples of tap water collected from eight cities covering six hydrologic areas of the United States. The UF-based procedure included hollow-fiber UF as the primary step for concentrating microbes and then used membrane filtration for bacterial culture assays, immunomagnetic separation for parasite recovery and quantification, and centrifugal UF for secondary concentration of viruses. Water samples were tested for nine water quality parameters to investigate whether water quality data correlated with measured recovery efficiencies and molecular detection levels. Average total method recovery efficiencies were 71, 97, 120, 110, and 91% for phiX174 bacteriophage, MS2 bacteriophage, Enterococcus faecalis, Clostridium perfringens spores, and Cryptosporidium parvum oocysts, respectively. Real-time PCR and reverse transcription-PCR (RT-PCR) for seeded microbes and controls indicated that tap water quality could affect the analytical performance of molecular amplification assays, although no specific water quality parameter was found to correlate with reduced PCR or RT-PCR performance.

A cross-sectional study of bacterial contamination in plant-protein feed from feed stores in Northern Virginia and Maryland

The objectives of this study were to estimate the prevalence of the enteric microbes: Escherichia coli, Salmonella, Campylobacter and Enterococcus spp ., in plant-protein-based animal feed products and milk replacer and to identify risk factors associated with contamination by these bacteria. A cross-sectional survey of 158 plant-derived feed and milk replacer samples was conducted using eight animal feed stores in Northern Virginia and Maryland from January 2002 to September 2002. Overall, all samples were negative for Campylobacter; one sample (0.6%) was positive for Salmonella; nine samples (5.7%) were positive for E. coli and 80 samples (50.6%) were positive for Enterococcus. Positive samples included: whole maize, cracked peanut, cotton seed, mixed grain horse feed, and milk replacer. Samples collected during the month of January (winter) had a lower prevalence of contamination with enteric bacteria than samples collected in August and September (summer/autumn). Samples collected in January 2002 were negative for both E. coli and Salmonella. Samples collected in August and September 2002 included one Salmonella positive, and nine E. coli positive. Animal feed purchased during August and September had a 38-fold increase (95% CI: 15.60–95.16) in the risk of contamination by Enterococcus than those samples purchased during January and February of that same year (P<0.001). Enterococcus positive samples were eight times more likely to also be E. coli positive than Enterococcus negative samples (95% CI: 1.04–70.12). Non-pelleted feed was 13 times more likely to be contaminated with Enterococcus than pelleted feed (95% CI: 3.02–59.84).