The use of probiotics to enhance intestinal health has been proposed for many years. Probiotics are traditionally defined as viable microorganisms that have a beneficial effect in the prevention and treatment of specific pathologic conditions when they are ingested. There is a relatively large volume of literature that supports the use of probiotics to prevent or treat intestinal disorders. However, the scientific basis of probiotic use has been firmly established only recently, and sound clinical studies have begun to be published. Currently, the best-studied probiotics are the lactic acid bacteria, particularly Lactobacillus sp. and Bifidobacterium sp. However, other organisms used as probiotics in humans include Escherichia coli, Streptococcus sp., Enterococcus sp., Bacteroides sp., Bacillus sp., Propionibacterium sp. and various fungi. Some probiotic preparations contain mixtures of more than one bacterial strain. Probiotics have been examined for their effectiveness in the prevention and treatment of a diverse spectrum of gastrointestinal disorders such as antibiotic-associated diarrhea (including Clostridium difficile-associated intestinal disease), infectious bacterial and viral diarrhea (including diarrhea caused by rotavirus, Shigella, Salmonella, enterotoxigenic E. coli, Vibrio cholerae and human immunodeficiency virus/acquired immunodeficiency disorder, enteral feeding diarrhea, Helicobacter pylori gastroenteritis, sucrase maltase deficiency, inflammatory bowel disease, irritable bowel syndrome, small bowel bacterial overgrowth and lactose intolerance. Probiotics have been found to inhibit intestinal bacterial enzymes involved in the synthesis of colonic carcinogens. There are many mechanisms by which probiotics enhance intestinal health, including stimulation of immunity, competition for limited nutrients, inhibition of epithelial and mucosal adherence, inhibition of epithelial invasion and production of antimicrobial substances. Probiotics represent an exciting prophylactic and therapeutic advance, although additional investigations must be undertaken before their role in intestinal health can be delineated clearly.

Some cases of late-onset (regressive) autism may involve abnormal flora because oral vancomycin, which is poorly absorbed, may lead to significant improvement in these children. Fecal flora of children with regressive autism was compared with that of control children, and clostridial counts were higher. The number of clostridial species found in the stools of children with autism was greater than in the stools of control children. Children with autism had 9 species of Clostridium not found in controls, whereas controls yielded only 3 species not found in children with autism. In all, there were 25 different clostridial species found. In gastric and duodenal specimens, the most striking finding was total absence of non-spore-forming anaerobes and microaerophilic bacteria from control children and significant numbers of such bacteria from children with autism. These studies demonstrate significant alterations in the upper and lower intestinal flora of children with late-onset autism and may provide insights into the nature of this disorder.

In this study, we have isolated a temperate phage (⌽CD119) from a pathogenic Clostridium difficile strain and sequenced and annotated its genome. This virus has an icosahedral capsid and a contractile tail covered by a sheath and contains a double-stranded DNA genome. It belongs to the Myoviridae family of the tailed phages and the order Caudovirales. The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. The DNA sequence of this phage consists of 53,325 bp, which carries 79 putative open reading frames (ORFs). A function could be assigned to 23 putative gene products, based upon bioinformatic analyses. The ⌽CD119 genome is organized in a modular format, which includes modules for lysogeny, DNA replication, DNA packaging, structural proteins, and host cell lysis. The ⌽CD119 attachment site attP lies in a noncoding region close to the putative integrase (int) gene. We have identified the phage integration site on the C. difficile chromosome (attB) located in a noncoding region just upstream of gene gltP, which encodes a carrier protein for glutamate and aspartate. This genetic analysis represents the first complete DNA sequence and annotation of a C. difficile phage.Clostridium difficile, a gram-positive, spore-forming, anaerobic bacillus, is the leading cause of nosocomial diarrhea associated with antibiotic therapy (2). C. difficile causes a variety of diarrheal syndromes, including diarrhea, nonspecific colitis, and pseudomembranous colitis, all of which vary widely in severity (2). Pathogenic C. difficile can produce two major toxins, toxin A, an enterotoxin, and toxin B, a cytotoxin, that are causative agents of diarrhea and colitis (4). Variation in the severity of symptoms of C. difficile-associated disease has been attributed in part to the level of toxin production by the infecting strain(s) (4). The toxin genes, tcdA and tcdB, are part of a 19.6-kb pathogenicity locus (PaLoc), which is present at identical locations in the chromosomes of pathogenic C. difficile strains but is missing from the nontoxinogenic strains. This observation has led to the suggestion that the presence of the PaLoc may be associated with a transposable element (5). In other clostridial species, toxins are known to be encoded by mobile elements such as bacteriophages and plasmids (10, 11). However, while there is no direct evidence of lysogenic conversion in C. difficile strains, Tan et al. have demonstrated homology between tcdE, a gene located within the PaLoc of C. difficile, and phage holin genes (33). In another study, Goh et al. analyzed the effect of bacteriophage infection on toxin production and found an increased toxin B production in some lysogens (12). The evolutionary aspects of the PaLoc and its relationship with C. difficile phages are not known. Detailed characterization of C. difficile phages is necessary to understand their genetics and their potential relationship with the PaLoc of C. difficile. In this study, one of our g...

Toxigenic Clostridium dvflcile is isolated from a majority of healthy human infants. The exact mechanism of asymptomatic colonisation is unclear; however, previous studies in this laboratory have shown that components of both the immunoglobulin and nonimmunoglobulin fractions of human milk bind to toxin A and prevent its interaction with hamster intestinal brush border membranes (BBMs). Secretory IgA (sIgA) is the primary immunoglobulin found in human milk. As sIgA resists digestion in the infant stomach and passes at high levels into the colon, its ability to bind toxin A was the subject of this investigation. Purified sIgA in concentrations at and below those found in human milk inhibited the binding of toxin A to purified BBM receptors. Heating sIgA to 100°C for 5 min did not affect its inhibitory activity. IgM, IgG and serum IgA did not appreciably inhibit the binding of toxin A to BBM receptors. SDS-PAGE separated sIgA into three major bands: secretory component, heavy chains and light chains. Autoradiography with radiolabelled toxin A revealed that toxin A bound to the secretory component (SC) of sIgA. When the three purified subunits of sIgA were coated on to microtitration wells, SC bound significantly more toxin A than the heavy or light chains of sIgA. Purified SC also inhibited toxin binding to receptors in a dose-dependent fashion similar to sIgA. The heavy and light chains of sIgA did not inhibit toxin A receptor binding. Removing carbohydrates from sIgA and SC by enzymic digestion showed that toxin A binds much less to deglycosylated SC than to glycosylated SC. These data suggest that SC in human milk binds to toxin A and may function as a receptor analogue, protecting human infants against C. difJicile-associated disease.

Several species of anaerobic bacteria display variable Gram stain reactions which often make identification difficult. A simple, rapid method utilizing a 3% solution of potassium hydroxide to distinguish between gram-positive and gram-negative bacterial was tested on 213 strains of anaerobic bacteria representing 19 genera. The Gram stain reaction and KOH test results were compared with the antibiotic disk susceptibilities (vancomycin and colistin) the preliminary grouping of anaerobic bacteria. All three procedures were in agreement for the majority of strains examined. Some strains of clostridia, eubacteria, and bifidobacteria stained gram negative or gram variable; the KOH and antibiotic disk susceptibility tests correctly classified these strains as gram-positive. The KOH test incorrectly grouped some strains of Bacteroides sp., Fusobacterium sp., Leptotrichia buccalis, and Veillonella parvula, but all Gram stain results for these strains were consistent for gram-negative bacteria. The KOH test is a useful supplement to the Gram stain and antibiotic disk susceptibility testing for the initial classification of anaerobic bacteria.

Clostridium difficile has been identified as the most important single identifiable cause of nosocomial antibiotic-associated diarrhea and colitis. Virulent strains of C. difficile produce two large protein toxins, toxin A and toxin B, which are involved in pathogenesis. In this study, we examined the effect of lysogeny by ⌽CD119 on C. difficile toxin production. Transcriptional analysis demonstrated a decrease in the expression of pathogenicity locus (PaLoc) genes tcdA, tcdB, tcdR, tcdE, and tcdC in ⌽CD119 lysogens. During this study we found that repR, a putative repressor gene of ⌽CD119, was expressed in C. difficile lysogens and that its product, RepR, could downregulate tcdA::gusA and tcdR::gusA reporter fusions in Escherichia coli. We cloned and purified a recombinant RepR containing a C-terminal six-His tag and documented its binding to the upstream regions of tcdR in C. difficile PaLoc and in repR upstream region in ⌽CD119 by gel shift assays. DNA footprinting experiments revealed similarities between the RepR binding sites in tcdR and repR upstream regions. These findings suggest that presence of a CD119-like temperate phage can influence toxin gene regulation in this nosocomially important pathogen.Clostridium difficile, a gram-positive, anaerobic, spore-forming bacterium, has been identified as one of the major causative agents of antibiotic-associated diarrhea and pseudomembranous colitis. C. difficile produces toxins A and B that damage intestinal mucosa and cause fluid accumulation in the colon (1). The toxin genes tcdA and tcdB, along with accessory genes tcdR, tcdC, and tcdE, are part of a 19.6-kb pathogenicity locus (PaLoc). Toxin genes tcdA and tcdB are positively regulated by TcdR (previously TxeR) (27), and tcdC is involved in the negative regulation of toxin genes (16,29). In pathogenic C. difficile strains, the PaLoc is present at identical locations in the chromosome, whereas it is completely absent in nontoxinogenic strains. This observation has led to the suggestion that the presence of the toxin gene cluster may be associated with a transposable genetic element (3). In other clostridial species, toxins are known to be encoded by mobile genetic elements such as bacteriophages and plasmids (6,9,10,31).Following publication of the genome of ⌽CD119 (15), the genome of a second C. difficile temperate phage (⌽C2, a member of the Myoviridae) was published (13). More recently, eight temperate phages were characterized from six different C. difficile isolates, including the hypervirulent strain responsible for a multi-institutional outbreak (NAP1/027 or QCD-32g58) (11). In addition, the multidrug-resistant C. difficile strain CD630 was found to harbor two highly related prophages (13, 39) as part of its mosaic genome, where nearly 11% is made of mobile genetic elements. Thus, it appears that C. difficile strains often harbor temperate phage(s) as part of their genetic makeup. No direct evidence of lysogenic conversion of a nontoxinogenic C. difficile strain to toxin production was shown. However...

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The effect of atmospheric oxygen on the viability of 13 strains of anaerobic bacteria, two strains of facultative bacteria, and one aerobic organism was examined. There were great variations in oxygen tolerance among the bacteria. All facultative bacteria survived more than 72 h of exposure to atmospheric oxygen. The survival time for anaerobes ranged from less than 45 min for Peptostreptococcus anaerobius to more than 72 h for two Clostridium perfringens strains. An effort was made to relate the degree of oxygen tolerance to the activities of superoxide dismutase, catalase, and peroxidases in cell-free extracts of the bacteria. All facultative bacteria and a number of anaerobic bacteria possessed superoxide dismutase. There was a correlation between superoxide dismutase activity and oxygen tolerance, but there were notable exceptions. Polyacrylamide gel electropherograms stained for superoxide dismutase indicated that many of the anaerobic bacteria contained at least two electrophoretically distinct enzymes with superoxide dismutase activity. All facultative bacteria contained peroxidase, whereas none of the anaerobic bacteria possessed measurable amounts of this enzyme. Catalase activity was variable among the bacteria and showed no relationship to oxygen tolerance. The ability of the bacteria to reduce oxygen was also examined and related to enzyme content and oxygen tolerance. In general, organisms that survived for relatively long periods of time in the presence of oxygen but demonstrated little superoxide dismutase activity reduced little oxygen. The effects of medium composition and conditions of growth were examined for their influence on the level of the three enzymes. Bacteria grown on the surface of an enriched blood agar medium generally had more enzyme activity than bacteria grown in a liquid medium. The data indicate that superoxide dismutase activity and oxygen reduction rates are important determinants related to the tolerance of anaerobic bacteria to oxygen.