Rial D. Rolfe

The Role of Probiotic Cultures in the Control of Gastrointestinal Health

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.

Gastrointestinal Microflora Studies in Late-Onset Autism

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 Vitro and In Vivo Activities of Nitazoxanide against Clostridium difficile

ABSTRACT We have used the hamster model of antibiotic-inducedClostridium difficile intestinal disease to evaluate nitazoxanide (NTZ), a nitrothiazole benzamide antimicrobial agent. The following in vitro and in vivo activities of NTZ in the adult hamster were examined and compared to those of metronidazole and vancomycin: (i) MICs and minimum bactericidal concentrations (MBCs) againstC. difficile, (ii) toxicity, (iii) ability to preventC. difficile-associated ileocecitis, and (iv) propensity to induce C. difficile-associated ileocecitis. The MICs and MBCs of NTZ against 15 toxigenic strains of C. difficilewere comparable to those of vancomycin or metronidazole. C. difficile-associated ileocecitis was induced with oral clindamycin and toxigenic C. difficile in a group of 60 hamsters. Subgroups of 10 hamsters were given six daily intragastric treatments of NTZ (15, 7.5, and 3.0 mg/100 g of body weight [gbw]), metronidazole (15 mg/100 gbw), vancomycin (5 mg/100 gbw), or saline (1 ml/100 gbw). Animals receiving saline died 3 days post-C. difficile challenge. During the treatment period, NTZ (≥7.5 mg/100 gbw), like metronidazole and vancomycin, prevented outward manifestations of clindamycin-induced C. difficileintestinal disease. Six of ten hamsters on a scheduled dose of 3.0 mg of NTZ/100 gbw survived for the complete treatment period. Of these surviving animals, all but three died of C. difficiledisease by between 3 and 12 days following discontinuation of antibiotic therapy. Another group of hamsters received six similar daily doses of the three antibiotics, followed by an inoculation with toxigenic C. difficile. All of the NTZ-treated animals survived the 15-day postinfection period. Upon necropsy, all hamsters appeared normal: there were no gross signs of toxicity or C. difficile intestinal disease, nor was C. difficiledetected in the cultures of the ceca of these animals. By contrast, vancomycin and metronidazole treatment induced fatal C. difficile intestinal disease in 20 and 70% of recipients, respectively.

Genomic Organization and Molecular Characterization of Clostridium difficile Bacteriophage ΦCD119

In this study, we have isolated a temperate phage (ΦCD119) 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 ΦCD119 genome is organized in a modular format, which includes modules for lysogeny, DNA replication, DNA packaging, structural proteins, and host cell lysis. The ΦCD119 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.

Evidence that Clostridium difficile TcdC Is a Membrane-Associated Protein

Clostridium difficile produces two toxins, A and B, which act together to cause pseudomembraneous colitis. The genes encoding these toxins, tcdA and tcdB, are part of the pathogenicity locus, which also includes tcdC, a putative negative regulator of the toxin genes. In this study, we demonstrate that TcdC is a membrane-associated protein in C. difficile.

Clostridium difficile-associated ileocecitis in clindamycin-treated infant hamsters

Infant hamsters, four days of age or older, developed aClostridium difficile-associated ileocecitis following clindamycin administration, whereas non-antibiotic-treated infant hamsters and hamsters less than four days old given clindamycin were asymptomatically colonized withC. difficile. The incidence of lethality among clindamycin-treated infant hamsters increased with age of the animals, such that all adult hamsters given clindamycin died within 72 h of treatment. Adult hamsters given clindamycin had significantly higher titers of cytotoxin throughout the intestinal tract compared with infant hamsters given clindamycin.

The effect of local hematoma blocks on early fracture healing.

To test the effects of lidocaine and bupivacaine on early fracture healing, the tensile strength of five groups of femora from normal rats 14 and 35 days after production of a closed diaphyseal fracture were compared. Data indicate that, in experimental animals injected with local anesthetics at the fracture site, no measurable difference is noted in callus composition, tensile strength, or histological appearance during the early phases of fracture repair prior to callus remodeling.

Diagnosis of Clostridium Difficile-Associated Intestinal Disease

Toxigenic Clostridium difficile is the major cause of antimicrobial agent-associated pseudomembranous colitis and is the etiological agent of approximately 30% of cases of nonspecific colitis and diarrhea (without colitis) induced by antimicrobial agents. In addition, C. difficile has been implicated in certain intestinal diseases not related to prior antimicrobial administration. C. difficile has been reported to be one of the most common enteropathogens isolated from stool specimens submitted to hospital laboratories. Thus, diagnosis of C. difficile-associated intestinal disease should now be routinely performed in diagnostic clinical laboratories. The diagnosis of C. difficile-associated intestinal disease relies on the demonstration of either the organism or the toxin(s) in stool specimens or antibody response in serum to the toxin(s). Several selective medium are available for the recovery of C. difficile from stool specimens. The toxin(s) of C. difficile can be demonstrated using a variety of techniques, including biological assays as well as immunological assays. This article will review the techniques currently available to aid in the diagnosis of C. difficile-associated intestinal disease.