Search

Clostridium difficile is a nosocomial pathogen identified as the cause of antibiotic associated diarrhea and colitis. In this study, we have documented the lysogeny of a C. difficile bacteriophage in hamsters during C. difficile infection. The lysogens isolated from the hamsters were toxin typed and their phage integration site was confirmed by PCR. Through toxin ELISA it was found that the toxin production in the in vivo isolated lysogens was affected due to ΦCD119 lysogenization as in the case of in vitro isolated ΦCD119 lysogens. Together our findings indicate that a baceriophage can lysogenize its C. difficile host even during the infection process and highlights the importance of lysogeny of C. difficile phages as an evolutionary adaptation for survival.

A bacteriophage specific for Clostridium difficile was examined for its ability to prevent ileocecitis in a hamster model. This species- and strain-specific bacteriophage was isolated from a lysogenic strain of C. difficile . Hamsters were maintained in sterile isolation cages to prevent the acquisition of C. difficile from the environment. Bicarbonate neutralization of gastric acidity was necessary for bacteriophage survival in the hamster's gastrointestinal tract. Bacteriophage recovery from the hamster cecum was 2×10 4 plaque forming units/mL of cecal contents 24 h after orogastric challenge with 10 8 plaque forming units/mL of bacteriophage. However, there was no bacteriophage recovery 48 h post challenge, indicating dissipation of bacteriophage from the hamster intestinal tract within this time frame. Twenty-four hours after being challenged with clindamycin, one group of hamsters was challenged with C. difficile followed by a single dose of bacteriophage (10 8 plaque forming units/mL). Two additional groups of hamsters received phage doses immediately after C. difficile challenge and subsequently thereafter every 8 h up to 48 and 72 h, respectively. The gastric acidity was neutralized with bicarbonate buffer preceding every bacteriophage treatment. Control animals that received only clindamycin and C. difficile died within 96 h after challenge while the majority of bacteriophage treated hamsters survived. Two weeks after stopping bacteriophage treatment, the surviving hamsters were re-challenged with clindamycin and C. difficile . All the hamsters died within 96 h indicating susceptibility of the surviving hamsters to C. difficile disease in the absence of bacteriophage treatment.

A receptor for Clostridium difficile toxin A was purified from brush border membranes (BBMs) from the small intestine of infant hamsters. The BBMs were solubilized with Triton X-114, and the solubilized receptor was purified with use of a toxin A immobilized affinity-chromatography column and differential temperature elution. SDS-PAGE and silver staining of the purified receptor revealed numerous high-molecular-weight bands. However, ligand blotting analysis with 125I-toxin A used as the probe identified a 163-kD protein as the predominate toxin A-binding molecule. Toxin A bound to the purified receptor at physiological temperature, but the amount of toxin bound increased at lower temperatures. Bovine thyroglobulin bound to toxin A and inhibited its binding to the purified receptor. Preincubation of the receptor with lectins produced by Bandeirea simplicifolia or Datura stramonium reduced specific binding by 125I-toxin A. Our data indicate that the purified toxin A receptor from small intestine BBMs of infant hamsters is a galactose- and N-acetylglucosamine-containing glycoprotein.

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.

We have used the hamster model of antibiotic-induced Clostridium 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) against C. difficile , (ii) toxicity, (iii) ability to prevent C. difficile -associated ileocecitis, and (iv) propensity to induce C. difficile -associated ileocecitis. The MICs and MBCs of NTZ against 15 toxigenic strains of C. difficile were 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. difficile intestinal 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. difficile disease 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. difficile detected 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.

To detect and characterize Salmonella antigen in blood, outbred CF-1 female mice were inoculated intraperitoneally with S. typhimurium LT-2 and blood was assayed by ELISA for Salmonella common structural antigen. Plasma antigen was detectable early in the course of infection and increased in quantity later in the course of illness when animals showed high grade bacteremia and high counts of splenic bacteria. Antigen was associated with a cell-free plasma fraction of blood, passed through filters with cut-offs of 0.2 mu and molecular mass of 1000 kDa, and was enhanced in detectability after heating to 100 degrees C for 15 min. Antigen was concentrated by diluting plasma 1:4 in 0.1 M EDTA, heating to 100 degrees C, and concentrating the supernate with an ultrafiltration membrane with a molecular mass cut-off of 15 kDa. By gel filtration, antigen was associated with a peak at about molecular mass 300 kDa in heated plasma and a peak at about 380 kDa in unheated plasma. These results indicate that murine typhoid infection results in circulating soluble plasma antigen, which is heat-stable with a molecular mass of approximately 300 kDa.

With the significant increase in resistance of anaerobic bacteria to antimicrobial agents in recent years, susceptibility testing of these organisms becomes very important. In addition to survey studies, hospitals should be determining their local patterns, and therapy of at least the more serious infections should be guided by susceptibility tests. Of the various tests available, those most suitable for smaller hospitals for guidance of patient management are the broth disc elution procedure and the micro broth dilution tray test.

Daily oral administration of ampicillin to hamsters consistently resulted in fatal ileocecitis due to ampicillin-susceptible strains of Clostridium difficile. Ampicillin was not detected in the cecal contents of these hamsters once C. difficile appeared. Cecal contents obtained from hamsters with ampicillin-induced ileocecitis displayed beta-lactamase activity, whereas cecal contents obtained from untreated control hamsters did not. Colonization of the ceca with C. difficile corresponded to a decrease in the concentration of cecal ampicillin below the minimum inhibitory concentration effective against C. difficile in vitro. The concomitant administration of ampicillin and sulbactam, a nonabsorbable beta-lactamase inhibitor, protected hamsters from developing fatal ileocecitis. However, ileocecitis developed upon the discontinuation of treatment. beta-Lactamase produced by the cecal flora inactivates ampicillin present in the intestinal tract, thereby permitting ampicillin-sensitive C. difficile to multiply and cause disease.

The animal host and its intestinal microbial flora function together as a complex ecologic system in which there is a significant impact of the intestinal flora on the host as well as of components of the microbial flora on one another. Aerobic and anaerobic bacteria of the intestinal flora influence numerous anatomic, physiologic, and immunologic parameters of the host. Constituents of the indigenous intestinal flora also engage in a multitude of antagonistic and cooperative interactions. The normal bacterial intestinal flora represents an extremely important defense mechanism, which effectively interferes with the establishment of many important enteric pathogens. Mechanisms by which microorganisms suppress the growth of other microorganisms include modification of bile acids, stimulation of peristalsis, induction of immunologic responses, depletion of essential substrates from the environment, competition for attachment sites, creation of restrictive physiologic environments, and elaboration of antibiotic-like substances. Components of the intestinal microbial flora also interact synergistically in the induction of disease or the utilization of substrate.

Clostridium difficile has been shown to be the cause of virtually all cases of pseudomembranous colitis related to the administration of antimicrobial agents. It is possible that some antimicrobial agents alter the normal bacterial flora of the gastrointestinal tract so as to permit colonization and/or proliferation by C. difficile. The inhibitory activity of representative fecal bacteria from 23 anaerobic and aerobic genera against C. difficile was examined using two in vitro procedures. Strains of bacteria in six of the genera inhibited the multiplication of C. difficile, with Lactobacillus organisms and group D enterococci displaying the most antagonistic activity. C. difficile was examined for its ability to inhibit the multiplication of several fecal strains of anaerobic and aerobic bacteria. All eight strains of C. difficile tested inhibited the growth of particular strains of bacteria in the genera Bacteroides, Peptococcus, and Peptostreptococcus.

Several new beta-lactam antimicrobial agents have been introduced in the last few years. In this investigation, the in vitro activities of several recently introduced cephalosporins (cefoperazone, cefotaxime, ceftazidime, and ceftizoxime), moxalactam, and N -formimidoyl thienamycin were compared with those of cefoxitin, clindamycin, and metronidazole against 203 strains of anaerobic bacteria. At achievable serum levels, all of the antimicrobial agents were active against essentially 100% of the strains of anaerobic gram-positive cocci, Clostridium perfringens, Leptotrichia buccalis , and species of Selenomonas, Veillonella , and Eubacterium . Clindamycin, metronidazole, and N -formimidoyl thienamycin were the most active agents against the Bacteroides fragilis group, inhibiting all strains at concentrations which can be achieved in serum. Of the remaining agents tested against the B. fragilis group, cefoxitin (which required 64 μg/ml to inhibit 90% of the strains) was the most active, followed by cefoperazone (128 μg/ml), cefotaxime (128 μg/ml), moxalactam (128 μg/ml), ceftizoxime (256 μg/ml), and ceftazidime (>256 μg/ml). Important differences in cephalosporin susceptibility among species of the B. fragilis group were observed. Metronidazole and N -formimidoyl thienamycin were the most active drugs against species of clostridia other than C. perfringens ; the other antibiotics displayed poor activity, although this is partly due to inclusion of a relatively large number of strains of Clostridium difficile which were very resistant to several of the cephalosporins. Only metronidazole was active against all species of Fusobacterium . Clindamycin and N -formimidoyl thienamycin displayed excellent activity against gram-positive, non-spore-forming bacilli, requiring ≤8 μg/ml to inhibit 100% of the strains. Ceftazidime, cefoperazone, and moxalactam were bactericidal for 25 strains of B. fragilis at concentrations equal or close to those required for inhibition. On the basis of its activity in vitro, N -formimidoyl thienamycin appears to be the most promising of the new beta-lactam antibiotics for the treatment of infections involving anaerobic bacteria.

The in vitro susceptibility of Clostridium septicum was studied with a microtiter broth dilution method. Several antimicrobial agents demonstrated consistently good activity against the organism.