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.

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 .

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.

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.

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.

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 ΦCD119 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 ΦCD119 lysogens. During this study we found that repR , a putative repressor gene of ΦCD119, 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 ΦCD119 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.

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.

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.

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.

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.

Recent studies have shown that a toxin of Clostridium difficile is implicated as the cause of virtually all cases of antimicrobial agent-associated pseudomembranous colitis (PMC), 1 and of approximately one-fifth of cases of antimicrobial agent-associated diarrhea without colonic pseudomembrane. 1,2 Occasionally, PMC or diarrhea not related to antimicrobial exposure has also been attributed to C. diflicile . 2–6 A variety of modes of therapy for antimicrobialassociated diarrhea (AAD) have been employed including vancomycin, metronidazole, tetracycline, bacitracin, anion-binding resins, antiperistaltic agents, corticosteroids, and an oral Lactobacillus preparation. Although assessment of the efficacy of treatment is complicated by the variable course of AAD, 7,8 available data are sufficient to permit a rational approach.

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.

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.

Toxigenic Clostridium difficile is the major cause of antimicrobial agent-associated pseudomembranous colitis in humans and of ileocaecitis in golden Syrian hamsters. The pathogenicity of C. difficile is believed to be dependent on the production of two immunologically and biochemically distinct toxins: toxin A (enterotoxin) and toxin B (cytotoxin). In earlier investigations we demonstrated that hamsters immunised against toxins A and B are protected against clindamycin-induced C. difficile -associated ileocaecitis and that this protection is transferred to infant hamsters through maternal milk. In the present investigation, the class-specific immunoglobulin response in adult and infant hamsters immunised against C. difficile toxins A and B was examined using enzyme linked immunosorbent assays. Parenteral immunisation of adult hamsters against toxins A and B induced IgG, IgA and IgM antibodies in hamster sera specific for these two toxins. IgG antibodies to toxins A and B predominated in the milk and intestines of adult hamsters immunised against toxins A and B and in the sera and intestine of infants from immunised adult hamsters. No significant IgA or IgM antibodies to toxins A and B were detected in maternal milk and intestines or in infant sera and intestines. IgG antibodies to toxins A and B were detected in infant caecal contents up until 10 d and in sera up until approximately 24 d of age. The results from this investigation suggest that serum derived IgG antibody to C. difficile toxins is primarily responsible for protection of adult and infant hamsters against C. difficile -associated ileocaecitis when immunised parenterally against toxins A and B. Keywords: Clostridium difficile ; Toxin A; Toxin B: Antibody; Immunisation; Hamster; Ileocaecitis; Enterotoxin; Cytotoxin.

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.

Antimicrobial agent-induced ileocecitis of laboratory animals and colitis of man share common features. The significance of a newly described toxin in these two entities, the apparent source of the toxin (Clostridium d�(fldile) and characteristics of the toxin are reviewed. Methods of toxin detection, isolation and rapid identification of C. dfJicile, and possible modes of therapy for antimicrobial agent-associated colitis of man are discussed. Am. J. Clin. Nuir. 32: 251-257, 1979. Diarrhea associated with the use of certain antimicrobial agents is a well-recognized problem in clinical medicine. The severity of this diarrhea ranges from a mild, self-limited process to a ful.minant necrotizing or pseu- domembranous colitis (PMC). Clindamycin (perhaps because ofits utility in the treatment of anaerobic infections, and its popularity with the clinician) is the most intensively studied of the several antimicrobial agents capable of producing PMC. Hypotheses proposed for the etiology of dlindamycin colitis have been summarized in a recent review of antimicrobial agent-asso- ciated diarrhea (1); they include the devel- opment of a localized Shwartzman reaction, alterations of protein synthesis in the gut mucosa, increases in the quantity of primary bile salts in the colon as a consequence of antibiotic-induced alterations in gastrointcs- tinal flora, viral-induced necrosis of cobonic mucosa, vascular compromise ofthe intestine, and suppression of the normal gut flora, thereby permitting the proliferation of an antibiotic-resistant, toxin-producing bacte- rium.

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.

Clostridium difficile has been shown to be a cause of antimicrobial agent-associated diarrhea and colitis. The source from which this organism gains access to the gastrointestinal tract is not known. Cultures of the hospital environments of six of eight patients whose fecal cultures were positive for C. difficile yielded this organism, whereas cultures of control hospital sites were almost invariably negative. These data suggest that hospital environmental contamination may be a potential source of infection. Further studies are needed to determine the role of various possible sources of C. difficile in human diarrheal disease.

Bacteremia, including potentially pathogenic species of the fecal flora, was detected during colonoscopy in 6 of 22 patients when blood cultures were taken at frequent intervals during the first 15 minutes of the procedure. While transient bacteremia during colonoscopy may be insignificant for most patients, the implications are potentially more grave for those with valvular heart disease or compromised host defenses.

Clostridium difficile was recovered from a variety of environmental sites in three hospital rooms occupied by a patient who had colitis due to this organism.C. difficile was detected for 40 days after the patient was moved from one of these rooms. These findings suggest that the contaminated hospital environment may be a clinically significant reservoir forC. difficile and that this organism may be a nosocomial pathogen. Isolation of patients and adequate decontamination of rooms may be needed to minimize risk to other patients.

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.

METABOLIC acidosis caused by D-lactic acid, the optical isomer of L-lactic acid, was recently described by Oh et al. in a patient with a short-bowel syndrome.1 We encountered a similar condition in two patients with prior gut surgery. One patient had undergone an extensive small-bowel resection after mesenteric infarction, and the other had had a jejunoileal bypass for obesity. These patients presented with repeated episodes of stupor or coma, metabolic acidosis with an increased anion gap, normal plasma levels of L-lactate, and elevated plasma levels of D-lactate. Treatment with oral vancomycin resulted in normalization of the neurologic and metabolic status. . . .

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.

VANCOMYCIN given by mouth is effective for the treatment of pseudomembranous colitis,1 2 3 4 a disease now known to be caused by an exotoxin of Clostridium difficile.5 6 7 8 Only one relapse of colitis after treatment with vancomycin has been reported.9 This report documents the relapse of colitis in three patients after discontinuation of treatment with vancomycin by mouth. Case Reports Pertinent clinical data appear in Table 1. Pseudomembranous colitis and relapse of colitis in Cases 1 and 2 were documented by sigmoidoscopy and rectal biopsy, according to established criteria.10 The initial episode of colitis in Case 3 was revealed by air-contrast barium enema, . . .

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.

C. difficile (CD) and its cytotoxin (C) has been demonstrated to be responsible for antimicrobial induced diarrhea (AID) and pseudomembranous colitis. We determined the incidence and manifestations of CD and C in 3 groups (Gp) of children with suspected acute or chronic AID (Gp 1), children with chronic diarrhea prior to antimicrobial therapy (Gp II) and control children without diarrhea (Gp III). Antibiotics (n of patients) used in Gp I include: lincomycin (1), clindamycin (1), ampicillin (4), amoxicillin (1), in Gp II inciude penicillin (2), metronidazole (1), amoxicillin (2), ampicillin (2), Six of 6 patients in Gp I had mild proctocolitis confirmed by rectal biopsy without pseudomembranes on proctosigmoidoscopy. All 7 patients in Gp I became asymptomatic within 6 weeks of discontinuing antimicrobial therapy except 1 patient who received oral vancomycin (V) and became asymptomatic during V therapy but diarrhea recurred after V therany with stool culture + for CD and C for up to 6 months. Children may become chronic carriers of CD; the presence of CD and C can be used to distinguish acute/chronic colitis related to antimicrobial therapy from other causes of colitis in children.

Clostridium dfJlcile is the etiologic agent of virtually all cases of antimicrobial- associated pseudomembranous colitis and approximately 2tTh of cases of nonspecific colitis or diarrhea without colitis related to antimicrobial administration. Essentially all antimicrobial agents, with the exception of vancomycin and parenteral aminoglycosides, are capable of inducing C. d(/jlcile-associated intestinal disease. These agents may alter the normal bacterial flora of the gastrointestinal tract so as to permit colonization and/or proliferation, and toxin elaboration, by C. dqfficile. The inhibitory activity of I 16 strains of aerobic bacteria (representing 14 genera) and 285 strains ofanaerobic bacteria (representing nine genera) against C. dfficile was examined using two in vitro procedures. Strains of bacteria in the genera Staphylococcus. Pseudomonas, Streptococcus, Bacteroides, Bifidobacterium, and Lactobacillus inhibited the multiplication ofC. dqfflcile. Members of Lactobacillus and group D enterococci displayed the most antagonistic activity. Not all strains of C. dfJlcile were inhibited by an antagonistic bacterium and not all strains of a particular species possessed inhibitory activity. The antagonistic activity of eight strains of C. dfJlcile against nine genera of anaerobic bacteria (79 strains) and seven genera of aerobic bacteria (25 strains) was examined. Only some strains of bacteria in the genera Peptococcus, Peptostreptococcus. and Bacteroides were inhibited by C. dfflcile. For example, four of the I 7 strains of Bacteroidesfragilis and six of the seven strains of Bacteroides thetaiotaomicron were inhibited by C. dqfJlcile. In several instances, however, all strains of bacteria belonging to a particular genus and species were inhibited by C. d�(Jlcile (e.g.. Bacteroides vulgatus, Bacteroides distasonis, and Bacteroides ovatus). The data obtained in this investigation may help to characterize the mechanism that provides the resistance of the gastrointestinal tract to colonization by C. dfficile. A better understanding of interactions between C. dfficile and other elements of the intestinal flora may lead to more efficient therapy, diagnosis and prophylaxis of oolitis and diarrheal disease associated with antimicrobial therapy. Am. J. Clin. Nutr. 33: 2539, 1980.