Epidemiological aspects of Clostridium difficile-induced diarrhea and colitis.

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.

Diarrhea and colitis associated with antimicrobial therapy in man and animals.

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 difficile) and characteristics of the toxin are reviewed. Methods of toxin detection, isolation and rapid identification of C. difficile, and possible modes of therapy for antimicrobial agent-associated colitis of man are discussed.

Susceptibility testing of anaerobic bacteria.

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.

Immunoglobulin and non-immunoglobulin components of human milk inhibit Clostridium difficile toxin A-receptor binding.

Clostridium difficile is isolated from the intestinal tracts of > 50% of healthy infants. The mechanism by which intestinal colonisation of infants by toxigenic C. difficile is generally asymptomatic is unknown but may reflect the presence in human milk of neutralising activity against C. difficile toxin A. On this basis, the ability of human milk to inhibit the binding of toxin A to a purified hamster brush border membrane receptor was determined. Ten milk samples from healthy volunteers in various stages of lactation inhibited the binding of toxin A to the receptor by an average of 90%. Heating and dialysis did not significantly alter the inhibitory activity of any of the milk samples. Human milk protected adult hamsters against a lethal challenge with toxin A but had no effect on the cytotoxic activity of the toxin. SDS-PAGE and ligand blot analyses showed that there were at least four distinct factors in human milk that specifically bound toxin A. Thiophilic adsorption chromatography was used to separate immunoglobulin from non-immunoglobulin components of human milk. IgA was the only immunoglobulin detected in human milk and > 90% of this immunoglobulin was recovered after purification by thiophilic adsorption. Both the unbound non-immunoglobulin and bound immunoglobulin fractions of human milk inhibited the binding of toxin A to the purified receptor. These results suggest that human milk may be important in protecting infants against C. difficile-associated intestinal disease.

Binding of Clostridium difficile toxin A to human milk secretory component.

Toxigenic Clostridium difficile 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 non-immunoglobulin 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 degrees 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. difficile-associated disease.

Soluble plasma antigen in experimental Salmonella typhimurium infection in mice.

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.

Binding kinetics of Clostridium difficile toxins A and B to intestinal brush border membranes from infant and adult hamsters.

This study was undertaken to determine if the relative resistance of neonates and infants to Clostridium difficile-associated intestinal disease can be related to age-dependent differences in intestinal receptors for C. difficile toxins A and B. Brush border membranes (BBMs) from the small intestines of adult and infant hamsters were examined for their ability to bind radiolabeled toxins A and B. [125I]toxin A bound to both infant and adult hamster BBMs at physiological temperature, whereas [125I]toxin B did not bind to the BBMs under any of the conditions examined. The number of [125I]toxin A molecules bound at saturation was approximately 4 x 10(10) per micrograms of membrane protein for adult BBMs and 1 x 10(11) per micrograms of membrane protein for infant BBMs. Scatchard plot analysis suggested the presence of a single class of toxin A binding sites on both infant and adult hamster BBMs. Maximal binding capacity and Kd values were 0.63 pmol/mg of protein and 66.7 nM, respectively, for the infant BBMs, and 0.24 pmol/mg of protein and 27 nM, respectively, for the adult BBMs. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analyses of extracted BBM proteins revealed differences in the proteins of infant and adult BBMs. However, there were not any detectable differences in the protein bands which bound [125I]toxin A between infant and adult hamsters. The results from these investigations indicate that differences in the binding kinetics of toxins A and/or B to infant and adult hamster BBMs do not account for the observed differences in their susceptibility to C. difficile-associated intestinal disease.

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.

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.

Role of volatile fatty acids in colonization resistance to Clostridium difficile.

The in vitro inhibition of Clostridium difficile by volatile fatty acids was correlated with the pH and concentrations of volatile fatty acids in the ceca of hamsters of different ages. The concentrations of cecal volatile fatty acids increased with the age of the animals. Maximum concentrations of individual volatile fatty acids were attained when the animals were ca. 19 days old, with acetic, propionic, and butyric acids occurring in the highest concentrations (72, 16, and 32 microequivalents/g of cecum, respectively). The cecal pH was approximately the same in hamsters of all ages (pH 6.6 to 7.0). Only butyric acid reached a concentration in the ceca of hamsters which was inhibitory to the in vitro multiplication of C. difficile. This inhibitory concentration was attained when the animals were ca. 19 days of age. When mixtures of volatile fatty acids were prepared at concentrations equal to those present in the ceca of hamsters, there was a direct correlation between the in vitro inhibitory activity of the volatile fatty acids and the susceptibility of hamsters 4 days of age or older to C. difficile intestinal colonization. The resistance of hamsters less than 4 days of age to C. difficile intestinal colonization appears to be due to factors other than volatile fatty acids.

Interactions among microorganisms of the indigenous intestinal flora and their influence on the host.

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.

Purification of a functional receptor for Clostridium difficile toxin A from intestinal brush border membranes of infant hamsters.

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.

Comparative in vitro activity of ceftriaxone against anaerobic bacteria.

The in vitro activity of ceftriaxone was compared with those of other recently introduced beta-lactam antimicrobial agents (cefoperazone, cefotaxime, and moxalactam) and with those of cefoxitin, clindamycin, and metronidazole against 227 strains of anaerobic bacteria. The data obtained in this investigation suggest that ceftriaxone, like a majority of the new beta-lactam antimicrobial agents, may be of limited value in the treatment of serious infections involving anaerobic bacteria.

Purification and characterization of Clostridium difficile toxin.

Recent evidence indicates that toxigenic Clostridium difficile strains are a major cause of antimicrobial-associated ileocecitis in laboratory animals and pseudomembranous colitis in humans. C. difficile ATCC 9689 was cultivated in a synthetic medium to which 3% ultrafiltrated proteose peptone was added. Purification of the toxin from broth filtrate was accomplished through ultrafiltration (100,000 nominal-molecular-weight-limit membrane), precipitation with 75% (NH4)2SO4, and chromatographic separation using Bio-Gel A 5m followed by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-25 column. The purified toxin displayed only one band on polyacrylamide gel electrophoresis, and approximately 170 pg was cytopathic for human amnion cells. The isolated toxin was neutralized by Clostridium sordelli antitoxin, heat labile (56 degrees C for 30 min), and inactivated at pH 4 and 9; it had an isoelectric point of 5.0, increased vascular permeability in rabbits, and caused ileocecitis in hamsters when injected intracecally. Treatment of the toxin with trypsin, chymotrypsin, pronase, amylase, or ethylmercurithiosalicylate caused inactivation, whereas lipase had no effect. By gel filtration, its molecular weight was estimated as 530,000. Upon reduction and denaturation, the toxin dissociated into 185,000- and 50,000-molecular-weight components, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive dissociation yielded only the 50,000-molecular-weight component. The toxin appears to be protoplasmic and is released into the surrounding environment upon autolysis of the cells. Attempts to correlate specific enzymatic activity with the toxin have been unsuccessful. These studies will help delineate the role of C. difficile toxin in antimicrobial-associated colitis and diarrhea.

Comparative in vitro activity of new beta-lactam antibiotics against anaerobic bacteria.

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 mug/ml to inhibit 90% of the strains) was the most active, followed by cefoperazone (128 mug/ml), cefotaxime (128 mug/ml), moxalactam (128 mug/ml), ceftizoxime (256 mug/ml), and ceftazidime (>256 mug/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

Intestinal colonization of infant hamsters with Clostridium difficile.

Infant hamsters of different ages were examined for their susceptibility to enteric Clostridium difficile colonization. Intragastric administration of C. difficile to infant hamsters resulted in multiplication of the organism in the intestinal tracts of animals 4 to 12 days old; hamsters younger or older were resistant to C. difficile intestinal colonization. Toxicity to the colonized animals could not be demonstrated despite cytotoxin titers in some infant hamsters comparable to titers found in the intestinal tracts of adult hamsters with C. difficile-associated intestinal disease. When introduced into 4-day-old hamsters, C. difficile colonized the intestinal tract and remained at high levels until the animals were 13 days old, at which time the presence of intestinal C. difficile could no longer be demonstrated. The number of C. difficile required to colonize the intestinal tracts of 50% of 7-day-old hamsters was 18 viable cells. On the other hand, 10(8) viable cells of C. difficile failed to colonize the intestinal tracts of healthy, non-antibiotic-treated adult hamsters.

Intestinal beta-lactamase activity in ampicillin-induced, Clostridium difficile-associated ileocecitis.

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.

In vitro and in vivo activities of nitazoxanide against Clostridium difficile.

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.

Bacterial interference between Clostridium difficile and normal fecal flora.

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.