Cellular immunity to Bacteroides fragilis capsular polysaccharide.

The polysaccharide capsule of Bacteroides fragilis has been shown to be important in the virulence of the organism. The capsular polysaccharide (CP) of B. fragilis has been extensively purified. Using a murine model of intraabdominal abscess formation, we have been able to demonstrate cellular immunity to the capsular polysaccharide of B. fragilis. Immunization of C57BL/10J mice with the CP over 5 wk prevents abscess formation when the mice are challenged with B. fragilis intraperitoneally. This immunity can be transferred to naive mice with spleen cells from immune animals. The immune cells bear Thy-1.2 and Ly-2.2 antigens. The immune response has been shown to be antigen specific, but not H-2 restricted. The possibility that these immune cells are suppressor T cells is discussed. The experimental system presented provides a model for the examination of the cellular interactions responsible for abscess formation and the cellular response to bacterial pathogens.

Structural features of polysaccharides that induce intra-abdominal abscesses.

The capsular polysaccharide complex from Bacteroides fragilis promotes the formation of intra-abdominal abscesses--a pathologic host response to infecting microorganisms. This complex consists of two distinct polysaccharides, each with repeating units that have positively charged amino groups and negatively charged carboxyl or phosphate groups. Analysis of these polysaccharides as well as other charged carbohydrates before and after chemical modification revealed that these oppositely charged groups are required for the induction of intra-abdominal abscesses in a rat model.

Polysaccharide-mediated protection against abscess formation in experimental intra-abdominal sepsis.

Abscess formation is a major complication of intra-abdominal sepsis that causes significant morbidity and mortality. In such cases, Bacteroides fragilis is the predominant anaerobic isolate. In a rat model of intra-abdominal sepsis, the capsular polysaccharide complex (CPC) from B. fragilis promotes abscess formation and when administered sub-cutaneously, protects against this host response by a T cell-dependent immune mechanism. In the present study, the polysaccharide A (PS A) component of CPC protected animals against challenge with live heterologous bacterial species (mixtures of anaerobes and facultative organisms) that are most commonly isolated from intra-abdominal abscesses in humans. Protection against heterologous bacterial challenge was transferred by T cells. Administration of PS A shortly before or even after challenge with B. fragilis protected against this host response. In experiments designed to simulate fecal contamination of the human peritoneal cavity, PS A protected animals against abscess formation induced by a rat cecal contents inoculum. The surprisingly broad protective activity of PS A indicates that this molecule is likely suppressing a nonspecific host tissue reaction that forms in response to a variety of abscess-inducing organisms and that it might be useful in preventing abscess formation associated with intra-abdominal sepsis in the clinical setting.

A soluble suppressor T cell factor protects against experimental intraabdominal abscesses.

This paper describes a suppressor T cell factor which protects mice against intraabdominal abscesses caused by Bacteroides fragilis. This soluble cell-free factor (ITF) is derived from splenic T cells from mice immunized with capsular polysaccharide (CP) of B. fragilis. Mice receiving ITF are protected from developing abscesses caused by B. fragilis to the same degree as animals receiving intact immune splenic T cells. The factor appears to be small in molecular size as protective activity is dialyzable through a 12,000-mol wt exclusion dialysis membrane and is present in fractions intermediate between the bed and void volumes of a P2 Biogel column. The protective effect of ITF is antigen-specific to B. fragilis alone. Mice given a complex inoculum of B. fragilis, enterococcus, and another anaerobe develop abscesses even after receiving column-purified ITF. The activity of ITF also is eliminated after adsorption with B. fragilis CP coupled to sheep erythrocytes but not with an unrelated CP coupled to sheep erythrocytes. ITF, therefore, appears to have a binding site for B. fragilis CP. ITF is heat-labile and loses efficacy after protease digestion, suggesting that the active material is a protein. These studies define a suppressor cell factor with activity in a model system resembling human disease and offer promise for increased understanding of the diversity of cell-mediated immune systems.

Evidence for T cell-dependent immunity to Bacteroides fragilis in an intraabdominal abscess model.

It has been shown that active immunization of rats with the capsular polysaccharide of Bacteroides fragilis protects these animals against abscess development following intraperitoneal challenge with this species. Passive transfer of hyperimmune globulin from immunized animals to nonimmune recipients provided protection against B. fragilis bacteremia in challenged animals, but did not confer protection against abscess development. On the other hand, adoptive transfer of spleen cells from immunized animals to nonimmunized recipients resulted in protection against abscesses following challenge with B. fragilis. These data suggested that a T cell-dependent immune response was involved in protection against abscess development after immunization with B. fragilis capsular antigen. To determine the possible role of cell-mediated immunity prompted by the capsular antigen, inbred congenitally athymic OLA/Rnu rats and their phenotypically normal littermates were actively immunized. Despite the development of high titers of anti-B. fragilis capsular antibody, 100% of actively immunized athymic rats developed abscesses, as did 100% of unimmunized athymic control rats. However, no phenotypically normal littermate control rats that were actively immunized developed abscesses, while 100% of phenotypically normal unimmunized rats developed abscesses. Additional studies showed that adoptive transfer of T cell-enriched spleen cell preparations from Wistar/Lewis rats immunized with the capsular polysaccharide to nonimmune recipients also resulted in protection against B. fragilis-induced abscesses. We conclude that the protection afforded by immunization with B. fragilis capsule against intraabdominal abscesses caused by that organism is T cell-mediated and does not require the presence of serum antibody.

Rapid diagnosis of anaerobic infections by direct gas-liquid chromatography of clinical speciments.

Current methods to isolate and identify anaerobic bacteria are laborious and time consuming. It was postulated that the short-chain fatty acids (SCFA) produced by these organisms might serve as microbial markers in clinical material. 98 specimens of pus or serous fluid were analyzed by gas-liquid chromatography, and findings were compared with culture results. Good correlations were found for the recovery of anaerobic Gram-negative bacilli and the presence of isobutyric, butyric, and succinic acids. 19 of 20 specimens with significant amounts of these acids (greater than 0.01 mumol/ml) yielded bacteroides or fusobacteria. Culture of the single "false-positive" specimen failed to grow anaerobic Gram-negative bacilli, although clinical data and Gram-stain suggested their presence. 77 of 78 specimens which has insignificant concentrations of the marker acids failed to yield anaerobic, Gram-negative bacilli in culture. The single "false-negative" specimen yielded Bacteroides pneumosintes, an organism which does not ferment carbohydrates. It is concluded that direct gas-liquid chromatographic analysis of clinical specimens provides a rapid presumptive test for the presence of anaerobic, Gram-negative bacilli.

Microbial interactions in the vaginal ecosystem, with emphasis on the pathogenesis of bacterial vaginosis.

During bacterial vaginosis (BV), populations of lactobacilli which are generally dominant in the vagina of overtly healthy women are replaced by other facultative and anaerobic microorganisms. Some Lactobacillus strains produce hydrogen peroxide and all produce lactic acid; however, the antagonistic role of these metabolites in vivo remains controversial. Positive interactions among BV-associated organisms may contribute to the pathogenesis of BV and its sequelae.

A polymeric drug for treatment of inflammatory bowel disease.

Sulfasalazine (SASP) consists of salicylic acid azo linked at the 5-position to a pyridine-containing sulfonamide. This drug, currently used in inflammatory bowel disease treatment, is reductively cleaved by anaerobic bacteria in the lower bowel to 5-aminosalicylic acid (5-ASA) and sulfapyridine (SP). Recent reports indicate that 5-ASA is the active therapeutic moiety and that SP is responsible for a variety of adverse clinical side effects. Water-soluble polymer 7, which contains salicylate residues azo linked at the 5-position to an inert polymer backbone, has been synthesized for the site-specific reductive release of 5-ASA in the lower bowel. Preparations of 7 deliver (chemical reduction) greater than 1.96 mmol of 5-ASA/g of polymer. In vitro studies with the polymer in anaerobic rat cecal bacteria demonstrated a reduction rate of approximately 1 mu equiv of azo bond h-1 (mL of cecal content)-1. A pharmacokinetic comparison of polymer and SASP showed similar deliveries of 5-ASA and metabolites to the lower bowel, blood, and urine of orally dosed rats. Polymer 7 proved more active than SASP or 5-ASA in the guinea pig ulcerative colitis model. Potential therapeutic advantages of 7 include nonabsorption/nonmetabolism in the small intestine, direct 5-ASA release at the disease site, and nonabsorption/nonmetabolism of the reduction-released carrier polymer.

Evaluation of a new enzyme immunoassay for Clostridium difficile toxin A.

AIM: To evaluate a new enzyme immunoassay (EIA) method for detection of Clostridium difficile toxin by comparing it to cytotoxicity assay. To investigate the nature of false negative and false positive EIA results by evaluating clinical and therapeutic parameters. METHODS: 737 consecutive diarrhoeal specimens collected from patients clinically suspected of having C difficile colitis were tested for the presence of C difficile toxin by EIA for toxin A and by cytotoxicity assay. Clinical data were evaluated in all cases positive by either method. RESULTS: With the cytotoxicity assay as a gold standard, the specificity of EIA for toxin detection was 99.3% and the sensitivity was 62.2%. No false negative EIA specimens were obtained from patients already being treated for C difficile colitis. Among patients with cytotoxicity positive specimens, those with EIA positive samples had no clinical features distinguishing them from patients with EIA negative samples. CONCLUSIONS: Although specific, the new EIA method directed against toxin A lacks sensitivity compared to cytotoxicity. False negative EIA tests are not associated with concurrent treatment for C difficile colitis nor with any specific clinical features examined in our study.

Nugent score related to vaginal culture in pregnant women.

OBJECTIVE: To relate Gram-stained smears, using the Nugent criteria, to quantitative and qualitative vaginal cultures in pregnant women. METHODS: Two independent evaluators using the Nugent criteria, a standardized method of Gram-stain interpretation designed to detect bacterial vaginosis, scored 104 vaginal smears from pregnant women. The quantitative and qualitative vaginal cultures were assessed at the same time and the results expressed as log(10) colony-forming units per gram of vaginal secretion. The Nugent scores were compared with the microbiologic findings. RESULTS: The prevalence of normal, intermediate, or bacterial vaginosis vaginal flora as determined by Gram stain was 68%, 21%, and 11%, respectively. A comparison of the mean bacterial counts with the Nugent score showed a weak negative correlation for Lactobacillus species and a positive correlation for gram-variable and gram-negative rods. Additional analysis revealed a strong positive correlation between the mean bacterial counts analyses of Peptostreptococcus, a genus not included in the Nugent scoring system, and the Nugent score. In addition, the Prevotella counts correlated strongly with both the Nugent score and the Peptostreptococcus counts. The quantitative counts for Lactobacillus did not vary significantly among the three defined groups of vaginal microflora; however, significant increases in the concentrations of Gardnerella vaginalis and Prevotella were found as the Nugent score increased. CONCLUSION: A strong correlation was found among the gram-variable and gram-negative genera comprised by the Nugent score. Peptostreptococcus also correlated strongly with the Nugent score and with the Prevotella counts, suggesting that this genus may play a role in determining vaginal health.

Effect of Candida albicans infection and clotrimazole treatment on vaginal microflora in vitro.

OBJECTIVE: To determine the effect of Candida albicans infection and clotrimazole treatment on vaginal microflora. METHODS: Studies were conducted using a model simulating the healthy vaginal ecosystem. The model consisted of a mixed culture of Lactobacillus acidophilus, Staphylococcus epidermidis, Prevotella bivia, and group D Streptococcus sp grown in continuous culture in a chemically defined medium. The status of the model was assessed using a mathematical equation that determines the probability a microflora is normal or abnormal. RESULTS: Challenge of the model with C albicans was followed within 24 hours by the development of microbial populations representing an abnormal microflora. Treatment of the system with clotrimazole (100 micrograms/mL) resulted in a decrease in C albicans counts to 0 within 48 hours. However, treatment also altered other components of the vaginal microflora, which did not return to normal. Addition of clotrimazole (100 micrograms/mL) to the system in the absence of C albicans also resulted in an abnormal model by 24 hours. CONCLUSIONS: Candida albicans infection of the vaginal ecosystem, as represented by this in vitro model, has a deleterious effect on members of the normal microflora. Clotrimazole, although effective against C albicans infection, also has a deleterious effect on components of the normal vaginal microflora. One of the implications for women using clotrimazole for microbiologically undocumented vaginal yeast infections is an increased risk of infection or disease through the disruption of the protective microflora barrier.

Quantitative and qualitative effects of douche preparations on vaginal microflora.

OBJECTIVE: To determine the effect of douching on the quantitative and qualitative makeup of the vaginal microflora. METHODS: We first evaluated the effect of douching with a solution of physiologic saline to determine the effect of washing the vaginal surface. Two douche preparations, one containing 0.04% acetic acid and one containing 0.30% povidone-iodine, were evaluated subsequently to determine whether any effects occurred in addition to those noted with saline. Duplicate vaginal swab samples were obtained at predetermined intervals from ten healthy volunteers for three sampling cycles before and after use of the douche preparations for various periods of time. Samples were analyzed for total facultative and obligately anaerobic bacterial populations. RESULTS: The use of a douche preparation containing acetic acid caused a transient reduction of the total bacterial counts, with most of the change attributable to the effect of washing the surface of the vaginal vault as noted with physiologic saline. In contrast, the povidone-iodine preparation caused a significant reduction in total counts compared with those obtained after use of a physiologic saline solution by the same subjects (P = .02). Little change in the qualitative makeup of the vaginal microflora occurred. CONCLUSION: The use of povidone-iodine douches decreases the numbers of the dominant bacterial species beyond those expected with other douches. In some individuals, such changes may allow rapid proliferation of potential pathogens during this altered state, increasing the risk of associated infections.

A review. Lessons from an animal model of intra-abdominal sepsis.

Intra-abdominal sepsis that involves multiple aerobic and anaerobic bacteria derived from the colonic flora was studied in Wistar rats to determine the relative roles of various microbial species. The rats challenged with pooled colonic contents showed a biphasic disease. Initially, there was acute peritonitis, Escherichia coli bacteremia, and high mortality. In rats that survived this acute peritonitis stage, intra-abdominal abscesses developed, and anaerobic bacteria were the preponderant organisms. Subsequent experiments showed that antibiotics directed against coliforms prevented mortality, whereas agents active against anaerobes reduced the incidence of abscesses. Challenges with Escherichia coli alone produced bacteremia and death, whereas pure cultures of Bacteroides fragilis caused intra-abdominal abscesses. These observations suggest that both coliforms and anaerobes are important pathogens in intra-abdominal sepsis, although the different types of microbes appear to play distinctive roles in the sequence of pathological events.

Effect of surgical adhesion reduction devices on the propagation of experimental intra-abdominal infection.

HYPOTHESIS: The use of certain surgical adhesion reduction devices where there is a risk of concomitant bacterial contamination potentiates intra-abdominal infection. DESIGN: Evaluation of adhesion reduction devices in an experimental model of intra-abdominal infection. SETTING: Experimental animal model. INTERVENTIONS: Adhesion reduction devices were administered at the time of bacterial challenge. MAIN OUTCOME MEASURES: Animal mortality rate, abscess formation, and bacterial counts in peritoneal fluid and blood cultures. RESULTS: The use of bioresorbable membrane adhesion reduction devices in the presence or absence of antibiotic therapy did not alter the disease process as compared with appropriate control groups. However, adhesion reduction gels prepared from sodium hyaluronate and carboxymethylcellulose chemically modified with carbodiimide or ferric ion complexed sodium hyaluronate increased the incidence of peritonitis in treated animals. Gel formulations containing diimide-modified carboxymethylcellulose did not have this effect. CONCLUSIONS: The use of certain adhesion reduction devices resulted in the propagation of intra-abdominal infection in an experimental rat model. This outcome was dependent on the composition of the device employed. The use of adhesion reduction devices should be tested in appropriate models of infection where there is the risk of concomitant bacterial contamination.

Comparison of in vivo and in vitro efficacy of ceftizoxime.

Two hundred obligately anaerobic bacterial isolates from human clinical sources were tested for susceptibility to ceftizoxime using a standard National Committee for Clinical Laboratory Standards microwell system. In general, the minimal inhibitory concentrations (MIC50) ranged from less than 0.125 to 32 microgram/ml; however, the MIC50 for Bacteroides fragilis averaged greater than 128 microgram/ml. This finding is inconsistent with the results of in vivo testing of ceftizoxime in an animal model of intra-abdominal sepsis (B fragilis is a major contributor to the development of intra-abdominal abscesses). Various modifications of in vitro assay parameters, including basal media (brain-heart infusion [BHI] or Wilkins-Chalgren [WC]) and methods (microwell, broth, and agar dilutions), were compared. Ten B fragilis isolates from the original clinical study were used. Results indicate that the activity of ceftizoxime decreased between two- and fourfold after storage for 48 hours at -80 degrees C, regardless of methodology or basal media. When microwell was compared with broth dilution, there was a four- to 64-fold decrease in MIC values by the latter method using BHI but little variation using WC. No differences were observed when the incubation time was varied. Preliminary data indicate that MIC values from broth dilution using BHI correspond with those of agar dilution assays. These results suggest methodologic as well as environmental discrepancies with regard to susceptibility testing of ceftizoxime. These differences may lead to misinterpretation of the true susceptibility of organisms to this agent, particularly when the results are compared with in vivo observations.

A commensal symbiosis between Prevotella bivia and Peptostreptococcus anaerobius involves amino acids: potential significance to the pathogenesis of bacterial vaginosis.

In both batch and continuous culture, Peptostreptococcus anaerobius was able to grow in vaginal defined medium with Prevotella bivia, but not in pure culture. Growth of P. anaerobius was increased by 238% (P < 0.001) in peptone-supplemented vaginal defined medium conditioned by prior growth of P. bivia. Analysis of P. bivia culture supernatants showed a net accumulation of amino acids and subsequent growth of P. anaerobius in the conditioned supernatants resulted generally in amino acid utilization. Supplementation of peptone-supplemented vaginal defined medium with amino acids in concentrations similar to those available after prior growth with P. bivia were growth-stimulatory (246%, P=0.006) for P. anaerobius. Increased availability of amino acids by P. bivia is proposed as a mechanism to support the observed in vitro commensal symbiosis between P. bivia and P. anaerobius.

Pharmacodynamics and microbiology of trovafloxacin in animal models of surgical infection.

Trovafloxacin provides broad in vitro and in vivo coverage of the aerobic and anaerobic pathogens found frequently in surgical infections. In vitro susceptibility testing indicated that trovafloxacin inhibited gram-positive staphylococci and enterococci, numerous gram-negative organisms, including Escherichia coli, and anaerobic pathogens, such as Bacteroides fragilis. Trovafloxacin protected mice from lethal infections induced by gram-negative or gram-positive organisms, even when these organisms were inoculated in combination with B. fragilis. Trovafloxacin protected rats in models of intra-abdominal sepsis induced by inoculation with E. coli and B. fragilis or with multiple aerobic and anaerobic pathogens. In these experimental models, trovafloxacin protected rats from lethal infection, reduced intra-abdominal abscess formation, and inhibited bacterial growth. Drug concentrations were greater in intra-abdominal abscesses than in serum, reflecting the good tissue penetration of trovafloxacin. These results indicate that trovafloxacin may be effective in prophylaxis and treatment of mixed infections in surgical patients.

Antibiotic-induced lethal enterocolitis in hamsters: studies with eleven agents and evidence to support the pathogenic role of toxin-producing Clostridia.

Clindamycin-induced enterocolitis in hamsters was studied, using a tissue culture assay to detect clostridial toxin. It was found that animals with lethal enterocolitis had a cytopathogenic substance in cecal contents and blood that was neutralized by clostridial antitoxins. Cultures of the cecal flora yielded numerous species of clostridia, but only 1 organism was detected which produced a toxin which was cytopathic in tissue culture. This organism, Clostridium difficile, was consistently present in high concentrations, and the cell-free supernate of these strains caused enterocolitis if injected intracecally into hamsters. Ten additional antimicrobials were tested ih hamsters. Ampicillin, vancomycin, erythromycin, cephalosporins, and oral gentamicin caused lethal enterocolitis in most recipients, and all animals which died had evidence of clostridia toxin in cecal contents at necropsy. Tetracycline and metronidazole were well tolerated, and the animals given these antimicrobials had no evidence of the toxin. We conclude that toxin-producing clostridia are responsible for lethal enterocolitis due to a variety of antimicrobials in hamsters.