Quantitative detection of enterotoxigenic Bacteroides fragilis subtypes isolated from children with and without diarrhea.

A rapid real-time PCR (RT-PCR) approach was developed to detect the bft gene subtypes in Bacteroides fragilis isolated from fecal samples. DNA obtained from diarrhea (110) and nondiarrhea (150) samples was evaluated. Subtype 1 was observed in 9 (8.2%) diarrhea and 7 (4.7%) nondiarrhea samples. Subtype 2 was not detected in any DNA samples, and subtype 3 was observed in only 1 diarrhea sample. The presence of the bft-1 gene did not show any statistically significant differences between the groups of children. This technique could be used to evaluate a possible correlation between disease and the presence of B. fragilis enterotoxin.

Innate immune recognition of, and response to, Clostridium sordellii.

Clostridium sordellii, an anaerobic pathogen, has recently been associated with rapidly fatal infections following medically induced abortions and injecting drug use. Patients with C. sordellii infection display few signs of inflammation such as fever, or redness and pain at the site of infection. We hypothesized that this could be due to reduced recognition of the organism by Toll-like receptors (TLRs) of the innate immune system. An ELAM-NF-kappaB luciferase reporter system in TLR-transfected HEK cells was used to measure TLR-dependent recognition of washed, heat-killed C. sordellii and other pathogenic clostridial species. Results demonstrated that all clostridia were well recognized by TLR2 alone and that responses were greatest when TLR2 was co-expressed with TLR6. Further, isolated human monocytes produced the pro-inflammatory cytokine TNFalpha and the immunoregulator IL-10 in response to C. sordellii. In addition, C. sordellii-stimulated monocytes produced 30% less TNFalpha following treatment with an anti-TLR2 blocking antibody. These data demonstrate that innate immune recognition of, and response to, cell-associated components of C. sordellii and other clostridial pathogens are mediated by TLR2 in combination with TLR6. We conclude that the characteristic absence of inflammatory signs and symptoms in C. sordellii infection is not related to inadequate immune detection of the organism, but rather is attributable to a species-specific immune system dysfunction that remains to be elucidated.

Study of the in vitro activities of rifaximin and comparator agents against 536 anaerobic intestinal bacteria from the perspective of potential utility in pathology involving bowel flora.

Rifaximin, ampicillin-sulbactam, neomycin, nitazoxanide, teicoplanin, and vancomycin were tested against 536 strains of anaerobic bacteria. The overall MIC of rifaximin at which 50% of strains were inhibited was 0.25 microg/ml. Ninety percent of the strains tested were inhibited by 256 microg/ml of rifaximin or less, an activity equivalent to those of teicoplanin and vancomycin but less than those of nitazoxanide and ampicillin-sulbactam.

Evaluation of genotypic and phenotypic methods for differentiation of the members of the Anginosus group streptococci.

The terminology and classification of the Anginosus group streptococci has been inconsistent. We tested the utility of 16S rRNA gene and tuf gene sequencing and conventional biochemical tests for the reliable differentiation of the Anginosus group streptococci. Biochemical testing included Rapid ID 32 Strep, API Strep, Fluo-Card Milleri, Wee-tabs, and Lancefield antigen typing. Altogether, 61 Anginosus group isolates from skin and soft tissue infections and four reference strains were included. Our results showed a good agreement between 16S rRNA gene and tuf gene sequencing. Using the full sequence was less discriminatory than using the first part of the 16S rRNA gene. The three species could not be separated with the API 20 Strep test. Streptococcus intermedius could be differentiated from the other two species by beta-galactosidase (ONPG) and beta-N-acetyl-glucosaminidase reactions. Rapid ID 32 Strep beta-glucosidase reaction was useful in separating S. anginosus strains from S. constellatus. In conclusion, both 16S rRNA gene and tuf gene sequencing can be used for the reliable identification of the Anginosus group streptococci. S. intermedius can be readily differentiated from the other two species by phenotypic tests; however, 16S rRNA gene or tuf gene sequencing may be needed for separating some strains of S. constellatus from S. anginosus.

Bacteraemia caused by Anaerotruncus colihominis and emended description of the species.

BACKGROUND: Anaerotruncus colihomonis is a newly described bacterial genus and species isolated from the stool specimens of children. Its clinical significance, however, is unknown. AIMS: To describe a case of A colihominis bacteraemia identified by 16S ribosomal RNA (rRNA) gene sequencing and provide an emended description of the species. METHODS: An unidentified anaerobic bacillus (strain HKU19) that stains Gram negative was subjected to characterisation by 16S rRNA gene sequencing, G+C content determination and electron microscopy. RESULTS: Strain HKU19 was isolated from the blood culture of a 78-year-old woman with nosocomial bacteraemia. It was found to be an anaerobic, non-motile, pleomorphic, thin bacillus that stains Gram negative. It produces Indole and utilises glucose and mannose. Identifying the strain to the species level was not possible by conventional phenotypic tests and commercial identification systems. The G+C content of strain HKU19 was found to be 53.43 mol%. A similarity of 99.3% nucleotide identities was found between the 16S rRNA gene sequence of strain HKU19 and that of A colihominis WAL 14 565(T), which was isolated from a human faecal specimen. In contrast with the original description of A colihominis, HKU19 was found to produce occasional oval, terminal spores, although the other phenotypic characteristics matched. Spores were also occasionally observed when the two previously reported strains were re-examined. CONCLUSIONS: Although the source of the bacteraemia in the patient cannot be determined, this report suggests that A colihominis is of clinical significance. Spore formation is proposed as an emended description of A colihominis.

Fecal bacteriology of colonic polyp patients and control patients.

Feces from 25 subjects with colonic polyps (multiple adenomatous, large single, or single with atypia) and from 25 matched control subjects were studied by detailed quantitative aerobic and anaerobic techniques, using a large battery of culture media and several atmospheric conditions. Over 55% of organisms detected on microscopic count were recovered anaerobically. In several cases, there were significantly different numbers of organisms of specific types recovered from the two different populations studied. However, these differed from organisms with "statistical significance" noted in a previous study from this laboratory involving two different diet groups (Japanese Americans on either a Japanese or a Western diet). Specific differences in bacteriology between groups with different risks of bowel cancer, noted in earlier British studies, were not noted in our present or previous study.

Transformation of 4-androsten-3,17-dione by growing cultures and cell extracts of Clostridium paraputrificum.

Growing cultures of Clostridium paraputrificum transformed 4-androsten-3,17-dione to 3 alpha-hydroxy-5 beta-androstan-17-one in a sequential manner with 5 beta-androstan-3,17-dione as an intermediate. The addition of 1.5 mM menadione to log-phase cultures which had formed 5 beta-androstan-3,17-dione resulted in a partial reoxidation of this steroid to 4-androsten-3,17-dione. However, this treatment also resulted in transient inhibition of culture growth. Resumption of growth was accompanied by complete reduction of 4-androsten-3,17-dione to 5 beta-androstan-3,17-dione. Cell extracts of C. paraputrificum were capable of carrying out these reductive transformations in the absence of added cofactors. However, Sephadex G-25 treated extracts required NADH or NADPH for these reactions. A flavin nucleotide, either FAD (plus NADH or NADPH) or FMN (plus NADH) was highly stimulatory for 4-androsten-3,17-dione reduction to 5 beta-androstan-3,17-dione. NADH was the preferred reduced pyridine nucleotide for reduction of the C4-C5 double bond, while time-course measurements suggested that NADPH was the preferred donor for reduction of the 3-keto group.

Pathogenic anaerobes.

Anaerobes are prevalent on all mucosal surfaces and virtually all anaerobic infections are endogenous. Two thirds of anaerobic infections involve five anaerobic organisms or groups--the Bacteroides fragilis group, the Bacteroides melaninogenicus-Bacteroides asaccharolyticus group, Fusobacterium nucleatum, the anaerobic cocci, and Clostridium perfringens. Conditions that lower the oxidation-reduction potential and disrupt the mucosal surface (eg, vascular problems, malignant neoplasms, and surgery) lead to infection with anaerobes. Clues to anaerobic infection include foul odor, gas, tissue destruction, underlying malignant neoplasms, and the unique appearance of certain anaerobes on Gram's stain. Specimens must be collected to avoid normal flora and transported to the laboratory under anaerobic conditions. Therapy involves surgical débridement and drainage and the use of various antimicrobial agents. Antimicrobial agents must be used for extended periods to avoid relapse.

Bacteriologic findings associated with chronic bacterial maxillary sinusitis in adults.

An open-label, multicenter study was performed to assess bacteriologic findings associated with chronic bacterial maxillary sinusitis in adults. Seventy aerobic (52.2%) and 64 anaerobic (47.8%) pathogens were recovered from clinically evaluable patients at baseline (before therapy). The most commonly isolated anaerobes were Prevotella species (31.1%), anaerobic streptococci (21.9%), and Fusobacterium species (15.6%). The aerobes most frequently recovered included Streptococcus species (21.4%), Haemophilus influenzae (15.7%), Pseudomonas aeruginosa (15.7%), and Staphylococcus aureus and Moraxella catarrhalis (10.0% each). Recurrences of signs or symptoms of bacterial maxillary sinusitis associated with anaerobes were twice as frequent as were those associated with aerobes when counts of anaerobes were > or =10(3) cfu/mL. A pathogenic role for Granulicatella species in cases of chronic sinusitis was documented for the first time.

Multilaboratory comparison of growth characteristics for anaerobes, using 5 different agar media.

A multilaboratory study compared the growth of 30 fastidious anaerobes, using 5 different agar media: Wilkins-Chalgren (WC), WC with either whole or laked sheep blood, and Brucella supplemented with vitamin K(1) and hemin and either laked or whole sheep blood. The media were compared for quality and quantity of growth. Experiments were conducted either entirely in an anaerobic chamber or inoculated in ambient air with anaerobic incubation. The results showed that (1) any medium plus whole or laked blood was better than unsupplemented WC, (2) whole blood and laked blood additives gave similar results, (3) supplemented Brucella with whole or laked blood was superior to WC and WC with whole or laked blood, and (4) anaerobic and aerobic inoculation with anaerobic incubation gave similar results. Brucella agar supplemented with whole or laked blood supports the growth of fastidious anaerobic species better than the WC agars do.

Multilaboratory comparison of anaerobe susceptibility results using 3 different agar media.

A 5-laboratory study was performed that used the National Committee for Clinical Laboratory Standards (NCCLS) reference agar dilution method with 3 media formulations to determine whether the use of different media would affect minimum inhibitory concentration (MIC) results. Wilkins-Chalgren, Brucella-based blood agar (BRU), and Wilkins-Chalgren agar plus blood (WCB) and 6 antibiotics (clindamycin, cefoxitin, ceftizoxime, piperacillin, metronidazole, and trovafloxacin) were evaluated with 58 isolates. The MIC values were compared, and a significant correlation of >0.80 was demonstrated for all media and each antibiotic/organism group. The cumulative rate of errors for all antibiotics was 0.1%. These data indicate that a change in the NCCLS reference medium for testing of anaerobic bacteria susceptibility to either BRU or WCB will not affect the MIC results for the antibiotics and organisms evaluated.

National survey on the susceptibility of Bacteroides Fragilis Group: report and analysis of trends for 1997-2000.

The results of a multicenter US survey using the National Committee for Clinical Laboratory Standards currently recommended methodology for measuring in vitro susceptibility of 2673 isolates of Bacteroides fragilis group species were compared from 1997 to 2000. The test panel consisted of 14 antibiotics: 3 carbapenems, 3 beta-lactam-beta-lactamase inhibitors, 3 cephamycins, 2 fluoroquinolones, clindamycin, chloramphenicol, and metronidazole. Declines in the geometric mean minimum inhibitory concentrations were seen with imipenem, meropenem, ampicillin-sulbactam, and the cephamycins. Increased geometric means were observed with the fluoroquinolones and were usually accompanied by an increase in resistance rates. Bacteroides distasonis shows the highest resistance rates among beta-lactam antibiotics, whereas Bacteroides vulgatus shows the highest resistance levels among fluoroquinolones. B. fragilis shows the lowest resistance rates for all antibiotics. All strains were susceptible to chloramphenicol and metronidazole concentrations <8 microgram/mL. The data underscore the need for species identification and continued surveillance to monitor resistance patterns.

Bone infections involving anaerobic bacteria.

Over 700 cases of anaerobic osteomyelitis have been reported in the literature. Nonetheless, most reviews of osteomyelitis have paid little attention to the potential role of anaerobes in bone infections. There have, as yet, been no prospective studies of osteomyelitis utlizing optimal anaerobic transport and culture techniques. In a retrospective study of osteomyelitis at Wadsworth VA Hospital from 1973--1975, 39 percent of 58 patients with osteomyelitis had an infection involving anaerobes. Anaerobes were isolated from 81 percent of 27 patients whose specimens were cultured anaerobically. Anaerobes were isolated from nine of ten samples of bone. Anaerobic bacteria were part of a mixed flora involving facultative bacteria in all but two cases. All of the patients with anaerobic infection had non-hematogenous osteomyelitis. Non-hematogenous disease comprises 80--90 percent of the osteomyelitis seen in adults. Our experience at Wadsworth VA Hospital and a review of the literature lead us to believe that anaerobes play a much larger role in osteomyelitis than has been appreciated previously. Infections of the calvarium, mastoid, mandible, maxilla and the extremities are most likely to involve anaerobes. Predisposing conditions include paranasal sinusitis, otitis media, periodontal disease, trauma, peripheral vascular disease, peripheral neuropathy and/or chronic osteomyelitis. The presence of a foul odor is a valuable clinical clue to the presence of anaerobes. Bacteroides, fusobacteria and anaerobic cocci have been reported with almost equal frequency from anaerobic bone infections. While Bacteroides fragilis is the most common anaerobe isolated in infections of other organ systems, it does not appear to be a common pathogen in anaerobic bone infections. The role of anaerobes in osteomyelitis is not yet resolved. They have been isolated in pure culture from infected bone, and under those circumstances are clearly pathogenic. Anaerobes are found more frequently as part of a mixed flora with facultative streptococci, gram-negative bacilli, and less often with S. aureus. In this setting it is unclear which organism or organisms are the primary invaders, or whether there is a synergistic mechanism of infection. The reliability of sinus drainage cultures also remains to be determined. Our retrospective study suggests that certain anaerobes isolated from sinus drainage are not present in infected bone. Cultures of bone or an abscess adjacent to bone would be expected to give more reliable data. The majority of anaerobes other than B. fragilis are susceptible to levels of penicillin achievable with parenteral administration of the antibiotic. Anaerobic pathogens should be sought in the situations noted above. We feel that parenteral penicillin should be part of the initial antibiotic regimen in patients with suspected or documented anaerobic bone infection...

Legionnaires' disease: report of sixty-five nosocomially acquired cases of review of the literature.

Sixty-five cases of nosocomially acquired Legionnaires' disease are reported and the world literature is reviewed. The etiologic agent, Legionnella pneumophila, has been isolated from several environmental sources at outbreak sites. Legionnaires' disease appears to be acquired by inhalation and is primarily manifested by severe, potentially fatal, pneumonia. Characteristic clinical disease consists of high fever with relative bradycardia, dry cough, chills, diarrhea, and pleuritic pain. Although no single feature is pathognomonic, the clinical presentation is usually sufficiently characteristic to suggest the diagnosis. The diagnosis of Legionnaires' disease during acute illness may be established by culture of Legionella pneumophila, or by demonstration of the bacterium using special stains. However, in most instances, the physician must make a presumptive diagnosis based on the clinical presentation in order to institute appropriate antimicrobial therapy. Retrospective confirmation of the diagnosis may be made by serologic studies in most instances. Erythromycin is, at this time, the drug of choice for the treatment of Legionnaires' disease. A prompt salutory response following institution of erythromycin therapy is typical.

Fecal flora in different populations, with special reference to diet.

Although potent antimicrobial agents may effect rapid and significant changes in fecal microflora, changes in diet or dietary supplements usually produce modest changes by comparison. It may be, however, that even such minor changes may result in significant changes in metabolic activity, particularly in terms of production of possible carcinogens or cocarcinogens. It is clear that significant changes may occur in metabolic activity without major qualitative or quantitative changes in the fecal microflora. Other research that might help explain the epidemiological observations of the association between diet and colon cancer would be studies of the flora at different levels of the gastrointestinal tract (particularly the cecum and small bowel), studies of the mucosa-associated flora at different levels, and long-term dietary studies.

Bypass enteropathy: an inflammatory process in the excluded segment with systemic complications.

Evidence is presented that many of the enteric and systemic manifestations after jejunoileal bypass can be related to an inflammatory process within the bypassed small bowel rather than to the surgically induced sequelae of a short bowel syndrome with malabsorption. Invasion of the excluded segment by fecal flora was associated with a histologically demonstrable inflammatory response of the mucosa. The disorder was of variable severity and duration and occurred in the majority of 28 bypass patients. Progression to a clinical syndrome resembling an acute abdomen occurred in about 15% of the patients. Small bowel ileus and, in some patients, obstruction of the colon were suggested by physical signs and x-ray findings. Surgical exploration in such instances demonstrated an inflammaotry process of the excluded small bowel loops with severe distention of this segment and of the colon, but not organic obstruction. Pneumatosis cystoides intestinalis was a sequal in two patients. Exudative protein loss was documented in the severe cases. Most of the systemic sequelae are comparable to those seen with inflammatory diseases of the bowel such as Crohn's disease. Fever, excessive weight and lean tissue loss, and the involvement of skin, blood vessels, joints and possibly, the liver suggest an immune response as a common factor in the pathogenesis. The clinical improvement with antibiotics such as metronidazole or with restitution of normal bowel continuity indicates that the bacterial flora in the excluded small bowel segment or its byproducts are causally related to the systemic complications. Hyperoxaluria may be primarily the sequela of steatorrhea and not of the inflammatory process.

Microbial flora in the bypassed jejunum of patients with biliopancreatic bypass for obesity.

The microbial flora in the bypassed biliopancreatic intestinal segment was studied after obesity surgery. This procedure causes less diarrhea than jejunoileal bypass and appears to avoid extraintestinal complications. This report concerns type and quantity of bacteria colonizing the biliopancreatic segment and changes occurring after oral metronidazole treatment. Twelve specimens were aspirated in 10 patients via catheter inserted percutaneously during surgery. The specimens were plated immediately on selective and nonselective media under aerobic and anaerobic conditions. Essentially equal numbers of aerobes and anaerobes were recovered from the biliopancreatic segment with average counts of 10(4) cfu/mL and median counts of 10(5) cfu/mL. Four patients had counts of 10(7) cfu/mL. The most common aerobes were E. coli, Klebsiella, Gram-positive cocci, and Candida; among anaerobes, Clostridium and the Bacteroides fragilis group were most common. In three patients treated with metronidazole because of diarrhea, anaerobes were eliminated and diarrhea cleared.

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.

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.

Fecal microbial flora in Seventh Day Adventist populations and control subjects.

A comparison of 13 vegetarian Seventh Day Adventists with 14 nonvegetarian Adventists revealed relatively few statistically significant differences in fecal flora. A separate study involved a comparison of vegetarian Adventists (49 subjects), nonvegetarian Adventists (45), and non-Adventists on a conventional American diet (31) re: the incidence of the C. paraputrificum group in the fecal flora. The Adventist groups had significantly fewer C. septicum and C. tertium isolates than the non-Adventists. Reference to earlier diet studies done by our group revealed certain striking differences. Fusobacterium and C. perfringens counts were very low and lactobacillus counts very high in Adventists as compared with Japanese-Americans on either a Japanese or Western diet or Caucasian individuals on a conventional U.S. diet. Comparison of nonvegetarian Adventists with the other groups on a nonvegetarian Western diet also revealed several statistically significant differences. Finally, there were a number of significant differences in fecal flora when high risk groups (Japanese-Americans on Western diet and Caucasians on conventional U.S. diet) were compared with low risk groups (Japanese-Americans on a Japanese diet and Adventists).