Isolation, Detection, and Characterization of Enterotoxigenic Bacteroides fragilis in Clinical Samples
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Bacteroides fragilis is an extensively studied anaerobic bacterium comprising the normal flora of the human gut. B. fragilis is known to be one of the most commonly isolated species from clinical samples and has been shown to cause a wide range of pathologies in humans [1, 2]. As an opportunistic pathogen B. fragilis can cause abscess formation and bacteremia [2]. Additionally in its enterotoxigenic form, B. fragilis is a known cause of diarrheal illness, is associated with inflammatory bowel disease, and has been recently characterized in patients with colon cancer [3 - 5]. As research in the field of the gut microbiome continues to expand at an ever increasing rate due to advances in the availability of next generation sequencing and analysis tools it is important to outline various molecular methods that can be employed in quickly detecting and isolating relevant strains of B. fragilis. This review outlines methods that are routinely employed in the isolation and detection of B. fragilis, with an emphasis on characterizing enterotoxigenic B. fragilis (ETBF) strains.Keywords:
Bacteroides fragilis
Isolation
Bacteremia
The capsular polysaccharide (CP) of Bacteroides fragilis is an important virulence factor in the formation of experimental intraabdominal abscesses. Incubation of this organism with subinhibitory doses of clindamycin induced morphological changes in the bacteria, including elongation and loss of CP, detected by ferritin-labeled antibody to capsule. Pretreatment of bacteria with subinhibitory doses of clindamycin, however, did not affect the ability of live or heat-killed organisms to produce intraabdominal abscesses in a mouse model of intraabdominal sepsis. Dose-response experiments with purified CP as well as lipopolysaccharide (LPS) from B. fragilis ATCC strain 23745 mixed with sterile cecal contents as adjuvant revealed that both surface components of the organism were capable of causing abscesses in the mouse model. The dose of LPS required to induce abscesses was five times higher than the required dose of CP. Nevertheless, these studies suggested that B. fragilis LPS is another virulence factor in the formation of intraabdominal abscesses.
Bacteroides fragilis
Virulence factor
Bacteroidaceae
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Bacteroides fragilis plays a key role in the pathogenesis of anaerobic infections and is often found mixed with aerobic organisms. We explored the interactions of this organism with phagocytes in an attempt to discern additional information about its virulence factors. We confirm an earlier report that killing of aerobic organisms by polymorphonuclear leukocytes (PMN) is decreased in the presence of high numbers of Bact. fragilis but this effect could also be demonstrated with Bact. distasonis or Staphylococcus aureus. Our data support the concept that this phenomenon may be due to competition for opsonins. Virulence of Bact. fragilis has been associated with a polysaccharide capsule. We were unable to demonstrate any deleterious effect of the purified capsular polysaccharide of Bact. fragilis on phagocytosis, killing, or chemotaxis by PMN. We were not able to demonstrate any effect of subinhibitory levels of clindamycin on the interactions of neutrophils and Bact. fragilis.
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Nitroimidazoles, including metronidazole, tinidazole and ornidazole, are low molecular weight antimicrobial compounds with excellent activity against anaerobic microorganisms. These compounds are usually bactericidal at low concentrations and their spectrum of activity encompasses almost all the anaerobic bacteria and some capnophylic organisms. The few anaerobic bacteria known to be resistant to the nitroimidazoles include occasional anaerobic cocci, some nonsporing gram positive bacilli and Propionibacterium. Nitroimidazoles are the most active antimicrobial agents known against Bacteroides fragilis, the most resistant of anaerobic bacteria. Kill-curve studies demonstrate that there is a 2 to 5 log decrease in the number of colony forming units with Bacteroides fragilis and clostridium perfringens within one hour. The killing is unaffected by inoculum, growth rate or components of the medium. However, a metronidazole resistant isolate of B. fragilis has been shown to have decreased ability to take up 14C-metronidazole as well as lessened ability to reduce metronidazole. This is associated with a decrease in the nitroreductase activity. The in vitro observations have also been demonstrated in vivo. Clinical studies have shown nitroimidazoles to be efficacious in the therapy of a variety of anaerobic infections including non-traumatic brain abscess, intraabdominal abscesses, pelvic suppuration and necrotizing soft tissue infections. However, there have been disappointing results in the therapy of anaerobic pleuropulmonary infections, with a number of superinfections caused by aerobic bacteria.
Bacteroides fragilis
Clostridium perfringens
Tinidazole
Nitroimidazole
Bacteroides thetaiotaomicron
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Bacteroides fragilis
Antibody opsonization
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The predominant bacteria from 50 inflamed human appendices were isolated. Anaerobic bacteria were isolated more frequently than aerobic bacteria ( 141 versus 96 isolates). E. coli was the most common aerobic genus (47 out of 50 patients). Bacteria belonging to the B. fragilis groups (B. Fragilis, B. ovatus, B. distasonis, B. thetaiotaomicron, B. vulgatus and B. uniformis) were the most frequently isolated anaerobic microorganisms (87 of 141 strains), and B. fragilis predominated within this group (24 out of 87 strains). There were no significant differences in the numbers and types of bacteria found in three patients groups (Group A--19 phlegmonous; Group B--19 gangrenous; and Group C--12 perforative appendicitis cases). Serological investigations of the humoral antibody responses (IFL) against the predominant bacteria in the three patient groups showed that a doubling, or more, of the titer was observed in the following cases: against B. fragilis, 3 out of 8 patients (Group A); 5 out of 7 patients (Group B); and 7 out of 7 patients (Group C). The corresponding figures for E. coli were 4 out of 18, 3 out of 17, and 5 out of 12 patients. Only minor responses against the remaining aerobic and anaerobic isolates were measured. From the bacteriological and immunological results we conclude that (1) the B. fragilis group were the predominant bacteria in diseased appendices; (2) that significant antibody responses were only observed against B. fragilis; (3) that the response increased with the degree of organ destruction and the length of time elapsed from the onset of the symptoms; (4) that B. fragilis is likely to play an important role in the pathogenesis of acute appendicitis, provided that other predisposing factors, such as a deterioration of the blood circulation in the appendiceal wall, are present.
Bacteroides fragilis
Group B
Group A
Aerobic bacteria
Fusobacterium
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Endotoxin, a lipopolysaccharide (LPS), has for many years been recognized as a key effector molecule in the pathogenesis of gram-negative sepsis and septic shock. Seven strains of the Bacteroides fragilis group were studied for their ability to liberate endotoxin upon exposure to five anti-anaerobic antibiotics, trovafloxacin, cefoxitin, imipenem, meropenem and piperacillin/tazobactam, in an in-vitro experiment. The minimum inhibitory concentrations (MICs) of the antibiotics were determined by using the broth macrodilution technique. Thereafter, endotoxin liberation was detected in the filtered broth cultures of the anaerobic bacteria by the limulus amebocyte lysate (LAL) assay after exposing the organisms to four different concentrations of the antibiotics in supplemented Brucella broth. Aliquots of the broth cultures were also taken at intervals of 0, 6, 24 and 48 h for viable counts. All seven gram-negative anaerobic bacteria investigated liberated induced cell-free endotoxin in filtered broth culture many times higher than the control. There was noticeable variation in the propensity of some antibiotics to induce endotoxin liberation. At four times the MICs, cefoxitin and piperacillin/tazobactam induced negligible quantities of endotoxin after 48 h exposure, whereas the others induced high levels of endotoxin release. After exposure to all concentrations for 48 h, endotoxin activity in the test system was many times higher with the Bacteroides fragilis sensu stricto than with the rest of the species in the Bacteroides group. To varying degrees, all five antibiotics had the capacity to induce endotoxin liberation by gram-negative anaerobic bacteria. This differential endotoxin release by the B. fragilis group may, in part, explain why B. fragilis sensu stricto, more than the other Bacteroides spp., is usually associated with clinical infections and higher morbidity.
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Cefotetan
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Bacteroides fragilis
Aerobic bacteria
Bacteroidaceae
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Experimental anaerobic infections in mice are reviewed with a description of a model of pure Bacteroides fragilis infection. In this experimental situation, B. fragilis produces large subcutaneous abscesses in the groins of mice that can be diagnosed without autopsy. This infection was treated effectively with clindamycin in doses that produced levels of drug in blood similar to those attainable in humans.
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Attempts were made to study the pathogenicity of some strains of Bacteroides fragilis group in the rat intra-abdominal abscess model. Multiple intraabdominal abscesses were produced in 50 to 70% of animals when an inoculum containing 10(9) CFU/ml of any of the five species of Bacteroides fragilis group was injected. Rising homologous antibody titers determined by indirect fluorescent antibody test were observed till the 3rd week when tested last, indirectly confirming the multiplication of the organisms as also evident by viable count of bacteria in the abscesses. In some cases in addition to inoculated organisms some intestinal bacteria like Escherichia coli, Proteus mirabilis and Streptococcus spp. were also recovered from the abscess pus. Studies with the electron microscope showed presence of capsular polysaccharide only in Bacteroides fragilis and Bacteroides thetaiotaomicron. It was doubtful in Bacteroides distasonis and absent in Bacteroides ovatus and Bacteroides vulgatus, suggesting that virulence factor beside the capsular polysaccharide may be playing a role. Further studies are required to investigate the virulence factor responsible for the pathogenicity of noncapsulated species.
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Bacteroidaceae
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