Characterization of the Bacteroides fragilis pathogenicity island in human blood culture isolates

Bacteroides fragilis is an important anaerobic pathogen accounting for up to 10% of bacteremias in adult patients. Enterotoxin producing B. fragilis (ETBF) strains have been identified as enteric pathogens of children and adults. In order to further characterize the B. fragilis pathogenicity island (BfPAI) and using PCR assays for bft- and mpII-metalloprotease genes, we determined the frequency of B. fragilis strains with pattern I (containing the BfPAI and its flanking region), pattern II (lacking both the BfPAI and the flanking region), and pattern III (lacking the BfPAI but containing the flanking region) in 63 blood culture isolates. The results were compared to 197 B. fragilis isolates from different clinical sources. We found 19% of blood culture isolates were pattern I (ETBF), 43% were pattern II (NTBF) and 38% were pattern III (NTBF). Comparatively, B. fragilis isolates from other clinical sources were 10% pattern I, 47% pattern II and 43% pattern III. This suggests that the pathogenicity island and the flanking elements may be general virulence factors of B. fragilis.     

Recovery of Bacteroides fragilis group from clinical specimens following antimicrobial therapy.

Over a period of 14 years (1973-1987), 3165 specimens submitted to the microbiology laboratory demonstrated the recovery of anaerobic bacteria. A total of 988 Bacteroides fragilis group isolates were recovered (0.3 isolates per specimen). Bacteroides fragilis accounted for 62% of the total of all B. fragilis group isolates, Bacteroides thetaiotaomicron for 15%, Bacteroides vulgatus for 8%, Bacteroides ovatus for 7%, Bacteroides distasonis for 6%, and Bacteroides uniformis for 2%. Of the 988 B. fragilis group isolates, 310 (31%) were recovered after the administration of antimicrobial therapy, and 129 (13%) were the single isolate recovered from the infected site at that time. The recovery rate of all members of B. fragilis group after the administration of antimicrobial therapy, when isolated alone or when mixed with other bacteria, was similar. The data illustrate the equal ability of all members of the B. fragilis group to persist in and to contribute to the inflammatory process; and provide further support for their pathogenic role.     

Effect of Bacterial Contamination on Cecal Size and Cecal Contents of Gnotobiotic Rodents

In the present investigation the effect of various bacterial contaminations of gnotobiotic mice and rats on cecal size is presented. Of the species tested, Bacteroides oralis and Fusobacterium nucleatum did not establish in germ-free mice. Streptococcus mutans, Clostridium difficile, a Neisseria strain and two recent cecal isolates established, but failed to exert an effect upon the cecum of mice. A group K streptococcus and B. fragilis increased the cecal size apparently by increasing the levels of water-soluble protein, peptides, and carbohydrates in the cecal contents. Mixed ileal bacteria decreased the cecal size by preventing accumulation of soluble proteins and carbohydrates in the cecum. A Peptococcus strain caused a reduction by lowering the levels of insoluble material in the cecum. When this strain was combined with two Clostridium isolates and introduced into gnotobiotic rats, 50 to 65% cecal reduction was observed. This polycontamination did not decrease the per cent water of the cecal contents but caused lower levels of both soluble and insoluble material to accumulate in the cecum. No net nitrogen absorption from the distal small intestine occurred in either the germ-free or polycontaminated rats.     

Bacteroides and appendicitis (author's transl)]

Bacteroides fragilis is frequently recovered from cases of appendicitis with perforation and from infections developing secondary to appendicitis. In order to assess the part played by B. fragilis in the aetiology of appendicitis, quantitative aerobic and anaerobic culture studies of the contents of 49 inflammated appendices were performed. Anaerobic gram-negative non-sporing rods were cultivated from 43 appendices in the range 10(3)-10(9)/g. A total of 1,473 isolates was differentiated by biochemical methods, and 1,374 cultures were found to belong to the saccharolytic species of the genus Bacteroides (B. fragilis, B. thetaiotaomicron, B. vulgatus, B. distasonis etc.). B fragilis was detected in 31 appendices; the species predominated in 18 samples. B theraiotamicron, recovered from 27 samples, was prevalent in 4 appendices. In one sample, B. fragilis and B. thetaiotaomicron outnumbered the other appendicular bacterial. B. vulgatus was cultivated from 12 appendices, but did once constitute the prevalent group. It has been previously shown that B. vulgatus (43% of intestinal isolates) and B. thetaiomicron predominate in the normal narge bowel flora. On the other hand, approximately 80% of pyrogenic Bacteroides strains belong to B. fragilis, B. thetaiotaomicron accounting for 19% and B. vulgatus being virtually absent. From these striking differences in species distribution the conclusion was drawn that B. fragilis possesses the highest virulence for man. Species distribution within the 1,374 appendicular isolates of saccharolytic Bacteroides (percentages of 62, 19 and 4.3 for B. fragilis, B. thetaiotaomicron, and B. vulgatus, respectively) was very similar to that encountered in clinical specimens. From the results obtained it becomes evident that pyrogenic Bacteroides, in particular B. fragilis, plays an important role in nearly 50% of cases of appendicitis.     

Susceptibility trending of blood isolates of the Bacteroides fragilis group over a 12-year period to clindamycin, ampicillin-sulbactam, cefoxitin, imipenem, and metronidazole

Numerous reports have described a steady overall increase in resistance among clinical isolates of the Bacteroides fragilis group to several antimicrobial agents, particularly clindamycin. Determination of resistance rates is significantly influenced by the number of isolates of each species within the B. fragilis group tested. Historically, the B. fragilis species has remained the most susceptible to most antimicrobials when compared to non-B. fragilis species. This study compares the effect of a gradually changing ratio of blood isolates of B. fragilis to non-B. fragilis species tested by broth micro-dilution over a 12-year period on selected antimicrobial agents. In 1987, the ratio of blood isolates of B. fragilis to non-B. fragilis was 68% to 32%; in 1991 it was 59% to 41%; and in 1999 it was 51% to 49%. Both metronidazole and imipenem showed the least changes because of their inherent high activity against all species. For clindamycin, decreases in susceptibility ranged from 84% to 64% for B. fragilis compared to 58% to 67% for non-B. fragilis species. Ampicillin-sulbactam showed a decrease in susceptibility in B. fragilis and non-B. fragilis species, but was highest in 1999 when the ratio of non-B. fragilis species was the highest. Overall resistance rates to cefoxitin varied from 8% to 25% during the testing years and was consistently higher among the non-B. fragilis species. These comparisons indicate that the ratio of B. fragilis group species isolated from the blood has changed over the last 12 years and has appreciably affected the resistance rates to some commonly used anti-anaerobic agents. Whether the noted changes in species isolation rates are a result of selective antibiotic pressure or other factors is yet to be determined.     

In vitro activity of 11 antibiotics against 74 anaerobes isolated from pediatric intra-abdominal infections

The in vitro activity of 11 antimicrobials was tested against 74 recent anaerobic isolates obtained from pretreatment cultures in pediatric patients with complicated intra-abdominal infections using the CLSI M11-A-6 agar dilution method. Carbapenems, beta-lactamase inhibitor combinations and metronidazole retained good activity, while all Bacteroides fragilis group species produced beta-lactamase and were penicillin resistant and 43% were either intermediately susceptible or resistant to clindamycin. Cefoxitin had moderate activity against B. fragilis but poor activity against Bacteroides thetaiotaomicron and other B. fragilis group isolates.     

Susceptibility trends of Bacteroides fragilis group and characterisation of carbapenemase-producing strains by automated REP-PCR and MALDI TOF

Susceptibility testing of clinical isolates of anaerobic bacteria is not considered, often, mandatory in routine clinical practice and the treatments are empirically established. Thus, periodic monitoring of the susceptibility patterns of anaerobic bacteria is advisable. The aim of this study was to update on resistance of Bacteroides fragilis group in our Institution with special attention to carbapenems reporting metallo-beta-lactamase producing strains for the first time in Spain, and to compare fingerprinting analysis results obtained by using automated rep-PCR (DiversiLab System) and MALDI-TOF MS. A total of 830 non-duplicated clinical isolates of the B. fragilis group recovered from the years 2006 to 2010 were studied. B. fragilis was the most prevalent species (59.5%). The total susceptibility of B. fragilis group isolates were: penicillin, 13.3%; amoxicillin/clavulanic, 89.6%; piperacillin-tazobactam, 91.8%; cefoxitin, 65.8%; ertapenem, 95.9%; imipenem, 98.2%; clindamycin, 53.4% and metronidazole, 96.4%. The percentage of sensitive isolates did not change significantly over time for amoxicillin/clavulanic, cefoxitin, clindamycin and metronidazole. A slight increase in the rate of resistance to ertapenem and imipenem was observed. Imipenem resistance and carbapenemase production were detected for the first time in our laboratory in the year 2007. No other report of carbapenemase-producing B. fragilis in our country has been previously published. Six imipenem-resistant isolates were MBL-producing and PCR positive for cfiA gene. Four of them were PCR positive for IS-like immediately upstream cfiA gene and two of them were negative. Both, automated rep-PCR (DiversiLab) and MALDI-TOF MS, revealed a great genetic diversity among carbapenem-producing strains suggesting the acquisition of novel resistance genes more than clonal dissemination of them. Both methods seem to be useful tools for fast and accurate identification and strain typing of B. fragilis group in the daily laboratory routine. Because of the relevant increase observed in Bacteroides species isolated from blood cultures and the appearance of carbapenemase-producing strains in our Institution, we recommend to test the antimicrobial susceptibility of the isolates, at least in the most severe patients.     

Antimicrobial susceptibilities of Bacteroides fragilis and Bacteroides thetaiotaomicron strains isolated from clinical specimens and human intestinal microbiota

Species of Bacteroides fragilis group bacteria are the most prevalent pathogens and have the highest resistance rates to antimicrobial agents among anaerobic bacteria. Infections due to these micro-organisms often originate from patient's own intestinal microbiota. The objective of the study was to determine and compare the susceptibility profiles of clinical and intestinal B. fragilis and B. thetaiotaomicron strains against certain antimicrobials. Isolates were identified by conventional methods and API-20 A. Susceptibility tests were performed according to recommendations of NCCLS (M 11-A4) agar dilution methods. Beta-lactamase production was determined with nitrocefin discs. Forty-five clinical isolates (33 B. fragilis and 12 B. thetaiotaomicron) were from following sites: blood (n:8), intra-abdominal abscess (n:7), soft tissue (n:26), and miscellaneous foci of infection (n:4). Fifty B. fragilis and 60 B. thetaiotaomicron isolates from intestinal microbiota of individuals with no history of antimicrobial treatment within last 30 days were also examined. Beta-lactamase production was detected in 93% of clinical and 99% of intestinal isolates. The organisms including intestinal isolates were uniformly susceptible to metronidazole. The MIC90s of other antibiotics and resistance rates of all clinical isolates to those antibiotics were as follows: 256 microg/mL (93%) for ampicillin, 128 microg/mL (13%) for piperacillin, 64 microg/mL (11%) for cefoxitin, 1 microg/mL (2%) for amoxicillin-clavulanate, 0.5 microg/mL (2%) for imipenem, >256 microg/mL (36%) for clindamycin, 8 microg/mL (2%) for chloramphenicol. Intestinal isolates demonstrated similar resistance rates and MIC90s. Metronidazole, imipenem, amoxicillin-clavulanate seem to be effective drugs against these bacteria in Turkey.     

The bactericidal mechanisms of polymorphonuclear leukocytes against Bacteroides fragilis: significance of the oxygen-dependent system

The mechanism by which polymorphonuclear leukocytes (PMNs) kill ingested Bacteroides fragilis was examined using PMNs from patients with chronic granulomatous disease (CGD) which is an inherited disease characterized by the defect of their PMNs in oxygen-radical generation. The phagocytosis of B. fragilis by PMNs from CGD patients was comparable to that by normal PMNs. Although CGD cells killed B. fragilis to some extent, they did so less effectively than the normal PMNS. B. fragilis was killed by a xanthine oxidase system that generates oxygen radicals. When PMNs were incubated with opsonized B. fragilis, B. fragilis triggered the release of O2- and H2O2 from normal PMNs. Thus, normal PMNs appear to kill B. fragilis by both oxygen-dependent and oxygen-independent mechanisms.     

Comparison of the post-antibiotic effect (PAE) induced by ceftizoxime, ceftriaxone, cefoxitin, ampicillin-sulbactam, and ticarcillin-clavulanate against selected isolates of Bacteroides fragilis and B. thetaiotaomicron

The exposure of bacteria to various groups of antimicrobials at different concentrations can produce damage to the bacteria that persists even after removal of the antimicrobial agent. The post antibiotic effect (PAE) of beta-lactams on aerobic gram-negative bacilli is relatively short (<1 h), however, little information is available regarding anaerobic gram-negative bacilli. We studied the PAE of ceftizoxime, ampicillin-sulbactam, ticarcillin-clavulanate, cefoxitin, and ceftriaxone against strains of Bacteroides fragilis and B. thetaiotaomicron at antimicrobial concentrations 4x, 8x, and 16x the MIC values using colony count determinations of treated and untreated cultures. Against B. fragilis H931, ceftizoxime-induced PAE values were 2 h, 3 h 24 min, and 11 h 36 min at 4xMIC, 8xMIC, and 16xMIC while for the B. thetaiotaomicron isolates PAEs ranged from 2 h 27 min to 6 h 12 min at the same concentrations. Cefoxitin PAE values were 3 h 6 min and 2 h 18 min for the clinical isolates at 16xMIC and 3 h 24 min and 1 h 12 min against the laboratory strains at 16xMIC respectively, and for ceftriaxone 1 h 12 min and 5 h 12 min, respectively, for the B. thetaiotaomicron D933 and B. fragilis H931 strains at 16xMIC. With ampicillin-sulbactam, the longest PAE values were observed at 16xMIC with all the test isolates of B. fragilis and B. thetaiotaomicron. PAE values induced by ticarcillin-clavulanate overall were the shortest for the two clinical isolates. These studies indicate substantial PAE values for beta-lactams against selected anaerobes which may be an important factor in the dosing regimen of these test agents.     

Enumeration of enterotoxigenic Bacteroides fragilis in municipal sewage.

Of 237 isolates of Bacteroides fragilis from sewage influent at the Bozeman, Mont., wastewater treatment plant, 22 (9.3%) were enterotoxigenic, as indicated by the ability to elicit fluid accumulation in the lamb ileal loop test. It appears that enterotoxigenic B. fragilis is endemic in the human population at a moderately high level.     

Enumeration of enterotoxigenic Bacteroides fragilis in municipal sewage

Of 237 isolates of Bacteroides fragilis from sewage influent at the Bozeman, Mont., wastewater treatment plant, 22 (9.3%) were enterotoxigenic, as indicated by the ability to elicit fluid accumulation in the lamb ileal loop test. It appears that enterotoxigenic B. fragilis is endemic in the human population at a moderately high level.     

Rapid identification of the species of the Bacteroides fragilis group by multiplex PCR assays using group- and species-specific primers

We report a rapid and reliable two-step multiplex polymerase chain reaction (PCR) assay to identify the 10 Bacteroides fragilis group species - Bacteroides caccae, B. distasonis, B. eggerthii, B. fragilis, B. merdae, B. ovatus, B. stercoris, B. thetaiotaomicron, B. uniformis and B. vulgatus. These 10 species were first divided into three subgroups by multiplex PCR-G, followed by three multiplex PCR assays with three species-specific primer mixtures for identification to the species level. The primers were designed from nucleotide sequences of the 16S rRNA, the 16S-23S rRNA intergenic spacer region and part of the 23S rRNA gene. The established two-step multiplex PCR identification scheme was applied to the identification of 155 clinical isolates of the B. fragilis group that were previously identified to the species level by phenotypic tests. The new scheme was more accurate than phenotypic identification, which was accurate only 84.5% of the time. The multiplex PCR scheme established in this study is a simple, rapid and reliable method for the identification of the B. fragilis group species. This will permit more accurate assessment of the role of various B. fragilis group members in infections and of the degree of antimicrobial resistance in each of the group members.     

Survey of antimicrobial susceptibility patterns of the bacteria of the Bacteroides fragilis group isolated from the intestinal tract of children

The bacteria of the Bacteroides fragilis group are considered important clinical pathogens and they are the most common anaerobes isolated from human endogenous infections. In this study, the susceptibility patterns to antibiotics and metals of 114 species of the B. fragilis group isolated from children with and without diarrhea were determined. Susceptibility was assayed by using an agar dilution method with Wilkins-Chalgren agar. All B. fragilis strains were resistant to lead and nickel, but susceptible to metronidazole and imipenem. beta-lactamase production was detected by using biological and nitrocefin methods, respectively, in 50% and 90.6% of the isolates of children with diarrhea and in 60% and 90% of the isolates of children without diarrhea. Our results show an increase of antibiotics and metals resistance in this microbial group, and a periodic evaluation of the antimicrobial susceptibility is needed. In Brazil, the contamination for antibiotics or metal ions is often observed, and it is suggested an increase the antimicrobial resistance surveillance of this microbial group, mainly those isolated from children's diarrhea.     

Hybridization studies reveal homologies between pBF4 and pBFTM10, Two clindamycin-erythromycin resistance transfer plasmids of Bacteroides fragilis

Two clindamycin-erythromycin resistance transfer factors of Bacteroides fragilis, pBF4 and pBFTM10, were analyzed for regions of DNA homology. Although the plasmids were derived from different clinical isolates of B. fragilis and have different sizes, they showed homology in the clindamycin-erythromycin resistance region; no homology could be detected outside of this region.     

Occurrence of enterotoxigenic and nonenterotoxigenic Bacteroides fragilis in calves and evaluation of their antimicrobial susceptibility

Bacteroides fragilis is considered an important clinical pathogen and the most common anaerobe isolated from human and animal clinical specimens; enterotoxigenic strains produce diarrhea. The presence of enterotoxigenic (ETBF) and nonenterotoxigenic B. fragilis in stool samples from calves with or without acute diarrhea and the antimicrobial susceptibility of the strains were evaluated. The stool samples were plated onto a selective B. fragilis-bile-esculin agar, and incubated anaerobically (10% CO(2)/90% N(2)), at 37 degrees C, for 72 h. Species of the B. fragilis group were identified by using the API 32-A kit. Enterotoxigenic strains were detected by PCR and the cytotoxic assay. From 54 diarrhea and 54 nondiarrhea stools, 124 and 92 members of the B. fragilis group, respectively, were recovered. Only two ETBF strains were isolated from two different diarrhea samples and the bft gene was detected in both. Moreover, the bft gene was detected in DNA from four different diarrheal stools samples but no ETBF strain was recovered. All the bacteria were susceptible to chloramphenicol, imipenem, moxifloxacin, piperacillin/tazobactam, metronidazole and tigecycline. Most of the isolates from both calves with and without diarrhea were resistant to all metals. Our results are of concern, and suggest the need to increase the surveillance of antibiotic and metal resistance of this microbial group isolated from animal production such as calves.     

Neuraminidase production by Bacteroides species

Abstract 128 strains of Bacteroides isolated from clinical specimens were surveyed for their ability to produce neuraminidase. All strains of Bacteroides fragilis and the B. fragilis group were neuraminidase-positive, as were strains of B. oralis and B. bivius . All strains of B. capillosus, B. ruminicola, B. disiens, B. multiacidus and B. uniformis did not produce a detectable neuraminidase. When human erythrocytes were exposed to cell extracts of neuraminidase-producing Bacteroides , and then tested with peanut ( Arachis hypogeae ) lectin, agglutination occurred. It was concluded that the production of neuraminidase by clinical isolates of Bacteroides may be associated with the pathophysiology of severe Bacteroides infections.     

Update on resistance of Bacteroides fragilis group and related species with special attention to carbapenems 2006-2009

The susceptibility trends for the species of the Bacteroides fragilis group against various antibiotics were determined using data from 4 years [2006-2009] on 1957 isolates referred by 8 medical centers participating in a National Survey for the Susceptibility of B. fragilis. The antibiotic test panel included doripenem, ertapenem, imipenem, meropenem, ampicillin:sulbactam, piperacillin:tazobactam, cefoxitin, clindamycin, moxifloxacin, tigecycline, chloramphenicol and metronidazole. MICs were determined using agar dilution methods following CLSI recommendations. Genetic analysis of isolates from 2008 with elevated MICs (>2 μg/mL) to one or more of the carbapenems to detect presence of the cfiA gene was performed using PCR methodology. The results showed an increase in the resistance rates to the β-lactam antibiotics. High resistance rates were seen for clindamycin and moxifloxacin (as high as 60% for clindamycin and >80% for moxifloxacin), with relatively stable low resistance (5.4%) for tigecycline. For carbapenems, resistance in B. fragilis was 1.1%-2.5% in 2008-9. One isolate resistant to metronidazole (MIC 32 μg/mL) was observed as well as isolates with elevated MICs to chloramphenicol (16 μg/mL). Genetic analysis indicated that the cfiA gene was present in some but not all of the isolates with high MICs to the carbapenems. These data indicate that there continue to be changes in susceptibility over time, and that resistance can be seen among the carbapenems. High antibiotic resistance rates tend to be associated with specific species.     

Distribution of the human faecal bacterium Bacteroides fragilis, its bacteriophages and their relationship to current sewage pollution indicators in bathing water

Although several bacteria are currently used as possible indicators of human pathogens in sewage-polluted sea water, they are often viewed as inadequate and especially inadequate as indicators of viral pathogens. This study investigates the distribution of Bacteroides fragilis and closely related Bacteroides spp. and their associated bacteriophages in sea water frequently used for recreational purposes. These organisms may provide a potentially more appropriate indicator. Bacteroides fragilis is one of about 10 species which are loosely placed together in the 'B. fragilis' group. Samples down-current from a sewage outfall were examined for the presence of B. fragilis group organisms and associated bacteriophages. Numbers were correlated with current bacterial and possible viral indicators at these sites. These B. fragilis group isolates were used as hosts to successfully isolate bacteriophages. The host range of these bacteriophages was investigated. It is hoped to expand this study by using these B. fragilis group hosts and their bacteriophages to identify a more suitable, European-wide, indicator of bacterial pathogens which can also be used to detect bacteriophages which are suitable as viral indicators.     

Virulence markers and antimicrobial susceptibility of bacteria of the Bacteroides fragilis group isolated from stool of children with diarrhea in São Paulo, Brazil

Bacteroides fragilis has been isolated from several human and non-human monomicrobial and mixed infections. In this study, some virulence markers and the antimicrobial susceptibility of bacteria of the B. fragilis group isolated from children's stools were evaluated. All the 64 isolates showed the following characteristics: capsulated, beta-hemolytic, hydrophilic, and serum-resistant. Only, 24 (37.5%) strains were resistant at 60 masculine C, for 30 min, and among them, 12 (18.75%) were resistant at 60 masculine C, for 60 min. Also, none strain was resistant at 100 masculine C. Four strains were able to hemagglutinate erythrocytes and D-mannose, D-galactose, D-arabinose, and D-xylose inhibited hemagglutination in 2 B. fragilis strains (p76a, p76b). The hemagglutination in the strain B. uniformis p3-2 was inhibited by D-xylose and D-galactose. The bft gene detection and the enterotoxin production were observed only in 13 EF-enterotoxigenic species. Fragilysin activity was confirmed on HT-29 cells. The antimicrobial determination confirmed that both imipenem and metronidazole were efficient against B. fragilis species; all the strains were resistant to lead and nickel. Plasmids of 2.9, 4.4, 4.8, and 8.9 kb were observed in 6 tested strains. These results show the values of the species identification from clinical infections, as well as of the periodic evaluation of the resistance patterns of the B. fragilis group at Brazilian medical institutions.