Enterotoxigenic Bacteroides fragilis in Hungary

Bacteroides fragilis, which constitutes about 1% of the colonic microflora in humans, is the most frequent anaerobic species involved in abscesses, soft-tissue infections and bacteraemias. Additionally, enterotoxigenic strains of B. fragilis have been demonstrated to be associated with diarrhoea in domestic animals and humans. Enterotoxigenic strains of B. fragilis derived from stool specimens and from infectious processes produce a toxin which induces a cytotoxic response in HT-29 colon carcinoma cells. These findings prompted us to investigate the prevalence of enterotoxigenic strains of B. fragilis isolated from various clinical specimens in Hungary. A total of 134 strains were collected from different clinical settings: 74 from infectious processes, 20 from stools of healthy subjects and 40 from the faeces of patients with diarrhoea where no other enteric pathogen could be isolated. Cell culture assays with HT-29 cells were performed on the filtered culture supernatants of the isolated strains. Of the 134 strains, 34 (25.3%) proved toxin-positive. The presence of free toxin was also observed in 20 of 50 (40%) of the faeces of adults with diarrhoea.     

Enterotoxin-producing Bacteroides fragilis strains isolated from horses]

Seven Bacteroides fragilis strains were cultured from samples collected from horses. From all the tested strains, as well as from the reference B. fragilis strains: enterotoxigenic NCTC 11925 and nonenterotoxigenic IPL 323 strain, DNA was isolated using Genomic DNA PREP PLUS isolation kit manufactured by A&A Biotechnology (Poland). To detect the enterotoxin (fragilysin) gene, polymerase chain reaction (PCR) was applied, using the following starters: 404 (GAG CCG AAG ACG GTG TAT GTG ATT TGT) and 407 (TGC TCA GCG CCC AGT ATA TGA CCT AGT). DNA obtained from bacterial cells was amplified in a thermocycler (Techne). The temperature profile was as follows: 1 cycle (4 min. 94 degrees C), 40 cycles (1 min. 94 degrees C, 1 min. 52 degrees C, 1 min. 74 degrees C). Amplification products were detected by electrophoresis in agarose gel (1%) with ethidium bromide added. The presence of the fragilysin gene was detected in two strains. Among the strains isolated from horses enterotoxin gene-possessing Bacteroides fragilis strains (ETBF) can be detected.     

Transferable 5-nitroimidazole resistance in the Bacteroides fragilis group

We report the characterization of a strain of Bacteroides vulgatus, BV17, that exhibits a moderate resistance to 5-nitroimidazoles and carries plasmids of 4.5, 5, 7.7, and 56 kb. A genetic determinant involved in this resistance is carried by the 7.7 +/- 0.2-kb plasmid (pIP417). This plasmid can be introduced and replicated in a sensitive strain of B. fragilis 638R by transformation or by conjugation. In the latter case, the transfer may involve mobilization by the 56-kb conjugative plasmid (pIP418) regularly found in transconjugants but not in transformants.     

Enhanced pathogenicity of susceptible strains of the Bacteroides fragilis group subjected to low doses of metronidazole

Different concentrations of metronidazole are used widely to treat protozoan and fungal infections. As an antibacterial drug, metronidazole is mainly used against anaerobes, of which the Bacteroides fragilis group is the most important in terms of the frequency of recovery and antimicrobial resistance patterns. The objective of this study was to investigate (1) in vivo metronidazole-induced modifications in the B. fragilis group reflected by altered virulence, and (2) the interference of metronidazole in cellular viability of these samples when subjected in vitro to human polymorphonuclear leukocytes (PMNs). Strains adapted to low metronidazole concentrations were observed to be more virulent, as demonstrated experimentally in mice by weight loss, quantitative evidence of tissue damage, hemorrhage and anatomopathology of spleen, liver and small intestine samples. A significant increase (P < 0.05) in mean bacterial viability rate of about 2.62-fold was observed for all the drug-adapted strains after contact with human PMNs. However, the level of this phenomenon was quite different among the tested species. These results draw attention to the risk that prolonged therapy, even with low concentrations of metronidazole, may affect the pathogenicity of Bacteroides strains, producing changes in host-bacteria relationships.     

Pathogenicity of Bacteroides fragilis group in rat intra-abdominal abscesses.

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.     

Growth of Bacteroides fragilis group in agar and broth media

The rate of bacterial growth of four Bacteroides fragilis group organisms was determined in agar and broth media. Exponential bacterial growth occurred in agar media within 4 to 8 h, while such growth was delayed in broth media and occurred within 12-24 h after inoculation. This phenomenon may explain why antimicrobials which manifest an 'inoculum effect' may show increased resistance to antimicrobials when tested in agar media.     

Isolation and identification of strains of Bacteroides fragilis group from the digestive tract of Callithrix penicillata marmosets

Thirty-five strains of the Bacteroides fragilis group were isolated from oral and intestinal samples from 5 wild caught, captive Callithrix penicillata. Nine oral strains of Bacteroides fragilis (7) and Bacteroides distasonis (2), and 26 intestinal strains of Bacteroides fragilis (14) and Bacteroides distasonis (12) were identified.     

Detection of 2-keto-3-deoxyoctonate in endotoxins isolated from six reference strains of the Bacteroides fragilis group

1. Endotoxins isolated from six serotype specific reference strains of the Bacteroides fragilis group were dephosphorylated by treatment with aqueous 50% hydrofluoric acid. 2. Mild acidic hydrolysis of the dephosphorylated endotoxins released 2-keto-3-deoxyaldonic acid, the presence of which was demonstrated by the colorimetric thiobarbituric acid assay (TBA). 3. Thin layer chromatography of the dephosphorylated lipopolysaccharide of B. fragilis IPL E 323 (serotype E2), after acidic hydrolysis, revealed a TBA-positive substance with the same Rf-value as authentical 2-keto-3-deoxyoctolusonic acid (KDO). 4. Quantification of 2-keto-3-deoxyoctonate-in the lipopolysaccharide of B. fragilis IPL E 323 by means of the TBA resulted in a KDO content of 15 nM mg-1 lipopolysaccharide.     

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.     

Antigenic properties of LPS extracted from Bacteroides fragilis enterotoxin producing strains

Antigenic properties of enterotoxigenic Bacteroides fragilis (ETBF) strains isolated in Poland were compared with reference strains. The agglutination and passive hemagglutination, SDS-PAGE analysis and immunoblotting tests as well as analyses of sugars and fatty acids were performed with lipopolysaccharide (LPS) preparations obtained from water-phase of phenol-water extracts. Some differences in serological reactivity between ETBF antigens were observed. The antigen of the NTBF (nonenterotoxigenic) reference strain IPL E-323 expressed weak cross-reactivity with sera against whole cells of ETBF strains in serological tests. There were some differences observed between ETBF and NTBF strains in fatty acids and sugar composition. The LPS preparations probably possess a common core structure and the O-specific polysaccharides of variable chain length.     

Clinical Importance of Infections due to Bacteroides fragilis and Role of Antibiotic Therapy

Out of 200 infections due to Bacteroides fragilis occurring over a period of three years 133 were related to the intestinal tract, 55 to the genitourinary tract, and the remainder were in bedsores and ulcers; 56% occurred in patients undergoing major intestinal surgery.B. fragilis was isolated in pure culture from 56% of the infections. In mixed culture it was most commonly associated with Klebsiella and Enterobacter species. Other anaerobic bacteria were isolated in 9% of the mixed cultures.Altogether 131 (65·5%) of the patients recovered without antibiotic therapy or further surgery, but 59 (29·5%) developed complications and 10 (5%) died. The commonest complication was abscess formation, and the incidence was highest with infections associated with malignancy (44%) and lowest with obstetric infections (5%). The mortality was 5% overall but in the presence of bacteraemia it rose to 33%.Only 43 patients received appropriate chemotherapy. Clindamycin was the most effective antibiotic, having a recovery rate of 78%, but this rate was little better than in untreated patients (65%). The role of prophylactic antibiotic therapy in preventing bacteroides infection remains to be studied.The incidence of the isolation of bacteroides from wound infections after major intestinal surgery rose from 13% in 1970 to 81% in 1973. This increase was due to both the accurate collection and care of specimens while in transit to the laboratory and the use of selective media for the isolation of bacteroides in laboratory culture. The importance of these precautions is emphasized.     

In vitro antibiotic susceptibility of Bacteroides fragilis strains isolated from excised appendix of patients with phlegmonous or gangrenous appendicitis

Out of 34 studied after-appendectomy tissues of adult and child patients 86 different strains of anaerobes were isolated. The antibiotic susceptibility of 30 isolated B. fragilis strains was tested using E tests. All studied strains were sensitive to imipenem, clindamycin and penicillin/tazobactam. Sensitivity to penicillin and cefoxitin was variable among these strains. One strain resistant to metronidazole (MIC--256 mg/L) and 3 strains with increased MIC to metronidazole were detected. Most of isolated strains were beta-lactamase producers.     

Haemagglutination of Bacteroides fragilis

Abstract The relationship between the ability to cause haemagglutination (HA) and the presence of capsules and/or pili was examined for 50 clinical isolates of Bacteroides fragilis . HA was tested using a slide technique, and bovine, porcine, guinea pig, rat, rabbit, horse, human, chicken and pigeon erythrocytes. Chicken and pigeon erythrocytes were the best indicators for HA with 43 (86%) of the strains tested causing HA and 39 (78%) with strong reactions. Capsule staining showed that the same 43 strains causing HA also produced a demonstrable capsule. No pili were found on either encapsulated or non-encapsulated strains using transmission electron microscopy. These results suggest that adherence of B. fragilis is related to the presence of capsular material, not pili.     

Multiple forms of 7-alpha-hydroxysteroid dehydrogenase in selected strains of Bacteroides fragilis.

Multiple forms of 7-alpha-hydroxysteroid dehydrogenase were detected in six of nine strains of Bacteroides fragilis. The enzymes differed with respect to pyridine nucleotide specificity, thermal stability, divalent metal cation requirement, and elution profilies from Sephadex G-200 columns. The nicotinamide adenine dinucleotide phosphate (NADP)-dependent enzyme required divalent metal cations, preferentially Mn-2+ (Km, 57 muM), for maximum catalytic activity. The NADP-dependent enzyme was labile at 65 C for 10 min, whereas the nicotinamide adenine dinucleotide (NAD)-dependent enzyme was stable at 65 C for 10 min. The specific activity of both the NAD- and NADP-dependent enzymes in crude extracts increased markedly (15- and 7.5-fold, respectively) during the transition from exponential- to stationary-phase growth in glucose medium containing 0.5 mM sodium cholate. The time course of apparent enzyme induction correlated temporally with the transformation of the 7-alpha-hydroxy group of cholate in the culture supernatant fluid. Both NAD- and NADP-dependent 7-alpha-hydroxysteroid dehydrogenase activities were found to be widely, but not universally, distributed in different strains and subspecies of B. fragilis. No NAD- or NADP-dependent 7-alpha-hydroxysteroid dehydrogenase activity could be detected in B. fragilis subsp. vulgatus Virginia Polytechnic Institute (VPI) no. 4245, subsp. thetaiotaomicron VPI 0061-1, or subsp. distasonis VPI 4243.     

Neuraminidase in Bacteroides fragilis.

A neuraminidase from Bacteroides fragilis was purified 542-fold by isoelectric focusing, adsorption chromatography on Affi-Gel 202, and gel filtration chromatography on Sephadex G-200. On isoelectric focusing the neuraminidase was resolved into three differently charged fractions with pI values of 6.8, 7.1, and 7.4. The major component of pI 7.1 was used for further purification. The purified enzyme had optimal activity at pH 6.4 with N-acetylneuraminlactose as the substrate. Its molecular weight, determined by Sephadex G-200 gel filtration chromatography, was 92,000. The neuraminidase hydrolyzed terminal neuraminic acid residues from N-acetylneuraminlactose, fetuin, bovine submaxillary mucin, and porcine stomach lining mucin. A new method for the detection of neuraminidase activity is described which is based on rocket affinoelectrophoresis. It utilizes the differences in the interaction of sialylated and desialylated mucin with Helix pomatia lectin, enzymatic activity being detected by formation of affinorockets after incubation of the neuraminidase with bovine submaxillary mucin.     

Antibiotic resistance patterns and plasmids of ruminal strains of Bacteroides ruminicola and Bacteroides multiacidus

Summary Plasmids were recovered and are described from three ruminal Bacteroides strains — 23, 223/M2/7 and 46/5(2). Although all three were originally isolated as Bacteroides ruminicola, 46/5(2) is shown here to be a strain of Bacteroides multiacidus, a species not previously described from the rumen. An 11.7 kbp plasmid present in strain 46/5(2) gave the same digestion pattern with Sal I and Sma I as a plasmid in B. multiacidus subgroup b type strain P208-58. Strains 46/5(2) and P208-58 both showed resistance to tetracycline, as did B. ruminicola strain 223/M2/7. B. multiacidus P208-58, and, to a lesser degree, B. ruminicola 23, showed resistance to ampicillin. Four other strains of B. ruminicola and one of B. multiacidus in which plasmids were not detected were sensitive to both antibiotics. It has still to be established whether these resistance traits are plasmid or chromosomally coded.     

Nutritional Features of Bacteroides fragilis subsp. fragilis

Studies of three reference strains of Bacteroides fragilis subsp. fragilis showed that they grow well in a minimal defined medium containing glucose, hemin, vitamin B₁₂, minerals, bicarbonate-carbon dioxide buffer, NH₄Cl, and sulfide. The vitamin B₁₂ requirement of 0.1 ng/ml was replaced with 7.5 μg of methionine. Cysteine or sulfide was an excellent source of sulfur, thioglycolate was a poor source, and thiosulfate, methionine, β-mercaptoethanol, dithiothreitol, sulfate, or sulfite did not serve as sole sources of sulfur. Neither single amino acids, nitrate, urea, nor a complex mixture of L-amino acids or peptides effectively replaced ammonia as the nitrogen source. Comparative studies with a few strains of other subspecies of B. fragilis including B. fragilis subsp. vulgatus, B. fragilis subsp. thetaiotaomicron, and B. fragilis subsp. distasonis indicate that they exhibit similar growth responses in the minimal medium. A single strain of B. fragilis subsp. ovatus required other materials. The results indicate the great biosynthetic ability of these organisms and suggest that, in their ecological niche within the large intestine, many nutrients such as amino acids are in very low supply, whereas materials such as ammonia, heme, and vitamin B₁₂, or related compounds, must be available during much of the time.     

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.     

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.     

The enumeration of Bacteroides fragilis group organisms from sewage and natural waters

A membrane filtration technique has been developed for the enumeration of Bacteroides fragilis group (BFG) organisms from sewage and natural waters. The method uses the agar medium of Wilkins and Chalgren supplemented with gentamicin, penicillin, aesculin and ferric ammonium citrate. Membrane filters with 0.22 micron pores were significantly more efficient than those with 0.45 micron pores in the isolation of BFG. A preliminary incubation period of 4 h at 30 degrees C prior to 44 h at 37 degrees C yielded significantly higher numbers of BFG than direct incubation at 37 degrees C for 48 h.