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Localized sinus inflammation in a rabbit sinusitis model induced by Bacteroides fragilis is accompanied by rigorous immune responses.

We evaluated inflammatory and immune responses against Bacteroides fragilis in a rabbit sinusitis model. Bacteroides was inoculated into the left maxillary sinus, and inflammatory (histology, cell number/cytology, lactose dehydrogenase, and apoptosis) and immune responses in the sinus, airway, and peripheral blood (PB) were determined for up to 4 weeks. In the inflamed sinus, the lactose dehydrogenase level was markedly elevated, with neutrophilic infiltration, severe tissue inflammation, and increased apoptosis. Low-grade tissue inflammation was present in the contralateral and sham-operated sinuses, but other parameters remained unchanged, and so did those in the airway and PB in the inoculated rabbits. Serum IgG antibody levels increased rapidly, were highest at 3 weeks, and began to decline at 4 weeks. Cellular immune responses (proliferation and interferon-gamma mRNA expression) against Bacteroides were detected in the PB of all inoculated rabbits. Vigorous immune responses against Bacteroides may have localized but failed to terminate inflammation in the sinus, indicating importance of microenvironmental factors.

Animals↗

Comparison of oral immediate-release (IR) and extended-release (ER) metronidazole bactericidal activity against Bacteroides spp. using an in vitro model of infection.

We used an anaerobic infection model capable of simulating human serum pharmacokinetic parameters (C(max), C(min), half-life, and AUC) to compare the activity of oral immediate-release (IR) and extended-release (ER) MTZ formulations. Four oral regimens of MTZ plus a control regimen were simulated in the model: [i] (IR) 500 mg q8h, [ii] ER 750 mg q12h, [iii] ER 750 mg q24h, and [iv] ER 1500 mg q24h. Two clinical Bacteroides fragilis isolates (MICs 0.5, 4.0 microg/mL) and two non-fragilis Bacteroides isolates (MICs 0.5, 3 microg/mL) were studied. All four oral MTZ regimens exhibited rapid, bactericidal activity (> or =3-log(10) decrease from the starting inoculum) within 12 h against both fragilis and non-fragilis Bacteroides isolates. Overall, no appreciable difference in the rate of bacteria killing was noted among the four MTZ formulations against either B. fragilis isolates (P = 0.907, 0.737) or non-fragilis isolates (P = 0.809, 0.768). We conclude that ER MTZ dosed at 12 or 24-h intervals possesses equivalent bactericidal activity to standard IR MTZ dosed every eight hours against susceptible Bacteroides spp.

Administration, Oral↗

In vitro susceptibility and in vivo efficacy of antimicrobials in the treatment of Bacteroides fragilis-Escherichia coli infection in mice.

Cefamandole, cefoxitin, cefotetan, ceftizoxime, imipenem plus cilastatin, and ampicillin plus sulbactam were compared in the eradication of subcutaneous abscess in mice caused by Bacteroides fragilis group organisms and Escherichia coli alone or in combination. The abscesses were examined 5 d after inoculation. B. fragilis group reached log10.1-11.0 organisms per abscess and E. coli log11.6-12.5. Imipenem plus cilastatin significantly reduced (in 6.9-10.6 logs) the number of E. coli and all members of B. fragilis group alone or in all combinations. Ampicillin plus sulbactam reduced the numbers of all B. fragilis group (in 4.2-7.2 logs) but was less effective against E. coli (reduction of 1.8-4.2 logs). Cefoxitin was effective in significantly reducing (in 4.9-6.2 logs) the number of E. coli and all members of B. fragilis group alone or in all combinations. Cefotetan was effective against B. fragilis (reduction of 5.1-6.6 logs) and E. coli alone or in combination but did not reduce the number of Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Bacteroides ovatus. Ceftizoxime was effective against only B. ovatus (reduction of 3.7-5.8) and E. coli (reduction of 6.0-8.1 logs); it did not reduce the number of other organisms. Cefamandole was effective against only E. coli and was not effective against any member of the B. fragilis group. These in vivo data confirm the in vitro activity of these antimicrobials.

Abscess↗

In-vitro and in-vivo activities of a novel cephamycin MT-141 against the Bacteroides fragilis group in comparison with six cephem antibiotics.

MT-141 inhibited 90% of the clinical isolates of Bacteroides fragilis at 3.13 mg/l, whereas six reference cephem antibiotics inhibited them at 12.5- greater than 100 mg/l. MT-141 was stable to the Bacteroides beta-lactamases, but the stability alone could not explain the potent activity of MT-141 against the other Bacteroides spp., which produced only small amounts of beta-lactamase, but were resistant to other cephem antibiotics. MT-141 exhibited a higher killing rate and more potent in-vivo efficacy than the reference cephem antibiotics against Bact. fragilis No. 36 combined with Escherichia coli No. 29. In conclusion, the in-vitro and in-vivo anti-bacteroides activities of MT-141 ranked first among those of the cephem antibiotics compared.

Animals↗

Resistance to beta-lactam antibiotics in Bacteroides spp.

Bacteroides spp., particularly B. fragilis, are well-recognised bacterial pathogens. Production of the typical beta-lactamases of Bacteroides restricts the therapeutic use of beta-lactam agents mainly to the beta-lactamase inhibitor combinations and carbapenems. These compounds have the advantage of broad-spectrum activity and the ability to combat polymicrobial infections. Resistance of Bacteroides spp. to beta-lactam antibiotics appears to be increasing, largely because of an overall increase in beta-lactamase activity. There has been a rise in the prevalence of isolates showing high-level production of typical Bacteroides beta-lactamases and an increase in reports other potent beta-lactamase types. In the case of B. fragilis, metallo-enzymes are a particular threat to current therapeutic practice, as they are not inhibited by common beta-lactamase inhibitors and are able to hydrolyse carbapenems. The presence of permeability barriers may confer low-level beta-lactam resistance and supplement the effect of beta-lactamase activity. There are also sporadic reports of loss of beta-lactam activity because of reduced affinity of the penicillin-binding proteins.

Anti-Bacterial Agents↗

Deconjugation of bile salts by Bacteroids and Clostridium.

Deconjugation of bile salts by four strains of Bacteroides and four strains of Clostridium was studied by use of resting cells and cell-free culture supernatants. Bacteroids strains yielded active cells but showed relatively low bile salt hydrolase (BSH) activity in the culture supernatants while the reverse was the case for the spore-forming clostridial strains. BSH was formed constitutively and was oxygen insensitive. The optimum pH was between 4.5 and 5.0. Marked substrate specificity was found in two strains, one Clostridium and one Bacteroides, which showed restricted activity against taurine conjugates. Bacteroides in general attacked the taurine conjugates of dihydroxy bile acids more readily than the trihydroxy taurine conjugates. Deconjugated bile acid moieties were further modified by some resting cells, depending on the bacterial strain while no enzymatic activity other than that of BSH was found in the culture supernatants. Cells of B. fragilis 2536 performed 7 alpha-dehydrogenation when the pH of the medium allowed the reaction, and this oxidative process was markedly enhanced in the presence of an abundant supply of oxygen as a terminal electron acceptor. C. perfringens PB 6K produced the 3- keto product in addition to the 3 beta-hydroxy derivative of the liberated bile acids and the formation of the latter derivative seemed to take place without preliminary deconjugation.

Amidohydrolases↗

Genetic variation in 16S-23S rDNA internal transcribed spacer regions and the possible use of this genetic variation for molecular diagnosis of Bacteroides species.

The structural variation in 16S-23S rDNA internal transcribed spacer regions (ITS) among Bacteroides species was assessed by PCR amplification and sequencing analysis, and its possible use for molecular diagnosis of these species was evaluated. Ninety strains of the genus Bacteroides, including the species B. distasonis, B. eggerthii, B. fragilis, B. ovatus, B. thetaiotaomicron, B. uniformis and B. vulgatus, produced one to three ITS amplification products with sizes ranging from 615 to 810 bp. Some Bacteroides strains could be differentiated at species level on the basis of ITS amplification patterns and restriction fragment length polymorphism (RFLP) analysis using a four-nucleotide-recognizing enzyme, Msp I. The results of sequence analysis of ITS amplification products revealed genes for Ile-tRNA and Ala-tRNA in all strains tested. The nucleotide sequence, except for that in tRNA-coding regions, was highly variable and characteristic for each species, but a common sequence among B. fragilis, B. thetaiotaomicron and B. ovatus was observed. A digoxigenin-labeled oligonucleotide probe (named FOT1), which was designed from this conserved sequence, specifically hybridized to the ITS amplification products from B. fragilis, B. thetaiotaomicron and B. ovatus. These results suggest that the ITS region is a useful target for the development of rapid and accurate techniques for identification of Bacteroides species.

Bacteroides↗

The suppressive effect of bifidobacteria on Bacteroides vulgatus, a putative pathogenic microbe in inflammatory bowel disease.

Bacteroides, a predominant commensal bacteria in the gut, are thought to be responsible for the development of inflammatory bowel disease (IBD). In the present study, we examined whether or not bifidobacteria suppress B. vulgatus, a representative pathogenic Bacteroides species, in both the coculture system and the gnotobiotic murine model. As a result, Bifidobacterium infantis 1222 highly inhibited the growth of B. vulgatus in the coculture and also significantly suppressed the systemic antibody response raised by B. vulgatus colonizing the gut in gnotobiotic mice. Colonization of the mice by B. vulgatus increased the number of Peyer's patch (PP) cells bearing PNA (peanut agglutinin)+/anti-kappa+ phenotype, which represents plasma cell-like B cells. Moreover, treatment of those B. vulgatus-implanted mice with B. infantis 1222 abrogated such increase in the number of PNA+/anti-kappa+ cells. These results thus suggested that B. infantis 1222 protected the gut epithelial layer including the PP from being invaded by Bacteroides, thereby suppressing the systemic antibody response raised by Bacteroides.

Administration, Rectal↗

Rapid differentiation of the species of the genus Bacteroides sensu stricto by capillary gas chromatography of cellular carbohydrates.

Whole-cell hydrolysates of Bacteroides fragilis, the type species of the genus Bacteroides Castellani and Chalmers 1919, and the genetical closely related species B. vulgatus, B. ovatus, B. eggerthii, B. distasonis, B. uniformis, B. thetaiotaomicron, B. stercoris, B. merdae, and B. caccae were used to determine characteristic carbohydrate patterns by capillary gas chromatography. On the basis of the chemical derivatization of the carbohydrates seven characteristic peaks for peracetylated aldononitriles and nine characteristic peaks for peracetylated o-methyloximes were selected from the carbohydrate fingerprints of the reference strains to prepare a dichotomous identification key. The classification of an unknown strain supposed to belong to the formerly called 'Bacteroides fragilis group' is possible with this key. Some of the advantages of the technique were that the identification of Bacteroides fragilis-like strains requires only 4-5 h after primary isolation and that the bacteria can be exposed to oxygen because viability of the organisms is not necessary. Sophisticated anaerobic techniques can therefore be avoided for identification.

Bacterial Typing Techniques↗

In vitro activity of MK0787 (N-formimidoyl thienamycin) and other beta-lactam compounds against Bacteroides spp.

The susceptibilities of 82 strains of the Bacteroides fragilis group to eight beta-lactam compounds, lincomycin, and metronidazole were determined by using an agar dilution technique. MK0787 (N-formimidoyl thienamycin) was the most active compound, inhibiting all strains at a concentration of 1 microgram/ml. Metronidazole was the only other drug of similar activity. Of the beta-lactam compounds, cefoxitin and MK0787 showed uniform activity against all species, whereas most other compounds were relatively less active against Bacteroides distasonis and Bacteroides thetaiotaomicron than against B. fragilis and Bacteroides vulgatus. Using a well diffusion technique, we determined the relative stability of each beta-lactam compound to sonicated cultures of selected resistant strains. Whereas MK0787 was completely stable to inactivation--and with one exception, cefoxitin was also--ceftriaxone, cefotaxime, cephaloridine, and cefoperazone always showed some inactivation, often quite substantial. Moxalactam and ceftazidime were completely stable to some of the enzyme preparations.

Anti-Bacterial Agents↗

Sequence homology of clindamycin resistance determinants in clinical isolates of Bacteroides spp.

The sequence homology of clindamycin resistance (Clnr) determinants was studied in 16 Clnr Bacteroides strains. The isolates were surveyed for plasmid content, homology with the Clnr determinant of pBFTM10, and ability to transfer Clnr. The Clnr DNA probes used in the Southern hybridizations were pBFTM10 and a plasmid derivative containing an EcoRI fragment of pBFTM10 cloned into Escherichia coli. A total of 13 of 16 Clnr strains also carried tetracycline resistance, and 15 of 16 Clnr Bacteroides isolates showed homology with the Clnr determinant of pBFTM10. These data suggest that the previously characterized Clnr determinant of pBFTM10 is widely distributed in nature and may be found on either a plasmid or the chromosome. The Clnr Bacteroides fragilis strain which lacked homology with pBFTM10 also had different transfer properties; thus, more than one type of Clnr determinant may exist in Bacteroides spp.

Bacteroides↗

In vitro kinetic analysis of oligofructose consumption by Bacteroides and Bifidobacterium spp. indicates different degradation mechanisms.

The growth of pure cultures of Bacteroides thetaiotaomicron LMG 11262 and Bacteroides fragilis LMG 10263 on fructose and oligofructose was examined and compared to that of Bifidobacterium longum BB536 through in vitro laboratory fermentations. Gas chromatography (GC) analysis was used to determine the different fractions of oligofructose and their degradation during the fermentation process. Both B. thetaiotaomicron LMG 11262 and B. fragilis LMG 10263 were able to grow on oligofructose as fast as on fructose, succinic acid being the major metabolite produced by both strains. B. longum BB536 grew slower on oligofructose than on fructose. Acetic acid and lactic acid were the main metabolites produced when fructose was used as the sole energy source. Increased amounts of formic acid and ethanol were produced when oligofructose was used as an energy source at the cost of lactic acid. Detailed kinetic analysis revealed a preferential metabolism of the short oligofructose fractions (e.g., F2 and F3) for B. longum BB536. After depletion of the short fractions, the larger oligofructose fractions (e.g., F4, GF4, F5, GF5, and F6) were metabolized, too. Both Bacteroides strains did not display such a preferential metabolism and degraded all oligofructose fractions simultaneously, transiently increasing the fructose concentration in the medium. This suggests a different mechanism for oligofructose breakdown between the strain of Bifidobacterium and both strains of Bacteroides, which helps to explain the bifidogenic nature of inulin-type fructans.

Bacteroides↗

Isolation of plasmid deoxyribonucleic acid from two strains of Bacteroides.

Two clinical isolates of Bacteroides contained covalently closed circular deoxyribonucleic acid (DNA) as shown by sedimentation in an alkaline sucrose gradient, CsCl ethidium bromide equilibrium centrifugation, and electron microscopy. Bacteriodes fragilis N1175 contained a homogeneous species of plasmid DNA with a molecular weight of 25 x 10(6). Bacteroides ochraceus 2228 contained two distinct, covalently closed circular DNA elements. The larger cosedimented with the covalently closed circular DNA form of the R plasmid, R100, corresponding to a molecular weight of 70 x 10(6); the smaller sedimented as a 58S molecule with a calculated molecular weight of 25 x 10(6). The roles of these plasmids are unknown. Neither strain transferred antibiotic resistance to plasmid-negative Bacteroides or Escherichia coli, and neither produced bacteriocins active against other Bacteroides or sensitive indicator strains of E. coli.

Anti-Bacterial Agents↗

Conjugal transfer of the 5-nitroimidazole resistance plasmid pIP417 from Bacteroides vulgatus BV-17: characterization and nucleotide sequence analysis of the mobilization region.

Three small 5-nitroimidazole (5-Ni) resistance plasmids (pIP417, pIP419, and pIP421) from Bacteroides clinical isolates are transferable by a conjugative process during homologous or heterologous matings. The mobilization properties of pIP417 originated from strain BV-17 of Bacteroides vulgatus were studied. The plasmid was successfully introduced by in vitro conjugation into different strains of Bacteroides and Prevotella species and could be transferred back from these various strains to a plasmid-free 5-Ni-sensitive Bacteroides fragilis strain, indicating that in vivo spread of the resistance gene may occur. The transfer of plasmid pIP417 harbored by the Tc(r) strain BF-2 of B. fragilis was stimulated by low concentrations of tetracycline or chlorotetracycline. This suggests a possible role for coresident conjugative transposons in the dissemination of 5-Ni resistance among gram-negative anaerobes. The nucleotide sequence of the 2.1-kb DNA mobilization region was determined. It contains a putative origin of transfer (oriT) in an A+T-rich-region, including three inverted repeats, and two integration host factor binding sites. The two identified mobilization genes (mobA and mobB) are organized in one operon and were both required for efficient transfer. Southern blotting indicated that the mobilization region of plasmid pIP417 is closely related to that of both the erythromycin resistance plasmid pBFTM1O and the 5-Ni resistance plasmid pIP419 but not to that of the 5-Ni resistance plasmid pIP421.

Amino Acid Sequence↗

ELECTRON MICROSCOPIC STUDY OF SOME STRAINS OF BACTEROIDES.

Bladen, Howard A. (National Institute of Dental Research, Bethesda, Md.), and Judith F. Waters. Electron microscopic study of some strains of Bacteroides. J. Bacteriol. 86:1339-1344. 1963.-Cultures of some unidentified strains of Bacteroides were fixed, and embedded in Vestopal W. Thin sections were cut with a microtome and examined in an electron microscope. A triple-layered cell wall completely encircled the organisms and was separated from the cytoplasmic membrane by a space of 150 to 250 A. This space was crossed by frequent bridges connecting the cell wall to the cytoplasmic membrane. A five-layered cytoplasmic membrane was occasionally observed. However, the outer two dense layers were often difficult to resolve as separate structures and usually appeared as a single thick dense line. Cytokinesis of Bacteroides involved the inward annular growth of the cell wall-cytoplasmic membrane. No cell-plate formation was observed. Intracellular membranous elements occurred in Bacteroides and are reported.

Bacteroides↗

Isolation of metronidazole-resistant Bacteroides fragilis carrying the nimA nitroreductase gene from a patient in Washington State.

Members of the Bacteroides fragilis group are among the most common anaerobic bacterial isolates in clinical specimens. Metronidazole, a 5-nitroimidazole, is often used as empirical therapy for anaerobic infections. Susceptibility testing is not routinely performed because of nearly universal susceptibility of Bacteroides spp. to this agent. We report a case of metronidazole-resistant Bacteroides fragilis in the United States and demonstrate the presence of the nimA gene, encoding a nitroreductase previously shown to mediate resistance to 5-nitroimidazole antimicrobial agents in B. fragilis strains from Europe and Africa. Because clinical failures in Bacteroides infections have been associated with the use of inactive antimicrobial agents, clinicians need to be aware of the possibility of metronidazole-resistant B. fragilis strains in the United States and the importance of susceptibility testing in selected situations.

Ankle Joint↗

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.

Abscess↗

Tetrapyrrole utilization of Bacteroides fragilis.

During growth in a defined medium containing protoheme, Bacteroides fragilis strain 2044 nd Bacteroides "l" 7CM formed protoheme-containing b-type cytochromes. The major reduced versus oxidized spectral peaks found for strain 2044 were at 562 and 428 nm whereas the peaks observed for strain 7CM were at 559 and 427 nm. When protoheme was replaced by deuteroporphyrin, mesoporphyrin, or their manganese or magnesium chelates, both organisms formed deuteroheme- or mesoheme-containing cytochromes with difference spectral peaks at 546 to 547 nm and 411 to 413 nm. Pyridine hemochromogen spectra of cells grown in the presence of tetrapyrroles containing the deuteroporphyrin or mesoporphyrin moiety confirmed the presence of deuteroheme of mesoheme in the cells. Although capable of delayed and relatively scant growth in media devoid of added tetrapyrroles or in media containing the vanadium chelates of mesoporphyrin or deuteroporphyrin, neither organism formed cytochromes under these growth conditions. The functional nature of the cytochromes containing deuteroheme or mesoheme was confirmed by their reduction by cells in the presence of 56 mM glucose and carbon dioxide compared with cells exposed to air. The use of tetrapyrroles by strains 2044 and 7CM is similar to that previously found for Bacteroides ruminicola ssp. ruminicola. Versatility in tetrapyrrole utilization for cytochrome synthesis appears to be a common characteristic of heme-requiring gastrointestinal Bacteroides species.

Bacteroides↗