In vitro activities of tinidazole and metronidazole against Clostridium difficile, Prevotella bivia and Bacteroides fragilis

Tinidazole, a 5-nitroimidazole similar to metronidazole, was studied against 40 Clostridium difficile, 10 Prevotella bivia and 11 Bacteroides fragilis clinical isolates. The geometric mean MICs of tinidazole and metronidazole were, respectively: C. difficile, 0.31 and 0.28 microg/mL; P. bivia, 2.33 and 1.52 microg/mL; B. fragilis, 0.5 and 0.71 microg/mL.     

Anti-anaerobic activity of serum from patients treated with tigecycline for skin/soft tissue infections

To gain additional data concerning the anti-anaerobic activity of tigecycline in serum, we analyzed blood samples from six patients with a complicated skin/soft tissue infection who were receiving IV tigecycline 50 mg every 12 h. Venous blood samples were obtained after multiple doses of tigecycline at 1, 6 and 12 h after the initiation of a 1 h IV infusion. Sera from these samples were tested to determine serum inhibitory and bactericidal activity over time against 4 anaerobic bacteria (Bacteroides fragilis, Peptoniphilus asaccharolyticus, Prevotella bivia and Finegoldia magna). An analysis of serum titers found that tigecycline exhibited early (1 h) and prolonged (12 h) inhibitory activity against each study isolate. Moreover, it provided bactericidal activity for 12 h against these strains with the exception of F. magna. Tigecycline was found to exhibit antibacterial activity at serum concentrations below the MICs of the anaerobic bacteria tested. This finding further supports that the antimicrobial activity of tigecycline can be greater than that suggested by the free fraction of drug and that serum appears to enhance this antibacterial activity.     

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.     

Review of the in vitro activity and potential clinical efficacy of levofloxacin in the treatment of anaerobic infections

The activity of levofloxacin against aerobic bacteria has been well documented both in vitro and clinically, but its anaerobic activity has been infrequently studied. This new fluoroquinolone exhibits good in vitro activity (MIC(S) < or =2.0 microg/mL) against many anaerobic pathogens associated with acute sinusitis, bite wounds, and other soft-tissue infections. It is less active against Bacteroides fragilis (MIC (90)=2-4 microg/mL ) and has poor inhibitory activity against non-fragilis B. fragilis group species that are associated with gastrointestinal and genitourinary tract infections. Levofloxacin does not antagonize the in vitro activity of clindamycin and metronidazole and often provides additive or synergistic activity against anaerobic bacteria with these agents. In pharmacodynamic models, levofloxacin exhibits rapid bactericidal activity at 2-4 times the MIC of anaerobic bacteria. Prolonged killing is observed when the area-under-the concentration-time-curve to MIC ratio is greater than 40. In clinical efficacy trials, levofloxacin has been effective in the treatment of patients with gynecologic, skin and skin-structure, and bone infections involving anaerobic pathogens. Both micro-biologic and pharmacodynamic studies support further evaluations of levofloxacin in the treatment of selective mixed aerobic/anaerobic infections.     

Bacteroides and Staphylococcus glycocalyx: chemical analysis, and the effects on chemiluminescence and chemotaxis of human polymorphonuclear leucocytes.

Glycocalyx (or slime), which is an important virulence factor of many pathogenic bacteria, was isolated from Bacteroides fragilis, Bacteroides thetaiotaomicron and Staphylococcus epidermidis. Organisms were grown for 24 h in a chemically defined, dialysable liquid medium. Bacteria were centrifuged and the supernatant was concentrated and dialysed against distilled water. Total carbohydrate and protein were estimated using standard methods. Thin layer and gas-liquid chromatography of trifluoro acetic acid hydrolysed and non-hydrolysed samples provided evidence for the presence of polysaccharide, the absence of nucleic acids and lipopolysaccharide and for the identification of the individual sugar residues. Glucose, mannose and galactose (B. fragilis), glucose (B. thetaiotaomicron), and glucose and heptose (S. epidermidis) were the sugar residues detected. Uronic acid and hexosamine were detected in all species. Glycocalyx preparations (1 mg/ml) from Bacteroides and Staphylococcus significantly inhibited the chemiluminescence and chemotactic responses of viable human polymorphonuclear leucocytes (PMNL), but were not toxic for PMNL.     

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.     

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.     

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.     

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.     

Use of randomly cloned DNA fragments for identification of Bacteroides thetaiotaomicron.

Randomly cloned fragments of DNA from Bacteroides thetaiotaomicron were used as hybridization probes for differentiation of B. thetaiotaomicron from closely related Bacteroides species. HindIII digestion fragments of DNA from B. thetaiotaomicron (type strain) were inserted into plasmid pBR322 and labeled with [alpha-32P]dCTP by nick translation. These labeled plasmids were screened for hybridization to HindIII digests of chromosomal DNA from type strains of the following human colonic Bacteroides species: B. thetaiotaomicron, Bacteroides ovatus, reference strain 3452-A (formerly part of B. distasonis), Bacteroides uniformis, Bacteroides fragilis, Bacteroides vulgatus, Bacteroides distasonis, Bacteroides eggerthii, and reference strain B5-21 (formerly B. fragilis subsp. a). Two of the five cloned fragments hybridized only to DNA from B. thetaiotaomicron. Each of these two fragments hybridized to the same DNA restriction fragment in five strains of B. thetaiotaomicron other than the strain from which the DNA was cloned. One of the cloned fragments (pBT2) was further tested for specificity by determining its ability to hybridize to DNA from 65 additional strains of colonic Bacteroides.     

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.     

Identification and strain differentiation of 'Bacteroides fragilis group' species and Prevotella bivia by PCR fingerprinting

Using single consensus primers of genomic nucleotide sequences, PCR-generated fingerprints were used for identification and differentiation of the Bacteroides fragilis group (B. fragilis, B. thetaiotaomicron, B. ovatus, B. distasonis, B. vulgatus) and Prevotella bivia (B. bivius) by comparing the DNA profiles with those of reference strains from the American Type Culture Collection and German Culture Collection. When primed by a single primer phage M13 core sequence, intra-species specific differences and species-specific bands were detected. Using primers derived from the evolutionarily conserved tRNA gene sequence, species-specific patterns were produced. A computer program, GelManager, was used to analyze the profiles and generate dendrograms. The correlation coefficients determined from the DNA fingerprint profiles of the clinical isolates (using the M13 core primer) fell within a narrow range, reflecting a high level of homology within the species. Based on the dendrograms, strains of one species were clearly differentiated from strains of other species. For comparison, SDS-PAGE analysis of whole cell extracts was also performed to obtain protein band patterns of various strains. Because of the simplicity of the PCR fingerprinting method and the ease of performance of computerized evaluation of data, this technique is a useful method for both species and strain differentiation, as well as for characterization of Bacteroides species and Prevotella bivia.     

Therapeutic efficacy of moxifloxacin in a murine model of severe systemic mixed infection with Escherichia coli and Bacteroides fragilis

The therapeutic efficacy of moxifloxacin was studied in an experimental murine model of a systemic aerobic/anaerobic mixed infection and compared to therapies with either imipenem or ciprofloxacin plus metronidazole. Groups of 20 mice each were intravenously (iv) infected with approximately 2.5 x 10(6) colony forming units (CFU) of Escherichia coli and 2 x 10(7)CFU of Bacteroides fragilis. Iv therapy was started 24 h post-infection (pi) with either moxifloxacin, imipenem, or ciprofloxacin plus metronidazole, for 3 days. A control group of 20 mice was left untreated. Survival rate at day seven pi was recorded, mice were then sacrificed and bacterial organ contents of livers and kidneys were determined. All mice treated survived at day seven, while six animals of the untreated group died. B. fragilis was not detected in any of the treated mice. E. coli was found in two of the moxifloxacin-treated mice and in two and five of the ciprofloxacin plus metronidazole and imipenem-treated animals, respectively. The results indicate that a therapy of severe mixed aerobic/anaerobic infections with moxifloxacin might be feasible and possibly be as efficacious as current therapy regimens with ciprofloxacin plus metronidazole or imipenem.     

Toxins of Bacteroides fragilis and Bacteroides thetaiotaomicron rods as stimulators of adhesion molecule expression on the surface of vascular endothelial cells

Lipopolysaccharides from four Bacteroides fragilis strains: one nonenterotoxigenic (NTBF) and three enterotoxigenic (ETBF), and from three B. thetaiotaomicron strains were extracted by hot phenol-water method and purified. B. fragilis enterotoxin was prepared according to the procedure of van Tassell et al. (1992). The influence of the examined toxins on the expression of adhesion molecules: ICAM-1, VCAM-1 and E-selectin on HMEC-1 (human dermal microvascular endothelial cells) was assayed in ELISA test with monoclonal antibodies. Four concentrations of toxins were applied: 0.01, 0.1, 1.0 and 10.0 (micrograms/ml). Endothelial cells were activated for 24 hours (ICAM-1 and VCAM-1 expression) and for 4 hours (E-selectin expression). The coloured product of immunoenzymatic reaction was measured by reading the absorbance at wavelength 492 nm. Two controls were performed in each experiment: with resting HMEC-1 and E. coli O55:B5 LPS (Sigma, USA). Bacteroides fragilis and B. thetaiotaomicron lipopolysaccharides stimulated three adhesion molecules under investigation. Their activity was comparable, but weaker than the activity of E. coli O55:B5 LPS. ICAM-1 was the most stimulated molecule. B. fragilis enterotoxin induced two adhesion molecules: VCAM-1 and E-selectin demonstrating weaker stimulatory activity than E. coli LPS. Stimulation of adhesion molecules on vascular endothelial cells should be considered to be a biological activity of B. fragilis and B. thetaiotaomicron endotoxins and B. fragilis enterotoxin.     

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.     

Prospective survey on carriage of Neisseria meningitidis and protective immunity to meningococci in schoolchildren in Niamey (Niger): focus on serogroup W135

We investigated the carriage of serogroup W135 meningococci and its relationship with protective immunity in Niamey. Between February and May 2003, three oropharyngeal swabs and two serum samples were each taken from 287 school children. Serogroup W135 isolates were obtained from 8.9% of children. Specific IgG > or = 2 microg/ml using ELISA or serum bactericidal assay (SBA) titre > or = 8 were supposed to represent the protective immunity to a serogroup. The proportion of children with serogroup W135-specific IgG > or = 2 microg/ml increased significantly during follow-up (13.9% to 19.1%), but not the proportion of those with SBA titre > or = 8 (10.1% to 11.6%). At the end of the follow-up, we observed a significant association between carriage of serogroup W135 strains and presumed protective immunity to this serogroup, using either ELISA or SBA. Among 240 children having an initial SBA titre < 8, 20 carried serogroup W135 strains at least once. In May, 25% of carriers had an SBA titre > or = 8, vs. 2.3% of non-carriers. For ELISA, 230 children had specific IgG < 2 microg/ml in February, with 22 having at least one swab positive for serogroup W135 meningococci later. In May, 45.5% of them had specific IgG > or = 2 microg/ml vs. 5.3% among non-carriers.     

Plasmid transformation of Bacteroides spp. by electroporation

Transformation of Bacteroides spp. with a variety of plasmid DNAs was accomplished using electroporation. The standard transformation assay system used to deduce the optimal electroporation parameters employed a 50-to 100-fold concentrated cell suspension of mid-logarithmic phase Bacteroides fragilis strain 638 and the 5.4-kb clindamycin resistance (Ccr) vector, pBI191. A variety of electroporation buffers were used successfully in transformation experiments but of these, 1 mM MgCl2 in 10% glycerol was superior. The incorporation of MgCl2 was essential for optimum viability prior to electroporation and for optimum transformation. Transformants were routinely obtained using 5-ms pulses over a range of field strengths from 5 to 12.5 kV/cm, with a maximum of greater than 10(6) micrograms-1 DNA at 12.5 kV/cm. The number of transformants increased linearly with respect to DNA concentration over the range 0.01-2 micrograms tested. Recovery of transformants required an expression period of up to 2.5 h following exposure to the electric field. This period, however, was dependent on the antibiotic resistance marker used for selection of transformants, with a significantly shorter incubation required when chloramphenicol rather than clindamycin was used in the selective medium. The effect of the DNA source on transformation was tested using the shuttle vector pFD288. Plasmid DNA isolated from Bacteroides uniformis, Bacteroides ovatus, or Bacteroides thetaiotaomicron transformed B. fragilis 638 at frequencies 7.5- to 12.5-fold less than those observed for controls with homologous DNA. Further reductions were seen with Escherichia coli purified pFD288, which transformed at 1000-fold lower frequencies. Finally, using homologous pFD288 or pBI191 isolated from strain 638, several strains of B. fragilis, B. uniformis, and B. ovatus were transformed successfully without modification of the standard assay system. Two strains each of B. thetaiotaomicron and Bacteroides ruminicola were not transformed using the methods described here.     

Application of quantitative real-time PCR for rapid identification of Bacteroides fragilis group and related organisms in human wound samples

Our goal was to establish a quantitative real-time PCR (QRT-PCR) method to detect Bacteroides fragilis group and related organisms from clinical specimens. Compared to conventional anaerobic culture, QRT-PCR can provide accurate and more rapid detection and identification of B.?fragilis group and similar species. B.?fragilis group and related organisms are the most frequently isolated anaerobic pathogens from clinical samples. However, culture and phenotypic identification is quite time-consuming. We designed specific primers and probes based on the 16S rRNA gene sequences of Bacteroides caccae, Bacteroides eggerthii, B.?fragilis, Bacteroides ovatus, Bacteroides stercoris, Bacteroides thetaiotaomicron, Bacteroides uniformis, Bacteroides vulgatus, Odoribacter splanchnicus (Bacteroides splanchnicus), Parabacteroides distasonis (Bacteroides distasonis) and Parabacteroides merdae (Bacteroides merdae), and detected these species by means of QRT-PCR in 400 human surgical wound infection samples or closed abscesses. The target bacteria were detected from 31 samples (8%) by culture, but from 132 samples (33%) by QRT-PCR (p-value?相似文献   

Screening for the presence of the insertion sequence IS1 in the genus Bacteroides

Abstract A specific DNA probe, containing a conserved region of the insertion sequence IS1, was hybridised to dot blots of total genomic DNA from 2 oral and 5 intestinal Bacteroides spp. Using Escherichia coli K12 as a positive control and Pseudomonas aeruginosa as a negative control, DNA homologous to the probe could not be detected in Bacteroides corporis, Bacteroides intermedius, Bacteroides ovatus, Bacteroides vulgatus, Bacteroides thetaiotaomicron or 2 strains of Bacteroides fragilis . The total DNA included plasmid DNA of 30.2, 42.7 and 42.7 MDa from B. fragilis, B. intermedius and B. corporis , respectively.
IS1 is commonly found in members of the Enterobacteriaceae, and it was concluded that the 2 groups of bacteria are not closely related.