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Bacteroides fragilis ATCC25285から得られたアルカリ安定脂質は赤外吸収スペクトルでアミド結合に由来する特徴的な吸収がみられることや,スフィンゴシンを有することなどからスフィンゴリン脂質であることが示された。このスフィンゴリン脂質の薄層クロマトグラフィーによる分析からセラミドホスホリルエタノールアミン(CPE)とセラミドホスホリルグリセロール(CPG)の2種類のスフィンゴリン脂質が検出された。また,Bacteroides属の全10菌種についてスフインゴリン脂質の組成を分析して菌種間の分布を調べたところ,CPEとCPGは7菌種から見いだされた。さらに,薄層クロマトグラフ上にはスフィンゴリン脂質と考えられる未知スポットが見いだされた。各菌種で見いだされたスフィンゴリン脂質組成は,菌種について特徴的であり,菌株間での変異は認められなかった。

Bacteroides and related species formerly classified in the genus Bacteroides have phosphosphingolipids as their membrane lipid. To evaluate the possible involvement of bacterial sphingolipids in the infection process, the effects of Bacteroides sphingolipids on murine neutrophils were examined in this study. Observation by differential interference contrast microscopy showed morphological changes in murine neutrophils in the presence of the sphingolipids, indicating biological activity by the Bacteroides sphingolipids. The lipids dose-dependently inhibited superoxide release from the neutrophils stimulated with phorbol myristate acetate. This observation suggests that bactericidal activity of the neutrophils may be affected by Bacteroides sphingolipids. Taken together, these findings provide evidence that Bacteroides sphingolipids may contribute to the pathogenesis of mixed infections by direct inhibition of neutrophil function.

The bft gene encoding Bacteroides fragilis toxin (BFT) has been devided into two subtypes, bft-1 and bft-2. We found a novel subtype by sequencing a segment of the bft gene from 64 enterotoxigenic B. fragilis (ETBF) strains isolated in Japan. The 1548-bp nucleotide sequences of the new bft, the bft-1, and bft-2 genes were determined for five, four, and four ETBF strains, respectively; the nucleotide sequence was identical among each bft subtype and the degree of identity between each subtype was between 89 and 94%. Most of the variations between the three subtypes were detected in the region encoding mature toxin. A multiplex PCR was developed with a four-primer mix to subtype the bft sequences. The subtyping of 143 ETBF isolates from extraintestinal and stool specimens of humans and cows showed that the bft-1 was the most prevalent subtype, followed by bft-2 and a new bft subtype. No other subtype was found among the strains tested.

The regression coefficients affecting the pharmacokinetic parameters of the breakpoints of antimicrobial agents (ie, empiric breakpoints) obtained retrospectively from clinical trial data were considered, and the pharmacokinetic parameters that were thought to strongly correlate to the empiric breakpoints were selected. The regression coefficients of the selected pharmacokinetic parameters were estimated, based on the categoric regression analysis. Estimated regression coefficients were rounded, and breakpoints were constructed using these rounded values. The breakpoint for complicated cystitis was determined, and it was decided that to obtain the breakpoint of complicated pyelonephritis, one tube (ie, one-half value) would be subtracted from the breakpoint of complicated cystitis, as calculated using the formula.

OBJECTIVE: Prevotella bivia is one of the anaerobic bacteria that resides in the flora of the female genital tract. We studied the pathogenicity of P. bivia in a rat pyometra model. METHODS: The experimental animal (rat) model of pyometra was developed to investigate the pathogenicity of P. bivia in a rat pyometra model. RESULTS: In the groups inoculated with aerobes alone, the infection rate was 10% (1/10) in the Staphylococcus aureus- or Staphylococcus agalactiae-inoculated group and 20% (2/10) in the Escherichia coli-inoculated group. Infection was not established in the groups inoculated with anaerobes alone. High infection rates were observed in all the mixed-infection groups. In the S. agalactiae- and Bacteroides fragilis-, S. agalactiae- and P. bivia-, F. coli- and B. fragilis-, and E. coli- and P. bivia-inoculated groups, an infection rate of 100% (10/10) was demonstrated. The efficacy of antibiotics such as flomoxef (FMOX) could be determined using a rat pyometra model. In relation to the alteration of vaginal microbial flora during the menstrual cycle, estrogen increased the growth of P. bivia. CONCLUSION: Mixture of aerobic bacteria and P. bivia increased the pathogenicity of P. bivia. Estrogen would be useful for raising up the inflammatory change of the uterus in experimental models of genital tract infection due to P. bivia.

市販の魚介類における好塩性の嫌気性菌の分布を検討した.嫌気培養を用いた37℃ の増菌培養法によれば50.8%の陽性率を認めた.分離された好塩性の嫌気性菌はすべて無芽胞グラム陰性嫌気性桿菌であった.貝類からの好塩性の無芽胞グラム陰性嫌気性桿菌の分離の程度を季節別に検討したが, 夏期では31.0%であったが, 冬期では66.7%であり, 夏期よりも冬期のほうが有意 (p

The bacteriological epidemiology of bacterial vaginosis (BV) and the efficacy of vaginal or oral treatment of BV with clindamycin (CLDM) were investigated. The epidemiology of BV was investigated in 100 symptomatic women before CLDM therapy. Two groups consisting of 50 patients each with the diagnosis of symptomatic BV were treated with either oral administration of 450 mg CLDM three times daily or 2% CLDM phosphate in vaginal cream (self-made) 5 g once a day, for 7 days. There was no significant difference in efficacy among vaginal and oral therapies with CLDM. Vulvovaginal irritation occurred in 3 patients orally treated and in 1 patient vaginally treated. Gastrointestinal disturbances were observed in 4 orally treated patients. A slight abnormal elevation of the glutamine-oxaloacetic transaminase level was also found in 1 patient orally treated. Since there were no statistically significant differences in efficacy rates between vaginal and oral CLDM treatments, we favor vaginal treatment of BV, based on less adverse effects.

Polymerase chain reaction (PCR) assay was compared with cell culture assay performed with use of HT29/C1 (human colonic epithelial) cells for identifying strains of enterotoxin-producing Bacteroides fragilis (ETBF) isolated from extraintestinal specimens. A total of 188 unselected strains obtained over 2 years at a central clinical laboratory in Tokyo were tested. Overall, 35 strains (18.6%) were positive by cell culture and PCR assay, 152 strains were negative by both assays, and 1 strain was negative by cell culture assay but positive by the PCR assay; the same results were obtained in repeated assays. Among 64 strains from blood, 18 (28.1%) were ETBF, a rate that was significantly higher (P < .05) than the 17 ETBF (13.7%) among 124 strains from other sites. These results suggest that PCR assay is a simple and reliable tool for detecting ETBF and that enterotoxin may be a virulence factor in bacteremia caused by B. fragilis.

The in vitro activity of DU-6859a, a new fluoroquinolone, was compared with those of other fluoroquinolones against clinical isolates of anaerobic bacteria and Gardnerella vaginalis. DU-6859a was the most active agent; it inhibited 90% of isolates of almost all species tested, including Bacteroides fragilis ator = 0.39 micrograms/mL. Although the other quinolones tested were active against gram-positive anaerobes, inhibiting their growth ator = 1.56 micrograms/mL, these agents were less active against the B. fragilis group and Prevotella bivia (90% of which were inhibited ator = 6.25 micrograms/mL). Mobiluncus species and G. vaginalis, which are well associated with bacterial vaginosis, were inhibited by DU-6859a at 0.1 microgram/mL. These results suggest that DU-6859a is a promising oral agent for the treatment of bacterial infections due to anaerobic bacteria; however, further studies, including determination of vaginal levels of this compound, should be performed to study the role of DU-6859a in the treatment of bacterial vaginosis.

The vaginal flora of 118 Japanese and 208 Thai pregnant women were investigated for the presence of bacterial vaginosis (BV), BV-associated organisms, and BV-associated enzyme. A similar prevalence of BV was found among the Japanese (13.6%) and Thai women (15.9%). The microbial flora of women with BV were complex; the mean number of isolates recovered in the BV group was approximately 2 times more than that in a group of healthy women. Prevotella species, Porphyromonas species, Peptostreptococcus species, Mobiluncus species, Gardnerella vaginalis, and H2O2-nonproducing lactobacilli were significantly associated with BV. These organisms were less associated with H2O2-producing lactobacilli, which were predominant in women with normal flora, suggesting that H2O2-producing lactobacilli have antibacterial activity against BV-associated organisms. The vaginal sialidase assay by means of a filter-paper spot test was not proved to be a useful screening aid for diagnosis of BV because of the low sensitivity (69.4%) of this test.

In vitro antimicrobial activity of NM394 (6-fluoro-1-methyl-7-(1-piperazinyl)-4-oxo-4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylic acid) against clinical isolates collected from samples (uterine contents, ascites, pus, etc) obtained from patients with obstetric and gynecologic infections was investigated and compared with that of four other quinolones: norfloxacin, ofloxacin, ciprofloxacin, and tosufloxacin. Among 80 strains of aerobes and 60 strains of anaerobes determined, the minimum inhibitory concentrations (MICs) of NM394 for 50% inhibition against methicillin-sensitive Staphylococcus aureus (MSSA), methicillin-resistant S aureus (MRSA), Streptococcus agalactiae, Escherichia coli, Peptostreptococcus magnus, Bacteroides fragilis , and Prevotella bivia were 0.39, 25, 0.78, 0.0125, 0.2, 3.13, and 0.78 μg/mL, respectively, and the MICs for 90% inhibition against MSSA, MRSA, S agalactiae, E coli, P magnus, B fragilis , and P bivia were 1.56, > 100, 1.56, 0.025, 0.39, 6.25, and 1.56 μg/mL, respectively. The antimicrobial activity of NM394 against gram-positive bacteria was less than that of tosufloxacin but was comparable to that of ofloxacin and ciprofloxacin. Judging from its antibacterial activity, the efficacy of NM394 against infections caused by MRSA might be expected to be similar to that of the other four quinolones. The antibacterial activity of NM394 against aerobic gram-negative bacteria, especially E coli , was greater than that of the other new quinolones. However, against anaerobic gram-negative bacteria, especially B fragilis , the antibacterial activity of NM394 appeared to be less than that of the other agents. The clinical implications of these results, if any, remain unknown until further clinical therapeutic trials are conducted.

Lactose-positive bacteria facilitate the colonization in the intestine in mono-associated quails fed a lactose-containing diet, because the quail does not possess intestinal lactase. We examined the influence of the overgrowth of various human intestinal lactose-positive bacteria on pathological change of mono-associated quails fed a lactose-containing diet. The strains used in this study included Bacteroides fiagilis Bifldobacterium breve, Clostidium butyrwwn, Esherichia coll. Enterococcus faecaliss, and Lactobacillus casei. Gas cysts, hemorrhage, and erosion were observed in the cecum of the quails mono-associated with E. coil or C. butyricum Necrotic areas were not observed in the lesion. These lesions were diagnosed as typical pneumatosis cystoides intestinalis (PCI), and the findings were benign, neither fulminant nor fatal. Weak-gas-producing mutants (gasw) derived from C. butyricum were compared as to incidence of lesion with parent strain. The biochemical characters of the mutants were consistent with the parent strain except for less than half gas production. The incidence of lesion was significantly lower in the quails mono-associated with gasw mutant than in those of mono-associated with the parent strain. These results suggest that a large amount of gas produced by C. butyricum might be mainly related to the onset of cecal PCI.

The polymerase chain reaction (PCR) was used in an attempt to detect Bacteroides fragilis by amplifying a segment of the gene encoding B. fragilis neuraminidase. Forty-five reference strains representing 45 species and 113 clinical isolates were tested. Only B. fragilis was PCR positive, except for Bacteroides merdae ATCC 43184, which gave a band by ethidium bromide staining that showed no signal by Southern hybridization. Using a protocol that employed DNA extraction by Sepa Gene kit and a highly sensitive digoxigenin-chemiluminescence detection system, detection of B. fragilis by PCR was in complete agreement with culture results for 44 clinical specimens from which a wide range of aerobic and anaerobic organisms and fungi were recovered.

The inhibition of bacterial regrowth by flomoxef (FMOX), a beta lactam, was determined using bacteria identified in the puerperal uterine cavity. FMOX was administered to five women within the first postpartum day, and the effective regrowth time (ERT), the time required to return to pretreatment bacterial count, was measured. Among aerobic gram-positive cocci, ERT values were 10, 12, and 16 hours for Staphylococcus aureus, Staphylococcus epidermidis , and Streptococcus agalactiae , respectively. Among aerobic gram-negative bacilli, the ERT was 24 hours for Escherichia coli and 6 hours for Burkholideria cepacia . Among anaerobic gram-positive bacteria, the ERT was 12 hours for both Peptostreptococcus anaerobius and Peptostreptococcus micros . Among anaerobic gram-negative bacteria, the ERT was 12 hours for Bacteroides fragilis and 7 hours for Prevotella bivia . ERT values obtained in this study may be useful indicators for the effectiveness of chemotherapeutic agents and dosage regimens in the treatment of infectious diseases in obstetrics and gynecology. The puerperal uterine cavity may be a good in vivo site for testing of various antimicrobial therapies.

The inhibition of bacterial regrowth by cefbuperazone (CBPZ), a beta-lactam, was investigated using bacteria identified in the puerperal uterine cavity. CBPZ was administered to four puerperae, and the effective regrowth time (ERT)—the time required to return to pretreatment bacterial count—was measured. Among aerobic gram-positive cocci, the ERT was 9.0 hours for Streptococcus agalactiae . Among aerobic gram-negative bacilli, ERT values were 11.3 and 18.0 hours for Escherichia coli in two patients. Among anaerobic gram-positive bacteria, ERT values were 7.5 and 10.5 hours for Peptostreptococcus magnus in the same two patients. Among anaerobic gram-negative bacteria, ERT values were 12.0 and 9.0 hours for Bacteroides fragilis and Prevotella bivia , respectively. The ERT values obtained in this study may be a useful index for the selection of chemotherapeutic agents and dosage regimens for the treatment of infectious diseases in obstetrics and gynecology. Because some CBPZ-sensitive bacteria showed ERT of 8 hours, CBPZ would be more effective against infections with CBPZ-sensitive bacteria when administered three times daily. Using the puerperal uterine cavity may be the best way to evaluate the inhibition of bacterial regrowth of other antibiotics as well.

Comparison of cytokine stimulation by lipopolysaccharide (LPS) of Bacteroides fragilis and Salmonella typhimurium was done to study the early events occurring in vivo. Mice injected intraperitoneally with either LPS demonstrated endogenous production of all the cytokines studied (tumor necrosis factor-alpha, interferon-gamma and interleukin-6) within 6 hr in the bloodstream. However induction of all the cytokines by B. fragilis LPS (50 micrograms/mouse) was much weaker compared with S. typhimurium LPS (50 micrograms/mouse). Even a dose of S. typhimurium LPS 40 times smaller (1.2 micrograms/mouse) induced cytokines more strongly compared with B. fragilis LPS. Thus, a weak biological response to B. fragilis LPS as evidenced by chick embryo lethality, limulus lysate gelation, LD50 for mice and rabbit pyrogenicity could be due to weak induction of bioactive mediators by LPS.

Pulsed-field gel electrophoresis (PFGE) analysis of restriction pattern polymorphism was applied to type Clostridium difficile isolated from neonates hospitalized in a neonatal intensive care unit, and the results were compared with those of immunoblot analysis. C. difficile was isolated from fecal specimens of 41 (61%) of 67 neonates during a 5-month investigation. All of these neonates were asymptomatic. Fifty-five C. difficile isolates from 32 patients were analyzed by PFGE after digestion with SmaI and SacII endonucleases and by immunoblotting with 10 different antisera. Fifty-three of 55 isolates from 30 patients were identical by PFGE analysis after SmaI and SacII digestion and immunoblot analysis. Two isolates were different from each other and from the epidemic group by both PFGE and immunoblot analysis. All 53 epidemic isolates were nontoxigenic, while the two remaining isolates were toxigenic. These results suggest that nosocomial spread of nontoxigenic C. difficile infection in the neonatal intensive care unit and suggest that both PFGE and immunoblot are powerful typing tools for the epidemiological study of C. difficile.

Microbial contamination of 0.025% benzalkonium chloride (BAC), in which the tracheal suction catheter was soaked, was investigated. Six strains of Pseudomonas cepacia were isolated from 30 samples of 0.025% BAC which were used for 24 h. The bactericidal activity of BAC and povidone-iodine (PVP-I) against clinical isolates of P. cepacia was evaluated by logarithmic reduction factors (LRFs). Two strains of P. cepacia isolated from the disinfectant were resistant to BAC, and the LRFs of 0.025% BAC for these strains after 10min exposure were 0 and 0.12, respectively. The LRFs of 0.025% and 0.05% BAC for five strains of P. cepacia isolated from clinical specimens after 1 min exposure were less than 0.94 and 2.09, respectively. On the other hand, 0.1% BAC and 0.2% PVP-I showed antimicrobial activities against P. cepacia isolates tested, and the LRFs after 1 min exposure were more than 3 or 4. The LRFs of 0.025% BAC for P. cepacia were not significantly elevated by the presence of 10v/v% ethanol. These findings suggest that 0.1% BAC and 0.2% PVP-I are suitable for the disinfection of tracheal suction catheters.

In an attempt to elucidate the pathophysiological significance of intestinal flora in hepatic encephalopathy, the faecal flora as well as production of toxic substances and drug susceptibility of the isolates from faeces were serially examined in six patients with chronic recurrent hepatic encephalopathy. Significant changes in the faecal flora accompanied with those in toxic substances (ammonia, short-chain fatty acids) and clinical features were observed in three cases who had been administered antimicrobial agents. Administration of antimicrobial agents in the three cases was revealed to be one of the important precipitating factors of hepatic encephaiopathy, due mainly to the induction of overgrowth of toxic substance-producing organisms, such as Clostridiums pecies, which produced ammonia and short-chain fatty acids. Moreover, administration of polymyxin B brought about the overgrowth of Morganella morganii , which produced large amounts of ammonia and methanethiol. Clinical use of conventional antimicrobial agents should be carefully decided in patients with hepatic encephalopathy. Keywords: Hepatic encephalopathy; Faecal flora; Ammonia; Short-chain fatty acids; Antimicrobial agent; Clostridium species.

A study was undertaken in Japan to evaluate the susceptibility patterns of Bacteroides fragilis group species (849 strains) isolated from December 1986 through May 1991. All of the strains, which included B. fragilis (610 strains), Bacteroides thetaiotaomicron (201 strains), and Bacteroides distasonis (38 strains), were studied for susceptibility to imipenem and 16 other antimicrobial agents by an agar dilution method. Metronidazole was the most active agent; the minimal concentration for 90% inhibition (MIC90) was 0.78 micrograms/mL, and no isolate was noted to be resistant to it during the entire study period. Amongst the beta-lactam agents tested, imipenem was the most effective antimicrobial drug; the rate of resistance to this agent was only 3.3%. Cefoperazone/sulbactam (MIC90, 6.25 micrograms/mL) and cephamycins (MIC90, 25-50 micrograms/mL) were found to be more active against B. fragilis strains, whereas minocycline (MIC90, 6.25 micrograms/mL) showed better activity against B. thetaiotaomicron and B. distasonis strains. Increasing resistance to imipenem was observed in B. fragilis (2.0%-5.9%) and B. thetaiotaomicron (2.5%-6.1%) during the 4-year study period. Detailed investigation of beta-lactamases from imipenem-resistant strains demonstrated that, amongst them, six of the B. fragilis strains for which the MICs of imipenem were > or = 25 micrograms/mL were producing imipenem-hydrolyzing metallo-beta-lactamase.

The bacterial regrowth-inhibition effects (previously known as bacterial regrowth-inhibiting activity) of cefmetazole (CMZ; a β-lactam) and/or netilmicin (NTL; an aminoglycoside) were investigated in bacteria identified from the puerperal uterine cavity. CMZ, NTL or CMZ plus NTL were administered to 12 puerperae and the effective regrowth time (i.e. time required to return to pretreatment bacterial counts) was examined.

Patterns of antimicrobial susceptibility were determined for 155 clinical isolates of the Bacteroides fragilis group from patients with acute appendicitis in Bali, Indonesia. The transfer of drug resistance was also studied, and plasmid analyses were undertaken. Metronidazole and chloramphenicol were the most active drugs against these isolates (resistance rate, < or = 0.6%). Among the beta-lactam drugs, cefoxitin was the most active (resistance rate, 2%). Rates of resistance to tetracycline were high (16%). Resistance to clindamycin (rate, 10%) increased during the 2-year study period. Except in the case of beta-lactam agents, overall susceptibility patterns were comparable to those reported from other countries. Tetracycline resistance was more frequently transferred after tetracycline induction than without such induction (P < .05, chi 2 test). Resistance to tetracycline and clindamycin was co-transferred by five of 12 donor strains. In one of these five strains, transferability was constitutive, with a high transfer frequency (10(-5) per input donor). Plasmid analysis indicated that the transfer of resistance to tetracycline and clindamycin among the strains studied was not plasmid mediated.

Polymerase chain reaction (PCR) amplification of a segment of the toxin A gene was used to detect toxigenic Clostridium difficile directly from stool specimens of patients with antibiotic-associated diarrhea. Although PCR-inhibitory substances were recognized in DNA prepared from stool specimens, the inhibitory substances were eliminated by using an ion-exchange column after phenol-chloroform extraction. Eventually, 39 stool specimens were evaluated by PCR. PCR results for detection of toxigenic C. difficile were in complete agreement with cell culture assay results; all 12 PCR-positive stool specimens were positive by cytotoxin assay, and all 27 PCR-negative specimens were negative by cytotoxin assay. Toxigenic C. difficile was cultured from all PCR-positive specimens. These results suggest that PCR amplification may be an effective method for laboratory diagnosis of C. difficile-associated diarrhea and colitis.

Polymerase chain reaction was applied to identify enterotoxin-producing Clostridium perfringens by amplifying a segment of the C. perfringens enterotoxin gene. All of the four enterotoxin-positive reference strains tested were PCR positive while an enterotoxin-negative strain was PCR positive. All 17 clostridial strains (16 species) other than C. perfringens were PCR negative. With clinical strains isolated from various clinical specimens in Japan, Korea, and Thailand, all three enterotoxin-positive isolates were PCR positive and all 82 enterotoxin-negative isolates were PCR negative. PCR results for amplifying a region containing the initiation codon of the C. perfringens enterotoxin gene also demonstrated complete agreement with enterotoxin producibility. These results suggested that the PCR assay is a rapid and simple test for identifying the enterotoxin-producing C. perfringens without using any cultures and spore treatments.

Certain bacteria produce some carcinogens such as N-nitro compounds, n-butyric acid and n-valeric acid. From this point of view, the examination of intrauterine bacterial flora in patients with uterine endometrial cancer may provide important information. Twenty patients with the diagnosis of uterine endometrial cancer and 20 patients without complications other than myoma uteri were enrolled in the study. Enterobacteriaceae, Streptococcus agalactiae and anaerobic bacteria were mainly detected. The products of these bacteria might be considered to contribute to the initiation of endometrial carcinogenesis. Mixed abnormal flora between aerobic and anaerobic bacteria were detected in all patients with uterine endometrial cancer. It is suggested that uterine endometrial cancer provides favorable conditions for bacterial growth. Mixed abnormal bacterial flora also might influence the onset and growth of uterine endometrial cancer.

Western blotting using antisera against each of reference ten serogroups was evaluated as a typing system for Clostridium difficile. A total of 164 isolates of C. difficile (114 epidemiologically unrelated and 50 isolates from a hospital outbreak in New York) were tested. Blotting patterns for the ten reference strains showed serogroup-specific bands located in the 30-60 kDa when each homologous antiserum was used. At greater than 60 kDa, variations in each serogroups were observed; these variations were used for subserogrouping the isolates. Serogroup A, G, H, and K were most frequently recovered in the group of epidemiologically unrelated isolates. Subserogroup G-1 strains of serogroup G was isolated from 28 of 36 patients (78%) of the hospital outbreak. The result suggested that the subserogroup G-1 strain was the major cause of infection in the hospital outbreak. A total of 46 of the 114 unrelated isolates (40.4%) and 9 of 50 outbreak isolates (18%) did not react with any of reference antisera and classified as nontypable. The western blotting was found to be useful not only as an epidemiological tool but as a typing system for C. difficile.

A strain of Bacteroides fragilis, which produces a metallo-beta-lactamase, was inoculated into pouches on the backs of rats together with a beta-lactamase-negative Escherichia coli highly sensitive to beta-lactam antibiotics. The mixed infection rat pouch model was treated with either flomoxef (susceptible to hydrolysis by the beta-lactamase produced by B. fragilis), or cefmetazole (relatively resistant to hydrolysis). In this model of mixed infection flomoxef showed weak in-vivo activity against E. coli, although showing the same strong activity in a model of single infection with E. coli. On the other hand, cefmetazole showed strong activity against E. coli, even in the model of mixed infection. The concentrations of both drugs in the pouches were decreased in infections with the strain of B. fragilis. There was a greater decrease in the concentration of flomoxef than of cefmetazole. Flomoxef was unstable whereas cefmetazole was relatively stable in the pouch exudates that had been infected with B. fragilis. These experimental data suggest that bacteria that produce a metallo-beta-lactamase decrease the in-vivo efficacy of beta-lactam antibiotics against other co-infecting bacteria. Thus, it is suggested that it is important in the chemotherapy of mixed bacterial infections that include these highly resistant beta-lactamase-producing bacteria to use antibiotics that are stable to hydrolysis by these enzymes.

GTP-binding proteins with Mr values of 22 000 and 25 000 in bovine brain cytosol were ADP-ribosylated by an exoenzyme (termed C3) purified from Clostridium botulinum type C. The rate of C3-catalyzed ADP-ribosylation of the partially purified substrates was extremely low by itself, but was increased enormously when a protein factor(s) obtained from the cytosol was simultaneously added. The rate of the C3-catalyzed reaction was also stimulated by the addition of certain types of detergents or phospholipids even in the absence of the protein factors. The ADP-ribosylation appeared to be enhanced to an extent more than the additive effect of either the protein factors or the detergents (and phospholipids). Thus, ADP-ribosylation catalyzed by botulinum C3 enzyme was affected not only by cytoplasmic protein factors but also by detergents or phospholipids in manners different from each other.

We described plasmid mediated transfer of resistance to beta-lactam antibiotics between Bacteroides fragilis strains. Ampicillin-resistance was transferred from B. fragilis strain GAI-10150 to a B. fragilis strain JC-101 with a frequency of 10(-6)/input donor by a filter mating technique. A common plasmid band, named pBFKW1, was found in both the donor and the transconjugants. The plasmid was purified by an ethidium bromide-CsCl ultracentrifugation. The molecular size of the plasmid pBFKW1 which seemed to encode the beta-lactam resistance and beta-lactamase production was estimated ca. 40 kb by the analysis of endonuclease digest. Substrate profile of the enzymes derived from the donor and a transconjugant were of cephalosporinase character.

The present article describes the recent status of research on anaerobes in Japan by reviewing publications from the last few years in the Japanese Journal of the Association for Anaerobic Infection Research. Most studies were categorized into clinical infection, susceptibility and resistance mechanisms, epidemiology and typing systems, and pathogenesis and virulence factors; clinical infection and susceptibility studies were the most

Aerobic and anaerobic cultures as well as a Gram stain and wet mount preparations were made of vaginal swabs in twenty patients with clinical bacterial vaginosis. Mobiluncus spp. were detected in 7 cases (35%). Cultures appeared to indicate that mixed abnormal flora between aerobic and anaerobic bacteria are found in bacterial vaginosis, and that Mobiluncus spp. may play a role in bacterial vaginosis.

In age-matched controlled studies performed in Japan, enterotoxigenic Bacteroides fragilis was isolated from 14.9% of 114 children aged 1 to 14 years with antibiotic-unassociated diarrhea (AUD) and 6.5% of 108 children aged 1 to 6 years with antibiotic-associated diarrhea (AAD). The difference in comparison with control children, was significant for AUD children but not AAD children.

Biophila wadsworthia is a recently recognized nonspore-forming anaerobic gram-negative rod and is reported to be associated with various infections such as gangrenous perforated appendicitis, peritonitis, osteomyelitis and bacteremia. Although the isolation of B. wadsworthia seems to be facilitated by using Bacteroides bile esculin (BBE) agar, the reliable scheme of identification of this species has been published. This study was conducted to find simple, rapid, and reliable measures for identification of B. wadsworthia. A total of 32 B. wadsworthia-suspected clinical isolates as well as B. wadsworthia WAL 7959 and Desulfomonas pigra DSM 749, which is the species that shows bacteriological characteristics similar to B. wadsworthia, were used. Through the study of various biochemical and enzymatic tests and culture on selective media, it is indicated that a nonspore-forming, gram-negative anaerobic rod that is nonmotile, forms "black-eyed" colonies on BBE agar, and demonstrates strongly positive catalase test, positive acid phosphatase test, and no-growth on Bacterides medium or to be susceptible to colistin (10 micrograms disk) can be identified as B. wadsworthia.

We conducted a study to assess the precise incidence of anaerobic infections among patients with pulmonary diseases in Japan. To avoid false-negative results of anaerobic cultures, we used percutaneous transtracheal aspiration and subsequent immediate bedside anaerobic inoculation onto a set of plates with appropriately selected culture media. Fifty-six episodes of pulmonary disease occurred in 50 patients; anaerobes were isolated in 20 (36%) of these episodes. Bacteria were recovered in 30 (94%) of 32 episodes not associated with prior antimicrobial therapy, and anaerobes were isolated in 15 (47%) of these 32 episodes. Rates of anaerobic isolation in episodes of pneumonia (7 of 14), lung abscess (3 of 3), and acute exacerbation of chronic lower respiratory tract infection (5 of 15) that were not associated with prior antimicrobial therapy were 50%, 100%, and 33%, respectively.

Immunoblotting and pulsed-field gel electrophoresis of Clostridium difficile isolates were employed to differentiate reinfection by a newly acquired strain from relapse by an original strain in a 10-year-old patient with four episodes of C. difficile-associated colitis. Immunoblot typing demonstrated subserogroup K-1 of serogroup K for the first and second organisms, subserogroup A-1 of serogroup A for the third organism, and subserogroup G-4 of serogroup G for the fourth organism. PFGE analysis revealed consistent results with immunoblot analysis except that the strains from the fourth episode, whose DNA constantly degraded, were nontypable by this method. Five separate isolates of C. difficile from a specimen of each episode showed identical PFGE patterns, indicating that infections of multiple strains probably did not occur in this patient. These typing results suggested that the second episode after a 17-day course of vancomycin therapy represented a relapse by the strain causing the first episode, and that the third and fourth episodes after tapering vancomycin therapy were reinfections by other strains. Both immunoblot and PFGE typing systems are promising tools for analyzing recurrence of C. difficile infection.

One hundred symptomatic women with clinical bacterial vaginosis (BV) were enrolled in this study. The bacteriological epidemiology of BV and efficacy of oral or vaginal administration of antimicrobial agents for treatment of BV were investigated. The epidemiology of BV was investigated before antimicrobial therapy. Fifty patients were treated with oral administration of metronidazole (MTN), 500 mg twice a day, and 50 patients with 2% clindamycin (CLDM) phosphate in a vaginal cream, 5 g once a day for 7 days. The major organisms occurring were Streptococcus agalactiae, Escherichia coli, Gardnerella vaginalis, Peptostreptococcus species, Bacteroides species, Prevotella species, and Mobiluncus species and opportunistic pathogens such as Staphylococcus epidermidis and Enterococcus faecalis. The therapeutic efficacy of CLDM cream on BV appeared to be preferable to oral MTN from clinical and bacteriological aspects. The clinical and bacteriological effectiveness of MTN and CLDM in the treatment of BV suggests that anaerobes might play a major role in causing the clinical symptoms in patients with BV.

A bacteriologic study of cases of chronic otitis media, chronic sinusitis, and paranasal mucopyocele diagnosed at Gifu University Hospital was performed between 1989 and 1992. Bacteria were isolated from all of 31 samples of discharge from the ears of patients with chronic otitis media, all of 14 mucosal biopsy specimens from patients with chronic sinusitis, 8 of 10 aspirate samples from patients with chronic sinusitis, and 9 of 15 aspirates samples from patients with mucopyocele. Cultures often yielded polymicrobial growth, with an average of 3.1, 3.8, and 4.2 species per positive specimen from chronic otitis media, chronic sinusitis, and mucopyocele, respectively. The most commonly encountered anaerobes were Peptostreptococcus, Propionibacterium, and Prevotella species ; the most common aerobes were Staphylococcus and Streptococcus species. One strain of Prevotella melaninogenica highly resistant to ampicillin and two strains of Peptostreptococcus resistant to cefaclor were found.

Bacteroides fragilis, the organism most frequently isolated in anaerobic infections, is known to cause intraabdominal, wound, pelvic, perirectal, and bloodstream infections. Recent studies have documented the existence of enterotoxigenic strains of B. fragilis; these strains can cause diarrhea in livestock and humans [1-5]. In 1992 Weikel et al. demonstrated that a human colonic epithelial cell line, HT29/C1, was sensitive to B. fragilis enterotoxin [6]. The cell culture assay developed by these investigators allows us to study enterotoxigenic B. fragilis with much less difficulty than is encountered with the ligated intestinal loop assay [1-5]. In this study, we used the cell culture assay to survey the prevalence of enterotoxin-producing strains of B. fragilis among isolates from extraintestinal sites. Enterotoxigenic B.fragilis strain ATCC (American Type Culture Collection) 43858, nonenterotoxigenic B. fragilis strain ATCC 25285, and 141 clinical strains of B. fragilis isolated from extraintestinal sites in 1987-1989 at the Institute of Anaerobic

The antimicrobial susceptibility of clinical isolates of Bacteroides fragilis and Bacteroides thetaiotaomicron collected from December 1990 through November 1992 was determined by the agar dilution technique. Metronidazole, imipenem, and cefoperazone/sulbactam remained highly active against both organisms. Rates of resistance to those agents were 0, 2%, and 0.9% in B. fragilis and 0, 0.9%, and 3% in B. thetaiotaomicron, respectively. Cefoxitin and other cephamycins were active against B. fragilis; rates of resistance to these agents did not tend to increase. With the inclusion of these data, the variation of rates of resistance to several agents was summarized for each year from 1987 to 1992. Rates of resistance to imipenem decreased in 1991 and 1992 among isolates of B. fragilis (2.3% in 1991, 1.8% in 1992) and B. thetaiotaomicron (2.4% in 1991, 0 in 1992). Rates of resistance to cefoxitin in B. thetaiotaomicron varied from 10% to 38% during these 6 years, though the distributional peak of MIC values did not change. The rate of resistance to ofloxacin in B. fragilis increased from 42% in 1989 to 81% in 1992. The rate of resistance to ampicillin in B. thetaiotaomicron was 68% in 1992--approximately 30% lower than before. Mostly, however, the rates of resistance to the antimicrobial agents examined did not change significantly.

Sphingolipid profiles of strains from species of genus Bacteroides, and representative strains from Prevotella and Porphyromonas, were analyzed by thin-layer chromatography and infrared spectrophotometry. Two major types of phosphosphingolipid, ceramide phosphorylethanolamine and ceramide phosphorylglycerol, were detected in B. fragilis, B. ovatus, B. uniformis, B. caccae, B. eggerthii, B. thetaiotaomicron, and B. stercoris, but not in B. merdae, B. distasonis, and B. vulgatus. Strains from the genera Prevotella and Porphyromonas also contained these two sphingolipids. These sphingolipid profiles were conserved within the species tested, and may be useful for differentiation and recognition of relationships within the genera Bacteroides, Prevotella and Porphyromonas.

Bacterial vaginosis (BV) is considered to be one of the most common vaginal infections in women. Fifteen symptomatic women with BV were enrolled in this study. Ten patients with the diagnosis of BV were treated with 10 days oral administration of metronidazole (MTN), 500 mg twice a day, and five patients with cefdinir (CFDN), 300 mg three times a day. In the MTN therapy, the rate of abnormal vaginal discharge subjectively decreased from 100 to 60%, the rate of abnormal vaginal discharges objectively decreased from 100 to 20%, the rate of positive amine tests decreased from 100 to 20%, the rate of genital malodor and abnormal pH of vaginal discharges decreased substantially from 100 to 10%, and the rate of the presence of clue cells also decreased notably from 90 to 10%. However, in the CFDN therapy, none of these factors improved. With respect to susceptibility to CFDN and MTN, CFDN demonstrated good antibacterial activity against almost all bacteria isolated except Gardnerella vaginalis. On the other hand, MTN demonstrated excellent activity against anaerobic bacteria except Peptostreptococcus spp., and had no antibacterial activity against aerobic bacteria. Since the therapeutic effect of MTN in BV appeared to be better than that of CFDN, anaerobes may play a major role in causing clinical symptoms in patients with BV.

The antibacterial activity of a new parenteral cephalosporin, FK037 was assessed against recent aerobic and anaerobic strains isolated from patients in the fields of obstetrics and gynecology during the period between January 1992 and June 1993. The MICs of FK037 for 90% of the clinical isolates tested were 0.10 microgram/ml for Escherichia coli and Klebsiella pneumoniae, 0.20 microgram/ml for Streptococcus agalactiae, 0.39 microgram/ml for Gardnerella vaginalis, 0.78 microgram/ml for Staphylococcus epidermidis, Peptostreptococcus anaerobius and Mobiluncus spp., 1.56 micrograms/ml for Peptostreptococcus magnus, 3.13 micrograms/ml for methicillin-sensitive Staphylococcus aureus, 25 micrograms/ml for methicillin-resistant S. aureus (MRSA), Bacteroides fragilis and Prevotella disiens, 100 micrograms/ml for Bacteroides thetaiotaomicron and Prevotella bivia; and > 100 micrograms/ml for Enterococcus faecalis. FK037 was superior in potency to ceftazidime against all strains except E. faecalis, P. anaerobius and P. bivia. It was 4- to 16-fold more active than cefotaxime against aerobic gram-positive bacteria and P. disiens, and its activity was similar to that of cefotaxime against the other strains. FK037 had 4- to 16-fold stronger activity than flomoxef against MRSA, S. agalactiae and E. coli and a similar activity to flomoxef against the other strains except G. vaginalis and B. fragilis that were 4-fold more sensitive to flomoxef than to FK037.

To study the mechanism of synergism between Bacteroides fragilis and Escherichia coli, the effect of sublethal dose of E. coli lipopolysaccharide (LPS) (25 micrograms/mouse) was checked on B. fragilis abscess formation. LPS was administered prior or after inoculum injection. No significant difference in the abscess size was observed at necropsy on day 6. However, all the groups receiving LPS showed higher incidence of recovery of additional intestinal bacteria (23.5-45.5%) from the abscess pus. When LPS was given 4 hr prior to inoculum administration, 83-100% mortality was observed. Detailed investigation showed autoclaved cecal contents alone could also cause similar mortality. Studies with stimulation of endogenous cytokines by E. coli LPS demonstrated induction of all of them within 3 hr in the blood stream with TNF-alpha demonstrating peak at 1 hr, IL-1 alpha and IL-6 at 4 hr and IFN-gamma between 6-9 hr with moderately high levels at 4 hr. This E. coli LPS-triggered cytokine cascade possibly gets further stimulated by injection of autoclaved cecal contents containing high concentration of endotoxins (1.6 x 10(5) EU/ml) contributed by dead bacteria and lead to the mortality of animals.

Twenty-five patients with uterine cervical cancer (Two cases stage O; four cases stage Ia; five cases stage Ib; one case stage IIa; ten cases stage IIb; two cases stage IIIa; one case stage IVa) served as the subjects. The bacterial flora inside the cervical cancers was investigated using the optimal technique. There was mixed abnormal aerobic and anaerobic bacterial flora in all subjects. The average number of bacterial species isolated from inside the cervical cancers was 6.3. The predominant bacteria isolated were the aerobes, Staphylococcus haemolyticus, Streptococcus agalactiae, Escherichia coli and Gardnerella vaginalis, and Prevotella bivia. As the stages of cervical cancer progressed, G. vaginalis, B. distasonis and P. bivia were detected at higher rates and higher counts than other bacteria. These findings suggest that there is a relationship between G. vaginalis, B. distasonis and P. bivia and the onset and growth of cervical cancer.

Patients with diabetes frequently suffer from various postoperative complications, especially infection. Diabetic patients also have a high incidence of uterine endometrial cancer. The nature of the intrauterine bacterial flora may be related to both infection and carcinogenesis. Therefore, identification of the intrauterine bacterial flora in diabetic patients may be useful. Bacteria were detected in the uterine endometrial cavity of 100% of ten diabetic patients with myoma uteri. However, among 20 non-diabetic control patients with myoma uteri, only three 15% harbored bacteria. Members of the Enterobacteriaceae (Escherichia coli, Proteus spp., Enterobacter cloacae, and Klebsiella pneumoniae) were the predominant bacteria. We speculate that bacterial products contribute to carcinogenesis, as has been proposed for colon carcinoma. Antimicrobial agents active against Enterobacteriaceae should be used to prevent postoperative infections in gynecologic procedures in diabetic patients.