Warfarin treatment of mice bearing autochthonous tumors: effect on spontaneous metastases.

Long-term oral administration of sodium warfarin significantly reduced the incidence of spontaneous metastases in the lungs from 83 percent in controls to 8 percent in treated C57/BL/6N mice. The size and weight of primary tumors in mice treated with warfarin were less than in control mice. Length of survival was unaffected.

Genetic analysis of mechanisms of multidrug resistance in a clinical isolate of Bacteroides fragilis.

This study investigated the mechanisms of multidrug resistance (MDR) in an isolate of Bacteroides fragilis (WI1) from a patient with anaerobic sepsis. The MDR of WI1 affected susceptibility to beta-lactams, clindamycin, fluoroquinolones, metronidazole and tetracycline. In addition to its 5.31-Mb chromosome, WI1 possessed two low-copy-number plasmids, pHagl (5.6 kb) and pHag2 (9.9 kb), that were absent from B. fragilis NCTC 9343. Restriction digestion with EcoRV, HindIII and SstI, combined with DNA sequencing, revealed that pHAG2 contained a tet(Q) gene at base position 3689 that resided on the conjugative transposon CTn341. Genes cfiA (encoding a metallo-beta-lactamase) and erm(F) (encoding a macrolide-lincosamide-streptogramin B resistance determinant) were also found in WI1, but were absent from B. fragilis NCTC 9343. Nitrocefin hydrolysis revealed that WI1 had high beta-lactamase activity. Sequencing of the gyrA quinolone resistance-determining region revealed a mutation causing a Ser82 --> Phe substitution, and comparative quantitative real-time RT-PCR revealed that the cfiA, erm(F) and tet(Q) genes were all expressed in WI1. In addition, the resistance-nodulation-division efflux pump genes bmeB9 and bmeB15 were significantly over-expressed (12.30 +/- 0.42-fold and 3541.1 +/- 95.4-fold, respectively), and the efflux pump inhibitors carbonyl cyanide m-chlorophenylhydrazone and reserpine significantly increased the susceptibility of the isolate to several unrelated antibiotics (p <0.005). These data suggested that WI1 was highly multidrug-resistant because of the additive effects of chromosome- and plasmid-encoded resistance determinants.

Pulmonary metastases from autochthonous 3-methylcholanthrene-induced murine tumors.

In three experiments, 87%, 75%, and 85% of female C57BL/6N mice developed pulmonary metastases by 50 days after amputation of legs having autochthonous 3-methylcholanthrene-induced sarcomas. No difference in the incidence of pulmonary metastases was observed when autochthonous tumors of short (49--94 days) and longer (95--119 days) latency periods were compared. These metastasizing autochthonous tumors may provide a useful model for studies of metastases and for the evaluation of cancer therapy in a minimal residual disease setting.

Bacteriologic findings associated with chronic bacterial maxillary sinusitis in adults.

An open-label, multicenter study was performed to assess bacteriologic findings associated with chronic bacterial maxillary sinusitis in adults. Seventy aerobic (52.2%) and 64 anaerobic (47.8%) pathogens were recovered from clinically evaluable patients at baseline (before therapy). The most commonly isolated anaerobes were Prevotella species (31.1%), anaerobic streptococci (21.9%), and Fusobacterium species (15.6%). The aerobes most frequently recovered included Streptococcus species (21.4%), Haemophilus influenzae (15.7%), Pseudomonas aeruginosa (15.7%), and Staphylococcus aureus and Moraxella catarrhalis (10.0% each). Recurrences of signs or symptoms of bacterial maxillary sinusitis associated with anaerobes were twice as frequent as were those associated with aerobes when counts of anaerobes were > or =10(3) cfu/mL. A pathogenic role for Granulicatella species in cases of chronic sinusitis was documented for the first time.

Multilaboratory comparison of growth characteristics for anaerobes, using 5 different agar media.

A multilaboratory study compared the growth of 30 fastidious anaerobes, using 5 different agar media: Wilkins-Chalgren (WC), WC with either whole or laked sheep blood, and Brucella supplemented with vitamin K(1) and hemin and either laked or whole sheep blood. The media were compared for quality and quantity of growth. Experiments were conducted either entirely in an anaerobic chamber or inoculated in ambient air with anaerobic incubation. The results showed that (1) any medium plus whole or laked blood was better than unsupplemented WC, (2) whole blood and laked blood additives gave similar results, (3) supplemented Brucella with whole or laked blood was superior to WC and WC with whole or laked blood, and (4) anaerobic and aerobic inoculation with anaerobic incubation gave similar results. Brucella agar supplemented with whole or laked blood supports the growth of fastidious anaerobic species better than the WC agars do.

Multilaboratory comparison of anaerobe susceptibility results using 3 different agar media.

A 5-laboratory study was performed that used the National Committee for Clinical Laboratory Standards (NCCLS) reference agar dilution method with 3 media formulations to determine whether the use of different media would affect minimum inhibitory concentration (MIC) results. Wilkins-Chalgren, Brucella-based blood agar (BRU), and Wilkins-Chalgren agar plus blood (WCB) and 6 antibiotics (clindamycin, cefoxitin, ceftizoxime, piperacillin, metronidazole, and trovafloxacin) were evaluated with 58 isolates. The MIC values were compared, and a significant correlation of >0.80 was demonstrated for all media and each antibiotic/organism group. The cumulative rate of errors for all antibiotics was 0.1%. These data indicate that a change in the NCCLS reference medium for testing of anaerobic bacteria susceptibility to either BRU or WCB will not affect the MIC results for the antibiotics and organisms evaluated.

Microbial flora in the bypassed jejunum of patients with biliopancreatic bypass for obesity.

The microbial flora in the bypassed biliopancreatic intestinal segment was studied after obesity surgery. This procedure causes less diarrhea than jejunoileal bypass and appears to avoid extraintestinal complications. This report concerns type and quantity of bacteria colonizing the biliopancreatic segment and changes occurring after oral metronidazole treatment. Twelve specimens were aspirated in 10 patients via catheter inserted percutaneously during surgery. The specimens were plated immediately on selective and nonselective media under aerobic and anaerobic conditions. Essentially equal numbers of aerobes and anaerobes were recovered from the biliopancreatic segment with average counts of 10(4) cfu/mL and median counts of 10(5) cfu/mL. Four patients had counts of 10(7) cfu/mL. The most common aerobes were E. coli, Klebsiella, Gram-positive cocci, and Candida; among anaerobes, Clostridium and the Bacteroides fragilis group were most common. In three patients treated with metronidazole because of diarrhea, anaerobes were eliminated and diarrhea cleared.

Impact of imipenem/cilastatin therapy on normal fecal flora.

The impact of parenteral imipenem/cilastatin therapy on the bowel flora of six patients was evaluated. Stool samples were collected before and during therapy and qualitative and quantitative bacteriologic studies were performed. Imipenem had no effect on total microorganism counts. Two patients acquired Candida albicans during therapy, and three patients acquired Proteus species. Pseudomonas species in one patient acquired resistance. Imipenem appears to have a relatively modest effect on the bowel flora and apparently does not readily induce resistance in the resident flora as compared with other agents.

Applications of diagnostic ultrasound and radionuclides to cardiovascular diagnosis. Part II. Cardiovascular disease in the young.

Echocardiography (ECHO) and radionuclide cardiography have had a significant impact on pediatric cardiology because they have proved to be sensitive enough to permit early diagnosis of many forms of heart disease and in some cases to estimate its severity and to provide information concerning ventricular performance. An overview of the anatomic and functional information that can be obtained from these two methods will be presented first, followed by details concerning the indications for their use and their relative clinical value in various acquired and congenital heart diseases. We have stressed particularly those facets of pediatric cardiac disease that differ most from those in the adult.

Enflurane and halothane in status asthmaticus.

A patient with status asthmaticus deteriorated while receiving conventional therapy including mechanical ventilation. She failed to respond to the inhalation of enflurane but had a beneficial response to halothane. Her subsequent course was complicated by a prolonged metabolic encephalopathy which was associated with an elevated plasma bromide level from the metabolism of halothane.

Quantitation of lung water by nuclear magnetic resonance imaging. A preliminary study.

Pulmonary edema was produced in four anesthetized dogs by saline lavage. The animals were maintained by assisted ventilation with O2/halothane and examined by a nuclear magnetic resonance (NMR) 0.15T resistive-magnet imager. The distribution of edematous fluid was clearly observed. Image contrast increased with prolongation of the cycle time (TR). Tomographic maps of spin-lattice relaxation times (T1) of the lungs were calculated from the NMR images. Comparison of T1 values with gravimetric measurements of water content of lung samples showed significant correlation (r = .7, P less than .02, n = 12) suggesting a potential for in vivo lung water quantitation by NMR imaging. This in vivo correlation is qualitatively similar to the in vitro correlation. Accurate in vivo determinations of pulmonary T2 values may require respiratory gating.

Current perspectives on anaerobic infections: diagnostic approaches.

This article discusses when to look for anaerobes, anaerobic infections as clues to other problems in patients, and underlying clinical conditions as clues to the nature of anaerobic infections. Diagnostic approaches, identification methods, and susceptibility testing are reviewed.

In vitro activity of grepafloxacin (OPC-17116) against anaerobic bacteria.

In vitro activity of the quinolone grepafloxacin (OPC-17116) was compared with that of ciprofloxacin, fleroxacin, clindamycin, imipenem, and metronidazole by using the NCCLS-approved Brucella-base-laked blood agar dilution method and breakpoints, when available. Clindamycin, metronidazole, and imipenem inhibited > or = 98% of Bacteroides fragilis at the breakpoint; grepafloxacin, ciprofloxacin, and fleroxacin inhibited 83%, 6%, and 0, respectively, at 2 micrograms/ml. Grepafloxacin inhibited 39% of other B. fragilis group species isolated (80) at breakpoint (< or = 2 micrograms/ml) compared with 100% for metronidazole and imipenem, 83% for clindamycin, 6% for ciprofloxacin, and 1% for fleroxacin. Grepafloxacin demonstrated substantially better activity against B. fragilis than did ciprofloxacin or fleroxacin; overall activity against anaerobes was marginally better than that of ciprofloxacin or fleroxacin.

Comparative activity of SCH 34343 against anaerobic bacteria.

The in vitro activity of SCH 34343 was compared with that of imipenem, clindamycin, cefoxitin, and metronidazole against 78 strains of anaerobic bacteria in the presence and absence of blood. Wilkins-Chalgren agar was used. SCH 34343 and imipenem were the most active agents, inhibiting all strains at less than or equal to 8 micrograms/ml. The addition of blood had little effect on the activity of any of the agents. Seven strains were unable to grow on Wilkins-Chalgren agar. Even with the addition of blood, one strain each of Bacteroides asaccharolyticus and Bacteroides bivius and two strains of Fusobacterium were unable to grow. Sixty-eight of the strains were tested against SCH 34343 and imipenem on Brucella lysed blood agar. Minimal inhibitory concentrations tended to be somewhat higher on Brucella lysed blood agar than on Wilkins-Chalgren agar, and all strains were able to grow on Brucella lysed blood agar.

Six-year retrospective survey of the resistance of Bacteroides fragilis group species to clindamycin and cefoxitin.

Two hundred and forty-six strains of the Bacteroides fragilis group, all clinical isolates, collected at the Wadsworth Veterans Administration Medical Center from 1977 to 1982, were tested for susceptibility to clindamycin and cefoxitin. There was no significant change in resistance to either clindamycin or cefoxitin over the time period tested for any individual species, nor for the B. fragilis group in toto. Striking differences in susceptibility to the two drugs were seen among species of the B. fragilis group. B. fragilis displayed resistance to cefoxitin (32 micrograms/ml) and clindamycin (8 micrograms/ml) of 0.0% and 0.8%, respectively, whereas B. thetaiotaomicron showed resistances of 12.7% to cefoxitin (32 micrograms/ml) and 9% to clindamycin (8 micrograms/ml). B. thetaiotaomicron, B. distasonis, and B. ovatus are distinctly more resistant to cefoxitin than B. fragilis and B. vulgatus. Similarly B. thetaiotaomicron and B. distasonis are much more resistant to clindamycin than are the other B. fragilis group species. It is apparent that determination of species within the B. fragilis group is important in evaluating a potential therapeutic regimen.

The isolation and characterisation of a major outer-membrane protein from Bacteroides distasonis.

An outer-membrane protein (OMP) was isolated from a clinical strain of Bacteroides distasonis. Changes in growth media did not appreciably affect the appearance of this protein in crude outer-membrane preparations examined by SDS-PAGE. However, the proportion of the protein relative to other OMPs was greater in 24-h cultures than in 48-h cultures. The protein could not be readily solubilised by various conventional detergent extraction techniques but treatment of the insoluble material at 100 degrees C with SDS released the protein, as did overnight extraction at 37 degrees C with SDS. This OMP was heat-modifiable, and thus was similar to the OmpA protein of Escherichia coli, with a faster mobility on SDS-PAGE when solubilised at 25 degrees C than at 100 degrees C. The critical temperature for conversion was between 80 degrees C and 90 degrees C. Because of the characteristic heat-modifiability, the protein was called B. distasonis HMP-1 (heat modifiable protein-1). Overnight exposure to EDTA or NaCl at 37 degrees C favoured conversion of the 25 degrees C form to the 100 degrees C form. In intact cells, the protein was labelled by a cell-surface radio-iodination procedure, and thus is at least partially exposed at the cell surface. No reactions between the B. distasonis HMP-1 and antibodies to either E. coli OmpA or E. coli porin were found by Western blot analysis. A B. distasonis OM preparation containing predominantly HMP-1 had pore-forming ability in a liposome assay. This study is the first report of the isolation and characterisation of a heat-modifiable OMP in Bacteroides, and it is the first description of pore-forming activity in a Bacteroides OM fraction.

In vitro activity of selected antibiotics against anaerobes.

Testing antibiotics for their activity against microorganisms is fraught with problems. The various methods and media yield different results, and controversy exists as to which is the most reliable. The technique used in our laboratory has shown wide differences in the susceptibility patterns of Bacteroides strains and other anaerobes to different antibiotics. Particularly with respect to the third-generation cephalosporins, reliable clinical data are needed to determine which in vitro tests most accurately predict clinical efficacy.

Media- and method-dependent variation in MIC values of ceftizoxime for clinical isolates of the Bacteroides fragilis group.

Although ceftizoxime has been used effectively in several clinical trials for infections involving anaerobic bacteria, reports of its in vitro activity against anaerobes are contradictory and confusing. In an effort to clarify the discrepant reports, we tested 90 strains of Bacteroides fragilis group organisms from patients with perforated or gangrenous appendicitis using eight different susceptibility testing procedures. The minimal inhibitory concentration values were dependent on the technique used; agar dilution values were often four twofold dilutions higher than microbroth dilution values. Agar techniques (including spiral gradient end point) gave values of 36% to 61% susceptible at breakpoint (depending on the technique), while the microbroth dilution techniques gave values of 84% to 92% susceptible. When a 64 microgram/ml breakpoint for agar dilution testing was used, the methods were more comparable, with the agar methods giving values of 64% to 83% susceptible. The results of the broth disk elution procedure were difficult to read and often did not agree with other values.

Antibiotic susceptibility testing of anaerobic organisms using the agar dilution method: comparison of three techniques.

Three currently used anaerobic susceptibility testing methods were compared: (1) the technique used at the Wadsworth Microbial Diseases Research Laboratory, (2) the technique listed as the reference standard by the National Committee on Clinical Laboratory Standards, and (3) the technique used at the Tufts New England Medical Center. Four-hundred-seventy anaerobic microorganisms, isolated from clinical specimens, were tested against cefoxitin, cefotetan, ceftizoxime, cefotaxime, ceftazidime, imipenem, and clindamycin. Significant differences were noted in mean inhibitory concentrations and percent susceptible at breakpoint among the three techniques used and varied with the antimicrobial agent and species tested.

Effect of inoculum size and medium on activity of seven antimicrobial agents against Bacteroides fragilis strains.

Antimicrobial activity of cefoxitin, cefotetan, cefotaxime, ceftizoxime, ceftazidime, imipenem, and clindamycin against four inocula of Bacteroides fragilis strains was determined on three different media. The inoculum sizes were 10(4), 10(5), 10(6), and 10(7) colony forming units (CFU) per spot. On all three media, substantial effects of inoculum size on minimal inhibitory concentrations (MIC) of cefotaxime and ceftizoxime were found: the doubled dilution differences in MICs between inocula of 10(7) and 10(4) CFU/spot were 2.2, 2.3, and 2.1 micrograms/ml of cefotaxime and 1.8, 4.4, and 4.0 micrograms/ml of ceftizoxime on Brucella base-laked blood agar, Wilkins-Chalgren agar, and a brain-heart infusion medium, respectively. An inoculum difference found on all three media with ceftazidime may also be of practical significance. There was evidence of larger differences between inocula on the Wilkins-Chalgren agar and brain-heart infusion than on the Brucella agar.