Biomedical subjects
Publications and source records attributed to R D Rolfe.
The in vitro susceptibility of Clostridium septicum was studied with a microtiter broth dilution method. Several antimicrobial agents demonstrated consistently good activity against the organism.
Antibiotic-associated pseudomembranous colitis has been linked with Clostridium difficile toxin. We examined the effect of toxins from four strains of C. difficile isolated from patients with pseudomembranous colitis on colonic adenylate (EC 4.6.1.1) and guanylate cyclase (EC 4.6.1.2) activities. Partially purified toxins had a cytotoxic effect on hamster fibroblasts in culture at a concentration of 10 ng/ml. Likewise, these toxins enhanced colonic guanylate cyclase activity two- to threefold, with the maximal stimulation being at 10 ng/ml. These toxins also enhanced guanylate cyclase activity in ileum, cecum, and duodenum. Both the cytotoxic activity on hamster fibroblasts and the enhancement of hamster guanylate cyclase activity were inhibited by antiserum to C. difficile toxin. These same toxins inhibited adenylate cyclase activity at a 100-ng/ml concentration, but had no effect at 10 ng/ml. They also had no effect at any concentration on colonic Na+-K+ adenosine triphosphatase. To be sure that the findings were not due to a contaminant, a purified C. difficile cytotoxin was used, and the same findings were found with the pure cytotoxin (at a 100-fold-lower concentration). The data suggest that activation of guanylate cyclase may be a factor in the pathogenesis of antimicrobial-associated pseudomembranous colitis.
Several species of anaerobic bacteria display variable Gram stain reactions which often make identification difficult. A simple, rapid method utilizing a 3% solution of potassium hydroxide to distinguish between gram-positive and gram-negative bacterial was tested on 213 strains of anaerobic bacteria representing 19 genera. The Gram stain reaction and KOH test results were compared with the antibiotic disk susceptibilities (vancomycin and colistin) the preliminary grouping of anaerobic bacteria. All three procedures were in agreement for the majority of strains examined. Some strains of clostridia, eubacteria, and bifidobacteria stained gram negative or gram variable; the KOH and antibiotic disk susceptibility tests correctly classified these strains as gram-positive. The KOH test incorrectly grouped some strains of Bacteroides sp., Fusobacterium sp., Leptotrichia buccalis, and Veillonella parvula, but all Gram stain results for these strains were consistent for gram-negative bacteria. The KOH test is a useful supplement to the Gram stain and antibiotic disk susceptibility testing for the initial classification of anaerobic bacteria.
Ten liquid media were compared under standard conditions for their ability to support the growth of Legionella pneumophila. Modified gonococcal-ferric cysteine broth (without sodium chloride) supplemented with 1% yeast extract yielded the best overall growth of the one strain of L. pneumophila examined. Growth rates were independent of pH changes which occurred during incubation. The growth rates of 10 different strains of L.pneumophila were compared in this medium. There appeared to be little difference in the growth rates of strains passaged frequently or infrequently, or between environmental and clinical isolates. Moderate aeration resulted in a faster growth rate and in approximately a 1 log10 higher final cell concentration as compared to a static broth culture. These experiments demonstrate that there are moderate to marked differences among the various media described in the literature and that no liquid medium yet developed supports rapid growth of L. pneumophila incubated without shaking.
Clostridium difficile has been shown to be a cause of antimicrobial agent-associated diarrhea and colitis. The source from which this organism gains access to the gastrointestinal tract is not known. Cultures of the hospital environments of six of eight patients whose fecal cultures were positive for C. difficile yielded this organism, whereas cultures of control hospital sites were almost invariably negative. These data suggest that hospital environmental contamination may be a potential source of infection. Further studies are needed to determine the role of various possible sources of C. difficile in human diarrheal disease.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Antimicrobial agent-induced ileocecitis of laboratory animals and colitis of man share common features. The significance of a newly described toxin in these two entities, the apparent source of the toxin (Clostridium difficile) and characteristics of the toxin are reviewed. Methods of toxin detection, isolation and rapid identification of C. difficile, and possible modes of therapy for antimicrobial agent-associated colitis of man are discussed.
Explore the source record for details and available documents.
Recent evidence indicates that toxigenic Clostridium difficile strains are a major cause of antimicrobial-associated ileocecitis in laboratory animals and pseudomembranous colitis in humans. C. difficile ATCC 9689 was cultivated in a synthetic medium to which 3% ultrafiltrated proteose peptone was added. Purification of the toxin from broth filtrate was accomplished through ultrafiltration (100,000 nominal-molecular-weight-limit membrane), precipitation with 75% (NH4)2SO4, and chromatographic separation using Bio-Gel A 5m followed by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-25 column. The purified toxin displayed only one band on polyacrylamide gel electrophoresis, and approximately 170 pg was cytopathic for human amnion cells. The isolated toxin was neutralized by Clostridium sordelli antitoxin, heat labile (56 degrees C for 30 min), and inactivated at pH 4 and 9; it had an isoelectric point of 5.0, increased vascular permeability in rabbits, and caused ileocecitis in hamsters when injected intracecally. Treatment of the toxin with trypsin, chymotrypsin, pronase, amylase, or ethylmercurithiosalicylate caused inactivation, whereas lipase had no effect. By gel filtration, its molecular weight was estimated as 530,000. Upon reduction and denaturation, the toxin dissociated into 185,000- and 50,000-molecular-weight components, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive dissociation yielded only the 50,000-molecular-weight component. The toxin appears to be protoplasmic and is released into the surrounding environment upon autolysis of the cells. Attempts to correlate specific enzymatic activity with the toxin have been unsuccessful. These studies will help delineate the role of C. difficile toxin in antimicrobial-associated colitis and diarrhea.
The effect of atmospheric oxygen on the viability of 13 strains of anaerobic bacteria, two strains of facultative bacteria, and one aerobic organism was examined. There were great variations in oxygen tolerance among the bacteria. All facultative bacteria survived more than 72 h of exposure to atmospheric oxygen. The survival time for anaerobes ranged from less than 45 min for Peptostreptococcus anaerobius to more than 72 h for two Clostridium perfringens strains. An effort was made to relate the degree of oxygen tolerance to the activities of superoxide dismutase, catalase, and peroxidases in cell-free extracts of the bacteria. All facultative bacteria and a number of anaerobic bacteria possessed superoxide dismutase. There was a correlation between superoxide dismutase activity and oxygen tolerance, but there were notable exceptions. Polyacrylamide gel electropherograms stained for superoxide dismutase indicated that many of the anaerobic bacteria contained at least two electrophoretically distinct enzymes with superoxide dismutase activity. All facultative bacteria contained peroxidase, whereas none of the anaerobic bacteria possessed measurable amounts of this enzyme. Catalase activity was variable among the bacteria and showed no relationship to oxygen tolerance. The ability of the bacteria to reduce oxygen was also examined and related to enzyme content and oxygen tolerance. In general, organisms that survived for relatively long periods of time in the presence of oxygen but demonstrated little superoxide dismutase activity reduced little oxygen. The effects of medium composition and conditions of growth were examined for their influence on the level of the three enzymes. Bacteria grown on the surface of an enriched blood agar medium generally had more enzyme activity than bacteria grown in a liquid medium. The data indicate that superoxide dismutase activity and oxygen reduction rates are important determinants related to the tolerance of anaerobic bacteria to oxygen.
Explore the source record for details and available documents.
Because of the increased price of palladium chloride, we investigated the effectiveness of the addition of this ingredient to the culture medium used for the recovery of anaerobes. With our system, the addition of palladium chloride did not consistently improve the recovery rate of bacteria from guinea pig intestinal contents and human fecal material.
The animal host and its intestinal microbial flora function together as a complex ecologic system in which there is a significant impact of the intestinal flora on the host as well as of components of the microbial flora on one another. Aerobic and anaerobic bacteria of the intestinal flora influence numerous anatomic, physiologic, and immunologic parameters of the host. Constituents of the indigenous intestinal flora also engage in a multitude of antagonistic and cooperative interactions. The normal bacterial intestinal flora represents an extremely important defense mechanism, which effectively interferes with the establishment of many important enteric pathogens. Mechanisms by which microorganisms suppress the growth of other microorganisms include modification of bile acids, stimulation of peristalsis, induction of immunologic responses, depletion of essential substrates from the environment, competition for attachment sites, creation of restrictive physiologic environments, and elaboration of antibiotic-like substances. Components of the intestinal microbial flora also interact synergistically in the induction of disease or the utilization of substrate.