Biomedical subjects
Publications and source records attributed to R J Carman.
Clostridium difficile causes approximately 25% of nosocomial antibiotic-associated diarrheas and most cases of pseudomembranous colitis. We evaluated C. DIFF CHEK, a new screening test that detects glutamate dehydrogenase of C. difficile. Our results showed that this test was comparable to PCR in sensitivity and specificity and outperformed bacterial culture.
To study the utility of an in vitro model system for assessing the effect of low concentrations of a fluoroquinolone (FQ) drug on the ecology of the human intestinal microflora, chemostats containing human fecal flora were exposed to 0.43, 4.3, and 43microg of ciprofloxacin (CI) per milliliter. Prior to and during drug exposure, we assayed short-chain fatty acids (SCFA), bacterial populations, and the relative levels of susceptibility of these populations to CI and trovafloxacin (TV), a newer related FQ with increased activity against anaerobes. The degree to which CI affected the chemostat ecology was measured statistically by comparing observed data with the corresponding predicted "no effect" level. No changes in total SCFA were observed; only butyrate was significantly higher at the intermediate and high-dose levels. Enterococci counts and the levels of susceptibility to CI among enterococci were also unaffected. Escherichia coli counts decreased in a dose-dependent manner. Susceptibility levels in E. coli followed no interpretable pattern. Bacteroides fragilis group (BfG) counts decreased significantly following exposure to 43 and 4.3microg/mL CI. Ciprofloxacin susceptibility among the BfG in these chemostats was not determined because the BfG counts were too low (less than 30 colonies per plate) when undiluted chemostat samples were plated. However, within 2 days of exposure to 0.43microg/mL CI, the percentage of BfG resistant to 4microg/mL CI increased to over 95%. Before exposure, all BfG were susceptible to both CI (2microg/mL) and TV (0.25microg/mL). All BfG isolated during exposure were resistant to both CI (4microg/mL) and TV (2microg/mL). Resistance selection in the BfG was unexpected as the MIC(90) of CI for B. fragilis is 8microg/mL. Since the average colon flora is about 20% B. fragilis and other bacteroides, CI may impact the human gut flora even at subtherapeutic levels.
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Clostridium perfringens type A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD). Recent studies suggest that C. perfringens type A food poisoning is caused by C. perfringens isolates carrying a chromosomal cpe gene, while CPE-associated non-food-borne GI diseases, such as AAD, are caused by plasmid cpe isolates. Those putative relationships, obtained predominantly with European isolates, were tested in the current study by examining 34 cpe-positive, C. perfringens fecal isolates from North American cases of food poisoning or AAD. These North American disease isolates were all classified as type A using a multiplex PCR assay. Furthermore, restriction fragment length polymorphism and pulsed-field gel electrophoresis genotyping analyses showed the North American AAD isolates included in this collection all have a plasmid cpe gene, but the North American food poisoning isolates all carry a chromosomal cpe gene. Western blotting demonstrated CPE expression by nearly all of these disease isolates, confirming their virulence potential. These findings with North American isolates provide important new evidence that, regardless of geographic origin or date of isolation, plasmid cpe isolates cause most CPE-associated AAD cases and chromosomal cpe isolates cause most C. perfringens type A food poisoning cases. These findings hold importance for the development of assays for distinguishing cases of CPE-associated food-borne and non-food-borne human GI illnesses and also identify potential epidemiologic tools for determining the reservoirs for these illnesses.
Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.
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Segmented filamentous bacteria (SFB) are nonpathogenic bacteria that are commonly found attached to the intestinal walls of many animals. Until now, these bacteria have not been cultured in vitro. Recently, a 16S rRNA sequence analysis revealed that SFB isolated from mice represent a distinct subline within the Clostridium subphylum of the gram-positive bacteria. Since SFB isolated from mice, rats, and chickens are known to be host specific, we investigated the phylogenetic relationships among SFB obtained from these three hosts. Total DNAs from the intestinal floras of chickens and rats were used as templates for PCR amplification of 16S rRNA genes. PCR products were cloned and screened by a dot blot hybridization procedure to identify homologous sequences that cross-reacted with mouse SFB-specific oligonucleotide probes. A phylogenetic analysis of these 16S ribosomal DNA sequences revealed that SFB isolated from these three hosts form a natural group, which is peripherally related to the genus Clostridium sensu stricto (group I Clostridium). The SFB obtained from chickens, rats, and mice had closely related, albeit different, 16S rRNA gene sequences. The observed levels of 16S rRNA sequence divergence, ca. 1.5 to 3%, together with host specificity, suggest that SFB isolated from mice, rats, and chickens represent different species and that coevolution of the SFB and their hosts occurred. "Candidatus Arthromitus" is proposed as the provisional generic name for this group of organisms.
We have previously observed that trypsin-like activity in Porphyromonas gingivalis culture supernatants is inhibitable by the plasma arg-serpin antithrombin III (ATIII). This report demonstrates that a partially purified P. gingivalis trypsin-like enzyme (M(r) 47,000) is inhibited by ATIII with an association rate constant (k(ass)) of 5.65 x 10(4) M-1 s-1 but does not form SDS-stable complexes. Heparin enhances the k(ass) and stabilizes the complexes but in either case such inhibition is temporary and results in ATIII inactivation by reactive centre proteolysis between R393-S394. In the absence of heparin this is accompanied by N-terminal cleavage between K39-I40.
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The small-subunit rRNA (16S rRNA) sequence of Tyzzer's bacillus (also known as "Bacillus piliformis") was elucidated by using the polymerase chain reaction followed by reverse transcriptase sequencing. By using maximum-likelihood analysis, a phylogenetic tree was constructed from this and other 16S rRNA sequences available from the first release of the Ribosomal Database Project (G. J. Olsen, R. Overbeek, N. Larsen, T. L. Marsh, M. J. McCaughey, M. A. Maciukenas, W.-M. Kuan, T. J. Macke, Y. Xing, and C. R. Woese, Nucleic Acids Res. 20:2199-2200, 1992). Tyzzer's bacillus grouped with a specific set of anaerobic bacteria, most of which are Clostridium spp. The closest identified relatives are Clostridium coccoides, Clostridium oroticum, Clostridium clostridiiforme, Clostridium symbiosum, and Streptococcus hansenii. Clostridium amino-valericum and "Acetitomaculum ruminis" are also solidly allied with this ensemble. We propose that Tyzzer's bacillus be reclassified as Clostridium piliforme on the basis of its 16S rRNA sequence.
The composition and functions of the intestinal floras from humans and other animals is compared and contrasted. Intrinsic and extrinsic factors, such as anatomy, age, and diet, help define the nature of the flora typical to each species of animal. Stable differences, such as the ability to reduce cholesterol or not, between the flora of individuals of the same species are described. The difficulties of characterizing a flora by cultural taxonomy are discussed and alternatives are suggested. These alternatives, collectively known as microflora associated characteristics (MACs), are measures of the biochemical activity of the bacterial flora. How MACs can be used to measure the effects of subtherapeutic levels of antibiotics on a flora is described.
Ten healthy ponies from a single herd were found by repeated fecal culture to be free of Salmonella species and Clostridium cadaveris. In a preliminary study, four ponies administered a single oral dose of 10 mg/kg lincomycin did not develop idiopathic colitis when the drug was administered alone. Four other ponies were administered 10 mg/kg lincomycin by stomach tube together with 0.45 L of colonic content from a horse with idiopathic colitis induced earlier by lincomycin alone. Two of the four ponies were treated with 25 g oral zinc bacitracin premix (110 g/kg active ingredient) 24 h later. Forty-two hours after inoculation the two untreated ponies had severe signs of idiopathic colitis and were euthanized. Postmortem findings were typical of idiopathic colitis. The two treated ponies had milder illness but the more severely affected was also euthanized; the other was retreated at 42 h with bacitracin pre-mix and again 12 h later. Its illness and diarrhea resolved over the next 24 h. Clostridium cadaveris was isolated in large numbers from the cecum of the euthanized ponies and their cecal content contained mouse lethal and guinea pig dermonecrotic, but not cytotoxic, activity. Enterotoxins of Clostridium perfringens and Clostridium difficile could not be demonstrated. No toxin could be demonstrated in culture supernatants of C. cadaveris or in supernatants of cecal contents treated with ethanol prior to culturing in anaerobically incubated broth. No Salmonella spp. were isolated. A further two ponies were administered 10 mg/kg lincomycin orally with 0.45 L colonic content from a horse with idiopathic colitis, as described.(ABSTRACT TRUNCATED AT 250 WORDS)
Using an agar dilution method we measured the minimum inhibitory concentration (MIC) of 12 antimicrobial agents against 11 strains of iota-toxigenic strains of Clostridium spiroforme. Each strain was isolated from a separate outbreak of toxic diarrhoea of rabbits. Vancomycin and bacitracin, both agents used to treat intestinal clostridioses of humans and other animals, had a relatively high MIC (8 micrograms/ml or more). Metronidazole was uniformly active against C. spiroforme. With MIC of 8 micrograms/ml or more, both lincomycin (11 strains) and erythromycin (9 strains) were relatively inactive against C. spiroforme, conversely, penicillin G was active (MIC for 8 strains was 0.5 micrograms/ml or less). Exposure to any one of these drugs has been implicated as a predisposing factor for C. spiroforme mediated diarrhoea of rabbits. The greatest variation in MIC was seen for erythromycin (8-fold), penicillin G (8-fold) and tetracycline (16-fold).
The interaction between Porphyromonas gingivalis culture supernatant and human serum was examined. Hydrolysis of the major serum proteins was thiol-dependent and correlated with the trypsin-like activity of the sample. Transferrin and IgG light chains were less susceptible to degradation than albumin and IgG heavy chains and partially degraded IgG retained antigen-binding capability. Serum inhibited the trypsin-like activity in a fluorimetric assay. The inhibition was shown to be independent of the level of IgG antibody reactive with whole cells of P. gingivalis. Purified preparations of antithrombin III, a serine protease inhibitor, but not alpha 1-antitrypsin nor alpha 2-macroglobulin inhibited the trypsin-like activity in the fluorometric assay.
The identity of the major surface antigens of Porphyromonas gingivalis was investigated. Outer membranes of P. gingivalis strains W83, W50, 381 and NCTC 11843 were prepared following inactivation of the trypsin-like enzyme activity. Three proteins, molecular weight 115, 55 and 40 kDa, were major components of the outer membranes of strains W83 and W50 and were also present in strains 381 and NCTC 11834. Two proteins, 55 and 47 kDa, were released from the cells during the sonication step of the outer membrane preparation procedure. Immunoblots using preparations of P. gingivalis W83 and serum from a case-control study of adult periodontitis demonstrated higher mean antibody reactivity in the case population to all the major proteins except for the 115 kDa outer membrane protein, which was recognized equally well by both populations. We conclude that the 55, 47 and 40 kDa proteins are important surface antigens of P. gingivalis. Characterization of the structure and function of these components should lead to an improved understanding of the host-parasite interactions in adult periodontitis.
We have reviewed the recent literature on the humoral immune responses to a variety of subgingival plaque bacterial species in patients with destructive periodontal diseases. We do not feel that the information presently available on the specific antibody responses to proposed pathogens such as Bacteroides gingivalis and Actinobacillus actinomycetemcomitans allows antibody responses to be diagnostic. All control subjects without periodontal destruction have antibodies to candidate pathogens but the generally higher levels in patients are not sufficiently elevated to be diagnostic. Nor can they be used to predict the initiation of disease or the onset of new episodes of destruction where disease had previously occurred. Successful treatment of patients may lead to lower levels of antibodies to some organisms, including possible pathogens, and thus support a given species in the aetiopathogenesis of disease. It appears that unsuccessful treatment may be accompanied by continuing high antibody levels to some organisms and further studies may enable this observation to be used to monitor therapy. There is some evidence from serological studies that each destructive episode may be induced by a different bacterial species or consortium. The start of studies using single antigens and the techniques of molecular biology will provide not only antibody-based diagnostic methods but also allow us to determine which bacterial antigens are virulence factors and thus the role of the antibody responses, whether protective or damaging, in the periodontal diseases.
Porphyromonas gingivalis culture supernate was found to induce homotypic agglutination of human polymorphonuclear leukocytes (PMN). Pretreatment of PMN with P. gingivalis supernate inhibited both the rate and the degree of agglutination induced by the secretagogues PMA and FMLP. Lipopolysaccharide from P. gingivalis upregulated the CR3 (Mac-1, CD11b) receptors on PMN. Treatment of glass-adherent PMN with P. gingivalis supernate did not alter their phagocytic capacity for P. gingivalis cells but when PMN were pretreated in suspension the cells adhered less well to glass and phagocytosis of those PMN that did adhere was reduced. P. gingivalis supernate treatment of PMN induced lysozyme release but the amount released during phagocytosis when supernate was present did not change. Neither P. gingivalis supernate nor LPS were cytotoxic for PMN. The data suggest that P. gingivalis factors could interfere with PMN elimination of this organism at the site of infection by inappropriately stimulating PMN, depressing phagocytosis and causing enhanced CR3 expression. The consequent agglutination or enhanced adherence could also lead to decreased phagocytic capacity of the adherent or agglutinated cells.
Periodontal microbiology is reviewed with regard to the potential of certain characteristics to serve as markers of high risk groups or individuals for periodontal diseases. The generally accepted associations between particular organisms and the various periodontal diseases are discussed. The usefulness of various clinical study designs is reviewed. The ecology of the subgingival plaque microflora is discussed and a number of suggestions for future research are made. We have concluded that there is no monospecific aetiology to any of the various periodontal conditions. Nevertheless, we give particular attention to the role of the black-pigmented bacteroides based upon our belief that they, and Bacteroides gingivalis in particular, are fundamental to our understanding of the biology of periodontal diseases in humans and other animals. Consequently, the contribution of its various virulence factors and their potential as markers of disease susceptibility and activity is addressed.