Biomedical subjects
Publications and source records attributed to K H Wilson.
UV irradiation is widely used to inactivate contaminating DNA in PCR. Highly UV-absorbent deoxyribonucleoside triphosphates in PCR mixtures reduce the efficiency of UV decontamination. Optimal decontamination may be achieved by irradiating the PCR mixture without the deoxyribonucleoside triphosphates.
Efforts to culture and identify the intracellular bacteria associated with Whipple's disease have been unsuccessful. Nucleotide sequencing and amplification by the polymerase chain reaction was done on the bacterial 16 S ribosomal DNA present in a small-bowel biopsy specimen taken from a patient with Whipple's disease. A search by computer for similar rRNA sequences filed in databases showed the Whipple's-associated organism to be most similar to bacteria of the Rhodococcus, Streptomyces, and Arthrobacter genera, and more weakly related to mycobacteria. The biopsy specimen was estimated to contain around 10(7) cells of the organism. The probable aetiological agent for our patient's illness has not been identified previously in a patient with Whipple's disease.
A new disease was recognized in the United States in 1986. The etiologic agent, although not previously isolated from a human, appeared to be serologically related to Ehrlichia canis, a canine leukotropic rickettsia. We obtained blood specimens from 27 febrile patients with a history of tick exposure. Leukocytes from 24 patients not treated with tetracycline were placed onto a monolayer of DH82 cells. We performed indirect immunofluorescence on sera from all 27 febrile patients as well as sera from 12 patients with previously diagnosed ehrlichiosis. Intractoplasmic inclusions were first observed in culture 35 days after the addition of infected blood from one patient. Partial sequencing of the rRNAs from the human isolate and E. canis indicated that they are 98.7% related. Positive indirect immunofluorescence reactions to the human isolate were obtained for all 12 previously diagnosed patients and for 33% of the 27 febrile patients. Two patients were seropositive for the human isolate but not for E. canis. No sera were positive for E. canis and negative for the human isolate. We report the isolation of a previously unrecognized Ehrlichia sp. that appears to be the etiologic agent of human ehrlichiosis. Serologic data (range of antibody titers, 256 to 32,768) in combination with rRNA sequencing indicated that the newly isolated Ehrlichia sp. is similar, but not identical, to E. canis.
The bacterial 16S rRNA genes from blood samples of two patients with human ehrlichiosis and from an isolate recovered from one of the patients were amplified by using the polymerase chain reaction. The amplimers were then cloned and sequenced. The 16S rRNA gene sequence was also determined for Ehrlichia canis (two strains), E. equi, E. phagocytophila (two strains), and E. sennetsu (two strains). These sequences, along with a previously published 16S rRNA gene sequence of E. risticii, were compared. The 16S rRNA gene sequences were identical for all three sources of the human ehrlichiosis agent. The sequence comparisons indicate that the human ehrlichiosis agent is a new species most closely related to E. canis (98.2%) and more distantly related to other Ehrlichia spp. We propose that this species be named Ehrlichia chaffeensis sp. nov., with the Arkansas strain as the type strain.
Ribosomal RNA sequences are useful for establishing phylogenetic relationships, for oligonucleotide probes and for characterization of uncultured organisms. We describe rapid ribosomal DNA sequencing using PCR with transcript sequencing. Nucleic acid specificity at three steps (amplification, transcription and sequencing) eliminated the need for nucleic acid extraction or purification. Sequence was obtained from a crude lysate from a single colony of bacteria. The basic sequencing method should be adaptable to provide rapid sequence information in a wide variety of applications.
The sequence of small-subunit rRNA varies in an orderly manner across phylogenetic lines and contains segments that are conserved at the species, genus, or kingdom level. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, we amplified bacterial 16S ribosomal DNA sequences with the polymerase chain reaction. Priming sites were located at the extreme 5' end, the extreme 3' end, and the center of 16S ribosomal DNA. The isolates tested with these primers included members of the genera Staphylococcus, Coxiella, Rickettsia, Clostridium, Neisseria, Mycobacterium, Bilophila, Eubacterium, Fusobacterium, and Lactobacillus and the family Enterobacteriaceae. Initially, the yields from the reactions were erratic because the primers were self-complementary at the 3' ends. Revised primers that were not self-complementary gave more reproducible results. With the latter primers, 0.4 pg of Escherichia coli DNA consistently gave a visible band after amplification. This method should be useful for increasing the amounts of bacterial 16S ribosomal DNA sequences for the purposes of sequencing and probing. It should have a broad range of applications, including the detection and identification of known pathogens that are difficult to culture. This approach may make it possible to identify new, nonculturable bacterial pathogens.
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In an effort to explore a sensitive taxon-specific detection system for bacteria, we sequenced the 16S rRNA from two strains of Rickettsia rickettsii, five other rickettsiae, and Coxiella burnetti to find a probe site unique to R. rickettsii. We then synthesized a 16-mer that hybridized only to the rRNA of R. rickettsii. Using a primer complementary to a sequence found only in rickettsial rRNA, we then generated a cDNA. We amplified the probe site in a 180-base segment of the cDNA by using the cDNA primer and a second primer also unique to rickettsiae in a polymerase chain reaction. The segments of rRNA from each of the rickettsiae were amplified 10(6)- to 10(7)-fold, and the R. rickettsii probe hybridized only to the amplified segment from R. rickettsii. The rRNAs from Staphylococcus aureus, C. burnetii, and Neisseria meningitidis were not amplified and did not hybridize with the probe. The approach detailed below may prove clinically useful in the direct detection of pathogens that are difficult to cultivate.
An outbreak of antibiotic-associated colitis that occurred on a ward of a Michigan hospital during February-April, 1984, was studied by bacteriophage-bacteriocin typing. Stools from the seven involved patients yielded Clostridium difficile isolates of types B1537 or Cld7;B1537. C. difficile was recovered from 31.4% of environmental cultures obtained on the ward, and the majority of isolates were types B1537 or Cld7;B1537. When the ward was disinfected with unbuffered hypochlorite (500 parts per million (ppm) available chlorine), surface contamination decreased to 21% of initial levels and the outbreak subsequently ended. Phosphate buffered hypochlorite (1,600 ppm available chlorine, pH 7.6) was even more effective; its use resulted in a 98% reduction in surface contamination. These findings suggest that environmental contamination with C. difficile is important in the epidemiology of antibiotic-associated colitis, and that hypochlorite is effective in eliminating C. difficile from the hospital environment.
The cecal flora of mice is able to eliminate Clostridium difficile from the mouse cecum even when C. difficile is the first organism established. We used a continuous-flow (CF) culture model of the cecal flora to investigate the possibility that competition for nutrients is one mechanism for this antagonism. The medium for the CF cultures consisted of homogenates of fecal pellets from germfree mice. Carbohydrate analysis showed that mouse flora depleted 74 to 99.8% of the various carbohydrates from this environment-simulating medium. When inoculated into filtrates made from CF cultures of mouse flora, C. difficile multiplied slower than the dilution rate of the CF cultures unless glucose, N-acetylglucosamine, or N-acetylneuraminic acid was added. C. difficile did not synthesize hydrolytic enzymes able to cleave these monosaccharides from oligosaccharide side chains. As found previously, veal infusion broth did not support the growth of a microflora that could be transferred to gnotobiotic mice and fully suppress C. difficile. When mucin or monosaccharides found in mucin were added to veal infusion broth, the flora functioned normally in this regard. These data suggest that as yet unidentified organisms compete more efficiently than C. difficile for monomeric glucose, N-acetylglucosamine, and sialic acids found in colonic contents.
The large copy number of rRNA makes it an appealing target for oligonucleotide probes designed to identify microorganisms. Given that nucleotide sequences in rRNA are known to reflect phylogeny, species-specific rRNA probes should be feasible if the sequences found in closely related species are different. We sequenced portions of the 16S rRNA of three closely related clostridia found in the human colonic microflora: Clostridium bifermentans, C. sordellii, and C. difficile. The rRNAs of these three species showed 97 to 98% sequence similarity. Five oligonucleotide probes complementary to unique segments of the sequences were end labeled with 32P and hybridized on a nylon filter to the immobilized rRNA of each clostridium. Each probe efficiently hybridized only to the rRNA of the species to which it was directed. Complementary probes emitted a signal that exceeded by a factor of 100 to 1,000 the signal of probes that mismatched the target rRNA by 2 to 5 bases. Even a 1-base difference in rRNA sequence allowed a clear distinction between species. A systematic approach can efficiently yield taxon-specific oligonucleotide probes directed at rRNA.
At the Ann Arbor Veterans Administration Medical Center, 30 patients over a 6-month period became nosocomially infected or colonized by methicillin-resistant Staphylococcus aureus. Immediate institution of strict infection control measures, in conjunction with surveillance cultures of personnel and treatment of carriers, did not limit spread of the outbreak strain of MRSA. Multiple nonoutbreak strains, phenotypically exhibiting heteroresistance, were also uncovered. Thirteen hospital personnel were identified as MRSA carriers. Trimethoprim-sulfamethoxazole (TMP-SMX) and rifampin initially eradicated the carrier state, documented by anterior nares cultures in 13 courses of treatment in 11 employees. However, three employees were recolonized, one at one month, one at both one and four months, and one at four months. Treatment of the carrier state reservoir among personnel appeared to have no effect on the emergence and spread of nosocomial MRSA.
Five clusters of pseudobacteremias over a seven-month period were traced to a BACTEC radiometric device. Four episodes were due to enterococcus and one involved Staphylococcus aureus. Each cluster began with multiple positive blood cultures from a patient with clinical evidence of bacteremia. On subsequent days each bottle in sequence yielded growth of the identical organism, as shown by phage typing and antibiograms. Cultures from patients believed to be infected with the organisms tested positive earlier (mean = 1.4 days) than those patients believed to be pseudobacteremic (mean = 4.8 days). Investigation pointed toward the BACTEC device as the most likely source. Resin beads were found in the needle on several occasions and were shown to be transferred from bottle to bottle. We postulated that contaminated resin beads or liquid from a positive bottle remained in the needle above the portion that was heat-sterilized, contaminating the next bottle when it was sampled.
Hamster flora introduced into germfree mice reduced the cecum to conventional size, suppressed populations of Escherichia coli and Clostridium difficile to the same degree that mouse flora did, and corrected the hypocellularity that is characteristic of the small bowel of germfree mice. A highly toxigenic strain of C. difficile readily induced cecitis in germfree and antibiotic-treated conventional mice, and histological examination frequently revealed pseudomembranes. Toxins A and B were both detected in ceca of animals with colitis. Gnotobiotic mice provide a model in which to study the role of the indigenous microflora in protecting against antibiotic-associated colitis.
We studied the interactions between the entire cecal flora of hamsters and the pathogens Clostridium difficile and Escherichia coli in gnotobiotic mice and in a continuous-flow (CF) culture system in which the growth medium consisted of an extract of fecal pellets from germfree mice. CF cultures and germfree mice were colonized first with C. difficile and E. coli and then with the cecal flora of hamsters. Both in vivo and in vitro hamster flora markedly suppressed the potential pathogens. Contents of CF cultures inoculated with hamster flora were introduced into gnotobiotic mice previously colonized with C. difficile and E. coli. These mice were compared with mice given homogenates of hamster ceca. In both groups, the C. difficile population decreased by a factor of more than 10(6) and the E. coli population decreased by a factor of 10(4) to 10(5). CF culture contents also reduced the size of the dilated germfree mouse cecum to normal. When veal infusion broth was used as a medium, contents of CF cultures colonized with hamster flora failed to eliminate C. difficile from mice. Thus, the extract of fecal pellets appeared to contain a substance important for sustained colonization by important components of the cecal flora. We also studied the ability of collections of isolates to suppress the potential pathogens in both gnotobiotic mice and CF cultures. A total of 150 isolates obtained from predominant hamster flora at the ecologic climax stage (C flora) suppressed C. difficile and E. coli to 10 and 1 to 3%, respectively, of the population sizes attained in monoassociated mice. A total of 67 isolates obtained during ecologic succession combined with a C flora consisting of 100 isolates suppressed the potential pathogens to 0.3 and 0.03% of their original levels, respectively. Similar degrees of suppression occurred in CF cultures, further indicating that anaerobic CF cultures are promising models for investigation of the microbial ecology of C. difficile.
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The population dynamics of Clostridium difficile in the hamster gastrointestinal tract were studied after intragastric inoculation with organisms and a 51Cr tracer. Seventy-eight percent of spores germinated within the small intestine within 1 hr. Germinated spores and vegetative cells both showed two phases of elimination from the hamster cecum--an initial phase of rapid death that was not affected by antibiotic treatment followed by a phase of complete inhibition of multiplication. The latter phase of inhibition was not seen in antibiotic-treated animals and was thus attributable to the indigenous flora. The 51Cr tracer mixed well with cecal contents and was eliminated exponentially with a dilution rate constant ranging from -0.46/hr to -0.31/hr in normal hamsters. The hamster cecum was therefore dynamically analogous to a continuous flow system, a finding supporting the concept that anaerobic continuous flow cultures are useful in vitro models of the cecal ecosystem.
Although osteomyelitis of the foot in diabetic patients is generally considered a difficult infection to cure, the literature contains few reports comparing various treatment regimens. We have described two diabetic patients with osteomyelitis of the foot treated with oral antibiotics chosen on the basis of their broad activity against the pathogens commonly found in such infections. Both patients responded well to therapy and have shown no evidence of active osteomyelitis for 11 and four months, respectively.