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PubMed1964

USE OF A BACTERIAL CULTURE FILTRATE AS AN AID TO THE ISOLATION AND GROWTH OF ANEROBIC SPIROCHETES.

Hardy, Paul H., Jr. (The Johns Hopkins University School of Medicine, Baltimore, Md.), Young C. Lee, and E. Ellen Nell. Use of a bacterial culture filtrate as an aid to the isolation and growth of anaerobic spirochetes. J. Bacteriol. 87:1521-1525. 1964.-A previously described procedure that permits colonial growth of laboratory-adapted anaerobic spirochetes on surface streaked media was applied to the isolation of new spirochetal strains from human oral microflora. Primary isolates were readily cultivable as colonies, but they were found to be nutritionally dependent upon other microorganisms with which they are normally associated in vivo. Spirochetal growth was first noted around colonies of oral bacteria; pure cultures were then grown on media fortified with the culture filtrate of one such organism-a microaerophilic diphtheroid.

Bacteria↗
PubMed1964

ELECTRON MICROSCOPY OF AXIAL FIBRILS, OUTER ENVELOPE, AND CELL DIVISION OF CERTAIN ORAL SPIROCHETES.

Listgarten, M. A. (Harvard School of Dental Medicine and Forsyth Dental Center, Boston, Mass.), and S. S. Socransky. Electron microscopy of axial fibrils, outer envelope, and cell division of certain oral spirochetes. J. Bacteriol. 88:1087-1103. 1964.-The ultrastructure of axial fibrils and outer envelopes of a number of oral spirochetes was studied in thin sections and by negative contrast. The axial fibrils measured 150 to 200 A in diameter. Only one end of each fibril was inserted subterminally into the protoplasmic cylinder by means of a 400 A wide disc. The free ends of fibrils inserted near one end of the cylinder extended toward, and overlapped in close apposition, the free ends of fibrils inserted at the other end. In thin sections, some axial fibrils showed a substructure, suggestive of a dense central core. The outer envelopes of most spirochetes appeared to consist of 80 A wide polygonal structural subunits. However, in one large spirochete, the outer envelope demonstrated a "pin-striped" pattern. Cell division in a pure culture of Treponema microdentium was studied by negative contrast. Results suggested that this organism divides by transverse fission, the outer envelope being last to divide. During the course of division, new axial fibrils appeared to originate on either side of the point of constriction of the protoplasmic cylinder. Flagellalike extensions which were found in rapidly dividing organisms were due to protruding axial fibrils, and appeared to be the result of cell division. Some evidence is presented to support the concept of a homologous origin for axial fibrils and flagella.

Cell Division↗
PubMed1982

API ZYM system for identification of Bacteroides spp., Capnocytophaga spp., and spirochetes of oral origin.

A total of 80 oral strains of Bacteroides gingivalis, B. asaccharolyticus, B. melaninogenicus subsp. intermedius, B. melaninogenicus subsp. melaninogenicus, Capnocytophaga, Treponema denticola, and T. vincentii were characterized with the API ZYM system for 19 enzyme activities. Comparison of anaerobic and aerobic incubation with nine reference strains of these organisms showed no important differences. The key differential tests for black-pigmented Bacteroides strains and treponemes of oral origin were trypsin, alpha-glucosidase, and N-acetyl-beta-glucosaminidase. All Capnocytophaga strains produced distinctive aminopeptidase activities but varied in their glycosidic capabilities. The presence of a trypsin-like activity in B. gingivalis, T. denticola, and a group of Capnocytophaga strains may contribute to tissue destruction in periodontal disease.

Acetylglucosaminidase↗
PubMed1984

Lyme disease: a selective medium for isolation of the suspected etiological agent, a spirochete.

A simple procedure with a new selective culture medium for the isolation of the suspected etiological agent of Lyme disease from ticks is described. Live ticks (Ixodes dammini) were ground with a mortar and pestle, and the suspensions were inoculated into a selective and nonselective medium. The selective medium, which contained kanamycin and 5-fluorouracil, yielded positive spirochete cultures from 100% of the pooled ticks and from 79% of the single tick specimens. The isolation rate for the nonselective medium was 0% from the tick pools and 58% from the single tick specimens.

Animals↗
PubMed1986

Microbiological and biochemical characterization of spirochetes isolated from the feces of homosexual males.

Spirochetes were isolated from the feces of 11 homosexual males who had diarrhea. The anaerobic organisms were isolated from a selective medium that consisted of Trypticase soy agar supplemented with either 5% horse or human blood, 400 micrograms of spectinomycin per ml, and 5 micrograms of polymyxin B per ml. Nonselective media that permitted good growth of these fastidious organisms were developed, and selected biochemical tests were performed. The tests included carbohydrate utilization, detection of certain enzymes, and determination of volatile fatty-acid end products of metabolism. Two growth patterns were noted on solid media, a haze of growth and production of small colonies. Based on the results of biochemical tests, patterns of preformed enzymes, and volatile fatty-acid production, we believe that the 11 isolates represent a heterogeneous group of spirochetes. The data suggest that the human colon may harbor unique strains of cultivable spirochetes; additional study of the taxonomy of the organisms and assessment of their virulence for humans are needed.

Culture Media↗
PubMed1987

Comparison of three dispersion procedures for quantitative recovery of cultivable species of subgingival spirochetes.

Spirochetes are usually the predominant organisms observed microscopically in subgingival plaques removed from tooth sites associated with periodontitis, but these organisms are rarely isolated by cultural means, presumably because the media do not support their growth and/or because these fragile organisms are disrupted by the various procedures used to disperse plaque samples. In the present investigation, three dispersal procedures, sonification, mechanical mixing, and homogenization, were compared for their ability to permit the isolation of Treponema denticola, Treponema vincentii, Treponema socranskii, and Treponema pectinovorum from plaque samples on media that support the growth of these species. Plaque samples in which the spirochetes averaged 50% of the microscopic count were chosen. The highest viable recoveries of spirochetes were observed when the plaques were dispersed with a Tekmar homogenizer, and the lowest occurred with sonification. The highest recoveries averaged only about 1% of the total cultivable counts, indicating either that the sought-after species were minor members of the flora or that the dispersal procedures were still too harsh. A total of 91% of the isolates were T. denticola, 5% were T. socranskii, and 4% were a heretofore-undescribed species.

Bacteriological Techniques↗
PubMed1990

Description of a medium for isolating Anaerobiospirillum spp., a possible cause of zoonotic disease, from diarrheal feces and blood of humans and use of the medium in a survey of human, canine, and feline feces.

Anaerobiospirillum spp., motile, spiral anaerobic bacteria, have been implicated as a cause of diarrhea and bacteremia in humans. Anaerobiospirillumlike organisms and Anaerobiospirillum succiniciproducens were reported from 17 cases of diarrhea. Sixteen of the patients did not have any underlying disorders and recovered from the infection; the other one, who had a heart defect, did not. The formulation of a selective medium for Anaerobiospirillum spp. has enabled a survey of human, cat, and dog feces as possible sources for these anaerobic bacteria. Anaerobiospirillum spp. were not isolated from 527 "normal" human feces but were found in both cat and dog feces. We also describe biochemical tests and API ZYM results of A. succiniciproducens and anaerobiospirillumlike organisms.

Adult↗
PubMed1992

Molecular characterization of Serpulina (Treponema) hyodysenteriae isolates representing serotypes 8 and 9.

The study described here was carried out to further characterize reference strains of Serpulina (Treponema) hyodysenteriae representing serotypes 8 and 9. Results obtained from restriction fragment length polymorphism analysis, enteropathogenicity testing, and endotoxin profiles confirmed their identifications. Electron microscopy indicated that both strains were covered with a thin layer of capsule-like material. Immunoblot analysis indicated that an antigen in the 19-kDa region of proteinase K-digested whole cells reacted only with homologous antiserum. The serotype-specific antigens were sensitive to periodate oxidation but resistant to proteinase K digestion and migrated in the same region as purified lipopolysaccharides. Immunoblotting with proteinase K-digested whole cells appeared as useful as immunodiffusion with extracted lipopolysaccharide for the serological classification of S. hyodysenteriae. Immunogold labeling of whole cells and purified periplasmic flagella showed strong cross-reactions between S. hyodysenteriae and Serpulina innocens. Outer membrane preparations of strains representing serotypes 8 and 9 contained four major proteins which reacted with antisera against both species, and one major protein with a molecular mass of 46 kDa which reacted only with antisera against S. hyodysenteriae, irrespective of the serotype. Our findings suggest that periplasmic flagella and some outer membrane proteins are antigens common to both S. hyodysenteriae and S. innocens, whereas a 46-kDa outer membrane protein may be a species-specific antigen of S. hyodysenteriae. Finally, we propose immunoblotting as an alternative method to immunodiffusion for the serotyping of S. hyodysenteriae.

Animals↗
PubMed1992

Polymerase chain reaction for detection of Leptospira spp. in clinical samples.

A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema pallidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative.

Adolescent↗
PubMed1993

Human intestinal spirochetes are distinct from Serpulina hyodysenteriae.

Twenty-nine intestinal spirochetes isolated from Australian aboriginal children and six strains from Italian adults (HRM1, -2, -4, -5, -7, and -14) were genetically examined at 15 enzyme loci by using multilocus enzyme electrophoresis. Results were compared with those previously obtained for 188 porcine intestinal spirochetes. DNA from human strain HRM7 and porcine strain Serpulina hyodysenteriae P18A were also radioactively labeled and hybridized with DNA from 12 other human and porcine intestinal spirochetes. Both the multilocus enzyme electrophoresis and hybridization techniques demonstrated that the human spirochetes were not S. hyodysenteriae. They belonged to another distinct genetic group of spirochetes that included P43/6/78, the bacterium recovered from the first recorded case of porcine intestinal spirochetosis. Bacteria in this distinct group also differed from Serpulina spp. in possessing only four, five, or occasionally six axial filaments, being slightly thinner, and having more pointed ends. These findings add further weight to the possibility that human intestinal spirochetes may act as enteric pathogens.

Adult↗
PubMed1993

Genetic similarity of intestinal spirochetes from humans and various animal species.

The chromosomal DNA of spirochetes isolated from human, swine, dog, mouse, rat, and chicken intestine or feces was subjected to restriction enzyme analysis and hybridization with three different DNA probes, derived from a flagellin gene, a hemolysin gene, and the 16S rDNA sequence of the pathogenic swine intestinal spirochete Serpulina hyodysenteriae. This genetic analysis showed that intestinal spirochetes represent a heterogeneous but related population of bacteria. In general, unique genotypes were distinguished among isolates from the same host species; they were not present among isolates from other host species. This suggests the host specificity of some strains. An exception to this are isolates from humans and dogs suffering from gastrointestinal disorders; these isolates showed highly similar or even identical genotypes. None of them resembled any of the genotypes of isolates found in other host species without apparent disease.

Animals↗
PubMed1993

Characterization of two DNA probes specific for Serpulina hyodysenteriae.

Two DNA probes, one 1.1- and one 0.75-kb probe, specific for Serpulina hyodysenteriae were isolated from a genomic library generated from virulent S. hyodysenteriae 5380. These probes are highly specific and react with all S. hyodysenteriae strains tested. Under stringent conditions, the DNA probes did not react with the nonpathogenic species Serpulina innocens or with other species of enteric bacteria, including Escherichia coli. Both probes are able to detect S. hyodysenteriae in colony blot hybridizations, and when applied to fecal specimens, they can detect 10(4) S. hyodysenteriae cells in 0.1 g of seeded fecal matter. Both probes can detect S. hyodysenteriae in fecal specimens from swine with clinical signs of swine dysentery after experimental challenge and from swine from a herd with an acute outbreak of swine dysentery. These probes have application as a diagnostic tool in veterinary microbiology.

Animals↗
PubMed1994

Identification and characterization of Serpulina hyodysenteriae by restriction enzyme analysis and Southern blot analysis.

Chromosomal DNA restriction enzyme analysis and Southern blot hybridization were used to characterize Serpulina hyodysenteriae strains. When chromosomal DNAs from selected strains (reference serotypes) of S. hyodysenteriae were digested with the restriction endonuclease Sau3A and hybridized with a 1.1-kb S. hyodysenteriae-specific DNA probe, a common 3-kb band was always detected in S. hyodysenteriae strains but was absent from Serpulina innocens strains. When the chromosomal DNA was digested with the restriction endonuclease Asp 700 and hybridized with two S. hyodysenteriae-specific DNA probes (0.75 and 1.1 kb of DNA), distinct hybridization patterns for each S. hyodysenteriae reference strain and the Australian isolate S. hyodysenteriae 5380 were detected. Neither the 1.1-kb nor the 0.75-kb DNA probe hybridized with Asp 700- or Sau3A-digested S. innocens chromosomal DNA. The presence of the 3-kb Sau3A DNA fragment in S. hyodysenteriae reference strains from diverse geographical locations shows that this fragment is conserved among S. hyodysenteriae strains and can be used as a species-specific marker. Restriction endonuclease analysis and Southern blot hybridization with these well-defined DNA probes are reliable and accurate methods for species-specific and strain-specific identification of S. hyodysenteriae.

Animals↗