Detection of Borrelia burgdorferi DNA in museum specimens of Ixodes dammini ticks.

In order to investigate the potential for Borrelia burgdorferi infection before the recognition of Lyme disease as a clinical entity, the polymerase chain reaction (PCR) was used to examine museum specimens of Ixodes dammini (deer ticks) for the presence of spirochete-specific DNA sequences. One hundred and thirty-six archival tick specimens were obtained representing various continental U.S. locations; DNA sequences characteristic of modern day isolates of B. burgdorferi were detected in 13 1940s specimens from Montauk Point and Hither Hills, Long Island, New York. Five archival specimens of Dermacentor variabilis (dog tick) from the same collection and 118 Ixodes specimens from other endemic and nonendemic sites were negative. These data suggest that the appearance of the Lyme disease spirochete in suitable arthropod vectors preceded, by at least a generation, the formal recognition of this disease as a clinical entity in the United States.

Quantitative comparison of intracellular concentration and volume of Clara cell 10 KD protein in rat bronchi and bronchioles based on laser scanning confocal microscopy.

We used an antibody to rat Clara cell 10 KD secretory protein (CC10) to compare the abundance of CC10 in non-ciliated epithelium of bronchioles and bronchi by immunohistochemistry and laser scanning confocal microscopy (LSCM) in the reflectance mode. Three zones of reflectance intensity (high, medium, low), directly related to CC10 content, were used to distinguish subcellular compartments of differing densities. Two major compartments contained CC10: granules and endoplasmic reticulum. Bronchiolar cell granules had relatively even reflectance, a high CC10 concentration (0.92 mg/ml), and occupied 7.5% of the cell volume. Bronchial cell granules were bi-zonal, lower in CC10 concentration (0.83 mg/ml), and occupied less cell volume (2.3%). Low-reflecting endoplasmic reticulum occupied a greater cell volume in bronchioles than in bronchi (32.8% and 22.1%, respectively). An intermediate-density zone consisted of endoplasmic reticulum close to granules. CC10 abundance, expressed as ng stored per unit surface area of epithelial basal lamina, was more than three times greater in bronchioles than in proximal bronchi (1.50 ng/mm2 vs 0.42 ng/mm2). These data demonstrate that the process of CC10 secretion differs markedly in non-ciliated epithelium of bronchi and bronchioles. Identifiable mucous-goblet cells did not contain CC10. The LSCM provides a method for quantifying intracellular CC10 pools in intact lung cells and has the potential for quantifying other proteins in subcellular compartments.

Regulation of Clara cell 10 kD protein secretion by pilocarpine: quantitative comparison of nonciliated cells in rat bronchi and bronchioles based on laser scanning confocal microscopy.

Standard immunohistochemical techniques and laser scanning confocal microscopy were used to assess changes in the abundance of Clara cell 10 kD protein (CC10) within granules and endoplasmic reticulum of rat nonciliated cells after the administration of the secretagogue pilocarpine. Intracellular pools of CC10 were compared over time with time-matched controls at two airway levels, proximal bronchi and terminal bronchioles. Three zones of reflectance intensity (high, medium, and low), corresponding to different densities of CC10 were used to quantify changes in CC10 levels. We observed a shift in the abundance of CC10 from endoplasmic reticulum to granules 30 min after injection of pilocarpine. In addition, the depletion of granule-based CC10 occurred earlier in bronchial cells (30 min) than in bronchiolar cells (60 min). We also noted that the density of CC10 within the two cellular compartments remained steady even though the levels of CC10 dropped due to degranulation. Following degranulation, CC10 levels returned to near-control levels. In the absence of pilocarpine, the percentage of cell volume occupied by the granule-based CC10 indicated that two varieties of nonciliated cells exist in bronchial airways. However, administration of pilocarpine abolishes this difference in CC10 abundance. Our results demonstrate that bronchial cells respond sooner to the secretagogue than do their bronchiolar counterparts and/or possess biosynthetic capabilities that are more easily overwhelmed than those of the terminal bronchiolar cells. These differences suggest that proximal nonciliated cells are distinct from distal nonciliated cells.

Does blood of healthy subjects contain bacterial ribosomal DNA?

Real-time PCR methods with primers and a probe targeting conserved regions of the bacterial 16S ribosomal DNA (rDNA) revealed a larger amount of rDNA in blood specimens from healthy individuals than in matched reagent controls. However, the origins and identities of these blood-associated bacterial rDNA sequences remain obscure.

Dose-dependent tolerance to ozone. III. Elevation of intracellular Clara cell 10-kDa protein in central acini of rats exposed for 20 months.

Understanding factors that promote pulmonary tolerance to long-term oxidant injury is essential to evaluating health risks in humans. One such factor may be Clara cell 10-kDa protein (CC10), a protein secreted by nonciliated cells in distal conducting airways thought to have an anti-inflammatory action against inhaled xenobiotics. Using standard immunohistochemical techniques and laser scanning confocal microscopy, we assessed changes in CC10 abundance in the centriacinar region of the rat following 20 months' exposure to 0.12 and 1.00 ppm ozone. Three zones of reflectance intensity (high, medium, and low), directly related to CC10 density, were used to distinguish between the two major subcellular compartments where CC10 is distributed: granules and endoplasmic reticulum. Low levels of ozone (0.12 ppm) had no significant effect on the cellular distribution or abundance of CC10 in nonciliated epithelium in the centriacinar region. In contrast, 1.00 ppm ozone not only elevated cellular volume of granule-based CC10, but also elevated the protein's concentration within the granules and increased the number of granules per cell profile. The proportion of nonciliated cells in terminal bronchioles increased significantly at the expense of ciliated cells. This combination of factors led to a threefold increase in CC10 stored per unit surface area of epithelium in terminal bronchioles. The nonciliated cells in ozone-induced respiratory bronchioles contained a distribution of CC10 similar to that of bronchiolar nonciliated cells in control animals. We conclude that ozone-induced tolerance may be related to the increased abundance and wider distribution of CC10 in central acini of rats following long-term ozone exposure.

Comparison of phenotypic and genotypic techniques for identification of unusual aerobic pathogenic gram-negative bacilli.

Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology, but one that is difficult or impossible for many slow-growing and fastidious organisms. We used identification systems based on cellular fatty acid profiles (Sherlock; MIDI, Inc., Newark, Del.), carbon source utilization (Microlog; Biolog, Inc., Hayward, Calif.), and 16S rRNA gene sequence (MicroSeq; Perkin-Elmer Applied Biosystems Division, Foster City, Calif.) to evaluate 72 unusual aerobic gram-negative bacilli isolated from clinical specimens at the Mayo Clinic. Compared to lengthy conventional methods, Sherlock, Microlog, and MicroSeq were able to identify 56 of 72 (77.8%), 63 of 72 (87.5%), and 70 of 72 (97.2%) isolates to the genus level (P = 0.002) and 44 to 65 (67.7%), 55 of 65 (84.6%), and 58 of 65 (89.2%) isolates to the species level (P = 0.005), respectively. Four Acinetobacter and three Bordetella isolates which could not be identified to the species level by conventional methods were identified by MicroSeq. In comparison to the full 16S rDNA sequences, the first 527 bp provided identical genus information for all 72 isolates and identical species information for 67 (93.1%) isolates. These data show that MicroSeq provides rapid, unambiguous identification of clinical bacterial isolates. The improved turnaround time provided by genotypic identification systems may translate into improved clinical outcomes.

Differentiated bronchiolar epithelium in alveolar ducts of rats exposed to ozone for 20 months.

The effects of exposure to 1.0 ppm of ozone for twenty months were studied in male Fischer 344 rats. Light microscopic, morphometric, and immunohistological approaches were used to determine the distribution and degree of differentiation of ciliated and nonciliated bronchiolar epithelial (Clara) cells lining alveolar ducts of the central acinus, a primary target of ozone-induced lung injury. Alveolar duct pathways extending beyond the level of the most proximal alveolar outpocketing of terminal bronchioles were isolated in longitudinal profile. The distance that ciliated and nonciliated bronchiolar epithelial (Clara) cells projected down each alveolar duct pathway was determined by placing concentric arcs radiating outward from a single reference point at the level of the first alveolar outpocketing. A high degree of heterogeneity in the magnitude of bronchiolar epithelial cell extension into alveolar ducts was noted for each isolation and animal. Age-matched control animals also demonstrated variation in the degree of bronchiolar epithelial cell extension down alveolar ducts. In animals exposed to ozone, a striking similarity was noted by scanning electron microscopy in the surface characteristics of cells lining both terminal bronchioles and alveolar ducts. The presence of Clara cell secretory protein in cells of bronchioles and alveolar ducts was also detected immunohistochemically and visualized using confocal laser scanning microscopy in the reflectance mode. Well-differentiated ciliated and nonciliated bronchiolar epithelial cells were found lining alveolar septal tips and alveoli up to a depth of 1,000 mu into the pulmonary acinus after 20 months of exposure to ozone. No evidence of inflammation was present in alveolar ducts, suggesting that epithelial cell transformations in alveolar ducts is a natural consequence of lifetime exposures to oxidant gases.

Evaluation of central nervous system involvement in Lyme borreliosis patients with a solitary erythema migrans lesion.

To determine whether early dissemination of Borrelia burgdorferi to the central nervous system occurs in stage I of Lyme borreliosis, neurological and cerebrospinal fluid examination was performed in 48 consecutive patients in whom the only sign of infection was a solitary erythema migrans lesion. Long-term follow-up after treatment with tetracycline was carried out by telephone interview. At presentation, neurological findings were normal in all 48 patients. Cerebrospinal fluid samples were obtained from 29 (60%) patients. Mild pleocytosis and mild impairment of the blood-brain barrier were present in four and one of these patients, respectively. No significant amount of tumor necrosis factor or interleukin 6 was found in the cerebrospinal fluid samples. Culture results of 13 cerebrospinal fluid samples were negative. Borrelia burgdorferi DNA was only detected by the polymerase chain reaction in one of two aliquots of the cerebrospinal fluid sample of one patient. None of 46 patients who were interviewed 12 to 51 (median 25) months after antibiotic treatment developed manifestations consistent with disseminated or chronic Lyme borreliosis. Thus, no compelling evidence was found for the presence of asymptomatic central nervous system involvement in patients with clinically localized Lyme borreliosis.

Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains.

Three hundred and twenty isolates of Staphylococcus aureus were typed by DNA sequence analysis of the X region of the protein A gene (spa). spa typing was compared to both phenotypic and molecular techniques for the ability to differentiate and categorize S. aureus strains into groups that correlate with epidemiological information. Two previously characterized study populations were examined. A collection of 59 isolates (F. C. Tenover, R. Arbeit, G. Archer, J. Biddle, S. Byrne, R. Goering, G. Hancock, G. A. Hébert, B. Hill, R. Hollis, W. R. Jarvis, B. Kreiswirth, W. Eisner, J. Maslow, L. K. McDougal, J. M. Miller, M. Mulligan, and M. A. Pfaller, J. Clin. Microbiol. 32:407-415, 1994) from the Centers for Disease Control and Prevention (CDC) was used to test for the ability to discriminate outbreak from epidemiologically unrelated strains. A separate collection of 261 isolates form a multicenter study (R. B. Roberts, A. de Lencastre, W. Eisner, E. P. Severina, B. Shopsin, B. N. Kreiswirth, and A. Tomasz, J. Infect. Dis. 178:164-171, 1998) of methicillin-resistant S. aureus in New York City (NYC) was used to compare the ability of spa typing to group strains along clonal lines to that of the combination of pulsed-field gel electrophoresis and Southern hybridization. In the 320 isolates studied, spa typing identified 24 distinct repeat types and 33 different strain types. spa typing distinguished 27 of 29 related strains and did not provide a unique fingerprint for 4 unrelated strains from the four outbreaks of the CDC collection. In the NYC collection, spa typing provided a clonal assignment for 185 of 195 strains within the five major groups previously described. spa sequencing appears to be a highly effective rapid typing tool for S. aureus that, despite some expense of specificity, has significant advantages in terms of speed, ease of use, ease of interpretation, and standardization among laboratories.

Staphylococcus succinus sp. nov., isolated from Dominican amber.

Two bacterial isolates, designated AMG-D1T and AMG-D2, were recovered from 25-35-million-year-old Dominican amber. AMG-D1T and AMG-D2 biochemically most closely resemble Staphylococcus xylosus; they differ physiologically from other staphylococci. Fatty acid analysis and comparisons with extensive databases were unable to show relatedness to any specific taxon. Moreover, AMG-D1T and AMG-D2 contain tuberculostearic acid and meso-diaminopimelic acid, characteristic of the G + C-rich coryneform bacteria, as opposed to L-lysine characteristic of staphylococci. AMG-D1T and AMG-D2 have a G + C ratio of 35 mol%. Phylogenetic analysis with the 16S rRNA gene indicated that AMG-D1T and AMG-D2 were most closely related to Staphylococcus equorum, S. xylosus, Staphylococcus saprophyticus and other novobiocin-resistant staphylococci. Stringent DNA-DNA hybridization studies with AMG-D1T revealed similarities of 38% with S. equorum, 23% with S. xylosus and 6% with S. saprophyticus. The results indicate that AMG-D1T and AMG-D2 represent a novel species, which was named Staphylococcus succinus sp. nov. The type strain of the new species is AMG-D1 (ATCC 700337).

Rapid detection of Mycoplasma pneumoniae by an assay based on PCR and probe hybridization in a nonradioactive microwell plate format.

A new molecular assay, based on a rapid DNA extraction protocol, PCR, and hybridization to a specific probe in a nonradioactive microwell plate format was used to detect Mycoplasma pneumoniae in bronchoalveolar fluid specimens. The sensitivity of the assay was determined to be 10 to 100 organisms with M. pneumoniae reference strains. Specificity testing with different bacteria capable of producing pneumonia showed no cross-reactivity. In a prospective study, bronchoalveolar lavage fluids obtained from patients with pneumonia were investigated with the PCR assay and compared to culture. Twelve positive samples were detected with the PCR assay. Seven of them were subsequently confirmed by culture. All patients with positive PCR results seroconverted. Application of the PCR assay described may lead to safe and early diagnosis of M. pneumoniae in patients with pneumonia.

Entomoplasma freundtii sp. nov., a new species from a green tiger beetle (Coleoptera: Cicindelidae).

A mollicute (strain BARC 318T) isolated from gut tissue of a green tiger beetle (Coleoptera: Cicindelidae) was found by dark-field microscopy to consist of non-helical, non-motile, pleomorphic coccoid forms of various sizes. In ultrastructural studies, individual cells varied in diameter from 300 to 1200 nm, were surrounded by a cytoplasmic membrane and showed no evidence of cell wall. The organisms were readily filterable through membrane filters with mean pore diameters of 450 and 300 nm, with unusually large numbers of organisms filterable through 200 nm pore membrane filters. Growth occurred over a temperature range of 15-32 degrees C with optimum growth at 30 degrees C. The organism fermented glucose and hydrolysed arginine but did not hydrolyse urea. Strain BARC 318T was insensitive to 500 U penicillin ml-1 and required serum or cholesterol for growth. It was serologically distinct from all currently described sterol-requiring, fermentative Mycoplasma species and from 12 non-sterol-requiring Mesoplasma species, 13 non-sterol-requiring Acholeplasma species and 5 previously described sterol-requiring Entomoplasma species. Strain BARC 318T was shown to have a G + C content of 34 mol% and a genome size of 870 kbp. The 16S rDNA sequence of strain BARC 318T was compared to 16S rDNA sequences of several other Entomoplasma species and to other representative species of the genera Spiroplasma and Mycoplasma, and to other members of the class Mollicutes. These comparisons indicated that strain BARC 318T had close phylogenetic relationships to other Entomoplasma species. On the basis of these findings and other similarities in morphology, growth and temperature requirements and genomic features, the organism was assigned to the genus Entomoplasma. Strain BARC 318T (ATCC 51999T) is designated the type strain of Entomoplasma freundtii sp. nov.