Brevinema andersonii gen. nov., sp. nov., an Infectious Spirochete Isolated from the Short-Tailed Shrew (Blarina brevicauda) and the White-Footed Mouse (Peromyscus leucopus)

Abstract

. Internet address: leland@lenti.med.umn.edu.Brevinema for the single species that has been identified so far, Brevinema andersonii. MATERIALS AND METHODSBacterial strains and culture conditions. The strains of Brevinema andersonii which we used are shown in Table 1. The spirochetes were cultured in a modified BSK medium (2) which is referred to below as shrew-mouse spirochete medium. Shrew-mouse spirochete medium was prepared as follows. To 900 ml of distilled water we added 3 g of N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) (Sigma Chemical Co., St. Louis, Mo.), 5 g of dextrose, 3 g of neopeptone (Difco Laboratories, Detroit, Mich.), 1 g of tryptone (Difco), 3 g of TC yeastolate (Difco), 0.8 g of sodium citrate, 2.2 g of sodium bicarbonate, 0.2 g of catalase (Sigma), and 0.4 g of cysteine (Sigma). After these components were fully dissolved, 100 ml of 10 X 1066 CMRL tissue culture medium (Gibco, Grand Island, N.Y.) was added, and the pH was adjusted to 7.6. The medium was then filter sterilized, and 35 ml of sterile fetal calf serum (Gibco) and 35 ml of bovine embryonic fluid (Sigma) were added. Finally, 200 ml of sterile 7.5% gelatin (Difco) was added before dispensing. The final pH was 7.4. This medium was dispensed into 7-ml tubes; each tube was filled to 95% of its capacity and tightly capped to provide a microaerophilic environment. Cultures were incubated at 30°C. The numbers of cells were determined by using a Petroff-Hausser counting chamber.Electron microscopy. For transmission microscopy, spirochetes were harvested by centrifugation at 10,000 X g, washed twice in phosphate-buffered saline (PBS) (pH 7.4) at 4"C, and resuspended in a small volume of PBS. Specimens were placed on Formvar-coated grids and stained for 1 min with 1% phosphotungstic acid (pH 7.4). Specimens for scanning microscopy were prepared by pretreating 10-mm glass coverslips with 1% bovine serum albumin and coating the preparations with cells for 20 min at 37°C. The cell preparations on coverslips were then immersed in 0.2% glutaraldehyde, washed in Hanks balanced salt solution, immersed in 1% glutaraldehyde, washed, and finally treated with 1% osmium tetroxide; in each fixation step the preparation was incubated at 4°C for 1 h. The cells were then dehydrated by using 15-min incubations in serially increasing concentrations of ethanol from 70% to absolute. The specimens were removed from ethanol and immediately subjected to critical point drying. Samples were then sputter coated and examined by conventional methods.Gas chromatographic analysis of cellular fatty acids. Cells were harvested at the mid-exponential growth phase by centrifugation at 10,000 X g and were washed once in PBS (pH 7.4). Fatty acids were extracted, separated, and analyzed as described previously (19) by workers at Microbial ID, Newark, Del.

Genetic Diversity of Borrelia burgdorferi Sensu Lato Isolated in Far Eastern Russia

“…From these results, 62 and 12% of the tick isolates, and 47 and 13% of the rodent isolates were identified as B. garinii and B. afzelii, respectively. However, some isolates showed unique and unclassified ribotyping patterns which might have resulted from multiple Borrelia culturing or unknown spirochetes such as Brevinema andersonii (2,9). In this study, we attempted to characterize the unclassified isolates from the far eastern part of Russia by analysis of the 5S-23S rRNA intergenic spacer and on the basis of reactivity with MAbs.…”

Phylogenetic analysis of cultivable oral treponemes from the Smibert collection

“…By comparison of 16s rRNA or 16s rDNA sequences, spirochaetes are divided into two major phylogenetic groupings (7,13,31). The first grouping, which corresponds to the family Spirochaetaceae, contains species of the genera Treponema, Spirochaeta, Burrelia, Serpulina (and Brachyspira) and Brevinema.…”

Exilispira thermophila gen. nov., sp. nov., an anaerobic, thermophilic spirochaete isolated from a deep-sea hydrothermal vent chimney

“…Differential characteristics between strain RASEN T and other genera in the phylum Spirochaetes Data for strain RASEN T are from this study. Data for other genera were taken from Holt et al (1994), Paster et al (1991) and the following sources: Treponema (Norris et al, 2006;Smibert, 1984;Wyss et al, 2004;Nordhoff et al, 2005;, Spirochaeta (Leschine et al, 2006;Canale-Parola, 1984a, b;Hoover et al, 2003;Dröge et al, 2006), Borrelia (Caimono, 2006;Kelly, 1984;Güner et al, 2004;Masuzawa et al, 2001;Fukunaga et al, 1996;Richter et al, 2004;Le Fleche et al, 1997;Baranton et al, 1992;Canica et al, 1993;Wang et al, 1997;Karimi et al, 1979), Cristispira (Breznak, 1984), Brevinema (Defosse et al, 1995), Brachyspira (Stanton, 2006;Hampson & La, 2006) and Leptospira/Leptonema (Adler & Faine, 2006;Faine, 1984;Levett et al, 2006;Perolat et al, 1998;Ramadass et al, 1992). +, Positive; 2, negative; V, dependent on the species; ND, no data available.…”

“…DTreponema pallidum may be microaerophilic or a facultative anaerobe (Smibert, 1984). dBrevinema andersonii may be pathogenic (Defosse et al, 1995).…”