INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, July 1997, p. 627-634 0020-7713‘07/$04.00+0 Copyright 0 1997, International Union of Microbiological Societies Vol. 47, No. 3 Helicobacter rodentium sp. nov., a Urease-Negative Helicobacter Species Isolated from Laboratory Mice Z. SHEN,’ J. G. FOX,’* F. E. DEWHIRST,2 B. J. PASTER,2 C. J. FOLTZ,’ L. YAN,’ B. SHAMES,’ AND L. PERRY~ Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139,‘ and Department of Molecular Genetics, Forsyth Dental Center, Boston, Massachusetts 021 I 52 A spiral-shaped bacterium with bipolar, single, nonsheathed flagella was isolated from the intestines of laboratory mice. The organism grew at 37 and 42°C under microaerobic and anaerobic conditions, did not hydrolyze urea, was weakly positive for catalase and oxidase, reduced nitrate to nitrite, did not hydrolyze indoxyl acetate or hippurate, and was resistant to cephalothin and nalidixic acid. This is the first urease- negative, murine Helicobacter spp. isolated from intestines. Also, Helicobacter pullorum and this bacterium are unique among the genus Helicobacter in having nonsheathed flagella. The new bacterium appears to be part of the normal intestinal flora; although its pathogenic potential is unknown, this organism was also isolated from scid mice with diarrhea that were co-infected with Helicobacter bilis. On the basis of 16s rRNA gene sequence analysis data and biochemical and phenotypic criteria, the new organism is classified as a novel helicobacter, for which we propose the name Helicobacter rodentium. The type strain is MIT 95-1707 (= ATCC 700285). Helicohacter spp. that possess different ultrastructural char- acteristics are common inhabitants of the gastrointestinal tracts of both humans and animals (8). The type species of the genus, Helicobacter pylori, causes chronic gastritis and peptic ulcer disease in humans and has recently been linked to the development of gastric adenocarcinoma and gastric mucosa- associated lymphoma (4, 17, 18). Other nonhuman Helicobac- ter spp., namely, Helicobacter felis, Helicobacter mustelae, and Helicohacter acinonyx, have been associated with gastritis in their respective hosts (3, 5, 6). Helicobacters infect several animal hosts, as well as colonize different anatomical regions of the gastrointestinal system. Six formally named Helicobacter spp. capable of colonizing the intestinal tracts of rodents have been characterized by pheno- typic, biochemical, and molecular analyses. Helicobacter rnuri- darum colonizes the cecum and ileum and induces a gastritis following colonization of the gastric mucosa in older rodents (15, 20). “Flexispira rappini,” a helicobacter based on 16s rRNA data but formally unnamed, which has been linked to abortion in sheep, necrotic liver foci in aborted sheep fetuses, and diarrheal disease in humans, has also been isolated re- cently from the feces of mice (1, 22). Helicobacter cinaedi, a normal intestinal inhabitant of hamsters, also has been isolated from homosexual men with enteritis, proctocolitis, and asymp- tomatic rectal infections (12, 25). Two other Helicobacter spp., Helicohacter bilis and Helicobacter hepaticus, have been iso- lated from livers, ceca, and colons of mice, and both of these species have also been isolated from the livers of animals with hepatitis (7, 9, 10). Most recently, Helicobacter trogontum has been isolated from intestines of asymptomatic rats (16). During routine health surveillance for H. hepaticus in mice, we isolated a urease-negative, helicobacter-like bacterium that differed from the previously described Helicobacter species in other phenotypic characteristics. Although other urease-nega- tive helicobacters have been described, this was the first ure- ase-negative helicobacter-like organism isolated from the mouse intestine. Consequently, we hypothesized that this or- ~ * Corre4ponding author. Mailing address: MIT-Division of Compar- ative Medicine, 77 Massachusetts Avenue, 45-106, Cambridge, MA 02139. Phone: (617) 253-1757. Fax: (617) 258-5708. ganism was a novel species. In this paper, we provide biochem- ical, phenotypic, and phylogenetic data which confirm that this bacterium is distinct from previously recognized Helicobacter species, and we propose the name Helicobacter rodentium for it. MATERIALS AND METHODS Animals. The novel murine Helicohacter species was recovered from 74 mice housed in an Association for Accreditation and Assessment of Laboratory An- imal Care-accredited animal facility. The mice either were housed in a barrier facility and originated from a single vendor or were rederived by embryo transfcr. Somc mice were animals that came from other academic institutions and thcre- fore were housed in a quarantine facility. Although thc bacterium was originally identified in asymptomatic mice, the organism was subsequently recognized in diarrhetic scid mice infccted with H. bilis. Mice housed in the barrier facility were free of all other recognized murine Helicohacter species. Bacterial culture. Freshly pooled fecal samples and cecum, colon, liver, and uterine specimens were aseptically collected from each mousc. Samples from each site were homogenized in 1.0 ml of phosphate-buffered saline. Each slurry was gently passed through a 0.45-pm-pore-size syringe filter, and the homoge- nate was streaked onto a Columbia blood agar (S% sheep blood) plate (Remel Laboratories, Lenexa, Kans.). The cultures were then incubated at 37°C under microaerophilic conditions in vented jars containing N,, H,, and COz (Y0:S:S). Electron microscopy. The following two isolates were examined by electron microscopy: type strain MIT 95-1707 (= ATCC 700285) and strain MIT 96-2160 (= ATCC 700286). Cells grown in Trypticase soy broth for 24 h were centrifuged and gently suspended in 10 mM Tris-HCI buffer (pH 7.4) at a concentration of about 10‘ cells per ml. Samples were negatively stained with 1% (wt/vol) phos- photungstic acid (pH 6.5) for 20 to 30 s. Specimens were examined with a JEOL model JEM-1200EX transmission electron microscope operating at 100 kV. Biochemical characterization. A detailed biochemical characterization analy- sis was performed with 9 of 74 isolates as previously described by Fox et al. (10). The nine isolates represented mice from five different sources. For thc remaining 65 strains, motility, Gram staining, oxidase, catalase, and urease assays were performed, and sensitivities to nalidixic acid and cephalothin were determined. Extraction of DNA for sequencing. Bacteria were cultured on blood agar plates, and cells were harvested and washed twice with 1 ml of double-distilled H,O. The pellets were suspended in STET buffer (8% sucrose, SO mM EDTA, 0.1% Triton X-100, 50 mM Tris-HCI [pH KO]), and lysozyme (hen egg white; Boehringer Mannhcim Biochemicals, Indianapolis. Ind.) was added to a final concentration of 3 mgiml. The suspension was incubated for 12 min at 37°C and was then lysed with 1% sodium dodecyl sulfate. RNase A (bovine pancreas; Boehringer Mannheim) was added to a final concentration of 0.05 mg/ml, and the solution was incubated for 1 h at 37°C. Then 0.1 volume of a 5% CTAB (cetyltrimcthylammonium bromide)-O.S M NaCl solution (Sigma Chemical Co., St. Louis, Mo.) was added, and the solution was gently mixed and incubated at 65°C for 10 min. DNA was extracted with an equal volume of phenol-chloroform (l:l, vol/vol), precipitated overnight in 0.3 M sodium acetate with 2 volumes of absolute ethanol at -20”C, and pelleted by centrifugation at 13,000 X g for 1 h at 4°C. The ethanol was decanted, and the pellet was air dried and suspended in sterile distilled water. 627