INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, July 1993, p. 490-499 Vol. 43, No. 3 0020-7713/93/030490-10$02.00/0 Copyright 0 1993, International Union of Microbiological Societies Phylogeny of Species in the Family Neisseriaceae Isolated from Human Dental Plaque and Description of Kingella orale sp. nov. FLOYD E. DEWHIRST,l* C.-K. CASEY CHEN,,? BRUCE J. PASTER,' AND JOSEPH J. -ZAMBON2 Department of Molecular Genetics, Forsyth Dental Center, 140 Fenway, Boston, Massachusetts 021 15, and Department of Oral Biology and Penodontology, School of Dental Medicine, State University of New York at Buflalo, Buflalo, New York 14214, Fourteen human periodontal isolates recovered from a purported Eikenella corrodens-selective medium containing 1 pg of clindamycin per ml displayed biochemical traits which differed from those described for E. corrodens. These organisms were gram-negative rods which corroded agar. The isolates were oxidase positive and urease, indole, and esculin negative. They differed from E. corrodens in catalase, nitrate reduction, lysine decarboxylase, and ornithine decarboxylase activities. One isolate, strain UB-294, was presumptively identified as Kingella denitnjkans. A second isolate, strain UB-204, differed from E. corrodens by being catalase positive and nitrate reduction negative. Twelve isolates, including strain UB-3ST (T = type strain), were phenotypically similar to Kingella kingae except that they did not produce acid from maltose and were not beta-hemolytic. Essentially complete (1,480-base) 16s rRNA sequences were determined for strains UB-3ST, UB-204, and UB-294 and the type strains of Neisseria animalis, Neisseria canis, Neisseria denitr@cans, Neisseria elongata, Neisseria JEavescens, Neisseria macaca, and Neisseriu polysaccharea. These sequences were compared with the previously published sequences of six other species belonging to the family Neisseriaceae. On the basis of the results of the comparative sequence analysis, UB-294 was confirmed as a K. denitrificans strain, UB-204 was identified as a member of a new species which may belong in the genus Eikenella, and UB-38T was identified as a member of a new species of the genus Kingelk, for which we propose the name Kingella orale. Since strain UB-204 was the only representative of a new species, it was not named. DNA probes for identification of E. corrodens, K. denitrificans, and K. orale based on 16s rRNA sequence information are described. In a previous study examining the prevalence of Eikenella MATERIALS AND METHODS corrodens in the human oral cavity, we frequently encoun- tered gram-negative bacteria phenotypically similar to, but Bacterial strains. Isolation of strains, characterization of distinguishable from, Eikenella corrodens (2). These non- the periodontal status of patients, and partial biochemical Eikenella corrodens isolates grew well on selective medium characterization of the 14 non-Eikenella corrodens, agar- containing 1 kg of clindamycin per ml, corroded agar, and corroding strains have been described previously (2, 3). The did not produce acid from sugars as determined by standard Neisseria species used for 16s rRNA sequencing were broth tests. In contrast to Eikenella corrodens, these isolates obtained from the American Type Culture Collection. The lacked one or more of the following biochemical traits: bacterial strains used, their sources, and their accession nitrate reduction activity, lysine decarboxylase activity, and numbers are shown in Table 1. ornithine decarboxylase activity. In a subsequent study of Biochemical characterization. Partial biochemical charac- six of these isolates, we reported that they exhibited less terization of six of the strains was described previously (3). than 33% DNA homology with Eikenella cowodens refer- Bacteria were grown on Trypticase soy agar plates supple- ence strains (3). Four of these strains exhibited more than mented with 5% sheep blood, 5 mg of hemin per liter, and 0.5 70% DNA homology with one another, indicating that they mg of menadione per liter at 37°C in a humidified anaerobic belonged to a single species. In this study, we further chamber containing 5% CO,. Catalase activity, esculin hy- characterized these six isolates, as well as eight additional drolysis, indole production, reduction of nitrate to nitrite, strains. We found that the 14 isolates fell into three groups and urease activity were determined by using the methods on the basis of biochemical characteristics and protein described in the Anaerobe Laboratory Manual (11). Oxidase profiles determined by sodium dodecyl sulfate (SDS)-poly- activity was determined by using a 1% aqueous solution acrylamide gel electrophoresis (PAGE). 16s rRNA se- of N,Nfl,"-tetramethyl-p-phenylenediamine dihydrochlo- quences were determined for representative isolates belong- ride (Becton Dickinson Reagent Droppers). Acid production ing to each of these groups and for a number of Neissena from glucose, xylose, mannitol, lactose, sucrose, and mal- species. The 16s rRNA sequence information was used to tose was determined by using broth cultures as described in determine the phylogenetic position of each of these groups the Manual of Clinical Microbiology (25). Strains UB-38= of non-Eikenella corrodens organisms within the family (T = type strain), UB-204, and UB-294 and the type strains Neisseriaceae and to design DNA probes. of Kingella and Neissen'a species were further characterized by using RapID/NH System (Innovative Diagnostics Sys- tems, Inc., Atlanta, Ga.) and Microscan HIND (Baxter * Corresponding author. Diagnostics Inc., Deerfield, Ill.) panels. For these tests, the t Present address: Department of Periodontology, School of strains were grown on brain heart infusion-blood-agar plates Dentistry, University of Southern California, Los Angeles, CA aerobically in an atmosphere containing 76% N,, 9% H,, 9% 90089. CO,, and 6% 02.The RapID/NH System and Microscan 490 VOL.43, 1993 KINGELLA ORALE SP. NOV. 491 TABLE 1. Strains, sources, and accession numbers Strain or species" Site of oral sample' Clinical diagnosis' Culture collection designation(s)d GenBank accession no.= Eikenella corrodens strains ATCC 23834T Sputum ATCC 23834T M22512 UB-344 Sub-GP UP Eikenella-like strain UB-204 Sub-GP AP CCUG 28283 LO6169 Kingella denitri cans strains ATCC 33394 zti ATCC 33394T M22516 UB-294 Supra-GP LJP CCUG 28284 L06166f Kingella orale strains UB-13 Sub-GP PH UB-25 Sub-GP PH UB-38T Supra-GP AP ATCC 51147T, CCUG 30450T LO6166 UB-75 Sub-GP AP UB-107 Sub-GP PH UB-149 Supra-GP AP UB-180 Supra-GP AP UB-209 Supra-GP AP UB-220 Supra-GP PH UB-2369 Sub-GP PH UB-245 Sub-GP PH UB-295 Supra-GP LJP Kingella kingae ATCC 23330T M22517 Neisseria gonowhoeae NCTC 8375T X07714 Neisseria polysaccharea ATCC 43768T LO6167 Neisseria jiavescens ATCC 13120T M6168f Neisseria macaca ATCC 33926T LO6169 Neisseria canis ATCC 14687T LO6176 Neisseria elongata ATCC 25295= LO617lf Neissek animalis ATCC 19573T LO6172 Neisseria denitnjicans ATCC 14686T L06173f Wtreoscilla stercoraria ATCC 15218T LO6176 UB, State University of New York at Buffalo School of Dental Medicine, Buffalo, N.Y.; ATCC, American Type Culture Collection, Rockville, Md. 'Sub-GP, subgingival plaque; supra-GP, supragingival plaque. ' Clinical diagnosis of patient from which the strain was isolated. PH, periodontally healthy; AP, adult periodontitis; UP, localized juvenile periodontitis. The criteria used for diagnosis have been described previously (2). Culture collections in which strains have been deposited or from which strains were obtained. ATCC, American Type Culture Collection, Rockville, Md.; CCUG, Culture Collection of the University of Goteborg, Goteborg, Sweden; NCTC, National Collection of Type Cultures, London, United Kingdom. 16s rRNA sequences are available for electronic retrieval from GenBank under the accession numbers shown. Through cross-distribution, these sequences should also be available from European (EMBL) and Japanese (DDBJ) data bases. Sequence determined in this study and deposited by us. This strain did not react with the K orak DNA probe. HIND panels contain 10 and 18 tests, respectively, including blood-agar plates were gently suspended in 10 mM Tris tests for alkaline phosphatase, ornithine decarboxylase, pro- buffer (pH 7.4) at a concentration of approximately lo8 cells line aminopeptidase, nitrate and nitrite reduction, resazurin per ml. Samples were negatively stained with 1% (wtkol) reduction, and production of acid from glucose. These phosphotungstic acid (pH 6.5) for 15 to 20 s. Specimens were panels were incubated in air at 37°C for 4 h. Strains were examined with a JEOL model JEM-1200EX transmission examined for growth on MacConkey agar (Difco) by incu- electron microscope operating at 100 kV. bation at 37°C in a humidified anaerobic chamber containing 16s rRNA sequencing. rRNA was isolated and partially 5% CO, for 7 days. purified by a modification of the procedure of Pace et al. (19) Protein profiles. Bacteria grown on blood-agar plates for 2 as previously described (20). rRNA sequences were deter- days were harvested into sterile saline and pelleted by mined by using a modification of the standard Sanger centrifugation. The cells were resuspended in distilled water, dideoxy chain termination technique (17). Seven primers and the protein concentration was determined by the method of Lowry et al. (18). The cell suspension was then diluted complementary to conserved regions of the 16s rRNA with an equal volume of 2 x sample buffer (0.125 M Tris-HC1 sequence were elongated by using avian myeloblastosis [pH 6.81, 8% SDS, 20% glycerol, 10% 2-mercaptoethanol, virus reverse transcriptase (9). 0.006% bromophenol blue) and boiled for 10 min before use. 16s rRNA data analysis. A program set for data entry, SDS-PAGE was performed by using a Laemmli gel system editing, sequence alignment, secondary structure compari- (16) with a 3.8% stacking gel and a 12% separating gel. A son, similarity matrix generation, and dendrogram construc- 20-kg portion of protein was loaded per lane. The protein tion for 16s rRNA data was written in Microsoft Quick- bands were visualized by Coomassie brilliant blue (Sigma) BASIC for use on IBM PC-AT and compatible computers.