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Alloiococcus Otitis Gen INTERNATIONALJOURNAL OF SYSTEMATICBACTERIOLOGY, Jan. 1992, p. 79-83 Vol. 42, No, 1 0020-7713/92/010079-05$02.OO/O Copyright 0 1992, International Union of Microbiological Societies Phylogenetic Analysis of Alloiococcus otitis gen. nov. sp. nov. an Organism from Human Middle Ear Fluid M. AGUIRRE AND M. D. COLLINS* Department of Microbiology, AFRC Institute of Food Research, Reading Laboratory, Shinjield, Reading RG2 9AT, United Kingdom The partial 16s rRNA sequence of an unknown bacterium that was originally isolated from middle ear fluids of children with persistent otitis media was determined by reverse transcription. A comparison of this sequence with sequences from other gram-positive species having low guanine-plus-cytosine contents revealed that this bacterium represents a new line of descent, for which the name Alloiococcus otitis gen. nov., sp. nov,, is proposed. The type strain is strain NCFB 2890. Faden and Dryja (7) recently reported the isolation of an MATERIALS AND METHODS unknown organism from typanocentesis fluid collected from middle ears of young children who were suffering from Cultures and biochemical tests. Strains 3621, 4491, 7213, chronic otitis media. The cells of this bacterium were large and 7760T (T = type strain) were received from D. H. Batts gram-positive cocci (often present as diplococci or tetrads) and R. R. Hinshaw, Upjohn Laboratories, Kalamazoo, which phenotypically most closely resembled aerococci and Mich. Cultures were grown on blood agar plates and Todd- streptococci (7). However, the organism differed from aero- Hewitt broth (Oxoid Ltd., Basingstoke, United Kingdom) cocci and streptococci in being catalase positive. Despite supplemented with 5% horse serum at 37°C. Biochemical possessing catalase, the bacterium could not be identified by tests were performed by using the API 20 STREP system using the Staph-Ident system (Analytab Products), and its (API-Biomerieux, Basingstoke, United Kingdom) according taxonomic position remained unresolved (7). to the instructions of the manufacturer. Test preparations 16s rRNA sequence analysis has assumed a central role in were incubated at 37"C, and tests were read after 4 and 24 h ascertaining the phylogenetic relationships of bacteria (17). and 7 days. GAGUUUGAUCCUGGCUCAGGACGAACGCUGGCGGCAUGCCUAAUA~UGCAAGUCGAACGCCAAAUCWCCUGCWG~GGAAGAGGCGGAGUGGCGMC GGGUGAGUAACACGUGAGGAACWGCCCAUAAGAGGGGGAUAACACCCGGAAACGGGUGCUAAUACCGCAUAAG~CCCAGUG~UG~G~~UA AAAGGUGGCUC~ACCGCWAUGGAU~UCGCGGCGCA~AGCUAGWGGUAGGGU~AGGCCUAC~GGC~UGAUG~GUAGCCGACWGAGAGGG UGAUCGGCCACACUGGAACUGAGACACGGUCCAGACUCCUCCUACGGGAGGCAG~GUAGGG~UCWCCA~UGGGUGCAAGC~GAUGGAGC~UGCCGC GUGAGUGAAGAAGGACWCGGWCGUAAAGCUCUGWGWGGGGAAG~CAC~AUAGGAGUCACUGCCUAUCCCWGACGGUACCC~CCAGAAAGUCA CGGCUAACUACGUGCCAGCAGCCGCGGUAAUACGUAGGUGACAAGC~WGUCCGGA~AWGGGCGUAAAGCGAGCGCAGGCGGUCCGGUAAGUCUGAU GUGAAAGCCCACGGCUCAACCGUGGAACGGCAW~~CUGGCGGAC~G~UGUAGCAGAGGAAAGUGGAAWCCAUGUGUAGCGGUGG~U~CGUAG AUAUAUGGAGGAACACCAGUGGCGAAAGCGACUUUCUGGGGCUAUGA~GACGCUGAGGCUCG~GCGUGG~AGCGAAC~~A~AGAUACCCUG~UAG UCCACGCCGUAAACGAUGAGUGCUAAGUGWGGAGGG~CCGCCCW~~UGCUGGAG~~CGCAW~GCACUCCGCCUGGGGA~ACGACCG~G GWGAAACUCAAAAGAAWGACGGGGGACCGCACCGCA~GCGGUGGAGCAUGUGG~AAWCGAAGCAACGCGAAG~C~ACCAGC~GACAUCWCU GCUAGCUUCAGAGAUGAAGCGUUCCCCWCGGGGACAGAAUGACAGGUGGUGCAUGGWGUCGUCAGCUCGUGUCGUGAGAUGWG~W~GUCCCGU~ CGAGCGCAACCCWAUUGWAG~GCCAGCAWGAGWGGGCACUCUAGCAAGACUGCCGGUGAUAAACCGGAGGAA~CG~AUGACGU~U~UC AUGCCCCWAUGAGCUGGGCUACA~CGUGCUA~UGGAUGGUACAACGAGUGGCGAGACCGCGAGG~AGCUMUCUCC~GC~WCU~GWC GGAUUGCAGGCUGCAACUCGCCUGCAUGAAGUCGGAAUCGCUAGUAAUCGCGGAUCAGAAUGCCGCGGUG~WCGWCCCG~UCWGUACA~CCGCC CGUCACACCACGAGAGUCUGAAACACCCCGAAGCCGGCCGGGCAACCA~GGAGCCAGCCGUCGAA FIG. 1. Primary structure of 16s rRNA from Alloiococcus otitis. The first nucleotide and the last nucleotide in the sequence correspond to positions 9 and 1469, respectively, of the E. coli numbering system (2). In recent years this molecule has done much to clarify the DNA base composition. DNA was prepared as described inter- and intrageneric relationships of low-guanine-plus- by Garvie (9). DNA base composition was determined by cytosine (G +C)-content gram-positive cocci (viz., the gen- subjecting the preparations to thermal denaturation in stan- era Aerococcus, Enterococcus, Lactococcus, Leuconostoc, dard saline citrate as described by Garvie (9), using DNA Pediococcus, and Streptococcus) (1, 3-5, 10, 12-16). Be- from Escherichia coli NCDO 1984 as the standard. cause of the possible role of the unknown coccus of Faden Analysis of 16s rRNA. Cells were mechanically broken by and Dryja in middle ear infections (7), we determined the using a Braun homogenizer, and rRNA was purified by using partial 16s rRNA sequence of this organism in order to the procedure of Embley et al. (6). Nucleotide sequences ascertain its precise phylogenetic position. were determined by the Sanger dideoxynucleotide chain termination method directly from rRNA by using avian myeloblastosis virus reverse transcriptase (6). The se- quences generated were aligned, and homology values were determined by using the Beckman Microgenie program (11). Nucleotide substitution rates (K,,, values) were calculated, and an unrooted phylogenetic tree was produced by using * Corresponding author. the algorithm of Fitch and Margoliash (8). 79 80 AGUIRRE AND COLLINS INT. J. SYST.BACTERIOL. TABLE 1. Homology values for a 1,340-nucleotide region of 16s rRNAs of Alloiococcus otitis and other taxa % Homology with rRNA from: Aerococcus viridans 88.7 NCDO 1225T Bacillus subtilis rrNB 87.8 87.6 Brochothrix 85.7 89.0 89.4 thermosphacta NCDO 1676T Camobacterium 89.9 92.5 90.1 90.2 divergens NCDO 2763T Camobacterium 89.5 91.9 90.5 90.7 97.5 piscicola NCDO 2762T Enterococcus cecomm 89.9 92.3 90.6 91.3 94.6 94.1 NCDO 2674T Enterococcus hira e 89.1 91.6 90.3 91.7 94.8 94.6 97.0 NCDO 1258T Lactobacillus 87.2 86.6 88.3 86.2 87.8 87.9 89.3 89.1 acidophilus NCDQ 174gT Lactobacillus brevk 89.0 90.7 88.8 88.1 91.7 92.9 91.6 92.9 89.8 NCDO 1749T Lactobacillus casei 88.9 90.0 88.5 88.5 91.9 92.0 90.7 91.1 89.7 93.5 NCDO 161T Lactobacillus delbmeckii 86.5 87.7 87.2 85.7 87.2 87.2 88.9 89.3 94.3 90.0 89.7 NCDO 213T Lactobacillus plantmum 87.5 90.1 89.1 87.3 91.1 92.1 90.8 91.6 88.9 95.4 93.5 89.0 NCDO 1752T Lactobacillus reuterii 88.6 89.9 87.7 87.3 90.5 90.4 90.6 91.0 91.5 93.1 93.6 91.2 NCDO 2589T Lactococcus gawieae 85.0 88.4 86.1 86.7 87.7 88.1 90.6 89.4 85.1 88.1 87.3 87.1 NCDO 21ST Lactococcus lactis 86.5 88.7 86.9 87.6 88.7 88.8 90.4 90.3 87.1 89.4 88.5 87.9 NCDO 21HT Leuconostoc 85.4 87.5 85.3 85.2 86.6 85.7 87.8 87.2 88.1 87.0 86.9 87.2 mesenteroides NCDO 523T Leuconostoc paranzesen- 85.9 89.3 86.8 87.9 90.1 89.9 90.7 90.7 87.8 90.4 90.4 87.5 teroides NCDO 803T Listeria innocua NCTC 87.8 90.2 91.8 93.1 92.0 91.9 92.2 92.0 87.0 88.3 88.4 86.9 1128gT Pediococcus acidihactici 89.9 89.4 88.7 88.8 92.1 92.0 92.1 92.4 89.5 94.6 93.8 86.9 NCFB 2767T Pediococcus damnosus 89.0 89.9 89.6 88.1 91.8 92.0 91.3 91.6 89.9 94.6 94.9 89.5 NCDO 1832T Staphylococcus xylosus 86.7 90.6 91.6 89.8 89.8 89.6 90.2 90.5 85.8 87.2 87.5 86.1 NCTC 11043T Streptococcus sanguis 86.9 89.4 89.7 88.2 89.6 89.9 91.1 91.2 86.5 88.8 88.9 86.7 NCTC 7863= Streptococcus 86.7 88.9 87.0 87.8 89.3 90.0 90.6 90.9 86.5 88.6 88.4 86.2 parauberis NCDO 651 Streptococcus porcinus 86.2 88.6 86.9 87.8 89.0 89.7 90.4 90.4 87.0 87.8 88.3 86.6 NCDO 600 Streptococcus pyogenes 86.6 88.7 86.6 87.8 89.3 89.6 90.4 90.4 87.1 87.8 88.6 86.5 NCDO 2381T Te tragenococcus 89.2 90.8 91.2 90.0 92.4 93.1 93.5 93.4 88.4 89.9 90.1 88.4 halophilus NCDQ 1635= Vagococcusfluvialis 88.9 92.7 89.5 91.2 93.7 93.2 94.3 94.3 88.3 91.3 90.0 88.3 NCDO 2497T Vagococcus 88.5 91.7 90.0 90.1 94.4 93.9 94.1 94.6 87.0 90.2 90.5 86.9 salmoninarum NCFB 2777= €'96 8'16 E'68 9-88 0'68 8'68 6'68 1'16 9'06 8'06 6-88 0'98 E'68 9'88 $238 9'06 L'16 0'68 P'88 9-88 6'68 9'68 S'T6 €76 1-16 E'88 O'L8 9'68 8'L8 1'68 0'16 0'68 2'06 2-16 S'06 5-68 8'68 0'16 E'06 S'68 9'L8 2-88 2-18 S-88 0.68 9'96 1'16 0'96 0'98 L'88 L'88 6'L8 9-18 S-98 L-26 S-16 6-88 8'L8 L'16 S'S6 6'S8 7-88 S'L8 S'L8 8'LS 9-98 S.26 8-16 0'88 S'L8 L'96 L's8 P'88 0'88 P'l8 P'88 8'98 S'Z6 9-16 2-88 9'L8 6'98 E'68 9'68 P'88 2'68 8'18 0'26 8'06 9'68 P'88 1'88 E'88 9'06 6'98 6'P8 0'58 8'98 6'S8 1'L8 9'96 0-68 9'68 I'L8 9'68 0'88 L'E6 9'96 1'68 2'06 9'98 0'68 6'88 2-96 E'96 1'88 1-98 8'18 6'98 6'L8 9'88 0'16 €38 O'L8 8'06 9'06 L'S8 9'S8 S'88 2'28 O'S6 6'L8 S'88 0'88 S'L8 1-26 82 AGUIRRE AND COLLINS INT.
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