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Leptotrichia Sanguinegens”: Description of Sneathia Sanguinegens Sp

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Leptotrichia Sanguinegens”: Description of Sneathia Sanguinegens Sp System. Appl. Microbiol. 24, 358–361 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam Characterization of some Strains from Human Clinical Sources which resemble “Leptotrichia sanguinegens”: Description of Sneathia sanguinegens sp. nov., gen. nov.* MATTHEW D. COLLINS1, LESLEY HOYLES1, EVA T ÖRNQVIST2, ROBERT VON ESSEN3, and ENEVOLD FALSEN4 1School of Food Biosciences, University of Reading, Whiteknights, Reading, UK 2Kliniskt Mikrobiologiska avd., Regionsjukhuset, Örebro, Sweden 3Department of Laboratory Medicine Sunderby Hospital, Luleå, Sweden 4Culture Collection, Department of Clinical Bacteriology, University of Göteborg, Göteborg, Sweden Received June 12, 2001 Summary Three strains of a Gram-negative, blood or serum requiring, rod-shaped bacterium recovered from human clinical specimens were characterised by phenotypic and molecular taxonomic methods. Compar- ative 16S rRNA gene sequencing showed the unknown rod-shaped strains are members of the same species as some fastidious isolates recovered from human blood specimens and previously designated “Leptotrichia sanguinegens”. Based on phylogenetic and phenotypic evidence, it is proposed that the iso- lates from human sources be classified in a new genus Sneathia, as Sneathia sanguinegens gen. nov., sp. nov. The type strain of Sneathia sanguinegens is CCUG 41628T. Key words: Taxonomy – Phylogeny – Sneathia sanguinegens – 16S rRNA Introduction Materials and Methods HANFF et al. (1995) reported the isolation of an unusu- Cultures and phenotypic characterisation al Gram-negative anaerobic rod-shaped organism from Strains CCUG 38322 and CCUG 41628T were isolated from postpartum and neonatal bacteremia. This fastidious, human blood (from a 35 year old male and 32 year old women, respectively) whereas strain CCUG 42621 was recovered from serum-requiring bacterium, was considered by HANFF et amniotic fluid (from a 28 year old woman). The strains were al. (1995) to be a member of the genus Leptotrichia and biochemically characterised by using the API Rapid ID32A, was designated “Leptotrichia sanguinegens”. The species Rapid ID32S, and ZYM systems according to the manufactur- was, however, not validly published and no type strain er’s instructions (API bioMérieux). Fermentation products were was designated. During the course of a study of taxonom- determined as described by HOLDEMAN et al. (1977). Alcohols ically problematic Gram-negative anaerobic bacteria and volatile acids were extracted with terbutyl ethyl ether in- from human clinical sources we have isolated 3 strains stead of diethyl ether and analysed using a Shimadzu GC-14A which resemble the bacterium described by HANFF et al. gas chromatograph. (1995). Based on both phenotypic and phylogenetic evi- DNA base composition dence, we conclude our isolates correspond to “Lepto- The mole % G+C content of DNA was determined by high trichia sanguinegens” and propose a new genus and performance liquid chromatography after digestion of DNA species, Sneathia sanguinegens to accomodate these or- with P1 nuclease and alkaline phosphatase as described by MES- ganisms. BAH et al. (1989). Abbreviations: CCUG – Culture Collection of the University of *The GenBank/EMBL accession number for the 16S rRNA se- Göteborg; CIP – Collection of Bacterial Strains of the Institute quence of CCUG 41628T is AJ344093. Pasteur. 0723-2020/01/24/03-358 $ 15.00/0 Sneathia sanguinegens gen. nov., sp. nov. 359 Phylogenetic analysis 41628T was phylogenetically almost identical (99.9% The 16S rRNA genes of the isolates were amplified by PCR similarity) to a sequence (L37789) deposited as “Lep- and directly sequenced using a Taq Dye-Deoxy terminator cycle totrichia microbii” and which is synonymous to the in- sequencing kit (Applied Biosystems) and an automated DNA se- valid species “Leptotrichia sanguinegens”. The next clos- quencer (model 373A, Applied Biosystems). The closest known relatives of the unknown isolates were determined by perform- est rRNA sequences derived from an uncultured eubac- ing database searches. These sequences and those of other terium (designated E1–K6) and from an unnamed fish known related strains were retrieved from GenBank and aligned pathogen (strain AVG2115). Other organisms phyloge- with the newly determined sequences using the program DNA netically related to the unidentified clinical isolates were tools (RASMUSSEN, 1995). The resulting multiple sequence align- Streptobacillus moniliformis, Sebaldella termitidis (for- ment was corrected manually and a distance matrix was calcu- merly Bacteroides termitidis), Leptotrichia buccalis, Pro- lated using the programs PRETTY and DNADIST (using the pionigenium modestum and Fusobacterium species. A Kimura-2 correction parameter). A phylogenetic tree was con- tree constructed by the neighbour joining method depict- structed using the neighbour joining method with the program ing the phylogenetic relationships of the clinical rod- NEIGHBOR (FELSENSTEIN, 1989). The stability of the groupings was estimated by bootstrap analysis (500 replications), using the shaped bacterium is shown in Fig. 1. The unknown bac- programs SEQBOOT, DNADIST, NEIGHBOR and CON- terium and “Leptotrichia sanguinegens” formed a dis- SENSE (FELSENSTEIN, 1989). Parsimony analysis was also per- tinct line, which clustered with the rDNA clone derived formed using the same package (FELSENSTEIN, 1989). from an uncultured eubacterium. This association was supported by a bootstrap re-sampling value of 90%. The fish pathogen, designated AVG2115, was the next nearest Results and Discussion relative to this cluster, with Streptobacillus moniliformis, Sebaldella termiditis and other taxa such as Leptotrichia The unknown strains recovered from human clinical buccalis and Propionigenium modestum being more re- specimens were highly fastidious requiring serum for motely related (Fig. 1). All associations showing boot- growth and consisted of Gram-negative rod-shaped or- strap re-sampling values of 90% or more in the neigh- ganisms. The 3 isolates were anaerobic, although strain bour joining tree were confirmed by parsimony analysis. CCUG 41628T was capable of growing, albeit poorly, on It is evident from both phenotypic and phylogenetic chocolate or blood agar in CO2. The isolates were cata- evidence that the unidentified Gram-negative fastidious lase and oxidase negative. Using the API Rapid ID32A anaerobic isolates recovered from human clinical sources system, all isolates produced positive reactions for alka- are members of the same species as strains described by line phosphatase arginine arylamidase and β-glu- HANFF et al. (1995). These latter workers considered the curonidase. Variable reactions were observed for acid organisms to represent a novel species of the genus Lep- production from mannose and raffinose and for the fol- totrichia, and designated these “Leptotrichia sanguine- lowing enzymes: glutamyl arylamidase, glycine arylami- gens” (HANFF et al., 1995). This species was however not dase, histidine arylamidase, phenyl alanine arylamidase, validly described. In addition it is very clear from the pre- leucine arylamidase, serine arylamidase and tyrosine ary- sent study that our isolates and “Leptotrichia sanguine- lamidase. All other tests in this system were negative. The gens” are phylogenetically not members of the Lep- 3 API Rapid ID32A codes obtained were 0046410000 totrichia genus. Phylogenetically our clinical (CCUG 38322), 0040412000 (CCUG 41628T), and isolates/“Leptotrichia sanguinegens” represent a distinct 0042413507 (CCUG 42621). The 3 isolates were rela- line and display >10% sequence divergence with Lep- tively un-reactive using the API Rapid ID32S system pro- totrichia buccalis, the type species of the genus (HOFSTAD, ducing positive reactions for hippurate hydrolysis and for 1984). The rDNA sequence (AJ289183) displaying high- alkaline phosphatase and β-glucuronidase activity. All est relatedness with that from our clinical isolates/“Lep- other tests were negative giving rise to the code totrichia sanguinegens” corresponded to that of an uncul- 05000010000 for this system. Using the API ZYM test tured eubacterium (93.5% sequence similarity). This se- system the 3 strains displayed activity for acid phos- quence was derived from rDNA analysis of an anaerobic phatase, alkaline phosphatase, phosphoamidase and β- community associated with corneal ulcer (Gurtner, Maca, glucuronidase. Variable results were observed for ester li- Kaminski, Roelleke, Lubitz, and Barisani-Asenbauer, un- pase C8 and esterase C-4. No activity was detected for published data cited in EMBL under accession number chymotrypsin, α-glucosidase, β-glucosidase, α-fucosi- AJ289183). This affinity was confirmed by treeing analy- dase, α-galactosidase, β-galactosidase, cystine arylami- sis, with the clustering of these sequences supported by a dase, leucine arylamidase, lipase C14, α-mannosidase, N- bootstrap value of 90% (Fig. 1). An rDNA sequence acetyl-β-glucosaminidase, valine arylamidase or trypsin. (X83517) derived from a fish pathogen (strain AVG2115) All of the isolates were indole negative and did not reduce was more distantly related to the clinical isolates/“Lep- nitrate to nitrite. To assess the phylogenetic relationships totrichia sanguinegens” (91.5% sequence similarity). of the unidentified rods their 16S rRNA gene sequences Nevertheless this organism, a causative agent of disease were determined and subjected to comparative analyses. in farmed Atlantic salmon (PALMER et al., 1994; MAHER The 3 strains
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