Leptotrichia Sanguinegens”: Description of Sneathia Sanguinegens Sp

System. Appl. Microbiol. 24, 358Ð361 (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 isolates 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 were found to be genetically highly related et al., 1995), displayed a relatively high bootstrap re-sam- to each other displaying >99.8% sequence similarity. pling value of 91% with the “Leptotrichia sanguinegens” Searchers of GenBank/EMBL sequence libraries revealed and uncultured eubacterium rRNA cluster. The next that the unknown bacterium as exemplified by CCUG species to join the cluster was Streptobacillus monili- 360 M. D. COLLINS et al.

(AJ344093)

Fig. 1. Unrooted tree showing the phylogenetic relationship of Sneathia sanguinegens gen. nov., sp. nov. and its close relatives. The tree, constructed using the neighbour-joining method, was based on a comparison of approx. 1350 nucleotides. Bootrap values, ex- pressed as a percentage of 500 replications, are given at branching points. formis followed by Sebaldella termiditis and Leptotrichia guished from Leptotrichia buccalis in requiring serum or buccalis. In terms of taxonomic rank, it is evident from blood for growth, by producing β-glucuronidase and its sequence divergence and tree branching considerations, negative α-glucosidase and β-glucosidase reactions. that the 3 clinical isolates and “Leptotrichia sanguinegens” merit classification in a new genus. This distinct bacterium from human sources is phylogenetically far re- Description of Sneathia gen. nov. moved from its closest named relatives such as Strepto- bacillus moniliformis, Sebaldella termiditis and Lep- Sneathia (Sneath.ia M. L. n. Sneathia, named after the totrichia buccalis. It is pertinent to note that the uncul- British microbiologist Peter H. A. Sneath, in recognition tured eubacterium associated with corneal ulcer may be a of his outstanding contributions to microbial systematics) candidate for inclusion in the same genus, but to our consists of Gram-negative, anaerobic (although some knowledge, currently no culture of this organism is avail- strains may show poor growth in CO2), asporogenous, able. The relationships of the fish pathogen AVG2115 non-motile, rod-shaped bacteria. Cells may display pleo- have been studied by MAHER et al. (1995). These authors morphism and filaments may be observed. Fastidious, re- concluded that it possibly represents a new species and quiring serum or blood for growth. Optimum tempera- genus. Based on a 16S rRNA sequence divergence value ture for growth 35–37 °C. Catalase and oxidase negative. of 8.5%, it is certainly very different from the clinical iso- Fermentative metabolism. Acid but no gas is produced lates/“Leptotrichia sanguinegens” and probably repre- from glucose. Acid is not produced from ribose or mal- sents the nucleus of another new genus. The aforemen- tose. Lactic acid and formic acid and minor amounts of tioned fish bacterium is a slow growing, facultatively acetic acid are the end products of glucose metabolism; anaerobic, Gram-negative rod which requires serum or succinic acid may be produced. Aesculin and hippurate blood for growth, but it differs markedly from the clinical are hydrolysed but not starch. β-Glucuronidase is pro- isolates/“Leptotrichia sanguinegens” in growing at low duced. Indole is not produced. Voges-Proskauer negative. temperatures. Therefore based on the phenotypic and Nitrate is not reduced to nitrite. The G+C content of phylogenetic distinctiveness of the clinical isolates and or- DNA is 22–25 mol%. The type species is Sneathia san- ganisms previously designated “Leptotrichia sanguine- guinegens. gens”, we propose this bacterium be assigned to a new genus Sneathia, as Sneathia sanguinegens sp. nov. It is pertinent to note that Sneathia sanguinegens can be readi- Description of Sneathia sanguinegens sp. ly identified in the routine clinical laboratory on the basis nov. of its cellular morphology and fastidious growth require- ments, combined with its API Rapid ID32A and ZYM Sneathia sanguinegens (sanguin.e.gens L. n. sanguis biochemical profiles. In particular, using these systems it blood; L. part adj. egens needing; n. L. adj. sanguinegens can be readily distinguished from Streptobacillus monili- needing blood; because the organism needs blood or formis by its positive β-glucuronidase reaction and by serum) consists of Gram-negative anaerobic or faculta- failing to produce chymotrypsin and proline arylamidase. tively anaerobic, non-sporeforming, non-motile rod- Similarly, Sneathia sanguinegens can be easily distin- shaped cells. Colonies on chocolate or blood agar are pin- Sneathia sanguinegens gen. nov., sp. nov. 361 point and convex after 72 hours. Fastidious requiring References blood or serum for growth. Catalase and oxidase negative. Lactic acid and formic acid and minor amounts of FELSENSTEIN, J.: PHYLIP-phylogeny inference package (version acetic acid are the end products of glucose metabolism; 3.2). Cladistics 5, 164–166 (1989). succinic acid may be produced. Acid but no gas is pro- HANFF, P. A., ROSOL-DONOGHUE, J.-A., SPIEGEL, C. A., WILSON, duced from glucose. Acid may or may not be produced K. H., MOORE, L. H.: Leptotrichia sanguinegens sp. nov., a from mannose and raffinose. Acid is not produced from new agent of postpartum and neonatal bacteremia. Clin. In- L-arabinose, D-arabitol, cyclodextrin, glycogen, lactose, fect. Dis. 20, S237–239 (1995). β HOLDEMAN, L. V., CATO, E. P., MOORE, W. E. C.: Anaerobe Lab- mannitol, maltose, melebiose, melezitose, methyl- -D- oratory Manual, 4th edn. Blacksburg, Virginia. Virginia Poly- glucopyranoside, pullulan, ribose, sorbitol, sucrose, technic Institute and State University (1977). tagatose, or trehalose. Alkaline phosphatase, acid phos- HOFSTAD, T.: Genus Leptotrichia, pp. 637–641. In: Bergey’s phatase, arginine arylamidase, phosphoamidase, and β- Manual of Systematic Bacteriology, vol. 1. Edited by N. R. glucuronidase are detected. Arginine dihydrolase, alanine KRIEG and J. G. HOLT. Baltimore, Williams and Wilkins Co. arylamidase, alanine phenylalanine proline arylamidase, (1984). α-arabinosidase, chymotrypsin, cystine arylamidase, α- MAHER, M., PALMER, R., GANNON, F., SMITH, T.: Relationship of fucosidase, α-galactosidase, β-galactosidase, β-galactosi- a novel bacterial fish pathogen to Streptobacillus monili- dase-6-phosphate, α-glucosidase, β-glucosidase, glutamic formis and the fusobacteria group, based on 16S ribosomal RNA analysis. Syst. Appl. Microbiol. 18, 79–84 (1995). acid decarboxylase, glycyl tryptophan arylamidase, py- MESBAH, M., PREMACHANDRAN, U., WHITMAN, W. B.: Precise roglutamic acid arylamidase, leucine glycin arylamidase, measurement of the G+C content of deoxyribonucleic acid by lipase C14, α-mannosidase, β-mannosidase, N-acetyl-β- high-performance liquid chromatography. Int. J. Syst. Bacte- glucosaminidase, proline arylamidase, trypsin, valine ary- riol. 39, 159–167 (1989). lamidase and urease are not detected. Activity may or PALMER, R., DRINAN, E., MURPHY, T.: A previously unknown dis- may not be detected for ester lipase C8, esterase C4, glu- ease of farmed Atlantis salmon: pathology and establishment tamyl glutamic acid arylamidase, glycine arylamidase, of bacterial aetiology. Dis. Aquat. Org. 19, 7–14 (1994). histidine arylamidase, phenyl alanine arylamidase, RASMUSSEN, S. W.: DNA Tools, a software package for DNA se- leucine arylamidase, serine arylamidase and tyrosine ary- quence analysis. Carlsberg Laboratory, Copenhabgen (1995). lamidase. Indole negative and Voges-Proskauer test negative. Aesculin and hippurate are hydrolysed but not Corresponding author: starch. Nitrate is not reduced to nitrite. Isolated from MATTHEW D. COLLINS, School of Food Biosciences, human clinical specimens (blood, amniotic fluid). Habitat Whiteknights, PO Vox 226, Reading, RG6 6AP, UK is not known. The G+C content of DNA is 22–25 mol%. Tel.: ++41-118-9357226; Fax: ++41-118-9357222; The type strain is CCUG 41628T (= CIP 106906T). e-mail: [email protected]