JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1999, p. 1161–1164 Vol. 37, No. 4 0095-1137/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved. Facklamia languida sp. nov., Isolated from Human Clinical Specimens PAUL A. LAWSON,1 MATTHEW D. COLLINS,1 ENEVOLD FALSEN,2 2 3 BERIT SJO¨ DEN, AND RICHARD R. FACKLAM * Department of Food Science and Technology, University of Reading, Reading, United Kingdom1; Culture Collection, Department of Clinical Bacteriology, University of Go¨teborg, Goteborg, Sweden2; and Centers for Disease Control and Prevention, Atlanta, Georgia3 Received 27 October 1998/Returned for modification 7 December 1998/Accepted 22 December 1998 Three strains of a gram-positive catalase-negative, facultatively anaerobic coccus-shaped organism origi- nating from human clinical samples were characterized by phenotypic and molecular taxonomic methods. Sequencing of genes encoding 16S rRNA showed that the strains are phylogenetically closely related (99.9 to 100% sequence similarity) and represent a new subline within the genus Facklamia. The unknown bacterium was readily distinguished from all currently described species of the genus Facklamia (viz., Facklamia hominis, Facklamia ignava, and Facklamia sourekii) by biochemical tests and electrophoretic analysis of whole-cell pro- teins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium be classified as Facklamia languida sp. nov. The type strain of F. languida is CCUG 37842. Over the past few years there has been a considerable ex- Collection of the University of Go¨teborg, CCUG 37842T) was pansion in the number of genera and species of aerobic or recovered from a blood culture of a 5-year-old female child facultatively anaerobic catalase-negative gram-positive cocci with bacteremia in Ohio. Strain 1664-95 (5CCUG 37420) was identified from human clinical sources. Much of this change received from Marguerite Lovegren (Edmonton, Canada) and has stemmed from the increased use of molecular genetic (e.g., originated from blood cultures of a 74-year-old woman with a 16S rRNA gene sequencing) and molecular chemical (e.g., urinary tract infection. Strain CCUG 30940 was submitted by whole-cell protein analysis) methodologies for the identifica- the Public Health Laboratory Service in Go¨teborg (Sweden) to tion of these organisms from clinical samples. In particular, the Culture Collection of the University of Go¨teborg for iden- 16S rRNA gene sequencing has resulted in phylogenetically tification and originated from cerebrospinal fluid in a 40-year- based descriptive frameworks, which, together with rapid se- old woman. No additional clinical information is available on quencing technology and readily accessible sequence libraries, the three strains. The unidentified organisms were cultured on are providing diagnostic laboratories with immensely powerful Columbia agar (Difco, Detroit, Mich.) supplemented with 5% technology for identifying not only atypical or problematic defibrinated horse blood (Oxoid, Unipath Ltd., Basingstoke, members of existing taxa but also a wealth of new and diverse United Kingdom) at 37°C, in air plus 5% CO2. The strains organisms. Examples of new catalase-negative gram-positive were biochemically characterized by using the API rapid cocci from human sources include Abiotrophia elegans (16), ID32S and API ZYM systems according to the manufacturer’s Aerococcus urinae (1), Alloiococcus otitis (2), Dolosigranulum instructions (API bioMe´rieux, Marcy l’Etoile, France). Conven- pigrum (3), Facklamia hominis (5), Facklamia ignava (10), Ge- tional physiological tests were also conducted as described by mella bergeri (9), Gemella sanguinis (8), Globicatella sanguinis Facklam and Elliott (13). Polyacrylamide gel electrophoresis (4), Helcococcus kunzii (6), and Ignavigranum ruoffiae (11). (PAGE) analysis of whole-cell proteins was performed as de- During an investigation of atypical gram-positive catalase-neg- scribed by Pot et al. (15). For densitometric analysis, normal- ative chain-forming cocci from human sources, we have en- ization, and interpretation of protein patterns the GelCompar countered three Facklamia-like isolates from clinical materials. GCW 3.0 software package (Applied Maths, Kortrijk, Bel- Preliminary biochemical studies indicated that these strains did gium) was used. The similarity between all pairs of traces was not conform to any of the currently recognized species of this expressed by the Pearson product moment correlation coeffi- genus, viz., F. hominis (5), F. ignava (10), and F. sourekii (7). In cient converted for convenience to a percentage similarity value. this paper, we report the phenotypic characteristics of these Phylogenetic analysis was conducted by 16S rRNA gene se- clinical isolates and the results of a molecular genetic and quencing. A large fragment of the 16S rRNA gene was ampli- molecular chemical analysis. Based on the findings presented fied by PCR using conserved primers close to the 39 and 59 ends here, we consider the unknown chain-forming coccus to rep- of the gene and directly sequenced with a Taq Dye-Deoxy ter- resent a new species of the genus Facklamia, for which the minator cycle sequencing kit (Applied Biosystems, Foster City, name F. languida sp. nov. is described. Calif.) and an automatic DNA sequencer (model 373A; Ap- Two strains (1144-97 and 1664-95) from human sources plied Biosystems). The closest known relatives of the new iso- were referred to the Centers for Disease Control and Preven- late were determined by performing database searches. These tion (Atlanta, Ga.) for identification. Strain 1144-97 (Culture sequences and those of other known related strains were re- trieved from the GenBank or Ribosomal Database Project li- braries and aligned with the newly determined sequence by us- * Corresponding author. Mailing address: Centers for Disease Con- ing the program PILEUP (12). The resulting multiple-sequence trol and Prevention, 1600 Clifton Rd., NE, Atlanta, GA 30333. Phone: alignment was corrected manually, and a distance matrix was (404) 639-1379. Fax: (404) 639-3123. E-mail: [email protected]. calculated by using the programs PRETTY and DNADIST 1161 1162 NOTES J. CLIN.MICROBIOL. FIG. 1. Similarity dendrogram based on whole-cell protein pattern of F. languida sp. nov. and related species. Levels of correlation are expressed as percentages for convenience. (using the Kimura-2 correction parameter) (14). A phyloge- 00020500000 with the API rapid ID32S strip and corresponded netic tree was constructed according to the neighbor-joining to a doubtful profile. Significant taxa were Gemella morbillo- method with the program NEIGHBOR, and the stability of rum (four tests against) and Gemella haemolysans (three tests the groupings was estimated by bootstrap analysis (500 rep- against). When commercial API systems were used, the iso- lications) by using the programs DNABOOT, DNADIST, lates produced acid from trehalose but failed to produce acid NEIGHBOR, and CONSENSE (14). from D-arabitol, L-arabinose, cyclodextrin, glycogen, lactose, On Trypticase soy agar containing 5% sheep blood (TSA- maltose, mannitol, melibiose, melezitose, pullulan, raffin- SB) (Becton Dickinson Co., Cockeysville, Md.), after 24 h ose, ribose, sorbitol, sucrose, tagatose, or methyl-b-D-glucopy- of incubation in an atmosphere of increased CO2, the isolates ranoside. Alkaline phosphatase, pyroglutamic acid arylami- formed small gray to colorless colonies with little hemolytic dase, leucine arylamidase, and glycyl-tryptophan arylamidase activity on the sheep blood cells. After 48 h of incubation the activities were detected. No activity was detected for arginine colonies were surrounded by a small zone of alpha hemolysis. dihydrolase, acid phosphatase, alanine-phenylalanine-proline All three isolates were ovoid and most commonly formed pairs, arylamidase, b-galacturonidase, a-galactosidase, b-galacto- but single cells and short chains were also observed. They were sidase, b-glucuronidase, a-glucosidase, b-glucosidase, lipase gram positive, non-spore forming, catalase negative, oxidase C14, a-fucosidase, a-mannosidase, b-mannosidase, N-acetyl- negative, and facultatively anaerobic. The strains grew in 6.5% glucosaminidase, cysteine arylamidase, chymotrypsin, trypsin, NaCl at 37°C but not at all at 10 or 45°C and were pyrroli- or urease. Esterase C4 and ester lipase C8 activity was found to donyl arylamidase and leucine aminopeptidase positive in be variable. The strains did not hydrolyze hippurate and were conventional tests (13). The three isolates gave the profile Voges-Proskauer negative. A numerical analysis of the whole- VOL. 37, 1999 NOTES 1163 FIG. 2. Unrooted tree showing the phylogenetic relationships of F. languida sp. nov. and some other low-G1C gram-positive bacteria. The tree was constructed by the neighbor-joining method and was based on a comparison of approximately 1,320 nucleotides. Bootstrap values, expressed as percentages of 500 replications, are given at branching points. Abbreviations: ATCC, American Type Culture Collection; DSM, Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH; IAM, Institute of Applied Microbiology; JCM, Japanese Collection of Microorganisms; NCDO, National Collection of Dairy Organisms; NCFB, National Collection of Food Bacteria; NCMIB, National Collection of Industrial and Marine Bacteria. cell protein patterns of the three clinical strains along with a It is evident from comparative 16S rRNA gene sequencing comprehensive range of other