Antonie van Leeuwenhoek (2016) 109:603–610 DOI 10.1007/s10482-016-0663-z

ORIGINAL PAPER

Kroppenstedtia pulmonis sp. nov. and Kroppenstedtia sanguinis sp. nov., isolated from human patients

Melissa E. Bell . Brent A. Lasker . Hans-Peter Klenk . Lesley Hoyles . Catherine Spro¨er . Peter Schumann . June M. Brown

Received: 30 September 2015 / Accepted: 2 February 2016 / Published online: 24 February 2016 Ó Springer International Publishing Switzerland (outside the USA) 2016

Abstract Three human clinical strains (W9323T, relatedness was 94.6 %, confirming that X0209T and X0209T and X0394) isolated from a lung biopsy, blood X0394 belong to the same species. Chemotaxonomic and cerebral spinal fluid, respectively, were charac- data for strains W9323T and X0209T were consistent terised using a polyphasic taxonomic approach. Com- with those described for the members of the genus parative analysis of the 16S rRNA gene sequences Kroppenstedtia: the peptidoglycan was found to con- showed the three strains belong to two novel branches tain LL-diaminopimelic acid; the major cellular fatty within the genus Kroppenstedtia: 16S rRNA gene acids were identified as iso-C15 and anteiso-C15; and sequence analysis of W9323T showed close sequence the major menaquinone was identified as MK-7. similarity to Kroppenstedtia eburnea JFMB-ATET Differences in endospore morphology, carbon source (95.3 %), Kroppenstedtia guangzhouensis GD02T utilisation profiles, and cell wall sugar patterns of (94.7 %) and strain X0209T (94.6 %); sequence anal- strains W9323T and X0209T, supported by phyloge- ysis of strain X0209T showed close sequence similarity netic analysis, enabled us to conclude that the strains to K. eburnea JFMB-ATET (96.4 %) and K. each represent a new species within the genus Krop- guangzhouensis GD02T (96.0 %). Strains X0209T penstedtia, for which the names Kroppenstedtia pul- and X0394 were 99.9 % similar to each other by 16S monis sp. nov. (type strain W9323T = DSM rRNA gene sequence analysis. The DNA–DNA 45752T = CCUG 68107T) and Kroppenstedtia

& M. E. Bell ( ) C. Spro¨er Á P. Schumann Centers for Disease Control and Prevention, Building 17, Leibniz-Institute DSMZ-German Collection of Room 2209, Mailstop D-11, 1600 Clifton Road, Atlanta, Microorganisms and Cell Cultures, 38124 Brunswick, GA 30333, USA Germany e-mail: [email protected] L. Hoyles B. A. Lasker Á J. M. Brown Department of Biomedical Sciences, Faculty of Science & Bacterial Special Pathogens Branch, Division of High- Technology, University of Westminster, 115 New Consequence Pathogens and Pathology, National Center Cavendish Street, London W1W 6UW, UK for Emerging and Zoonotic Infectious Disease, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA

H.-P. Klenk Head of School, School of Biology, Newcastle University, Ridley Building 2, Newcastle upon Tyne NE1 7RU, UK 123 604 Antonie van Leeuwenhoek (2016) 109:603–610 sanguinis sp. nov. (type strain X0209T = DSM Special Bacteriology Reference Laboratory (SBRL), 45749T = CCUG 38657T) are proposed. Atlanta, GA. Genotypic and phenotypic data suggest that strain W9323T represents a new species, for which Keywords Kroppenstedtia species Á Kroppenstedtia we propose the name Kroppenstedtia pulmonis sp. pulmonis Á Kroppenstedtia sanguinis Á Polyphasic nov., and that strains X0209T and X0394 both taxonomy Á 16S rRNA gene Á Thermoactinomycetes represent another new species, for which we propose the name Kroppenstedtia sanguinis sp. nov.

Introduction Materials and methods

The family Thermoactinomycetaceae was established to Bacterial strains accommodate new taxa (Mechercharimyces spp.) and the previously described genera Thermoactinomyces, Strain X0209T was obtained from the Culture Collec- Laceyella, Thermoflavimicrobium, Seinonella and tion University of Go¨teborg (CCUG), Go¨teborg, Swe- Planifilum (Matsuo et al. 2006). The family now den as ‘Thermoactinomyces sanguinis’(CCUG encompasses 19 genera, with representatives isolated 38657T). Two strains, W9323T and X0394, were sent from clinical specimens (e.g. Desmospora, Marinither- to the SBRL, Centers for Disease Control and Preven- mofilum, Hazenella) or environmental sources (e.g. tion for identification. K. eburnea DSM 45196T was Mechercharimyces, Lihuaxuella, Geothermomicrobium used as a phenotypic, chemotaxonomic and genetic and Risungbinella) (Matsuo et al. 2006; Yassin et al. control and K. guangzhouensis GD02T and Melghir- 2009;Yuetal.2012;Bussetal.2013;Zhouetal.2014; imyces algeriensis NariEXT were used as chemotaxo- Kim et al. 2015; Zhang et al. 2015). Salient properties of nomic and genetic controls throughout this study. The the family Thermoactinomycetaceae include thermo- GenBank accession numbers for the 16S rRNA gene tolerant growth up to 60 °C, Gram positivity, non-acid sequences and the patients’ data associated with these fastness and formation of single spores that may be strains are given in Table 1. sessile or on simple sporophores with the structure and properties of endospores. The description of the family Phenotypic analyses was emended by establishing the 16S rRNA gene sequence signature nucleotides appropriate for supra- Morphological, cultural, physiological generic assignment (Yassin et al. 2009). Von Jan et al. and biochemical analyses (2011) further emended the description of the family Thermoactinomycetaceae (to contain either LL-di- To examine morphologic features, strains were grown aminopimelic acid or meso-diaminopimelic acid) when aerobically using brain heart infusion (BHI) broth, they described a new genus and species, Kroppenstedtia heart infusion agar (HIA; Becton, Dickinson and Co, eburnea, that contains the isomer LL-diaminopimelic Sparks, MD) supplemented with 5 % rabbit blood, acid in its peptidoglycan. HIA slants, trypticase soy agar (TSA) supplemented Kroppenstedtia species have been isolated from with 5 % sheep blood (Becton, Dickinson and Co) at environmental and clinical sources. Although the type 35 and 45 °C for 3 to 7 days and then examined for strain of K. eburnea was isolated from an environ- microscopic and macroscopic features. Gram and mental source (a plastic surface in a contract manu- modified Kinyoun acid-fast staining were conducted facturing company in Germany), Barker et al. (2012) as described previously (Berd 1973). Cultures were identified 14 strains of this species in clinical samples examined for optimal growth at 35, 45, 50 and 60 °C (blood, skin, peritoneal fluid, cerebral spinal fluid) in for 7 days on HIA slants with 5 % rabbit blood. the United States. Another species, Kroppenstedtia Optimal growth was determined by comparative guangzhouensis, was isolated from soil in south China observation of the amount of cell mass produced at (Yang et al. 2013). Three human clinical strains of each temperature. All phenotypic studies were per- Kroppenstedtia species were identified in a retrospec- formed under optimal growth conditions (at 45 °Cin tive evaluation of 16S rRNA gene sequences at the air). 123 Antonie van Leeuwenhoek (2016) 109:603–610 605

Table 1 Strains used in this study Strain Date received or Source Geographic GenBank accession reference source number of 16S rRNA gene

Kroppenstedtia eburnea (DSM 45196T) von Jan et al. (2011) Plastic surface Germany FN665656 K. guangzhouensis (GD02T) Yang et al. (2013) Soil China KC311557 Melghirimyces algeriensis (NariEXT) Addou et al. (2012) Soil from salt lake Algeria HQ383683 W9323T 2008 Lung, 78-year-old male New York, USA KC875234 X0209T 1997 Blood, 59-year-old male Ga¨vle, Sweden AJ251778 X0394 2010 CSF, 16-year-old female Quebec, Canada KC875233

Decomposition tests for adenine, casein, esculin, analysed by the method of Minnikin et al. (1984). hypoxanthine, tyrosine, urea and xanthine, utilisation Analysis of isoprenoid quinones by HPLC was of 22 carbohydrates as sole source of carbon, utilisa- performed as described by Kroppenstedt (1982, tion of acetamide and citrate, arylsulfatase production 1985). Cellular fatty acids were prepared by the and nitrate reduction and growth in the presence of method of Klatte et al. (1994) and the fatty acid methyl lysozyme were performed as described previously esters were then separated as described by Sasser (Conville and Witebsky 2007; Conville et al. 2008; (1990) using the Microbial Identification System Weyant et al. 1996; Yassin et al. 1995). The decom- (MIDI, Inc., Sherlock version 6.1). Standardisation position of casein was compared with casein plus of the physiological age of the study and reference 0.5 % NaCl as described by von Jan et al. (2011). strains was obtained by choosing the sector from a quadrant streak of culture plates. Antimicrobial susceptibility testing Genetic analyses Since no guidelines were available for the genus Kroppenstedtia, the MICs to 10 antimicrobial agents 16S rRNA gene sequence analysis were determined following interpretative breakpoints as recommended for aerobic actinomycetes (Clinical Purification of whole-cell DNA, amplification of near and Laboratory Standards Institute 2011) for amikacin, full-length 16S rRNA gene fragments, primers and amoxicillin-clavulanate, ceftriaxone, ciprofloxacin, nucleotides for PCR, purification of amplicons and clarithromycin, imipenem, linezolid, minocycline, DNA cycle sequencing were described previously moxifloxacin and trimethoprim-sulfamethoxazole; the (Lasker et al. 2011). Consensus 16S rRNA gene interpretive breakpoint for ampicillin used was that sequences were assembled and edited using recommended for Enterobacteriaceae (Clinical and Sequencher version 4.10.1 software. To identify Laboratory Standards Institute 2015). The preparation related gene sequences in the GenBank database, of the inoculum suspension followed the guidelines as consensus sequences were submitted to GenBank described previously (Clinical and Laboratory Stan- using BLASTn software (https://www.ncbi.nlm.nih. dards Institute 2003). gov/blast/). A multiple sequence alignment was cre- ated using Clustal W (within Geneious 8.1.4), from Chemotaxonomic analyses which gaps and 50 and 30 ends were trimmed. The distance matrix was calculated using DNADIST Assays of diaminopimelic acid stereoisomers and (Kimura 2-correction parameter) (Felsenstein 1989). whole cell sugars were performed by thin-layer Phylogenetic trees were constructed using the neigh- chromatography using methods described previously bor-joining (NEIGHBOR), maximum likelihood (Lechevalier and Lechevalier 1970; Rhuland et al. (DNAML with global rearrangements) and maximum 1955; Staneck and Roberts 1974). Isoprenoid quinones parsimony (DNAPARS with global rearrangements) and polar lipids were extracted and purified and methods available in the PHYLIP package

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(Felsenstein, 1989). Stability of groupings within the unbranched sporophores of strain W9323T, the micro- neighbor-joining tree was estimated by bootstrap scopic morphology is consistent with those of the type analysis (1000 replications) using the programs strains of K. eburnea and K. guangzhouensis.The SEQBOOT, DNADIST, NEIGHBOR and CON- macroscopic morphology of strains W9323T,X0209T SENSE (Felsenstein 1989). The phenograms were and X0394 is consistent with that described for the type visualised by using the program DRAWGRAM strains of K. eburnea and K. guangzhouensis, however, (Felsenstein 1989). The sequence of the type strain of none of the three strains produced aerial hyphae as Bacillus subtilis was used as the outgroup. reported by von Jan et al. (2011) and Yang et al. (2013). Table 2 gives the differential phenotypic, chemo- DNA–DNA hybridization and DNA G?C content taxonomic and genetic characteristics of the study strains and their close phylogenetic relatives. Strains T Cells for hybridization and G?C mol% determination X0209 and X0394 both are able to utilise D-mannitol; T were disrupted using a Constant Systems TS 0.75 KW W9323 is the only strain able to utilise D-glucose and (IUL Instruments, Germany). DNA in the crude sucrose. Strain X0209T is able to utilise cellobiose and lysates was purified on hydroxyapatite by chromatog- salicin but the related strain X0394 does not. raphy as described by Cashion et al. (1977). DNA– Results of antimicrobial susceptibility testing DNA hybridization was carried out in duplicate as showed strains W9323T,X0209T and X0394 were described by De Ley et al. (1970) as modified by Huss resistant (MIC, C8 lg/ml) to clarithromycin but were et al. (1983) using a model Cary 100 Bio UV/VIS- susceptible to amikacin (MIC, B8), amoxicillin-clavu- spectrophotometer equipped with a Peltier-thermostat lanate (MIC, B8/4 lg/ml), ampicillin (MIC, B4 lg/ 6 9 6 multicell changer and a temperature controller ml), ceftriaxone (MIC, B8 lg/ml), ciprofloxacin with in situ temperature control. The method of (MIC, B1 lg/ml), imipenem (MIC, B4 lg/ml), line- Mesbah et al. (1989) was performed to determine the zolid (MIC, B8 lg/ml), minocycline (MIC, B1 lg/ G?C content of the novel type strains. ml), moxifloxacin (MIC, B1 lg/ml) and trimetho- prim-sulfamethoxazole (MIC, B2/38 lg/ml). Except for resistance to ampicillin of K. eburnea JFMB- Results and discussion ATET, susceptibility results of the three study strains were consistent with those of K. eburnea JFMB-ATET. Strains W9323T,X0209T and X0394 were found to be The antimicrobial susceptibility test results in our study aerobic, mesophilic to thermophilic, Gram-positive are consistent with the MIC results of 14 clinical strains bacteria but are not acid fast. Substrate hyphae were of K. eburnea reported by Barker et al. (2012); for observed to be filamentous and branched, and could be example, all their strains were susceptible to all seen as fringes around the colony margins; no aerial antimicrobial agents tested except for clarithromycin. hyphae were observed. Rare elongated (paddle shaped) Cell wall hydrolysates were found to contain LL- endospores on long, unbranched sporophores were seen diaminopimelic acid, ribose and traces of galactose on Gram-stained smears of strain W9323T;single (X0209T and X0394); K. eburnea JFMB-ATET con- globose endospores on unbranched sporophores were tains ribose and glucose. The predominant menaqui- seen on Gram-stained smears of strains X0209T and nones of strain W9323T were identified as MK-7 X0394. Colonies of all three strains showed beta (95 %) and MK-8 (5 %); the predominant menaqui- hemolysis on TSA with 5 % sheep blood at 45 °C. nones of strains X0209T and X0394 were identified as Growth was found to occur at 35 and 45 °C but not at MK-7 (97 %) and MK8 (3 %). Polar lipids were 50 °C, with optimum growth at 45 °C; the optimal identified as phosphatidylglycerol, diphosphatidyl- growth at 45 °C was consistent with that of the type glycerol, phosphatidylethanolamine, phosphatidyl- strain of K. eburnea but differed from the optimal methylethanolamine, two unidentified phospholipids growth at 50 °CoftheK. guangzhouensis type strain. (W9323T and X0394; X0209T) and one unidentified Pale yellow colonies were observed on 7-day TSA glycolipid (W9323T, X0209T and X0394). The fatty plates with 5 % sheep blood that had irregular margins acid profiles of the three strains were found to consist with random surface ridges at 35 and 45 °C. Except for predominantly of iso-C15:0 (74–75 %) and anteiso- the production of paddle-shaped endospores on long, C15:0 (11–13 %). 123 Antonie van Leeuwenhoek (2016) 109:603–610 607

Table 2 Phenotypic, chemotaxonomic and genotypic characteristics that differentiate the study strains from other Kroppenstedtia species Characteristic K. pulmonis W9323T K. sanguinis K. sanguinis K. eburnea K. guangzhouensis X0209T X0394 DSM 45196T GD02Ta

Endospore morphology Elongated paddle shaped Globose Globose Globose Globose Aerial hyphae - ---(?b)NT(?c) Optimal growth on TSA sheep blood 45 °C45°C45°C45°C50°C at 7 days Beta hemolysis on TSA sheep blood ? ??? NT Utilization of Cellobiose - ?-? - Dulcitol - --? -

D-Fructose - --? -

D-Glucose ? --- -

D-Mannitol - ??- - Mannose - ?-- NT Salicin - ?-? NT Sucrose ? --- - Whole cell-wall Ribose Ribose Ribose Ribose NT sugars Glucose Trace of galactose Trace of galactose

Cellular fatty acids iso-C15 (75.0 %) iso-C15 (74.0 %) iso-C15 (75.0 %) iso-C15(73.0 %) iso-C15 (63.5 %)

([ 5%) anteiso-C15 (11.0 %) anteiso-C15 (12.0 %) anteiso-C15 (13.0 %) anteiso-C15 (13.0 %) anteiso-C15 (7.0 %)

iso-C17 (8.0 %) iso-C17 (7.0 %) iso-C17 (8.0 %) iso-C17 (–) iso-C17 (8 %)

iso-C16 (2.5 %) iso-C16 (1.4 %) iso-C16 (-) iso-C16 (4.5 %) iso-C16 (12.6 %) Menaquinones MK-7 (95.0 %) MK-7 (97.0 %) MK-7 (97.0 %) MK-7 (97. 0 %) MK-7 (98.6 %) MK-8 (5.0 %) MK-8 (3.0 %) MK-8 (3.0 %) MK-8 (3.0 %) MK-8 (1.4 %) Polar lipids PG, DPG, PE, PME PG, DPG, PE, PME PG, DPG, PE, PME, PG, DPG, PE, PME, PG, DPG, PE, PME 4 unknown PL 5 unknown PL 4 unknown PL 2 unknown PL 2 unknown L 1 unknown GL 1 unknown GL 1 unknown GL DNA G?C mol % 45.9 50.5 50.6 54.6 56.3

All strains tested were negative for utilization of acetamide, adonitol, L-arabinose, citrate, i-erythritol, D-galactose, glycerol, i-myo- inositol, lactose, maltose, melibiose, raffinose, L-rhamnose, D-sorbitol, trehalose and D-xylose; production of arylsulfatase and urease, hydrolysis of adenine, aesculin by fluorescence, hypoxanthine, tyrosine, xanthine and reduction of nitrate; all strains tested were positive for browning of aesculin, growth in lysozyme and hydrolysis of casein with 0.5 % NaCl. No growth on casein PG phosphatidylglycerol, DPG diphosphatidylglycerol, PE phosphatidylethanolamine, PME phosphatidylmethylethanolamine, PL phospholipid, GL glycolipid, L lipid, - negative, ? positive, NT not tested, TSA trypticase soy agar a All phenotypic data for K. guangzhouensis from Yang et al. (2013). All other data including cellular fatty acids, menaquinones, polar lipids and DNA G?C content were generated from present study b Aerial hyphae reported by von Jan et al. (2011) c Aerial hyphae reported by Yang et al. (2013)

Based on 16S rRNA gene sequence analysis, the sequence similarity), M. algeriensis DSM 45474T clinical strains were assigned within the subclade for (94.3 % similarity) and Desmospora activa DSM the genus Kroppenstedtia, within the family Ther- 45169T (94.2 % similarity). The 16S rRNA gene moactinomycetaceae (Fig. 1). Strain W9323T was sequences for strains X0209T and X0394 are 99.9 % found to show high sequence similarity to K. eburnea identical to each other. Strain X0209T was found to JFMB-ATET (95.3 % sequence similarity), K. show high sequence similarity to K. eburnea JFMB- guangzhouensis GD02T (94.7 % sequence similarity), ATET (96.4 % similarity), K. guangzhouensis GD02T Melghirimyces thermohalophilus Nari11AT (94.5 % (96.0 % similarity), M. algeriensis DSM 45474T

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T 97 Bacillus subtilis DSM 10 (AJ276351) ML, MP Aneurinibacillus thermoaerophilus DSM 10154T (X94196) T 100 Calditerricola yamamurae YMO722 (AB308475) ML, MP Calditerricola satsumensis YM081T (AB250968) T 100 Planifilum fulgidum JCM 12508 (AB088362) 100 ML, ML, Planifilum yunnanense CCTCC AA206002T (DQ119659) MP MP Planifilum fimeticola JCM 12507T (AB088364) Polycladomyces abyssicola JIR-001T (AB688114) T 100 Marininema mesophilum SCSIO 10219 (JN006758) ML, MP 88 T ML, MP Marininema halotolerans YIM11385 (KC684888) 99 ML, Salinithrix halophila R4S8T (KJ206288) MP Desmospora activa DSM 45169T (AM940019)

100 Kroppenstedtia sanguinis X0394 (KC875233) ML, MP X0209T (AJ251778) 81 Kroppenstedtia sanguinis ML, MP T 76 Kroppenstedtia eburnea JFMB-ATE (FN665656) ML, MP ML, 88 Kroppenstedtia guangzhouensis GD02T (KC311557) MP 81 Kroppenstedtia pulmonis W9323T (KC875234) Melghirimyces thermohalophilus Nari11AT (JX861508) 80 Melghirimyces algeriensis DSM 45474T (HQ383683) T 100 Mechercharimyces mesophilus MBIC06230 (AB239529) ML, MP T ML Mechercharimyces asporophorigensis MBIC06487 (AB239532) Novibacillus thermophilus SG-1T (KF958346) Risungbinella pyongyangensis MC210T (KJ729272) Shimazuella kribbensis KCTC 9933T (AB049939) Geothermomicrobium terrae YIM 77562T (AB859256) 95 Baia soyae NEAU-gxj18T (KM359702) Seinonella peptonophila KCTC 9740T (AF138735) 84 ML 1 % Hazenella coriacea 23436T (JQ798970) T 100 Laceyella sacchari DSM 43356 (AJ251779) ML, MP Laceyella putida KCTC 3666T (AF138736) T 100 Thermoactinomyces intermedius KCTC 9646 (AF138734) ML, MP T ML Thermoactinomyces vulgaris KCTC 9076 (AF138739) T ML Thermoflavimicrobium dichotomicum KCTC 3667 (AF138733) Lihuaxuella thermophia YIM 77831T (JX045707)

Fig. 1 Neighbor joining tree showing the positions of Krop- ML nodes common to the neighbor joining and maximum penstedtia sanguinis sp. nov. and Kroppenstedtia pulmonis sp. likelihood analyses; MP nodes common to the neighbor joining nov. within the family Thermoactinomycetaceae. The tree was and maximum parsimony analyses (Felsenstein 1989). The constructed based on an analysis of *1480 nt. Bootstrap values sequence of the type strain Bacillus subtilis was used as the shown at the nodes are expressed as a percentage of 1000 outgroup replications (neighbor joining); only values [ 70 % are shown.

(94.3 % similarity), M. thermohalophilus Nari11AT inferred from 16S rRNA gene similarities observed and D. activa DSM 45169T (93.7 % similarity). The (Meier-Kolthoff et al. 2013). The genomic DNA G?C 16S rRNA gene sequences of strains X0209T and K. content of strains W9323T and X0209T were deter- eburnea JFMB-ATET differ by 59 bp; the sequences mined to be 45.9 and 50.6 mol%, respectively. These of strains W9323T and K. eburnea JFMB-ATET differ values fall within the range of genomic DNA G?C by 66 bp. content reported for members of the genus Kroppen- Consistent with their high 16S rRNA sequence stedtia as it exists presently (46 to 56 mol%) (von Jan similarity, DNA–DNA hybridization studies between et al. 2011, Yang et al. 2013). the clinical strains X0209T and X0394 confirmed they From the results of our phenotypic, chemotaxo- belong to the same genomic species with a relatedness nomic and genetic studies, it is proposed that strains value of 93.6 % ± 6.5 %. DNA–DNA hybridization W9323T and X0209T be classified in the genus studies between X0209T and W9323T and their Kroppenstedtia as type strains of the species Krop- respective close phylogenetically related neighbours penstedtia pulmonis sp. nov. and Kroppenstedtia were not conducted because of the low probability sanguinis sp. nov., respectively.

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Description of Kroppenstedtia pulmonis sp. nov salicin are utilised but i-adonitol, L-arabinose, citrate, dulcitol, i-erythritol, D-fructose, D-galactose, D-glucose, Kroppenstedtia pulmonis (N. L. fem. adj. pul.mo’nis. glycerol, i-myo-inositol, lactose, maltose, melibiose, L. n. pulmo-onis, lung; L. gen. n. pulmonis of a lung, raffinose, L-rhamnose, D-sorbitol, sucrose, trehalose and isolated from a lung). D-xylose are not utilised. Grows in the presence of Cells are Gram-positive, non-acid-fast and filamen- lysozyme but has no arylsulfatase activity and does not tous. Elongated spores (paddle shaped) are formed reduce nitrate. Hydrolyses casein with 0.5 % NaCl but singly on sessile sporophores on substrate hyphae. does not hydrolyse acetamide, adenine, casein, hypox- Pale yellowish-gray colonies are wrinkled with ran- anthine, tyrosine, urea or xanthine. Esculin hydrolysis is dom ridges after 3 to 7 days at 35 and 45 °Con positive by browning but negative by UV light absorp- trypticase soy agar with 5 % sheep blood. Colonies are tion. The predominant menaquinone is MK-7. Whole beta hemolytic but do not form aerial hyphae. Growth cell hydrolysates contain LL-diaminopimelic acid, the occurs at 35 and 45 °C but not at 50 °C. D-Glucose and sugar ribose, and traces of the sugar galactose. Major sucrose are utilised but i-adonitol, L-arabinose, cel- fatty acids ([10 %) are iso-C15:0 and anteiso-C15:0. lobiose, citrate, dulcitol, i-erythritol, D-fructose, D- Polar lipids are phosphatidylglycerol, diphosphatidyl- galactose, glycerol, i-myo-inositol, lactose, maltose, D- glycerol, phosphatidylethanolamine, phosphatidyl- mannitol, mannose, melibiose, raffinose, L-rhamnose, methylethanolamine, one unidentified phospholipid salicin, D-sorbitol, trehalose and xylose are not and one unidentified glycolipid. The G?Ccontentof utilised. Grows in the presence of lysozyme but has the genomic DNA of the type strain is 50.6 mol%. no arylsulfatase activity and does not reduce nitrate. The type strain (X0209T = DSM 45749T Hydrolyses casein with 0.5 % NaCl but does not = CCUG 38657T) was isolated from the blood of a hydrolyse acetamide, adenine, casein, hypoxanthine, 59-year-old male from Ga¨vle, Sweden. tyrosine, urea or xanthine. Esculin hydrolysis is positive by browning but negative by UV light Acknowledgments We thank Jean Euze´by for nomenclatural advice. absorption. Major fatty acids ([10 %) are iso-C15:0 and anteiso-C15:0. Whole cell hydrolysates contain LL-diaminopimelic acid and the sugar ribose. The predominant menaquinones are MK-7 and MK-8. References Polar lipids are phosphatidylglycerol, diphosphatidyl- glycerol, phosphatidylethanolamine, phosphatidyl- Addou AN, Schumann P, Spro¨er C, Hacene H, Cayol JL, Far- methylethanolamine, one unidentified phospholipid deau ML (2012) Melghirimyces algeriensis gen. nov., sp. nov., a member of the family Thermoactinomycetaceae, and one unidentified glycolipid. The G ? C content of isolated from a salt lake. Int J Syst Evol Microbiol the genomic DNA of the type strain is 45.9 mol%. 62:1491–1498 The type strain (W9323T = DSM 45752T Barker AP, Simmons KE, Cohen S, Slechta ES, Fisher MA, = CCUG 68107T) was isolated from a lung biopsy Schlaberg R (2012) Isolation and identification of Krop- penstedtia eburnea isolates from multiple patient samples. of a 78-year-old male patient from New York, USA. J Clin Microbiol 50:3391–3394 Berd D (1973) Laboratory identification of clinically important Description of Kroppenstedtia sanguinis sp. nov aerobic actinomycetes. Appl Microbiol 25:665–681 Buss SN, Cole JA, Hannett GE, Nazarian EJ, Nazarian L, Coorevits A, Van Landschoot A, De Vos P, Schumann P, Kroppenstedtia sanguinis (san’gui.nis L. n. sanguis- Musser KA, Wolfgang WJ (2013) Hazenella coriacea gen. inis, blood; L. gen. n. sanguinis, of blood). nov., sp. nov., isolated from clinical specimens. Int J Syst Cells are Gram-positive, non-acid-fast and filamen- Evol Microbiol 63:4087–4093 tous. Globose spores are formed singly on sessile Cashion P, Hodler-Franklin MA, McCully J, Franklin M (1977) A rapid method for base ratio determination of bacterial sporophores on substrate hyphae. Pale yellowish gray DNA. Anal Biochem 81:461–466 colonies are wrinkled with random ridges after 3 to Clinical and Laboratory Standards Institute (2003) Suscepti- 5 days at 35 and 45 °C on trypticase soy agar with 5 % bility testing of mycobacteria, nocardiae, and other aerobic sheep blood. Colonies are beta hemolytic but do not actinomycetes; approved standard M24-A. National Committee for Clinical Laboratory Standards, Wayne form aerial hyphae. Growth occurs at 35 and 45 °Cbut Clinical and Laboratory Standards Institute (2011) Suscepti- not at 50 °C. Cellobiose, D-mannitol, mannose and bility testing of mycobacteria, nocardiae, and other aerobic 123 610 Antonie van Leeuwenhoek (2016) 109:603–610

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