SGM Journals
Bacteroidetes

Epilithonimonas lactis sp. nov., isolated from raw cow'smilk

Tamar Shakéd, Elionora Hantsis-Zacharov, Malka Halpern

  • 1Department of Evolutionary and Environmental Biology, Faculty of Science and Science Education, University of Haifa, Mount Carmel, Haifa 31905, Israel
  • 2Department of Biology Education, Faculty of Science and Science Education, University of Haifa, Oranim, Tivon 36006, Israel
  • Correspondence
    Malka Halpern
    mhalpern{at}research.haifa.ac.il

    • International Journal of Systematic and Evolutionary Microbiology 2010; 60(3):675–679 · https://doi.org/10.1099/ijs.0.012575-0

    View at publisher PubMed

    Abstract

    A Gram-staining-negative, rod-shaped, oxidase-positive, aerobic, non-motile and yellow-pigmented bacterial strain containing flexirubin type pigments, designated H1T, was isolated from raw cow'smilk in Israel. 16S rRNA gene sequence analysis indicated that the isolate should be placed in the genus Epilithonimonas (family Flavobacteriaceae, phylum Bacteroidetes). The level of 16S rRNA gene sequence similarity between strain H1T and the type strain of Epilithonimonas tenax was 97.6 %. Strain H1T grew at 5–33 °C and with 0–2.0 % NaCl. The dominant cellular fatty acids of strain H1T were iso-C15 : 0, iso-C17 : 0 3-OH and summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 1ω7c), and the DNA G+C content was 38.0 mol%. On the basis of phenotypic properties and phylogenetic distinctiveness, the milk isolate is classified as a new species in the genus Epilithonimonas, for which the name Epilithonimonas lactis sp. nov. (type strain H1T =LMG 24401T =DSM 19921T) is proposed.

    • The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain H1T is EF204460.

    The genus Epilithonimonas (O'Sullivan et al., 2006) is a member of the family Flavobacteriaceae, phylum Bacteroidetes. At the time of writing, it only consists of the type species, Epilithonimonas tenax, which was isolated from river epilithon (River Taff, Cardiff, UK).

    Strain H1T was isolated in April 2004, in the course of a study on the diversity of culturable psychrotolerant bacteria in raw cow'smilk (Hantsis-Zacharov & Halpern, 2007). Raw milk samples were serially diluted and plated on sterile standard plate count agar (SPC, Oxoid CM0463). Plates were incubated at 7 °C for 10 days and colonies displaying distinct morphologies were subcultured to obtain pure cultures. A comparative analysis of 16S rRNA gene sequences indicated that the strain was a member of the genus Epilithonimonas. To determine its exact taxonomic position, a polyphasic taxonomic study was carried out.

    The universal bacterial primers 8f and 1512r (based on Escherichia coli positions) were used to amplify internal fragments of the 16S rRNA gene according to Felske et al. (1997). The PCR products (approx. 1.5 kb) were purified by means of an AccuPrep PCR purification kit (Bioneer). Purified PCR products were sequenced at the Technion Medical School, Haifa, Israel, directly by the dideoxynucleotide chain-termination method using a DNA sequencer (ABI PRISM 3100) with BigDye terminator reagents (Applied Biosystems) according to the manufacturer's instructions. Sequencing was performed by use of the 8f (5′-GGATCCAGACTTTGATYMTGGCTCAG-3′), 534r (5′-ATTACCGCGGCTGCTGG-3′), 968f (5′-AACGCGAAGAACCTTAC-3′) and 1512f (5′-GTGAAGCTTACGGYTAGCTTGTTACGACTT-3′) primers.

    Phylogenetic neighbours were initially identified by the blast (Altschul et al., 1997) and fasta (Pearson & Lipman, 1988) programs against the database of type strains with valid prokaryotic names (Chun et al., 2007). The 50 sequences with the highest scores were then selected for the calculation of pairwise sequence similarity using the global alignment algorithm, which was implemented at the EzTaxon server (; Chun et al., 2007). Sequence alignment was performed with the clustal w program, and a phylogenetic tree (Fig. 1⇓) was generated by the neighbour-joining and by maximum-parsimony methods in the mega version 4.1 software (Tamura et al., 2007). The 1497 bp sequence of strain H1T shared the highest sequence similarity of 97.6 % with Epilithonimonas tenax DSM 16809T, 96.0 % sequence similarity with Chryseobacterium arothri DSM 19326T and Chryseobacterium hominis CCUG 52711T, and <95.9 % similarity with the type strains of all other type species.

    Figure image not available in archive
    Fig. 1.

    Neighbour-joining tree based on 16S rRNA gene sequences showing the phylogenetic positions of strain H1T, Epilithonimonas tenax and representatives of related taxa. Bootstrap values (>50 %) resulting from 1000 replicates are indicated at branching nodes. Bacteroides fragilis was used as an outgroup. Asterisks indicate branches of the tree that were also formed by using the maximum-parsimony method. Bar, 0.02 substitutions per nucleotide position.

    Together with Epilithonimonas tenax, strain H1T formed a branch distinctly separate from the Chryseobacterium/Sejongia cluster (Fig. 1⇑). This result suggests that strain H1T may represent a separate species within the genus Epilithonimonas. As previous studies have reported DNA–DNA reassociation values well below 70 % between Chryseobacterium strains sharing high values of 16S rRNA gene sequence similarity [e.g. 28 % (Weon et al., 2006), 33.9 % (Kim et al., 2005) and 27.1 % (Young et al., 2005)], we refrained from performing DNA hybridization experiments and considered strain H1T to represent a distinct genospecies (Stackebrandt & Ebers, 2006).

    For electron microscopy, bacteria grown on SPC agar (48 h, 32 °C) were suspended in saline, fixed to a carbon-coated grid, stained with 2 % uranyl acetate, and photographed under a JEM-1200EX electron microscope (JEOL, Japan). No flagella were observed.

    For phenotypic characterization, Luria–Bertani agar (LB) was used as the basal growth medium unless otherwise stated. Salt tolerance was determined at 30 °C on LB agar containing 0–5 % (w/v) NaCl at 1 % intervals. Growth was measured on SPC agar at 5, 7, 10, 14, 17, 25, 30, 33, 37, 40 and 45 °C. The pH range for growth was determined in LB broth adjusted to pH 4.0–10.0 at 2 pH unit intervals. The pH was adjusted prior to sterilization by the addition of HCl or NaOH and measured again after sterilization. Growth under anaerobic conditions (approx. 4–10 % CO2) was determined after incubation in a BBL GasPak anaerobic jar (Becton Dickinson) on LB agar supplemented with 0.5 % (w/v) glucose or 0.1 % (w/v) potassium nitrate. Biochemical tests were performed in parallel on strains H1T and Epilithonimonas tenax DSM 16809T by using API 20E, API 20NE, API 50CH and API ZYM identification systems (bioMérieux), according to the manufacturer's instructions, except that the incubation temperature was 25 °C. Catalase activity was evidenced by bubble production in a 3 % (v/v) hydrogen peroxide solution. Oxidase activity was determined with 1 % N,N,N′,N′-tetramethyl-p-phenylenediamine dihydrochloride (Sigma-Aldrich, T3134). Growth was tested on MacConkey agar (Difco) and cetrimide agar (Himedia). Hydrolysis of casein and tributyrin was determined on 1 % skimmed milk agar supplemented with 0.5 % yeast extract and tributyrin agar (Hylabs), respectively. For detection of flexirubin-type pigments, the cell mass of strain H1T was subjected to the KOH test as described by Fautz & Reichenbach (1980) and Bernardet et al. (2002). The phenotypic traits of the isolate are given in the species description and in Table 1⇓.

    Table 1.

    Differentiating characteristics of species of the genus Epilithonimonas

    Strains: 1, Epilithonimonas lactis sp. nov. H1T; 2. E. tenax EP105T. Data for both strains are from the current study except where indicated. Both strains are positive for the following characteristics: production of flexirubin-type pigments; presence of alkaline and acid phosphatases, esterase (C4), esterase lipase (C8), leucine and valine arylamidases and naphthol-AS-BI-phosphohydrolase activities; hydrolysis of aesculin; assimilation of glucose and maltose; acid production from glucose, amygdalin, aesculin, maltose, trehalose, starch and gentiobiose. Both strains are negative for the following characteristics: presence of α-galactosidase, β-glucuronidase, β-glucosaminidase, α-mannosidase and α-fucosidase activities; assimilation of l-arabinose, mannitol, N-acetylglucosamine, potassium gluconate, capric acid, adipate, malate, citrate and phenylacetic acid; acid production from glycerol, erythritol, d- and l-arabinose, d-ribose, d- and l-xylose, d-adonitol, methyl β-d-xylopyranoside, d-galactose, d-fructose, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl α-d-mannopyranoside, methyl α-d-glucopyranoside, N-acetylglucosamine, melibiose, inulin, melezitose, raffinose, xylitol, turanose, d-lyxose, d-tagatose, d- and l-fucose, d- and l-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. +, Positive; w, weakly positive; −, negative.

    For cellular fatty acid analysis, strain H1T was cultured on tryptic soy agar (Difco) for 24 h at 28 °C and the fatty acids were extracted and methylated (Ben-Ze'ev et al., 2005). The fatty acid methyl esters were analysed by gas chromatography with the MIDI/Hewlett Packard microbial identification system (Analytical Services). The major fatty acids (>20 %) of strain H1T were iso-C15 : 0 and summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 1ω7c) (Table 2⇓).

    Table 2.

    Cellular fatty acid compositions (%) of species of the genus Epilithonimonas

    Strains: 1, Epilithonimonas lactis sp. nov. H1T; 2, E. tenax EP105T. Cultivation on tryptic soy agar at 28 °C (strain H1T, this study) or 25 °C (E. tenax EP105T, O'Sullivan et al., 2006), for 24 h. Fatty acids amounting to <1 % of the total fatty acids in both strains are not shown. tr, Trace (<1 %); nd, not detected.

    For determination of the DNA G+C content, genomic DNA of strain H1T was prepared according to a modified version of the procedure of Wilson (1987). The G+C content of the DNA sample was determined in three independent analyses using the HPLC technique (Mesbah et al., 1989) by the BCCM/LMG Bacteria Collection Identification Service (Laboratory of Microbiology, Ghent University, Ghent, Belgium). The DNA G+C content of strain H1T was 38.0 mol%.

    Strain H1T was strictly aerobic, catalase- and oxidase-positive and non-motile; iso-C15 : 0 and summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 1ω7c) were the dominant fatty acids, the presence of Na+ was not required and some carbohydrates could be utilized for growth. Hence, strain H1T shared all characteristics listed in the description of the genus Epilithonimonas (O'Sullivan et al., 2006). A colour change of the cell mass was observed when the KOH test was performed, demonstrating that flexirubin-type pigments are produced, in accordance with Epilithonimonas tenax (O'Sullivan et al., 2006). However, strain H1T differed from Epilithonimonas tenax in several phenotypic traits that are listed in Tables 1⇑ and 2⇑.

    On the basis of phenotypic characterization and phylogenetic analysis we propose that isolate H1T be classified as a new species in the genus Epilithonimonas, for which the name Epilithonimonas lactis sp. nov. is proposed.

    Description of Epilithonimonas lactis sp. nov.

    Epilithonimonas lactis (lac′tis. L. gen. n. lactis of milk, referring to the isolation of the type strain from raw milk).

    Cells are Gram-staining-negative rods, 0.7–0.9 μm in width and 1.1–2.2 μm in length, devoid of flagellar motility. After 48 h of incubation on standard plate count agar at 30 °C in the dark, colonies are circular with entire edges, opaque, smooth and orange-coloured. Flexirubin-type pigments are produced. Good growth occurs under aerobic conditions. Growth is not observed after five days under anaerobic conditions on LB agar supplemented with 0.1 % (w/v) potassium nitrate or 0.5 % glucose. Grows at 5–33 °C (optimum, 25–30 °C), with 0–2.0 % NaCl (optimum, 0–0.5 % NaCl) and at pH 4.0–8.0 (optimum, pH 6.0–8.0). Growth is observed on standard plate count, nutrient and trypticase-soy agars but not on MacConkey and cetrimide agars. Catalase and oxidase activities are present. The following results were obtained from API 20NE strips after 48 h of incubation at 25 °C: indole is produced; β-galactosidase activity is present; aesculin is hydrolysed; glucose and maltose are assimilated. The following results were obtained from API 20E strips after 24 h of incubation at 25 °C: acetoin and indole are produced; H2S is not produced; gelatin and urea are not hydrolysed; citrate is not utilized; β-galactosidase activity is present; arginine dihydrolase, lysine and ornithine decarboxylases, and tryptophan deaminase activities are absent. In API 50CH strips incubated for 48 h at 25 °C, oxidative acid production occurs for glucose, amygdalin, arbutin, aesculin, salicin, cellobiose, maltose, lactose, trehalose, starch, glycogen and gentiobiose, while oxidative acid production does not occur for glycerol, erythritol, d- and l-arabinose, d-ribose, d- and l-xylose, d-adonitol, methyl β-d-xylopyranoside, d-galactose, d-fructose, d-mannose, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl α-d-mannopyranoside, methyl α-d-glucopyranoside, N-acetylglucosamine, melibiose, sucrose, inulin, melezitose, raffinose, xylitol, turanose, d-lyxose, d-tagatose, d- and l-fucose, d- and l-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. In API ZYM strips incubated for 4.5 h at 25 °C, alkaline and acid phosphatases, esterase (C4), esterase lipase (C8), leucine and valine arylamidases, naphthol-AS-BI-phosphohydrolase and α- and β-glucosidase activities are present; lipase (C14), cystine arylamidase, α-chymotrypsin and β-galactosidase activities are weakly present. Trypsin, α-galactosidase, β-glucuronidase, β-glucosaminidase, α-mannosidase and α-fucosidase activities are absent. The dominant (>10 %) cellular fatty acids are summed feature 3 (comprising iso-C15 : 0 2-OH and/or C16 : 1ω7c), iso-C15 : 0 and iso-C17 : 0 3-OH. The detailed fatty acid composition of strain H1T is given in Table 2⇑. The DNA G+C content of the type strain is 38.0 mol%.

    The type strain is H1T (=LMG 24401T =DSM 19921T), isolated from raw cow'smilk in Israel.

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