International Journal of Systematic and Evolutionary Microbiology (2015), 65, 11–14
DOI 10.1099/ijs.0.066837-0
Faucicola mancuniensis gen. nov., sp. nov., isolated from the human oropharynx Gavin J. Humphreys, Angela Oates, Ruth G. Ledder and Andrew J. McBain Correspondence
Manchester Pharmacy School, Oxford Road, University of Manchester, Manchester M13 9PT, UK
Gavin J. Humphreys [email protected]. uk
An aerobic, Gram-stain-negative, non-motile coccus, designated strain GVCNT2T, was isolated from the tonsils of a healthy adult female. Cells were oxidase- and catalase-positive, positive for the production of esterase (C4), esterase lipase (C8) and leucine arylamidase, and weakly positive for naphthol-AS-BI-phosphohydrolase and alkaline phosphatase. Cells were also capable of hydrolysing DNA. Growth was observed at 20–37 6C and in the presence of up to 1.5 % NaCl. Phylogenetic analysis of near full-length 16S rRNA gene sequences indicated that the strain exhibited closest sequence similarity to Moraxella boevrei ATCC 700022T (94.68 %) and an uncultured, unspeciated bacterial clone (strain S12-08; 99 %). The major fatty acids were C18 : 1v9c, C18 : 0, C16 : 0 and C16 : 1v6c/C16 : 1v7c. The DNA G+C content of strain GVCNT2T was 40.7 mol%. The major respiratory quinone identified was Q-8. Strain GVCNT2T exhibited a comparable phenotypic profile to other members of the genus Moraxella but could be distinguished based on its ability to produce acid (weakly) from D-glucose, melibiose, L-arabinose and rhamnose and on its ability to hydrolyse DNA. On the basis of phenotypic and phylogenetic differences from other members of the family Moraxellaceae, strain GVCNT2T is considered to represent a novel species of a new genus, for which the name Faucicola mancuniensis gen. nov., sp. nov. is proposed. The type strain of Faucicola mancuniensis is GVCNT2T (5DSM 28411T5NCIMB 14946T).
The family Moraxellaceae was first described by Rossau et al. (1991) to accommodate the genera Moraxella (Lwoff, 1939), Acinetobacter (Brisou & Prevot, 1954) and Psychrobacter (Juni & Heym, 1986). Later, this family was expanded to include Enhydrobacter (Staley et al., 1987), Alkanindiges (Bogan et al., 2003), Perlucidibaca (Song et al., 2008) and Paraperlucidibaca (Oh et al., 2011). Members of the family Moraxellaceae are found to occupy an array of ecological niches and have been isolated from a variety of environmental sources, including the upper respiratory tracts of mammals (Kodjo et al., 1995; Vandamme et al., 1993; Vela et al., 2009). In the present study, we describe a novel member of the family Moraxellaceae. Strain GCVNT2T was isolated from the pharynx of a healthy adult female during a survey of the healthy human oropharynx (Humphreys & McBain, 2013). Cells were recovered on Thayer Martin medium (Oxoid) supplemented with vancomycin (1.5 mg l21), colistin sulphate (3.7 mg l21) and nystatin (6250 IU l–1) following 5 days of aerobic incubation at 37 uC. Further subcultivation was achieved using Columbia blood agar (5 %, v/v, defibrinated horse blood; Oxoid) at 37 uC for 48 h. Using this agar, strain GVCNT2T exhibited growth at 20–37 uC, but not at 4, 10 or 45 uC. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GVCNT2T is KC688888.
066837 G 2015 IUMS
Printed in Great Britain
Gram staining was achieved as described by Gregersen (1978). Morphology, cell size and motility were determined using phase-contrast microscopy following 48 h of incubation on Columbia blood agar plates. In addition, motility was further investigated through stab inoculation of soft nutrient agar (0.4 % agar) and assessed following 7 days of incubation (37 uC). Salinity requirements were determined by growth in brain heart infusion broth supplemented with 0.5, 1.5, 3.0 and 5.0 % NaCl. Anaerobic growth was assessed at 37 uC for up to 7 days in a Mark II anaerobic chamber (H2, 10 %; CO2, 10 %; N2, 80 %). Oxidase and catalase production were determined according to standard methods (Cowan & Steel, 1974), whilst DNA hydrolysis was assessed using DNase agar (Oxoid). Additional biochemical analyses were performed using the API20E, API20NE and API ZYM systems (bioMe´rieux). Detailed descriptions of the biochemical and morphological characteristics of strain GVCNT2T are given in the species description. A summary is also shown in Table 1. Chemotaxonomic analysis was carried out by the German Collection of Microorganisms and Cell Cultures Identification Service (DSMZ; Braunschweig, Germany). Fatty acid methyl esters were determined for cells of both strain GVCNT2T and Moraxella boevrei ATCC 700022T (40 mg) following culture on Columbia blood agar (5 % defibrinated horse blood; 37 uC, 48 h) as described by Kuykendall 11
G. J. Humphreys and others
Table 1. Differential phenotypic characteristics between strain GVCNT2T and other members of the family Moraxellaceae Strains: 1, GVCNT2T (data from this study); 2, Acinetobacter calcoaceticus DSM 30006T (A´lvarez-Pe´rez et al., 2013; Song et al., 2008), 3, Alkanindiges illinoisensis DSM 15370T (Bogan et al., 2003; Song et al., 2008); 4, Enhydrobacter aerosaccus ATCC 27094T (Staley et al., 1987); 5, M. boevrei ATCC 700022T (data from this study and Kodjo et al., 1997); 6, Moraxella lacunata ATCC 17967T (Lwoff, 1939; Rossau et al., 1991); 7, Paraperlucidibaca baekdonensis CCUG 59307T (Oh et al., 2011); 8, Perlucidibaca piscinae NBRC 102354T (Song et al., 2008); 9, Psychrobacter immobilis ATCC 43116T (Bowman et al., 1996; Juni & Heym, 1986; Yoon et al., 2005). +, Positive; 2, negative; V, variable reaction; ND, no data available; W, weakly positive; C, cocci; R, rods; SR, short rods. All strains grow at 30 uC and in the presence of 0.5 % NaCl. Strains do not grow at 42 uC. All strains are Gram-stain-negative. Characteristic
1
2
3
4
5
6
7
8
9
Morphology Motility Growth at/with: 37 uC 5 % NaCl Catalase Oxidase Reduction of nitrate Urease DNA hydrolysis Gelatin hydrolysis Aesculin hydrolysis Acid production from D-glucose Enzyme activityD Alkaline phosphatase Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Naphthol-AS-BI-phosphohydrolase Utilization ofD D-Glucose L-Arabinose D-Mannose D-Mannitol Maltose DNA G+C content (mol%)
C 2
SR 2
C/SR 2
R 2
SR 2
R 2
R 2
SR +
SR 2
+ 2 + + 2 2 + 2 2
+ 2 + 2 2 + 2 2
+
+ 2 + + +
+ 2* + + + 2 2 + 2* 2
+ 2 + + + 2 2 + 2D 2
2 + 2 + 2 2
+ 2 2 +
ND
ND
2 2
2
ND
2
2 + + + + + 2 2 2 +
+ 2 2 2
2
W
2
+ 2 + + 2
2 2 2 2 2
2 2 2 2 2 43.9
+ 2 +
+ + + + + 63.1
2 2 2 2 2 45
ND
+ 2 + +
ND
W
2 2
W
W
ND
+
2
+
2 2 2 2 +
ND
W
+ 2 2 2 +
ND
2* +* 2* 2* +*
2 2 2 2 2 40.7
2 2 + 2 2 40–42
2 2 2 2 2 46.2
+ + + 2 + 66
W* 2* 2* 2* 2* 41–41.5
W
W
2 2 2
ND
+ + +
ND ND ND
ND
+ 61.3
W
2
W
*Data generated from this study. DData pertaining to enzyme activity and carbohydrate assimilation for Acinetobacter calcoaceticus, M. lacunata and Psychrobacter immobilis are taken from the CCUG database (http://www.ccug.se/) based on API ZYM and API 20NE assays.
et al. (1988). Mixtures of fatty acid methyl esters were separated using the Sherlock Microbial Identification System (MIDI). For the analysis of respiratory quinones, cultures of strain GVCNT2T were pelleted and freeze-dried following incubation in nutrient broth (37 uC, 48 h). Lipoquinones were differentiated using TLC and analysed by HPLC (Tindall, 1990). The major fatty acid components (.10 %) of strain GVCNT2T were C18 : 1v9c, C18 : 0, C16 : 0 and C16 : 1v6c/C16 : 1v7c. The complete fatty acid composition of strain GVCNT2T is given in Table 2. Subsequent to HPLC, the major respiratory quinone identified was Q-8. The G+C content of strain GVCNT2T was determined by HPLC following DNA extraction using a French pressure cell (Tamaoka & Komagata, 1984). The G+C content of the genomic DNA of strain GVCNT2T was 40.7 mol%.
Near full-length 16S rRNA gene sequences were used to delineate the phylogenetic position of strain GVCNT2T (1430 bp). The 16S rRNA gene was PCR-amplified using the universal primer set 27F/1492R (O’Sullivan et al., 2004). The amplified 16S rRNA gene was purified using a QIAquick PCR purification kit (Qiagen) and sequenced in both directions to generate a consensus of overlapping sequences. A search of the GenBank nucleotide database using the BLAST program (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) suggested that strain GVCNT2T was related most closely to M. boevrei ATCC 700022T (94.68 %) and an uncultured clone (strain S12-08; GenBank accession no. AY880059; 99 %). 16S rRNA gene sequences were analysed using MEGA5 (Tamura et al., 2011) in comparison with other known related strains. The multiple alignment of
12
International Journal of Systematic and Evolutionary Microbiology 65
Faucicola mancuniensis gen. nov., sp. nov.
Table 2. Complete fatty acid contents of strain GVCNT2T and M. boevrei Data are expressed as the percentage of total fatty acids. 2, Not detected. All data shown were generated in this study. Fatty acid C10 : 0 C12 : 0 C16 : 0 C17 : 0 C18 : 0 C16 : 1v9c C17 : 1v8c C18 : 1v9c C18 : 3v6,9,12c C20 : 4v6,9,12,15c C12 : 0 3-OH Summed features C16 : 1v6c/C16 : 1v7c C18 : 0 ante/C18 : 2v6, 9c C18 : 1v6c
GVCNT2T
M. boevrei ATCC 700022T
3.1 2 14.6 1.9 15.1 2.0 2.7 34.0 0.6 1.1 6.4
2.5 1.6 9.6 1.9 10.4 1.8 3.0 36.0 2.2 1.2 6.1
10.2 4.8 2.2
11.3 7.1 1.7
Description of Faucicola gen. nov. Faucicola [Fau.ci9co.la. L. pl. n. fauces the throat; L. suff. -cola (from L. masc. or fem. n. incola) inhabitant or dweller; N.L. fem. n. Faucicola throat dweller]. Cells are Gram-stain-negative, and catalase- and oxidasepositive. Cells are coccoid in shape and aerobic. Cells are not motile. The major respiratory quinone is Q-8. The major fatty acids are C18 : 1v9c, C18 : 0, C16 : 0 and C16 : 1v6c/ C16 : 1v7c. The type species is Faucicola mancuniensis. Description of Faucicola mancuniensis sp. nov. Faucicola mancuniensis (man.cu.ni.en9sis. N.L. fem. adj. mancuniensis pertaining to Manchester where the first isolate was cultured).
sequence data was performed using CLUSTAL X (Thompson et al., 1997). Phylogenetic trees were reconstructed using the neighbour-joining (Jukes & Cantor, 1969), maximumparsimony and maximum-likelihood (Tamura et al., 2011) algorithms. Bootstrap analyses were performed and expressed as a percentage of 1000 replications. These data suggested that strain GVCNT2T shared ,95 % sequence similarity to recognized members of the Moraxellaceae, including its closest phylogenetic relative, M. boevrei ATCC 700022T (Fig. 1). Overall, the phylogenetic data generated in this study suggest that isolate GVCNT2T represents a novel species of
Exhibits the following properties in addition to those described for the genus. Colonies are circular, nonpigmented, entire and non-haemolytic following 48 h of incubation at 37 uC on Columbia blood agar (5 % defibrinated horse blood; Oxoid). Growth occurs at 20– 37 uC and in the presence of 0–1.5 % NaCl. Cells do not reduce nitrates or nitrites. Growth occurs on nutrient agar, brain heart infusion media and Muller Hinton agar (Oxoid). Acid is produced (weakly) from D-glucose, rhamnose, melibiose and L-arabinose but not sucrose, amygdalin, D-sorbitol, inositol or D-mannitol (API 20E). Cells do not produce indole or urease and are not capable of hydrolysing aesculin or gelatin. Cells do not assimilate
Faucicola mancuniensis GVCNT2T (KC688888)
99 91
0.02
a new genus within the family Moraxellaceae, for which the name Faucicola mancuniensis gen. nov., sp. nov. is proposed. Whilst strain GVCNT2T is phenotypically similar to members of the genus Moraxella, it can be distinguished based on its ability to produce acid (weakly) in the presence of certain carbohydrates.
Moraxella boevrei ATCC 700022T (NR_043585)
82
Enhydrobacter aerosaccus ATCC 27094T (AJ550856)
99
Psychrobacter immobilis ATCC 43116T (NR_118808) Moraxella lacunata ATCC 17967T (NR_114416) Acinetobacter calcoaceticus DSM 30006T (AJ633632)
94
Alkanindiges illinoisensis DSM 15370T (AF513979)
93
Perlucidibaca piscinae NBRC 102354T (NR_114062)
100
Paraperlucidibaca baekdonensis CCUG 59307T (NR_117543) Alteromonas macleodii DSM 6062T (Y18228)
Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences of selected members of the family Moraxellaceae. The evolutionary history was inferred using the neighbour-joining method and distances were calculated using the Jukes and Cantor method. Bootstrap values (percentages of 1000 replicates) are given at branch points. Evolutionary analyses were conducted in MEGA5. Open circles indicate that the corresponding node was obtained using both the maximum-likelihood and the maximumparsimony algorithms. Bar, 0.02 changes per nucleotide position. http://ijs.sgmjournals.org
13
G. J. Humphreys and others
glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate or phenylacetic acid. Cells do not produce arginine dihydrolase, lysine decarboxylase or ornithine decarboxylase, do not utilize citrate and are negative for the production of both acetoin and H2S. Cells are positive for the production of esterase (C4), esterase lipase (C8) and leucine arylamidase and are weakly positive for naphthol-AS-BI-phosphohydrolase and alkaline phosphatase. Cells are also capable of hydrolysing DNA. Cells do not produce lipase (C14), valine arylamidase, crystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, a-galactosidase, b-galactosidase, b-glucoronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase or a-fucosidase. T
T
The type strain, GVCNT2 (5DSM 28411 5NCIMB 14946T), was isolated from the oropharynx of a healthy adult female. Its clinical significance is unknown. The DNA G+C content of the type strain is 40.7 mol%.
Acknowledgements We thank Sarah Tennant for support with the Latin etymology of the new genus name.
References A´lvarez-Pe´rez, S., Lievens, B., Jacquemyn, H. & Herrera, C. M. (2013). Acinetobacter nectaris sp. nov. and Acinetobacter boissieri sp.
nov., isolated from floral nectar of wild Mediterranean insectpollinated plants. Int J Syst Evol Microbiol 63, 1532–1539. Bogan, B. W., Sullivan, W. R., Kayser, K. J., Derr, K. D., Aldrich, H. C. & Paterek, J. R. (2003). Alkanindiges illinoisensis gen. nov., sp. nov., an
obligately hydrocarbonoclastic, aerobic squalane-degrading bacterium isolated from oilfield soils. Int J Syst Evol Microbiol 53, 1389– 1395. Bowman, J. P., Cavanagh, J., Austin, J. J. & Sanderson, K. (1996).
Novel Psychrobacter species from Antarctic ornithogenic soils. Int J Syst Bacteriol 46, 841–848. Brisou, J. & Prevot, A. R. (1954). [Studies on bacterial taxonomy. X.
The revision of species under Acromobacter group]. Ann Inst Pasteur (Paris) 86, 722–728. Cowan, S. T. & Steel, K. J. (1974). Manual for the Identification of
Medical Bacteria. London: Cambridge University Press. Gregersen, T. (1978). Rapid method for distinction of Gram-negative
from Gram-positive bacteria. Eur J Microbiol Biotechnol 5, 123– 127. Humphreys, G. J. & McBain, A. J. (2013). Continuous culture of sessile
Juni, E. & Heym, G. (1986). Psychrobacter immobilis gen. nov., sp.
nov.: genospecies composed of Gram-negative, aerobic, oxidase positive coccobacilli. Int J Syst Bacteriol 36, 388–391. Kodjo, A., Tønjum, T., Richard, Y. & Bøvre, K. (1995). Moraxella
caprae sp. nov., a new member of the classical Moraxellae with very close affinity to Moraxella bovis. Int J Syst Bacteriol 45, 467–471. Kodjo, A., Richard, Y. & Tønjum, T. (1997). Moraxella boevrei sp. nov., a
new Moraxella species found in goats. Int J Syst Bacteriol 47, 115–121. Kuykendall, L. D., Roy, M. A., O’Neill, J. J. & Devine, T. E. (1988). Fatty
acids, antibiotic resistance and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum. Int J Syst Bacteriol 38, 358–361. Lwoff, A. (1939). Re´vision et de´membrement des Hemophileae, le genre Moraxella nov. gen. Ann Inst Pasteur (Paris) 62, 168–176. O’Sullivan, L. A., Fuller, K. E., Thomas, E. M., Turley, C. M., Fry, J. C. & Weightman, A. J. (2004). Distribution and culturability of the
uncultivated ‘AGG58 cluster’ of the Bacteroidetes phylum in aquatic environments. FEMS Microbiol Ecol 47, 359–370. Oh, K. H., Lee, S. Y., Lee, M. H., Oh, T. K. & Yoon, J. H. (2011).
Paraperlucidibaca baekdonensis gen. nov., sp. nov., isolated from seawater. Int J Syst Evol Microbiol 61, 1382–1385. Rossau, R., Van Landschoot, A., Gillis, M. & De Ley, J. (1991).
Taxonomy of Moraxellaceae fam. nov., a new bacterial family to accommodate the genera Moraxella, Acinetobacter, and Psychrobacter and related organisms. Int J Syst Bacteriol 41, 310–319. Song, J., Choo, Y. J. & Cho, J. C. (2008). Perlucidibaca piscinae gen.
nov., sp. nov., a freshwater bacterium belonging to the family Moraxellaceae. Int J Syst Evol Microbiol 58, 97–102. Staley, J., Irgens, R. & Brenner, D. J. (1987). Enhydrobacter aerosaccus
gen. nov., sp. nov., a gas vacuolated, facultatively anaerobic, heterotrophic rod. Int J Syst Bacteriol 37, 289–291. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
composition by reversed-phase high-performace liquid chromatography. FEMS Microbiol Lett 25, 125–128. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882. Tindall, B. J. (1990). A comparative study of the lipid composition of
Halobacterium saccharovorum from various sources. Syst Appl Microbiol 13, 128–130. Vandamme, P., Gillis, M., Vancanneyt, M., Hoste, B., Kersters, K. & Falsen, E. (1993). Moraxella lincolnii sp. nov., isolated from the
human respiratory tract, and reevaluation of the taxonomic position of Moraxella osloensis. Int J Syst Bacteriol 43, 474–481. Vela, A. I., Arroyo, E., Arago´n, V., Sa´nchez-Porro, C., Latre, M. V., Cerda`-Cue´llar, M., Ventosa, A., Domı´nguez, L. & Ferna´ndezGarayza´bal, J. F. (2009). Moraxella pluranimalium sp. nov., isolated
human oropharyngeal microbiotas. J Med Microbiol 62, 906–916.
from animal specimens. Int J Syst Evol Microbiol 59, 671–674.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In
Yoon, J. H., Yeo, S. H., Oh, T. K. & Park, Y. H. (2005). Psychrobacter
Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
alimentarius sp. nov., isolated from squid jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 55, 171–176.
14
International Journal of Systematic and Evolutionary Microbiology 65
DOI 10.1099/ijs.0.066837-0
Faucicola mancuniensis gen. nov., sp. nov., isolated from the human oropharynx Gavin J. Humphreys, Angela Oates, Ruth G. Ledder and Andrew J. McBain Correspondence
Manchester Pharmacy School, Oxford Road, University of Manchester, Manchester M13 9PT, UK
Gavin J. Humphreys [email protected]. uk
An aerobic, Gram-stain-negative, non-motile coccus, designated strain GVCNT2T, was isolated from the tonsils of a healthy adult female. Cells were oxidase- and catalase-positive, positive for the production of esterase (C4), esterase lipase (C8) and leucine arylamidase, and weakly positive for naphthol-AS-BI-phosphohydrolase and alkaline phosphatase. Cells were also capable of hydrolysing DNA. Growth was observed at 20–37 6C and in the presence of up to 1.5 % NaCl. Phylogenetic analysis of near full-length 16S rRNA gene sequences indicated that the strain exhibited closest sequence similarity to Moraxella boevrei ATCC 700022T (94.68 %) and an uncultured, unspeciated bacterial clone (strain S12-08; 99 %). The major fatty acids were C18 : 1v9c, C18 : 0, C16 : 0 and C16 : 1v6c/C16 : 1v7c. The DNA G+C content of strain GVCNT2T was 40.7 mol%. The major respiratory quinone identified was Q-8. Strain GVCNT2T exhibited a comparable phenotypic profile to other members of the genus Moraxella but could be distinguished based on its ability to produce acid (weakly) from D-glucose, melibiose, L-arabinose and rhamnose and on its ability to hydrolyse DNA. On the basis of phenotypic and phylogenetic differences from other members of the family Moraxellaceae, strain GVCNT2T is considered to represent a novel species of a new genus, for which the name Faucicola mancuniensis gen. nov., sp. nov. is proposed. The type strain of Faucicola mancuniensis is GVCNT2T (5DSM 28411T5NCIMB 14946T).
The family Moraxellaceae was first described by Rossau et al. (1991) to accommodate the genera Moraxella (Lwoff, 1939), Acinetobacter (Brisou & Prevot, 1954) and Psychrobacter (Juni & Heym, 1986). Later, this family was expanded to include Enhydrobacter (Staley et al., 1987), Alkanindiges (Bogan et al., 2003), Perlucidibaca (Song et al., 2008) and Paraperlucidibaca (Oh et al., 2011). Members of the family Moraxellaceae are found to occupy an array of ecological niches and have been isolated from a variety of environmental sources, including the upper respiratory tracts of mammals (Kodjo et al., 1995; Vandamme et al., 1993; Vela et al., 2009). In the present study, we describe a novel member of the family Moraxellaceae. Strain GCVNT2T was isolated from the pharynx of a healthy adult female during a survey of the healthy human oropharynx (Humphreys & McBain, 2013). Cells were recovered on Thayer Martin medium (Oxoid) supplemented with vancomycin (1.5 mg l21), colistin sulphate (3.7 mg l21) and nystatin (6250 IU l–1) following 5 days of aerobic incubation at 37 uC. Further subcultivation was achieved using Columbia blood agar (5 %, v/v, defibrinated horse blood; Oxoid) at 37 uC for 48 h. Using this agar, strain GVCNT2T exhibited growth at 20–37 uC, but not at 4, 10 or 45 uC. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain GVCNT2T is KC688888.
066837 G 2015 IUMS
Printed in Great Britain
Gram staining was achieved as described by Gregersen (1978). Morphology, cell size and motility were determined using phase-contrast microscopy following 48 h of incubation on Columbia blood agar plates. In addition, motility was further investigated through stab inoculation of soft nutrient agar (0.4 % agar) and assessed following 7 days of incubation (37 uC). Salinity requirements were determined by growth in brain heart infusion broth supplemented with 0.5, 1.5, 3.0 and 5.0 % NaCl. Anaerobic growth was assessed at 37 uC for up to 7 days in a Mark II anaerobic chamber (H2, 10 %; CO2, 10 %; N2, 80 %). Oxidase and catalase production were determined according to standard methods (Cowan & Steel, 1974), whilst DNA hydrolysis was assessed using DNase agar (Oxoid). Additional biochemical analyses were performed using the API20E, API20NE and API ZYM systems (bioMe´rieux). Detailed descriptions of the biochemical and morphological characteristics of strain GVCNT2T are given in the species description. A summary is also shown in Table 1. Chemotaxonomic analysis was carried out by the German Collection of Microorganisms and Cell Cultures Identification Service (DSMZ; Braunschweig, Germany). Fatty acid methyl esters were determined for cells of both strain GVCNT2T and Moraxella boevrei ATCC 700022T (40 mg) following culture on Columbia blood agar (5 % defibrinated horse blood; 37 uC, 48 h) as described by Kuykendall 11
G. J. Humphreys and others
Table 1. Differential phenotypic characteristics between strain GVCNT2T and other members of the family Moraxellaceae Strains: 1, GVCNT2T (data from this study); 2, Acinetobacter calcoaceticus DSM 30006T (A´lvarez-Pe´rez et al., 2013; Song et al., 2008), 3, Alkanindiges illinoisensis DSM 15370T (Bogan et al., 2003; Song et al., 2008); 4, Enhydrobacter aerosaccus ATCC 27094T (Staley et al., 1987); 5, M. boevrei ATCC 700022T (data from this study and Kodjo et al., 1997); 6, Moraxella lacunata ATCC 17967T (Lwoff, 1939; Rossau et al., 1991); 7, Paraperlucidibaca baekdonensis CCUG 59307T (Oh et al., 2011); 8, Perlucidibaca piscinae NBRC 102354T (Song et al., 2008); 9, Psychrobacter immobilis ATCC 43116T (Bowman et al., 1996; Juni & Heym, 1986; Yoon et al., 2005). +, Positive; 2, negative; V, variable reaction; ND, no data available; W, weakly positive; C, cocci; R, rods; SR, short rods. All strains grow at 30 uC and in the presence of 0.5 % NaCl. Strains do not grow at 42 uC. All strains are Gram-stain-negative. Characteristic
1
2
3
4
5
6
7
8
9
Morphology Motility Growth at/with: 37 uC 5 % NaCl Catalase Oxidase Reduction of nitrate Urease DNA hydrolysis Gelatin hydrolysis Aesculin hydrolysis Acid production from D-glucose Enzyme activityD Alkaline phosphatase Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Naphthol-AS-BI-phosphohydrolase Utilization ofD D-Glucose L-Arabinose D-Mannose D-Mannitol Maltose DNA G+C content (mol%)
C 2
SR 2
C/SR 2
R 2
SR 2
R 2
R 2
SR +
SR 2
+ 2 + + 2 2 + 2 2
+ 2 + 2 2 + 2 2
+
+ 2 + + +
+ 2* + + + 2 2 + 2* 2
+ 2 + + + 2 2 + 2D 2
2 + 2 + 2 2
+ 2 2 +
ND
ND
2 2
2
ND
2
2 + + + + + 2 2 2 +
+ 2 2 2
2
W
2
+ 2 + + 2
2 2 2 2 2
2 2 2 2 2 43.9
+ 2 +
+ + + + + 63.1
2 2 2 2 2 45
ND
+ 2 + +
ND
W
2 2
W
W
ND
+
2
+
2 2 2 2 +
ND
W
+ 2 2 2 +
ND
2* +* 2* 2* +*
2 2 2 2 2 40.7
2 2 + 2 2 40–42
2 2 2 2 2 46.2
+ + + 2 + 66
W* 2* 2* 2* 2* 41–41.5
W
W
2 2 2
ND
+ + +
ND ND ND
ND
+ 61.3
W
2
W
*Data generated from this study. DData pertaining to enzyme activity and carbohydrate assimilation for Acinetobacter calcoaceticus, M. lacunata and Psychrobacter immobilis are taken from the CCUG database (http://www.ccug.se/) based on API ZYM and API 20NE assays.
et al. (1988). Mixtures of fatty acid methyl esters were separated using the Sherlock Microbial Identification System (MIDI). For the analysis of respiratory quinones, cultures of strain GVCNT2T were pelleted and freeze-dried following incubation in nutrient broth (37 uC, 48 h). Lipoquinones were differentiated using TLC and analysed by HPLC (Tindall, 1990). The major fatty acid components (.10 %) of strain GVCNT2T were C18 : 1v9c, C18 : 0, C16 : 0 and C16 : 1v6c/C16 : 1v7c. The complete fatty acid composition of strain GVCNT2T is given in Table 2. Subsequent to HPLC, the major respiratory quinone identified was Q-8. The G+C content of strain GVCNT2T was determined by HPLC following DNA extraction using a French pressure cell (Tamaoka & Komagata, 1984). The G+C content of the genomic DNA of strain GVCNT2T was 40.7 mol%.
Near full-length 16S rRNA gene sequences were used to delineate the phylogenetic position of strain GVCNT2T (1430 bp). The 16S rRNA gene was PCR-amplified using the universal primer set 27F/1492R (O’Sullivan et al., 2004). The amplified 16S rRNA gene was purified using a QIAquick PCR purification kit (Qiagen) and sequenced in both directions to generate a consensus of overlapping sequences. A search of the GenBank nucleotide database using the BLAST program (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) suggested that strain GVCNT2T was related most closely to M. boevrei ATCC 700022T (94.68 %) and an uncultured clone (strain S12-08; GenBank accession no. AY880059; 99 %). 16S rRNA gene sequences were analysed using MEGA5 (Tamura et al., 2011) in comparison with other known related strains. The multiple alignment of
12
International Journal of Systematic and Evolutionary Microbiology 65
Faucicola mancuniensis gen. nov., sp. nov.
Table 2. Complete fatty acid contents of strain GVCNT2T and M. boevrei Data are expressed as the percentage of total fatty acids. 2, Not detected. All data shown were generated in this study. Fatty acid C10 : 0 C12 : 0 C16 : 0 C17 : 0 C18 : 0 C16 : 1v9c C17 : 1v8c C18 : 1v9c C18 : 3v6,9,12c C20 : 4v6,9,12,15c C12 : 0 3-OH Summed features C16 : 1v6c/C16 : 1v7c C18 : 0 ante/C18 : 2v6, 9c C18 : 1v6c
GVCNT2T
M. boevrei ATCC 700022T
3.1 2 14.6 1.9 15.1 2.0 2.7 34.0 0.6 1.1 6.4
2.5 1.6 9.6 1.9 10.4 1.8 3.0 36.0 2.2 1.2 6.1
10.2 4.8 2.2
11.3 7.1 1.7
Description of Faucicola gen. nov. Faucicola [Fau.ci9co.la. L. pl. n. fauces the throat; L. suff. -cola (from L. masc. or fem. n. incola) inhabitant or dweller; N.L. fem. n. Faucicola throat dweller]. Cells are Gram-stain-negative, and catalase- and oxidasepositive. Cells are coccoid in shape and aerobic. Cells are not motile. The major respiratory quinone is Q-8. The major fatty acids are C18 : 1v9c, C18 : 0, C16 : 0 and C16 : 1v6c/ C16 : 1v7c. The type species is Faucicola mancuniensis. Description of Faucicola mancuniensis sp. nov. Faucicola mancuniensis (man.cu.ni.en9sis. N.L. fem. adj. mancuniensis pertaining to Manchester where the first isolate was cultured).
sequence data was performed using CLUSTAL X (Thompson et al., 1997). Phylogenetic trees were reconstructed using the neighbour-joining (Jukes & Cantor, 1969), maximumparsimony and maximum-likelihood (Tamura et al., 2011) algorithms. Bootstrap analyses were performed and expressed as a percentage of 1000 replications. These data suggested that strain GVCNT2T shared ,95 % sequence similarity to recognized members of the Moraxellaceae, including its closest phylogenetic relative, M. boevrei ATCC 700022T (Fig. 1). Overall, the phylogenetic data generated in this study suggest that isolate GVCNT2T represents a novel species of
Exhibits the following properties in addition to those described for the genus. Colonies are circular, nonpigmented, entire and non-haemolytic following 48 h of incubation at 37 uC on Columbia blood agar (5 % defibrinated horse blood; Oxoid). Growth occurs at 20– 37 uC and in the presence of 0–1.5 % NaCl. Cells do not reduce nitrates or nitrites. Growth occurs on nutrient agar, brain heart infusion media and Muller Hinton agar (Oxoid). Acid is produced (weakly) from D-glucose, rhamnose, melibiose and L-arabinose but not sucrose, amygdalin, D-sorbitol, inositol or D-mannitol (API 20E). Cells do not produce indole or urease and are not capable of hydrolysing aesculin or gelatin. Cells do not assimilate
Faucicola mancuniensis GVCNT2T (KC688888)
99 91
0.02
a new genus within the family Moraxellaceae, for which the name Faucicola mancuniensis gen. nov., sp. nov. is proposed. Whilst strain GVCNT2T is phenotypically similar to members of the genus Moraxella, it can be distinguished based on its ability to produce acid (weakly) in the presence of certain carbohydrates.
Moraxella boevrei ATCC 700022T (NR_043585)
82
Enhydrobacter aerosaccus ATCC 27094T (AJ550856)
99
Psychrobacter immobilis ATCC 43116T (NR_118808) Moraxella lacunata ATCC 17967T (NR_114416) Acinetobacter calcoaceticus DSM 30006T (AJ633632)
94
Alkanindiges illinoisensis DSM 15370T (AF513979)
93
Perlucidibaca piscinae NBRC 102354T (NR_114062)
100
Paraperlucidibaca baekdonensis CCUG 59307T (NR_117543) Alteromonas macleodii DSM 6062T (Y18228)
Fig. 1. Phylogenetic tree based on 16S rRNA gene sequences of selected members of the family Moraxellaceae. The evolutionary history was inferred using the neighbour-joining method and distances were calculated using the Jukes and Cantor method. Bootstrap values (percentages of 1000 replicates) are given at branch points. Evolutionary analyses were conducted in MEGA5. Open circles indicate that the corresponding node was obtained using both the maximum-likelihood and the maximumparsimony algorithms. Bar, 0.02 changes per nucleotide position. http://ijs.sgmjournals.org
13
G. J. Humphreys and others
glucose, arabinose, mannose, mannitol, N-acetylglucosamine, maltose, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate or phenylacetic acid. Cells do not produce arginine dihydrolase, lysine decarboxylase or ornithine decarboxylase, do not utilize citrate and are negative for the production of both acetoin and H2S. Cells are positive for the production of esterase (C4), esterase lipase (C8) and leucine arylamidase and are weakly positive for naphthol-AS-BI-phosphohydrolase and alkaline phosphatase. Cells are also capable of hydrolysing DNA. Cells do not produce lipase (C14), valine arylamidase, crystine arylamidase, trypsin, a-chymotrypsin, acid phosphatase, a-galactosidase, b-galactosidase, b-glucoronidase, a-glucosidase, b-glucosidase, N-acetyl-b-glucosaminidase, a-mannosidase or a-fucosidase. T
T
The type strain, GVCNT2 (5DSM 28411 5NCIMB 14946T), was isolated from the oropharynx of a healthy adult female. Its clinical significance is unknown. The DNA G+C content of the type strain is 40.7 mol%.
Acknowledgements We thank Sarah Tennant for support with the Latin etymology of the new genus name.
References A´lvarez-Pe´rez, S., Lievens, B., Jacquemyn, H. & Herrera, C. M. (2013). Acinetobacter nectaris sp. nov. and Acinetobacter boissieri sp.
nov., isolated from floral nectar of wild Mediterranean insectpollinated plants. Int J Syst Evol Microbiol 63, 1532–1539. Bogan, B. W., Sullivan, W. R., Kayser, K. J., Derr, K. D., Aldrich, H. C. & Paterek, J. R. (2003). Alkanindiges illinoisensis gen. nov., sp. nov., an
obligately hydrocarbonoclastic, aerobic squalane-degrading bacterium isolated from oilfield soils. Int J Syst Evol Microbiol 53, 1389– 1395. Bowman, J. P., Cavanagh, J., Austin, J. J. & Sanderson, K. (1996).
Novel Psychrobacter species from Antarctic ornithogenic soils. Int J Syst Bacteriol 46, 841–848. Brisou, J. & Prevot, A. R. (1954). [Studies on bacterial taxonomy. X.
The revision of species under Acromobacter group]. Ann Inst Pasteur (Paris) 86, 722–728. Cowan, S. T. & Steel, K. J. (1974). Manual for the Identification of
Medical Bacteria. London: Cambridge University Press. Gregersen, T. (1978). Rapid method for distinction of Gram-negative
from Gram-positive bacteria. Eur J Microbiol Biotechnol 5, 123– 127. Humphreys, G. J. & McBain, A. J. (2013). Continuous culture of sessile
Juni, E. & Heym, G. (1986). Psychrobacter immobilis gen. nov., sp.
nov.: genospecies composed of Gram-negative, aerobic, oxidase positive coccobacilli. Int J Syst Bacteriol 36, 388–391. Kodjo, A., Tønjum, T., Richard, Y. & Bøvre, K. (1995). Moraxella
caprae sp. nov., a new member of the classical Moraxellae with very close affinity to Moraxella bovis. Int J Syst Bacteriol 45, 467–471. Kodjo, A., Richard, Y. & Tønjum, T. (1997). Moraxella boevrei sp. nov., a
new Moraxella species found in goats. Int J Syst Bacteriol 47, 115–121. Kuykendall, L. D., Roy, M. A., O’Neill, J. J. & Devine, T. E. (1988). Fatty
acids, antibiotic resistance and deoxyribonucleic acid homology groups of Bradyrhizobium japonicum. Int J Syst Bacteriol 38, 358–361. Lwoff, A. (1939). Re´vision et de´membrement des Hemophileae, le genre Moraxella nov. gen. Ann Inst Pasteur (Paris) 62, 168–176. O’Sullivan, L. A., Fuller, K. E., Thomas, E. M., Turley, C. M., Fry, J. C. & Weightman, A. J. (2004). Distribution and culturability of the
uncultivated ‘AGG58 cluster’ of the Bacteroidetes phylum in aquatic environments. FEMS Microbiol Ecol 47, 359–370. Oh, K. H., Lee, S. Y., Lee, M. H., Oh, T. K. & Yoon, J. H. (2011).
Paraperlucidibaca baekdonensis gen. nov., sp. nov., isolated from seawater. Int J Syst Evol Microbiol 61, 1382–1385. Rossau, R., Van Landschoot, A., Gillis, M. & De Ley, J. (1991).
Taxonomy of Moraxellaceae fam. nov., a new bacterial family to accommodate the genera Moraxella, Acinetobacter, and Psychrobacter and related organisms. Int J Syst Bacteriol 41, 310–319. Song, J., Choo, Y. J. & Cho, J. C. (2008). Perlucidibaca piscinae gen.
nov., sp. nov., a freshwater bacterium belonging to the family Moraxellaceae. Int J Syst Evol Microbiol 58, 97–102. Staley, J., Irgens, R. & Brenner, D. J. (1987). Enhydrobacter aerosaccus
gen. nov., sp. nov., a gas vacuolated, facultatively anaerobic, heterotrophic rod. Int J Syst Bacteriol 37, 289–291. Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
composition by reversed-phase high-performace liquid chromatography. FEMS Microbiol Lett 25, 125–128. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. & Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876–4882. Tindall, B. J. (1990). A comparative study of the lipid composition of
Halobacterium saccharovorum from various sources. Syst Appl Microbiol 13, 128–130. Vandamme, P., Gillis, M., Vancanneyt, M., Hoste, B., Kersters, K. & Falsen, E. (1993). Moraxella lincolnii sp. nov., isolated from the
human respiratory tract, and reevaluation of the taxonomic position of Moraxella osloensis. Int J Syst Bacteriol 43, 474–481. Vela, A. I., Arroyo, E., Arago´n, V., Sa´nchez-Porro, C., Latre, M. V., Cerda`-Cue´llar, M., Ventosa, A., Domı´nguez, L. & Ferna´ndezGarayza´bal, J. F. (2009). Moraxella pluranimalium sp. nov., isolated
human oropharyngeal microbiotas. J Med Microbiol 62, 906–916.
from animal specimens. Int J Syst Evol Microbiol 59, 671–674.
Jukes, T. H. & Cantor, C. R. (1969). Evolution of protein molecules. In
Yoon, J. H., Yeo, S. H., Oh, T. K. & Park, Y. H. (2005). Psychrobacter
Mammalian Protein Metabolism, pp. 21–132. Edited by H. N. Munro. New York: Academic Press.
alimentarius sp. nov., isolated from squid jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 55, 171–176.
14
International Journal of Systematic and Evolutionary Microbiology 65
