International Journal of Systematic and Evolutionary Microbiology (2014), 64, 2424–2430
DOI 10.1099/ijs.0.062786-0
Proposal of Vespertiliibacter pulmonis gen. nov., sp. nov. and two genomospecies as new members of the family Pasteurellaceae isolated from European bats Kristin Mu¨hldorfer,13 Stephanie Speck2 and Gudrun Wibbelt1 Correspondence Kristin Mu¨hldorfer [email protected]
1
Department of Wildlife Diseases, Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany
2
Institute for Animal Hygiene and Veterinary Public Health, University of Leipzig, Leipzig, Germany
Five bacterial strains isolated from bats of the family Vespertilionidae were characterized by phenotypic tests and multilocus sequence analysis (MLSA) using the 16S rRNA gene and four housekeeping genes (rpoA, rpoB, infB, recN). Phylogenetic analyses of individual and combined datasets indicated that the five strains represent a monophyletic cluster within the family Pasteurellaceae. Comparison of 16S rRNA gene sequences demonstrated a high degree of similarity (98.3–99.9 %) among the group of bat-derived strains, while searches in nucleotide databases indicated less than 96 % sequence similarity to known members of the Pasteurellaceae. The housekeeping genes rpoA, rpoB, infB and recN provided higher resolution compared with the 16S rRNA gene and subdivided the group according to the bat species from which the strains were isolated. Three strains derived from noctule bats shared 98.6–100 % sequence similarity in all four genes investigated, whereas, based on rpoB, infB and recN gene sequences, 91.8–96 % similarity was observed with and between the remaining two strains isolated from a serotine bat and a pipistrelle bat, respectively. Genome relatedness as deduced from recN gene sequences correlated well with the results of MLSA and indicated that the five strains represent a new genus. Based on these results, it is proposed to classify the five strains derived from bats within Vespertiliibacter pulmonis gen. nov., sp. nov. (the type species), Vespertiliibacter genomospecies 1 and Vespertiliibacter genomospecies 2. The genus can be distinguished phenotypically from recognized genera of the Pasteurellaceae by at least three characteristics. All strains are nutritionally fastidious and require a chemically defined supplement with NAD for growth. The DNA G+C content of strain E127/08T is 38.2 mol%. The type strain of Vespertiliibacter pulmonis gen. nov., sp. nov. is E127/08T (5CCUG 64585T5DSM 27238T). The reference strains of Vespertiliibacter genomospecies 1 and 2 are E145/08 and E157/08, respectively.
The family Pasteurellaceae currently comprises 18 recognized genera with more than 70 species of wide distribution and host range (http://www.bacterio.net/). An additional genus ‘Seminibacterium’ has been proposed recently by Vela et al. (2013). Members of the Pasteurellaceae commonly belong to 3Present address: Institute of Poultry Diseases, Freie Universita¨t Berlin, Ko¨nigsweg 63, 14163 Berlin, Germany. Abbreviation: GGT, gamma-glutamyltransferase. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains E127/08T, E146/08, E40/12, E145/08 and E157/08 are KF031240–KF031244, respectively. The accession numbers of the rpoA, rpoB, infB and recN gene sequences of these strains are detailed in Table S1. Two supplementary tables and four supplementary figures are available with the online version of this paper.
2424
the mucosal flora of their hosts, but some species are able to cause localized or systemic infections in animals and humans (Olsen et al., 2005). In chiropteran species, members of the genus Pasteurella have been identified as primary pathogens responsible for pneumonia, severe organ necroses and systemic infections in European bat species of the family Vespertilionidae (Simpson, 2000; Daffner, 2001; Hajkova & Pikula, 2007; Mu¨hldorfer et al., 2011a, b, c). Bite wounds from domestic cats appear to be the most likely source of Pasteurella infections in European bats (Mu¨hldorfer et al., 2011a, b, c; Mu¨hldorfer, 2013). However, the role of members of the Pasteurellaceae as commensal or opportunistic bacteria of bat hosts is unknown. The aim of the present study was to characterize five strains of the Pasteurellaceae obtained from the lung, heart or thoracic cavity of five bats of three different European species 062786 G 2014 IUMS Printed in Great Britain
Vespertiliibacter pulmonis gen. nov., sp. nov.
(Nyctalus noctula, n53; Pipistrellus pipistrellus, n51; Eptesicus serotinus, n51) to investigate their phenotypic and genetic characteristics as well as their phylogenetic relationships to other members of the family Pasteurellaceae. Between 2008 and 2012, 280 deceased free-ranging bats were subjected to post-mortem examination followed by histopathological and bacteriological investigations. The bat carcasses were provided by bat researchers and bat rehabilitation centres throughout Germany. Tissue samples from lung, liver, heart, kidney and spleen as well as swab samples from the thoracic cavity of these carcasses were plated onto Columbia agar (7 % sheep blood), chocolate agar (with Vitox, 5 % CO2) and MacConkey agar (all from Oxoid) and were incubated at 37 uC for 24–48 h. In five bats, colonies typical of the Pasteurellaceae were isolated on chocolate agar supplemented with Vitox. Single colonies of the five strains (Table S1, available in the online Supplementary Material) were subcultured and subsequently stored at 280 uC suspended in nutrient broth supplemented with 20 % glycerol. Primary identification of bacterial strains was based on Gram-staining, indole production and catalase and oxidase reactions. Phenotypic characterization was performed by conventional tube tests, as recommended by the minimal standards for the description of genera, species and subspecies of the Pasteurellaceae (Christensen et al., 2007). Additionally, all strains were characterized with the API NH and API ZYM systems (bioMe´rieux), according to the manufacturer’s instructions. Fermentation of carbohydrates, alcohols and glycosides, malonate utilization, methyl red, Voges–Proskauer, ornithine decarboxylase, lysine decarboxylase and arginine dihydrolase tests were performed by adding Vitox supplement (with a final concentration of 10 mg NAD ml21) dissolved in distilled water and adjusted to pH 7.4. The X-factor (haemin) and/or V-factor (NAD) requirement and haemolytic reactions were identified by using the Haemophilus ID Quad plate with growth factors (BD Diagnostics), according to the manufacturer’s instructions. Colonies of the strains grown on chocolate agar supplemented with Vitox (5 % CO2, 24 h at 37 uC) were 1 mm in diameter, smooth, greyish, opaque, circular and convex with an entire margin. The texture of single colonies was butyrous (E40/12, E145/08 and E157/08) or dry (E127/08T and E146/08). The five strains were V-factor-dependent but, instead of NAD alone, all strains required a nutritional supplement such as IsoVitaleX Enrichment (BD Diagnostics) or Vitox (Oxoid) for growth. Haemolysis was produced on 5 % horse blood agar supplemented with IsoVitaleX. All strains were catalase- and oxidase-positive and indolenegative and stained as Gram-negative coccobacilli. Phenotypic properties of the five strains are given in the description of the genus. Three to seven characteristics allowed differentiation from other genera of the family Pasteurellaceae, as listed in Table 1. Variable reactions were observed for Dfructose (strain E145/08 negative), D-mannitol (strains E146/ 08 and E157/08 negative) and trehalose (strains E146/08 and E157/08 positive). http://ijs.sgmjournals.org
Using the API NH system, positive reactions were obtained for D-glucose, D-fructose, alkaline phosphatase and gammaglutamyltransferase (GGT) with the exception of strain E145/08, which was negative for GGT. On API ZYM, all strains showed strong positive reactions for alkaline phosphatase, acid phosphatase, leucine arylamidase and naphthol-AS-BI-phosphohydrolase. Weakly positive reactions were further obtained for esterase, esterase lipase and a-glucosidase with the exception of strain E145/08, which tested negative for these enzymic reactions, and strain E40/12, which was negative only for esterase lipase. Phylogenetic analysis of bacterial strains was performed by sequencing segments of their 16S rRNA (1.5 kb), rpoB (520 bp), rpoA (1 kb), infB (1.3 kb) and recN (1.4 kb) genes as described previously (Mu¨hldorfer et al., 2011a; Korczak et al., 2004; Christensen et al., 2004; Kuhnert & Korczak, 2006; Kuhnert et al., 2004; Bisgaard et al., 2012), with the following modifications. The annealing temperatures for the 16S rRNA (56 uC) and recN gene (46 uC) PCRs had to be lowered; for recN, the two internal primers recN-3 (59-ACGTTATGGCAACACCACCA-39) and recN-4 (59-ATAATTGTGGGGCGTGCTGA-39) were used instead of primers recN-1 and recN-2 (Bisgaard et al., 2012) to obtain nearly full-length sequences. Phylogenetic trees were reconstructed with the Bionumerics software version 7.0 (Applied Maths). Genome similarity calculations based on recN gene sequences were performed as reported by Kuhnert & Korczak (2006) using the MEGA software version 5.1 (Tamura et al., 2011) and the formula of Zeigler (2003). This approach has been applied successfully to the prediction of genome similarities within the family Pasteurellaceae as an alternative to DNA–DNA hybridization (Christensen et al., 2007, 2011; Kuhnert et al., 2007, 2010; Bisgaard et al., 2007, 2009, 2012; Foster et al., 2011). The DNA G+C content of strain E127/08T was determined by HPLC at the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, according to the method of Mesbah et al. (1989). Stretches of up to 1462 (16S rRNA), 854 (rpoA), 520 (rpoB), 1389 (infB) and 1379 bp (recN) were used in a multiple sequence alignment for phylogenetic tree reconstruction and for pairwise similarity calculations. Sequences of the five novel strains were compared to those of the type strains of type species of genera of the Pasteurellaceae with validly published names available in nucleotide databases. In each tree (Figs 1, 2 and S1–S4), the five strains formed a distinct cluster clearly separated from other genera of the Pasteurellaceae. Within this cluster, strains E127/08T, E146/08 and E40/12 isolated from bats of the same species grouped more closely together and showed a high degree of sequence homogeneity. These strains were derived from noctule bats (N. noctula) and revealed pairwise sequence similarities of .99.6 % (16S rRNA gene), .99.7 % (rpoA), .99.0 % (rpoB), .98.6 % (infB) and .99.2 % (recN). Comparison of sequences of strain E145/08 isolated from a serotine bat (E. serotinus) or of strain E157/08 isolated 2425
K. Mu¨hldorfer, S. Speck and G. Wibbelt
Table 1. Phenotypic characteristics for differentiation of Vespertiliibacter gen. nov. from other genera of the family Pasteurellaceae Genera: 1, Vespertiliibacter gen. nov. (data from this study); 2, Otariodibacter (Hansen et al., 2012); 3, Haemophilus sensu stricto (Kilian, 2005; Nørskov-Lauritsen et al., 2005); 4, Actinobacillus sensu stricto (Christensen & Bisgaard, 2004); 5, Lonepinella (Osawa et al., 1995); 6, Mannheimia (Angen et al., 1999); 7, Pasteurella sensu stricto (Christensen & Bisgaard, 2006; Mutters et al., 1985); 8, Phocoenobacter (Foster et al., 2000); 9, Gallibacterium (Bisgaard et al., 2009); 10, Volucribacter (Christensen et al., 2004); 11, Histophilus (Angen et al., 2003); 12, Avibacterium (Blackall et al., 2005); 13, Nicoletella (Kuhnert et al., 2004); 14, Bibersteinia (Blackall et al., 2007); 15, Aggregatibacter (Nørskov-Lauritsen & Kilian, 2006; Patel et al., 2004); 16, Basfia (Kuhnert et al., 2010); 17, Chelonobacter (Gregersen et al., 2009); 18, Necropsobacter (Christensen et al., 2011); 19, Bisgaardia (Foster et al., 2011); 20, ‘Seminibacterium’ (Vela et al., 2013). +, Positive; 2, negative; V, variable; ND, no data available; ONPG, o-nitrophenyl b-galactoside. Test Catalase Oxidase Indole production X-/V-factor dependency b-Galactosidase (ONPG) Voges–Proskauer (37 uC) Methyl red Urease Acid from: L-Arabinose Dulcitol myo-Inositol Maltose Raffinose L-Rhamnose Sucrose D-Xylose
1 + + 2 2/+*
2
3
4
5
6
7
V V 2 + + + V 2 V V + + + V 2 + 2 + 2/2 +/+ 2/V 2/2 2/2 2/2
8
9
10
11
12
13
14
15
16
17
18
2 V V 2 V + V V 2 + + + V V + + + V 2 + + + 2 V 2 + 2 2 2 2 2 V 2 2/2 2/2 2/2 2/2 2/V 2/2 2/2 2/V 2/2 2/2 2/2
19
20
+ + + + 2 2 2/2 2/2
2
+
2
V
+
V
V
+
V
V
ND
ND
V
2
V
+
+
+
2
2
2
+
2
2
+
2
2
+
2
2
2
2
ND
2
ND
2
ND
2
2
ND
ND
ND
2 2
2 2
2 2
+
2 2
2 2
+ 2
ND
ND
V
2 2
ND
2
+ 2
ND
V
2 +
ND
V
2
2
+ 2
2 2 2
2 2 2 2 2 2 2 2
2 2
2 ND
2 2 + + 2 2 + 2
2 2 2 2 2 2 2 2
+ 2 + 2 + 2 2 + 2 +
V
+ 2 2 2 2
2
+
V
V
ND
V
V
ND
V
2 2 +
ND
2
V
V
2
ND
2
2
V
2
V
V
ND
V
ND
V
V
V
V
2
V
V
ND
ND
V
V
ND
+
2 +
ND
V
+ 2 +
V
2 +
+ 2 +
ND
V
2 +
V
V
ND
V
V
2 2 2 2 2 2 2
V
V
V
2 2
V
2 2
V
+
ND
2
V
ND
+
+
2 2 2 +
V
V
ND
+ +
+ + 2 + +
2 + 2
ND
V
ND
ND
+ +
+ +
+ +
V V
+ +
+ 2
ND
*Members of Vespertiliibacter gen. nov. are nutritionally fastidious bacteria that require a chemically defined supplement such as Vitox (Oxoid) or IsoVitaleX Enrichment (BD Diagnostics) as an additive to media for cultivation and conventional phenotypic tests.
from a pipistrelle bat (P. pipistrellus) with those of the other four strains showed sequence similarities of 98.3–99.2 % (16S rRNA gene), 97.8–99.3 % (rpoA), 93.7–96 % (rpoB), 91.8–95.8 % (infB) and 91.9–93 % (recN) (Table S2). According to the phylogenetic analysis of 16S rRNA gene sequences (Fig. 1), the five strains were most closely related to the type species Otariodibacter oris and Bisgaardia hudsonensis. A similar observation was made in the infB gene-based analysis (Fig. S3). Highest sequence similarity based on the 16S rRNA (94.5 %) and infB (84.6 %) genes was found with the type strain of O. oris. In the rpoA gene-based phylogenetic tree (Fig. S1), the five strains clustered in the proximity of the type strain of Nicoletella semolina, sharing 89 % sequence similarity. In the rpoB gene-based phylogenetic tree (Fig. S2), the most closely related type species was again O. oris, with 89 % sequence similarity. In the recN gene-based phylogenetic tree (Fig. S4), the five strains clustered in the proximity of the type species of the genera Nicoletella, Actinobacillus, Mannheimia, Otariodibacter and Bibersteinia; the highest sequence similarities were observed with the type strains of O. oris (75 %) and N. semolina (75.2 %). 2426
Genome similarity of strain E127/08T to members of the other genera of the Pasteurellaceae as well as to the other four strains was calculated based on recN gene sequences according to Zeigler (2003) (Table 2). Comparison of strain E127/08T with the type species of the Pasteurellaceae indicated similarity values below 0.4. The highest value (0.39) was observed with the type strains of N. semolina and O. oris, which corresponded well to the results of the phylogenetic analyses (Figs 1 and S1–S4). In the combined tree (Fig. 2), genera of a central cluster of the Pasteurellaceae (Actinobacillus, Bibersteinia, Mannheimia, Otariodibacter, Nicoletella, Phocoenobacter) that includes the five strains showed genome similarity values clearly above 0.2, while the remaining genera had values below 0.1 (Table 2). The calculated genome similarities among the five strains were clearly above the threshold of 0.4 for genus separation (Korczak & Kuhnert, 2008), indicating that they represent a new genus within the family Pasteurellaceae. Strains E127/08T, E146/08 and E40/12 isolated from noctule bats were genetically very homogeneous, with similarity values ranging from 0.93 to 0.94. The calculated similarity values International Journal of Systematic and Evolutionary Microbiology 64
Vespertiliibacter pulmonis gen. nov., sp. nov.
80 100 1%
95 89 97 93
60
Vespertiliibacter genomospecies 2
E157/08
KF031244
Vespertiliibacter genomospecies 1
E145/08
KF031243
Vespertiliibacter pulmonis
E146/08
KF031241
Vespertiliibacter pulmonis
E40/12
KF031242
Vespertiliibacter pulmonis
E127/08T
KF031240
Bisgaardia hudsonensis
CCUG 43067T
GU295084
Otariodibacter oris
CCUG 59994T
HM626613
Actinobacillus lignieresii
CCUG 41384T
AY362892
Mannheimia haemolytica
CCUG 12392T
M75080
Lonepinella koalarum
ATCC 700131T
Y17189
Bibersteinia trehalosi
CCUG 27190T
AY362927
Basfia succiniciproducens
CCUG 57335T
FJ463881
Nicoletella semolina
CCUG
43639T
AY508816
CCUG
36157T
AF549387
‘Seminibacterium arietis’
CCUG
61707T
HE797791
Gallibacterium anatis
CCUG 15563T
AF228001
Volucribacter psittacicida
CCUG 47536T
AF487723
Avibacterium gallinarum
CCUG 12391T
AY362921
Necropsobacter rosorum
CCUG 28028T
GU966652
Histophilus somni
75
71 100
54 66
Pasteurella multocida subsp. multocida
CCUG 17976T Aggregatibacter actinomycetemcomitans CCUG 13227T Haemophilus influenzae ATCC 33391T
AF294410
Chelonobacter oris
55632T
EU331064
47322T
X89379
Phocoenobacter uteri Escherichia coli
CCUG
CCUG MG1655
M75039 M35019
NC_000913
Fig. 1. Phylogenetic tree based on partial 16S rRNA gene sequences. Sequences of strains of Vespertiliibacter gen. nov. were compared to those of the type strains of type species of all genera of the Pasteurellaceae with validly published names. Escherichia coli MG1655 was included as an outgroup. The tree was reconstructed in Bionumerics version 7.0 using the Jukes–Cantor correction for matrix calculation and neighbour-joining for cluster analysis. Bootstrap values greater than 50 % (expressed as percentages of 1000 replications) are given. Open circles on branching nodes of the tree indicate bootstrap values lower than 75 %; closed circles indicate bootstrap values of 75 % (light grey) to 100 % (black). Bar, 1 % sequence divergence.
of strain E127/08T with the remaining two strains E145/08 (0.78) and E157/08 (0.77) as well as of strains E145/08 and E157/08 (0.79) were below the limit of 0.85 for species separation (Korczak & Kuhnert, 2008), indicating that the five strains probably represent three different species. Based on the results of phenotypic and genetic analyses, we propose that the strains isolated from bats represent a novel species of a new genus, Vespertiliibacter pulmonis gen. nov., sp. nov., within the family Pasteurellaceae. The monophyly of the novel genus is strongly supported by individual and combined sequence analyses of the 16S rRNA gene and four housekeeping genes. The intraspecies homogeneity is demonstrated by a very low phenotypic and genetic variance, with representatives sharing 99.6–99.9 % sequence similarity in the 16S rRNA gene. Due to the limited number of strains that are available for phenotypic and genetic separation of different species, we propose to classify strain E145/08, isolated from a serotine bat, as Vespertiliibacter genomospecies 1, and strain E157/08, isolated from a pipistrelle bat, as Vespertiliibacter genomospecies 2. With http://ijs.sgmjournals.org
respect to the high bat species richness, further bacterial strains are needed in order to determine whether host specificity (based on genus or species levels of bats) is a useful criterion to distinguish between different species of Vespertiliibacter gen. nov., which finally might increase the number of species combined within this genus. Description of Vespertiliibacter gen. nov. Vespertiliibacter (Ves.per9ti.li.i.bac9ter. N.L. pl. n. Vespertilionidae taxonomic name for the largest family of bats, consisting of insectivorous microbats; N.L. masc. n. bacter a rod; N.L. masc. n. Vespertiliibacter rod isolated from vespertilionid bats). Members are non-motile, Gram-negative coccoid rods that require a nutritional supplement with NAD such as Vitox or IsoVitaleX Enrichment for growth. They reduce nitrate to nitrite and are catalase-, oxidase- and methyl red- (37 uC) positive. Negative reactions occur for indole, Simmons’ citrate, malonate-base, H2S/TSI, urease, Voges–Proskauer 2427
K. Mu¨hldorfer, S. Speck and G. Wibbelt
10% 100
97
96
97 98 95
94
100 100 100
94
100 100
Basfia succiniciproducens
CCUG 57335T
Pasteurella multocida subsp. multocida
CCUG 17976T
Bisgaardia hudsonensis
CCUG 43067T
Gallibacterium anatis
CCUG 15563T
Chelonobacter oris
CCUG 55632T
Lonepinella koalarum
ATCC 700131T
Aggregatibacter actinomycetemcomitans
D11S1
Haemophilus influenzae
Rd KW20
Histophilus somni
CCUG 36157T
Volucribacter psittacicida
CCUG 47536T
Necropsobacter rosorum
CCUG 28028T
Avibacterium gallinarum
CCUG 12391T
Actinobacillus lignieresii
CCUG 41384T
Bibersteinia trehalosi
CCUG 27190T
Mannheimia haemolytica
CCUG 12392T
Otariodibacter oris
CCUG 59994T
Nicoletella semolina
CCUG 43639T
Vespertiliibacter genomospecies 1
E145/08
Vespertiliibacter genomospecies 2
E157/08
100
Vespertiliibacter pulmonis
E146/08
92
Vespertiliibacter pulmonis
E40/12
Vespertiliibacter pulmonis
E127/08T
Phocoenobacter uteri
CCUG 47322T
Escherichia coli
MG1655
Fig. 2. Combined phylogenetic tree based on partial 16S rRNA, rpoB, infB and recN gene sequences. Sequences of strains of Vespertiliibacter gen. nov. were compared to those of the type strains of type species of all genera of the Pasteurellaceae with validly published names. For genera Aggregatibacter and Haemophilus, whole-genome sequences of reference strains were included. The tree was reconstructed in Bionumerics version 7.0 using the Jukes–Cantor correction for matrix calculation and neighbour-joining for tree building. E. coli MG1655 was included as an outgroup. Cophenic correlations are given and indicated as closed circles on the branching nodes, showing the reliability of the branching compared with the actual genetic relatedness of the taxa. Bar, 10 % sequence divergence. Details of sequence accession numbers of the novel strains are given in Table S1.
(37 uC), arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and phenylalanine deaminase tests. Acid is produced from L-arabinose, D-glucose, myo-inositol, Lrhamnose and D-xylose, but not from dulcitol, maltose, raffinose or sucrose. On API ZYM strips, strong positive reactions are observed for alkaline phosphatase, acid phosphatase, leucine arylamidase and naphthol-AS-BI-phosphohydrolase, while b-galactosidase is not produced. The G+C content of DNA from the type strain of the type species is 38.2 mol%. The type species is Vespertiliibacter pulmonis.
Vespertiliibacter pulmonis (pul.mo9nis. L. n. pulmo the lung; L. gen. n. pulmonis of the lung).
smooth. The texture of single colonies is butyrous (E40/12) or dry (E127/08T and E146/08). Known strains grow on brain heart infusion agar and on 5 % horse blood agar (both supplemented with IsoVitaleX), producing a clear zone of haemolysis around colonies on horse blood agar. In addition to the phenotypic characteristics given in the genus description, acid is produced from D-fructose and D-mannose, but not from aesculin, cellobiose, D-galactose, inulin, lactose, melezitose, melibiose, salicin or D-sorbitol. Acid production from D-mannitol (strain E146/08 is negative) and trehalose is variable (type strain E127/08T is negative). On API NH, positive reactions are obtained for D-glucose, D-fructose, alkaline phosphatase and GGT. On API ZYM, weakly positive or variable reactions are further obtained for esterase, esterase lipase (strain E40/12 is negative) and a-glucosidase.
Bacterial colonies grown on chocolate agar (5 % CO2, 24 h at 37 uC) supplemented with Vitox are 1 mm in diameter, greyish, opaque, circular, convex with an entire margin and
The type strain is E127/08T (5CCUG 64585T5DSM 27238T), isolated from the lung of a noctule bat (Nyctalus noctula) from Germany. The pathogenic potential is unknown.
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International Journal of Systematic and Evolutionary Microbiology 64
Description of Vespertiliibacter pulmonis sp. nov.
Vespertiliibacter pulmonis gen. nov., sp. nov.
Table 2. Genome similarity of strain E127/08T to strains of the type species of the family Pasteurellaceae, and to Vespertiliibacter genomospecies 1 and 2, calculated based on recN gene sequences Strain(s) Vespertiliibacter pulmonis E146/08, E40/12 Vespertiliibacter genomospecies 1 E145/08 Vespertiliibacter genomospecies 2 E157/08 Nicoletella semolina CCUG 43639T Otariodibacter oris CCUG 59994T Bibersteinia trehalosi CCUG 27190T Actinobacillus lignieresii CCUG 41384T Phocoenobacter uteri CCUG 47322T Mannheimia haemolytica CCUG 12392T Haemophilus influenzae Rd KW20 Volucribacter psittacicida CCUG 47536T Pasteurella multocida CCUG 17976T Bisgaardia hudsonensis CCUG 43067T Basfia succiniciproducens CCUG 57335T Lonepinella koalarum ATCC 700131T Aggregatibacter actinomycetemcomitans D11S-1 Avibacterium gallinarum CCUG 12391T Histophilus somni CCUG 36157T Chelonobacter oris CCUG 55632T Gallibacterium anatis CCUG 15563T Necropsobacter rosorum CCUG 28028T
Similarity to strain 127/08T 0.93–0.94 0.78 0.77 0.39 0.39 0.34 0.32 0.29 0.28 0.09 0.08 0.07 0.07 0.04 0.04 0.03 0.03 0.03 0.00 0.00 0.00
Bisgaard, M., Nørskov-Lauritsen, N., de Wit, S. J., Hess, C. & Christensen, H. (2012). Multilocus sequence phylogenetic analysis of
Avibacterium. Microbiology 158, 993–1004. Blackall, P. J., Christensen, H., Beckenham, T., Blackall, L. L. & Bisgaard, M. (2005). Reclassification of Pasteurella gallinarum,
[Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov. Int J Syst Evol Microbiol 55, 353–362.
Blackall, P. J., Bojesen, A. M., Christensen, H. & Bisgaard, M. (2007).
Reclassification of [Pasteurella] trehalosi as Bibersteinia trehalosi gen. nov., comb. nov. Int J Syst Evol Microbiol 57, 666–674. Christensen, H. & Bisgaard, M. (2006). The genus Pasteurella. In The
Prokaryotes, 3rd edn, vol. 6, pp. 1062–1090. Edited by M. Dworkin, S. Lyons, E. Rosenberg, K. H. Schleifer & E. Stackebrandt. New York: Springer. Christensen, H. & Bisgaard, M. (2004). Revised definition of
Actinobacillus sensu stricto isolated from animals. A review with special emphasis on diagnosis. Vet Microbiol 99, 13–30. Christensen, H., Bisgaard, M., Aalbaek, B. & Olsen, J. E. (2004).
Reclassification of Bisgaard taxon 33, with proposal of Volucribacter psittacicida gen. nov., sp. nov. and Volucribacter amazonae sp. nov. as new members of the Pasteurellaceae. Int J Syst Evol Microbiol 54, 813– 818. Christensen, H., Kuhnert, P., Busse, H.-J., Frederiksen, W. C. & Bisgaard, M. (2007). Proposed minimal standards for the description
of genera, species and subspecies of the Pasteurellaceae. Int J Syst Evol Microbiol 57, 166–178. Christensen, H., Korczak, B. M., Bojesen, A. M., Kuhnert, P., Frederiksen, W. & Bisgaard, M. (2011). Classification of organisms
Acknowledgements The authors are grateful to Nicole Dinse for technical assistance and to Berliner Artenschutz Team – BAT-e.V., Elke Mu¨hlbach and Waltraud and Helmut Zoels for providing the bat carcasses. The study was supported by the Adolf and Hildegard Isler-Stiftung and the Klara Samariter-Stiftung.
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complex as evaluated by DNA–DNA hybridizations and 16S rRNA sequencing with proposal of Mannheimia haemolytica gen. nov., comb. nov., Mannheimia granulomatis comb. nov., Mannheimia glucosida sp. nov., Mannheimia ruminalis sp. nov. and Mannheimia varigena sp. nov. Int J Syst Bacteriol 49, 67–86. Angen, Ø., Ahrens, P., Kuhnert, P., Christensen, H. & Mutters, R. (2003). Proposal of Histophilus somni gen. nov., sp. nov. for the three
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testudinis and [P.] testudinis-like bacteria and proposal of Chelonobacter oris gen. nov., sp. nov. as a new member of the Pasteurellaceae. Int J Syst Evol Microbiol 59, 1583–1588.
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Hansen, M. J., Bertelsen, M. F., Christensen, H., Bojesen, A. M. & Bisgaard, M. (2012). Otariodibacter oris gen. nov., sp. nov., a member
Bisgaard, M., Christensen, J. P., Bojesen, A. M. & Christensen, H. (2007). Avibacterium endocarditidis sp. nov., isolated from valvular
taxon 3 complex of Bisgaard within Gallibacterium and description of Gallibacterium melopsittaci sp. nov., Gallibacterium trehalosifermentans sp. nov. and Gallibacterium salpingitidis sp. nov. Int J Syst Evol Microbiol 59, 735–744. http://ijs.sgmjournals.org
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K. Mu¨hldorfer, S. Speck and G. Wibbelt D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer. Korczak, B. & Kuhnert, P. (2008). Phylogeny of Pasteurellaceae. In
Pasteurellaceae: Biology, Genomics and Molecular Aspects, pp. 27–52. Edited by P. Kuhnert & H. Christensen. Norwich, UK: Caister Academic. Korczak, B., Christensen, H., Emler, S., Frey, J. & Kuhnert, P. (2004).
Phylogeny of the family Pasteurellaceae based on rpoB sequences. Int J Syst Evol Microbiol 54, 1393–1399. Kuhnert, P. & Korczak, B. M. (2006). Prediction of whole-genome
DNA–DNA similarity, determination of G+C content and phylogenetic analysis within the family Pasteurellaceae by multilocus sequence analysis (MLSA). Microbiology 152, 2537–2548. Kuhnert, P., Korczak, B., Falsen, E., Straub, R., Hoops, A., Boerlin, P., Frey, J. & Mutters, R. (2004). Nicoletella semolina gen. nov., sp. nov., a
new member of Pasteurellaceae isolated from horses with airway disease. J Clin Microbiol 42, 5542–5548. Kuhnert, P., Korczak, B. M., Christensen, H. & Bisgaard, M. (2007).
Emended description of Actinobacillus capsulatus Arseculeratne 1962, 38AL. Int J Syst Evol Microbiol 57, 625–632. Kuhnert, P., Scholten, E., Haefner, S., Mayor, D. & Frey, J. (2010).
Basfia succiniciproducens gen. nov., sp. nov., a new member of the family Pasteurellaceae isolated from bovine rumen. Int J Syst Evol Microbiol 60, 44–50. Mesbah, M., Premachandran, U. & Whitman, W. (1989). Precise
measurement of the G + C content of deoxyribonucleic acid by high performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.
basis of deoxyribonucleic acid homology, with proposals for the new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteurella anatis, and Pasteurella langaa. Int J Syst Bacteriol 35, 309– 322. Nørskov-Lauritsen, N. & Kilian, M. (2006). Reclassification of
Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis as Aggregatibacter actinomycetemcomitans gen. nov., comb. nov., Aggregatibacter aphrophilus comb. nov. and Aggregatibacter segnis comb. nov., and emended description of Aggregatibacter aphrophilus to include V factor-dependent and V factor-independent isolates. Int J Syst Evol Microbiol 56, 2135–2146. Nørskov-Lauritsen, N., Bruun, B. & Kilian, M. (2005). Multilocus
sequence phylogenetic study of the genus Haemophilus with description of Haemophilus pittmaniae sp. nov. Int J Syst Evol Microbiol 55, 449–456. Olsen, I., Dewhirst, F. E., Paster, B. J. & Busse, H.-J. (2005). Family I.
Pasteurellaceae Pohl 1981b, 382VP (Effective publication: Pohl 1979, 81). In Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 2B, pp. 851–912. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer. Osawa, R., Rainey, F. A., Fujisawa, T., Lang, E., Busse, H.-J., Walsh, T. & Stackebrandt, E. (1995). Lonepinella koalarum gen. nov., sp. nov.,
a new tannin-protein complex degrading bacterium. Syst Appl Microbiol 18, 368–373. Patel, S. M., Mo, J. H., Walker, M. T., Adley, B. & Noskin, G. A. (2004).
Mu¨hldorfer, K. (2013). Bats and bacterial pathogens: a review.
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Mu¨hldorfer, K., Schwarz, S., Fickel, J., Wibbelt, G. & Speck, S. (2011a). Genetic diversity of Pasteurella species isolated from
wildlife diseases in Britain. Res Vet Sci 69, 11–16.
European vespertilionid bats. Vet Microbiol 149, 163–171.
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011). MEGA5: molecular evolutionary genetics analysis using
Mu¨hldorfer, K., Speck, S., Kurth, A., Lesnik, R., Freuling, C., Mu¨ller, T., Kramer-Schadt, S. & Wibbelt, G. (2011b). Diseases and causes of
maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28, 2731–2739.
death in European bats: dynamics in disease susceptibility and infection rates. PLoS ONE 6, e29773.
Vela, A. I., Bueso, J. P., Domı´nguez, L., Busse, H.-J. & Ferna´ndezGarayza´bal, J. F. (2013). Seminibacterium arietis gen. nov., sp. nov.,
Mu¨hldorfer, K., Speck, S. & Wibbelt, G. (2011c). Diseases in free-
isolated from the semen of rams. Syst Appl Microbiol 36, 166–170.
ranging bats from Germany. BMC Vet Res 7, 61.
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of whole genomes in bacteria. Int J Syst Evol Microbiol 53, 1893– 1900.
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International Journal of Systematic and Evolutionary Microbiology 64
DOI 10.1099/ijs.0.062786-0
Proposal of Vespertiliibacter pulmonis gen. nov., sp. nov. and two genomospecies as new members of the family Pasteurellaceae isolated from European bats Kristin Mu¨hldorfer,13 Stephanie Speck2 and Gudrun Wibbelt1 Correspondence Kristin Mu¨hldorfer [email protected]
1
Department of Wildlife Diseases, Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany
2
Institute for Animal Hygiene and Veterinary Public Health, University of Leipzig, Leipzig, Germany
Five bacterial strains isolated from bats of the family Vespertilionidae were characterized by phenotypic tests and multilocus sequence analysis (MLSA) using the 16S rRNA gene and four housekeeping genes (rpoA, rpoB, infB, recN). Phylogenetic analyses of individual and combined datasets indicated that the five strains represent a monophyletic cluster within the family Pasteurellaceae. Comparison of 16S rRNA gene sequences demonstrated a high degree of similarity (98.3–99.9 %) among the group of bat-derived strains, while searches in nucleotide databases indicated less than 96 % sequence similarity to known members of the Pasteurellaceae. The housekeeping genes rpoA, rpoB, infB and recN provided higher resolution compared with the 16S rRNA gene and subdivided the group according to the bat species from which the strains were isolated. Three strains derived from noctule bats shared 98.6–100 % sequence similarity in all four genes investigated, whereas, based on rpoB, infB and recN gene sequences, 91.8–96 % similarity was observed with and between the remaining two strains isolated from a serotine bat and a pipistrelle bat, respectively. Genome relatedness as deduced from recN gene sequences correlated well with the results of MLSA and indicated that the five strains represent a new genus. Based on these results, it is proposed to classify the five strains derived from bats within Vespertiliibacter pulmonis gen. nov., sp. nov. (the type species), Vespertiliibacter genomospecies 1 and Vespertiliibacter genomospecies 2. The genus can be distinguished phenotypically from recognized genera of the Pasteurellaceae by at least three characteristics. All strains are nutritionally fastidious and require a chemically defined supplement with NAD for growth. The DNA G+C content of strain E127/08T is 38.2 mol%. The type strain of Vespertiliibacter pulmonis gen. nov., sp. nov. is E127/08T (5CCUG 64585T5DSM 27238T). The reference strains of Vespertiliibacter genomospecies 1 and 2 are E145/08 and E157/08, respectively.
The family Pasteurellaceae currently comprises 18 recognized genera with more than 70 species of wide distribution and host range (http://www.bacterio.net/). An additional genus ‘Seminibacterium’ has been proposed recently by Vela et al. (2013). Members of the Pasteurellaceae commonly belong to 3Present address: Institute of Poultry Diseases, Freie Universita¨t Berlin, Ko¨nigsweg 63, 14163 Berlin, Germany. Abbreviation: GGT, gamma-glutamyltransferase. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains E127/08T, E146/08, E40/12, E145/08 and E157/08 are KF031240–KF031244, respectively. The accession numbers of the rpoA, rpoB, infB and recN gene sequences of these strains are detailed in Table S1. Two supplementary tables and four supplementary figures are available with the online version of this paper.
2424
the mucosal flora of their hosts, but some species are able to cause localized or systemic infections in animals and humans (Olsen et al., 2005). In chiropteran species, members of the genus Pasteurella have been identified as primary pathogens responsible for pneumonia, severe organ necroses and systemic infections in European bat species of the family Vespertilionidae (Simpson, 2000; Daffner, 2001; Hajkova & Pikula, 2007; Mu¨hldorfer et al., 2011a, b, c). Bite wounds from domestic cats appear to be the most likely source of Pasteurella infections in European bats (Mu¨hldorfer et al., 2011a, b, c; Mu¨hldorfer, 2013). However, the role of members of the Pasteurellaceae as commensal or opportunistic bacteria of bat hosts is unknown. The aim of the present study was to characterize five strains of the Pasteurellaceae obtained from the lung, heart or thoracic cavity of five bats of three different European species 062786 G 2014 IUMS Printed in Great Britain
Vespertiliibacter pulmonis gen. nov., sp. nov.
(Nyctalus noctula, n53; Pipistrellus pipistrellus, n51; Eptesicus serotinus, n51) to investigate their phenotypic and genetic characteristics as well as their phylogenetic relationships to other members of the family Pasteurellaceae. Between 2008 and 2012, 280 deceased free-ranging bats were subjected to post-mortem examination followed by histopathological and bacteriological investigations. The bat carcasses were provided by bat researchers and bat rehabilitation centres throughout Germany. Tissue samples from lung, liver, heart, kidney and spleen as well as swab samples from the thoracic cavity of these carcasses were plated onto Columbia agar (7 % sheep blood), chocolate agar (with Vitox, 5 % CO2) and MacConkey agar (all from Oxoid) and were incubated at 37 uC for 24–48 h. In five bats, colonies typical of the Pasteurellaceae were isolated on chocolate agar supplemented with Vitox. Single colonies of the five strains (Table S1, available in the online Supplementary Material) were subcultured and subsequently stored at 280 uC suspended in nutrient broth supplemented with 20 % glycerol. Primary identification of bacterial strains was based on Gram-staining, indole production and catalase and oxidase reactions. Phenotypic characterization was performed by conventional tube tests, as recommended by the minimal standards for the description of genera, species and subspecies of the Pasteurellaceae (Christensen et al., 2007). Additionally, all strains were characterized with the API NH and API ZYM systems (bioMe´rieux), according to the manufacturer’s instructions. Fermentation of carbohydrates, alcohols and glycosides, malonate utilization, methyl red, Voges–Proskauer, ornithine decarboxylase, lysine decarboxylase and arginine dihydrolase tests were performed by adding Vitox supplement (with a final concentration of 10 mg NAD ml21) dissolved in distilled water and adjusted to pH 7.4. The X-factor (haemin) and/or V-factor (NAD) requirement and haemolytic reactions were identified by using the Haemophilus ID Quad plate with growth factors (BD Diagnostics), according to the manufacturer’s instructions. Colonies of the strains grown on chocolate agar supplemented with Vitox (5 % CO2, 24 h at 37 uC) were 1 mm in diameter, smooth, greyish, opaque, circular and convex with an entire margin. The texture of single colonies was butyrous (E40/12, E145/08 and E157/08) or dry (E127/08T and E146/08). The five strains were V-factor-dependent but, instead of NAD alone, all strains required a nutritional supplement such as IsoVitaleX Enrichment (BD Diagnostics) or Vitox (Oxoid) for growth. Haemolysis was produced on 5 % horse blood agar supplemented with IsoVitaleX. All strains were catalase- and oxidase-positive and indolenegative and stained as Gram-negative coccobacilli. Phenotypic properties of the five strains are given in the description of the genus. Three to seven characteristics allowed differentiation from other genera of the family Pasteurellaceae, as listed in Table 1. Variable reactions were observed for Dfructose (strain E145/08 negative), D-mannitol (strains E146/ 08 and E157/08 negative) and trehalose (strains E146/08 and E157/08 positive). http://ijs.sgmjournals.org
Using the API NH system, positive reactions were obtained for D-glucose, D-fructose, alkaline phosphatase and gammaglutamyltransferase (GGT) with the exception of strain E145/08, which was negative for GGT. On API ZYM, all strains showed strong positive reactions for alkaline phosphatase, acid phosphatase, leucine arylamidase and naphthol-AS-BI-phosphohydrolase. Weakly positive reactions were further obtained for esterase, esterase lipase and a-glucosidase with the exception of strain E145/08, which tested negative for these enzymic reactions, and strain E40/12, which was negative only for esterase lipase. Phylogenetic analysis of bacterial strains was performed by sequencing segments of their 16S rRNA (1.5 kb), rpoB (520 bp), rpoA (1 kb), infB (1.3 kb) and recN (1.4 kb) genes as described previously (Mu¨hldorfer et al., 2011a; Korczak et al., 2004; Christensen et al., 2004; Kuhnert & Korczak, 2006; Kuhnert et al., 2004; Bisgaard et al., 2012), with the following modifications. The annealing temperatures for the 16S rRNA (56 uC) and recN gene (46 uC) PCRs had to be lowered; for recN, the two internal primers recN-3 (59-ACGTTATGGCAACACCACCA-39) and recN-4 (59-ATAATTGTGGGGCGTGCTGA-39) were used instead of primers recN-1 and recN-2 (Bisgaard et al., 2012) to obtain nearly full-length sequences. Phylogenetic trees were reconstructed with the Bionumerics software version 7.0 (Applied Maths). Genome similarity calculations based on recN gene sequences were performed as reported by Kuhnert & Korczak (2006) using the MEGA software version 5.1 (Tamura et al., 2011) and the formula of Zeigler (2003). This approach has been applied successfully to the prediction of genome similarities within the family Pasteurellaceae as an alternative to DNA–DNA hybridization (Christensen et al., 2007, 2011; Kuhnert et al., 2007, 2010; Bisgaard et al., 2007, 2009, 2012; Foster et al., 2011). The DNA G+C content of strain E127/08T was determined by HPLC at the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany, according to the method of Mesbah et al. (1989). Stretches of up to 1462 (16S rRNA), 854 (rpoA), 520 (rpoB), 1389 (infB) and 1379 bp (recN) were used in a multiple sequence alignment for phylogenetic tree reconstruction and for pairwise similarity calculations. Sequences of the five novel strains were compared to those of the type strains of type species of genera of the Pasteurellaceae with validly published names available in nucleotide databases. In each tree (Figs 1, 2 and S1–S4), the five strains formed a distinct cluster clearly separated from other genera of the Pasteurellaceae. Within this cluster, strains E127/08T, E146/08 and E40/12 isolated from bats of the same species grouped more closely together and showed a high degree of sequence homogeneity. These strains were derived from noctule bats (N. noctula) and revealed pairwise sequence similarities of .99.6 % (16S rRNA gene), .99.7 % (rpoA), .99.0 % (rpoB), .98.6 % (infB) and .99.2 % (recN). Comparison of sequences of strain E145/08 isolated from a serotine bat (E. serotinus) or of strain E157/08 isolated 2425
K. Mu¨hldorfer, S. Speck and G. Wibbelt
Table 1. Phenotypic characteristics for differentiation of Vespertiliibacter gen. nov. from other genera of the family Pasteurellaceae Genera: 1, Vespertiliibacter gen. nov. (data from this study); 2, Otariodibacter (Hansen et al., 2012); 3, Haemophilus sensu stricto (Kilian, 2005; Nørskov-Lauritsen et al., 2005); 4, Actinobacillus sensu stricto (Christensen & Bisgaard, 2004); 5, Lonepinella (Osawa et al., 1995); 6, Mannheimia (Angen et al., 1999); 7, Pasteurella sensu stricto (Christensen & Bisgaard, 2006; Mutters et al., 1985); 8, Phocoenobacter (Foster et al., 2000); 9, Gallibacterium (Bisgaard et al., 2009); 10, Volucribacter (Christensen et al., 2004); 11, Histophilus (Angen et al., 2003); 12, Avibacterium (Blackall et al., 2005); 13, Nicoletella (Kuhnert et al., 2004); 14, Bibersteinia (Blackall et al., 2007); 15, Aggregatibacter (Nørskov-Lauritsen & Kilian, 2006; Patel et al., 2004); 16, Basfia (Kuhnert et al., 2010); 17, Chelonobacter (Gregersen et al., 2009); 18, Necropsobacter (Christensen et al., 2011); 19, Bisgaardia (Foster et al., 2011); 20, ‘Seminibacterium’ (Vela et al., 2013). +, Positive; 2, negative; V, variable; ND, no data available; ONPG, o-nitrophenyl b-galactoside. Test Catalase Oxidase Indole production X-/V-factor dependency b-Galactosidase (ONPG) Voges–Proskauer (37 uC) Methyl red Urease Acid from: L-Arabinose Dulcitol myo-Inositol Maltose Raffinose L-Rhamnose Sucrose D-Xylose
1 + + 2 2/+*
2
3
4
5
6
7
V V 2 + + + V 2 V V + + + V 2 + 2 + 2/2 +/+ 2/V 2/2 2/2 2/2
8
9
10
11
12
13
14
15
16
17
18
2 V V 2 V + V V 2 + + + V V + + + V 2 + + + 2 V 2 + 2 2 2 2 2 V 2 2/2 2/2 2/2 2/2 2/V 2/2 2/2 2/V 2/2 2/2 2/2
19
20
+ + + + 2 2 2/2 2/2
2
+
2
V
+
V
V
+
V
V
ND
ND
V
2
V
+
+
+
2
2
2
+
2
2
+
2
2
+
2
2
2
2
ND
2
ND
2
ND
2
2
ND
ND
ND
2 2
2 2
2 2
+
2 2
2 2
+ 2
ND
ND
V
2 2
ND
2
+ 2
ND
V
2 +
ND
V
2
2
+ 2
2 2 2
2 2 2 2 2 2 2 2
2 2
2 ND
2 2 + + 2 2 + 2
2 2 2 2 2 2 2 2
+ 2 + 2 + 2 2 + 2 +
V
+ 2 2 2 2
2
+
V
V
ND
V
V
ND
V
2 2 +
ND
2
V
V
2
ND
2
2
V
2
V
V
ND
V
ND
V
V
V
V
2
V
V
ND
ND
V
V
ND
+
2 +
ND
V
+ 2 +
V
2 +
+ 2 +
ND
V
2 +
V
V
ND
V
V
2 2 2 2 2 2 2
V
V
V
2 2
V
2 2
V
+
ND
2
V
ND
+
+
2 2 2 +
V
V
ND
+ +
+ + 2 + +
2 + 2
ND
V
ND
ND
+ +
+ +
+ +
V V
+ +
+ 2
ND
*Members of Vespertiliibacter gen. nov. are nutritionally fastidious bacteria that require a chemically defined supplement such as Vitox (Oxoid) or IsoVitaleX Enrichment (BD Diagnostics) as an additive to media for cultivation and conventional phenotypic tests.
from a pipistrelle bat (P. pipistrellus) with those of the other four strains showed sequence similarities of 98.3–99.2 % (16S rRNA gene), 97.8–99.3 % (rpoA), 93.7–96 % (rpoB), 91.8–95.8 % (infB) and 91.9–93 % (recN) (Table S2). According to the phylogenetic analysis of 16S rRNA gene sequences (Fig. 1), the five strains were most closely related to the type species Otariodibacter oris and Bisgaardia hudsonensis. A similar observation was made in the infB gene-based analysis (Fig. S3). Highest sequence similarity based on the 16S rRNA (94.5 %) and infB (84.6 %) genes was found with the type strain of O. oris. In the rpoA gene-based phylogenetic tree (Fig. S1), the five strains clustered in the proximity of the type strain of Nicoletella semolina, sharing 89 % sequence similarity. In the rpoB gene-based phylogenetic tree (Fig. S2), the most closely related type species was again O. oris, with 89 % sequence similarity. In the recN gene-based phylogenetic tree (Fig. S4), the five strains clustered in the proximity of the type species of the genera Nicoletella, Actinobacillus, Mannheimia, Otariodibacter and Bibersteinia; the highest sequence similarities were observed with the type strains of O. oris (75 %) and N. semolina (75.2 %). 2426
Genome similarity of strain E127/08T to members of the other genera of the Pasteurellaceae as well as to the other four strains was calculated based on recN gene sequences according to Zeigler (2003) (Table 2). Comparison of strain E127/08T with the type species of the Pasteurellaceae indicated similarity values below 0.4. The highest value (0.39) was observed with the type strains of N. semolina and O. oris, which corresponded well to the results of the phylogenetic analyses (Figs 1 and S1–S4). In the combined tree (Fig. 2), genera of a central cluster of the Pasteurellaceae (Actinobacillus, Bibersteinia, Mannheimia, Otariodibacter, Nicoletella, Phocoenobacter) that includes the five strains showed genome similarity values clearly above 0.2, while the remaining genera had values below 0.1 (Table 2). The calculated genome similarities among the five strains were clearly above the threshold of 0.4 for genus separation (Korczak & Kuhnert, 2008), indicating that they represent a new genus within the family Pasteurellaceae. Strains E127/08T, E146/08 and E40/12 isolated from noctule bats were genetically very homogeneous, with similarity values ranging from 0.93 to 0.94. The calculated similarity values International Journal of Systematic and Evolutionary Microbiology 64
Vespertiliibacter pulmonis gen. nov., sp. nov.
80 100 1%
95 89 97 93
60
Vespertiliibacter genomospecies 2
E157/08
KF031244
Vespertiliibacter genomospecies 1
E145/08
KF031243
Vespertiliibacter pulmonis
E146/08
KF031241
Vespertiliibacter pulmonis
E40/12
KF031242
Vespertiliibacter pulmonis
E127/08T
KF031240
Bisgaardia hudsonensis
CCUG 43067T
GU295084
Otariodibacter oris
CCUG 59994T
HM626613
Actinobacillus lignieresii
CCUG 41384T
AY362892
Mannheimia haemolytica
CCUG 12392T
M75080
Lonepinella koalarum
ATCC 700131T
Y17189
Bibersteinia trehalosi
CCUG 27190T
AY362927
Basfia succiniciproducens
CCUG 57335T
FJ463881
Nicoletella semolina
CCUG
43639T
AY508816
CCUG
36157T
AF549387
‘Seminibacterium arietis’
CCUG
61707T
HE797791
Gallibacterium anatis
CCUG 15563T
AF228001
Volucribacter psittacicida
CCUG 47536T
AF487723
Avibacterium gallinarum
CCUG 12391T
AY362921
Necropsobacter rosorum
CCUG 28028T
GU966652
Histophilus somni
75
71 100
54 66
Pasteurella multocida subsp. multocida
CCUG 17976T Aggregatibacter actinomycetemcomitans CCUG 13227T Haemophilus influenzae ATCC 33391T
AF294410
Chelonobacter oris
55632T
EU331064
47322T
X89379
Phocoenobacter uteri Escherichia coli
CCUG
CCUG MG1655
M75039 M35019
NC_000913
Fig. 1. Phylogenetic tree based on partial 16S rRNA gene sequences. Sequences of strains of Vespertiliibacter gen. nov. were compared to those of the type strains of type species of all genera of the Pasteurellaceae with validly published names. Escherichia coli MG1655 was included as an outgroup. The tree was reconstructed in Bionumerics version 7.0 using the Jukes–Cantor correction for matrix calculation and neighbour-joining for cluster analysis. Bootstrap values greater than 50 % (expressed as percentages of 1000 replications) are given. Open circles on branching nodes of the tree indicate bootstrap values lower than 75 %; closed circles indicate bootstrap values of 75 % (light grey) to 100 % (black). Bar, 1 % sequence divergence.
of strain E127/08T with the remaining two strains E145/08 (0.78) and E157/08 (0.77) as well as of strains E145/08 and E157/08 (0.79) were below the limit of 0.85 for species separation (Korczak & Kuhnert, 2008), indicating that the five strains probably represent three different species. Based on the results of phenotypic and genetic analyses, we propose that the strains isolated from bats represent a novel species of a new genus, Vespertiliibacter pulmonis gen. nov., sp. nov., within the family Pasteurellaceae. The monophyly of the novel genus is strongly supported by individual and combined sequence analyses of the 16S rRNA gene and four housekeeping genes. The intraspecies homogeneity is demonstrated by a very low phenotypic and genetic variance, with representatives sharing 99.6–99.9 % sequence similarity in the 16S rRNA gene. Due to the limited number of strains that are available for phenotypic and genetic separation of different species, we propose to classify strain E145/08, isolated from a serotine bat, as Vespertiliibacter genomospecies 1, and strain E157/08, isolated from a pipistrelle bat, as Vespertiliibacter genomospecies 2. With http://ijs.sgmjournals.org
respect to the high bat species richness, further bacterial strains are needed in order to determine whether host specificity (based on genus or species levels of bats) is a useful criterion to distinguish between different species of Vespertiliibacter gen. nov., which finally might increase the number of species combined within this genus. Description of Vespertiliibacter gen. nov. Vespertiliibacter (Ves.per9ti.li.i.bac9ter. N.L. pl. n. Vespertilionidae taxonomic name for the largest family of bats, consisting of insectivorous microbats; N.L. masc. n. bacter a rod; N.L. masc. n. Vespertiliibacter rod isolated from vespertilionid bats). Members are non-motile, Gram-negative coccoid rods that require a nutritional supplement with NAD such as Vitox or IsoVitaleX Enrichment for growth. They reduce nitrate to nitrite and are catalase-, oxidase- and methyl red- (37 uC) positive. Negative reactions occur for indole, Simmons’ citrate, malonate-base, H2S/TSI, urease, Voges–Proskauer 2427
K. Mu¨hldorfer, S. Speck and G. Wibbelt
10% 100
97
96
97 98 95
94
100 100 100
94
100 100
Basfia succiniciproducens
CCUG 57335T
Pasteurella multocida subsp. multocida
CCUG 17976T
Bisgaardia hudsonensis
CCUG 43067T
Gallibacterium anatis
CCUG 15563T
Chelonobacter oris
CCUG 55632T
Lonepinella koalarum
ATCC 700131T
Aggregatibacter actinomycetemcomitans
D11S1
Haemophilus influenzae
Rd KW20
Histophilus somni
CCUG 36157T
Volucribacter psittacicida
CCUG 47536T
Necropsobacter rosorum
CCUG 28028T
Avibacterium gallinarum
CCUG 12391T
Actinobacillus lignieresii
CCUG 41384T
Bibersteinia trehalosi
CCUG 27190T
Mannheimia haemolytica
CCUG 12392T
Otariodibacter oris
CCUG 59994T
Nicoletella semolina
CCUG 43639T
Vespertiliibacter genomospecies 1
E145/08
Vespertiliibacter genomospecies 2
E157/08
100
Vespertiliibacter pulmonis
E146/08
92
Vespertiliibacter pulmonis
E40/12
Vespertiliibacter pulmonis
E127/08T
Phocoenobacter uteri
CCUG 47322T
Escherichia coli
MG1655
Fig. 2. Combined phylogenetic tree based on partial 16S rRNA, rpoB, infB and recN gene sequences. Sequences of strains of Vespertiliibacter gen. nov. were compared to those of the type strains of type species of all genera of the Pasteurellaceae with validly published names. For genera Aggregatibacter and Haemophilus, whole-genome sequences of reference strains were included. The tree was reconstructed in Bionumerics version 7.0 using the Jukes–Cantor correction for matrix calculation and neighbour-joining for tree building. E. coli MG1655 was included as an outgroup. Cophenic correlations are given and indicated as closed circles on the branching nodes, showing the reliability of the branching compared with the actual genetic relatedness of the taxa. Bar, 10 % sequence divergence. Details of sequence accession numbers of the novel strains are given in Table S1.
(37 uC), arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and phenylalanine deaminase tests. Acid is produced from L-arabinose, D-glucose, myo-inositol, Lrhamnose and D-xylose, but not from dulcitol, maltose, raffinose or sucrose. On API ZYM strips, strong positive reactions are observed for alkaline phosphatase, acid phosphatase, leucine arylamidase and naphthol-AS-BI-phosphohydrolase, while b-galactosidase is not produced. The G+C content of DNA from the type strain of the type species is 38.2 mol%. The type species is Vespertiliibacter pulmonis.
Vespertiliibacter pulmonis (pul.mo9nis. L. n. pulmo the lung; L. gen. n. pulmonis of the lung).
smooth. The texture of single colonies is butyrous (E40/12) or dry (E127/08T and E146/08). Known strains grow on brain heart infusion agar and on 5 % horse blood agar (both supplemented with IsoVitaleX), producing a clear zone of haemolysis around colonies on horse blood agar. In addition to the phenotypic characteristics given in the genus description, acid is produced from D-fructose and D-mannose, but not from aesculin, cellobiose, D-galactose, inulin, lactose, melezitose, melibiose, salicin or D-sorbitol. Acid production from D-mannitol (strain E146/08 is negative) and trehalose is variable (type strain E127/08T is negative). On API NH, positive reactions are obtained for D-glucose, D-fructose, alkaline phosphatase and GGT. On API ZYM, weakly positive or variable reactions are further obtained for esterase, esterase lipase (strain E40/12 is negative) and a-glucosidase.
Bacterial colonies grown on chocolate agar (5 % CO2, 24 h at 37 uC) supplemented with Vitox are 1 mm in diameter, greyish, opaque, circular, convex with an entire margin and
The type strain is E127/08T (5CCUG 64585T5DSM 27238T), isolated from the lung of a noctule bat (Nyctalus noctula) from Germany. The pathogenic potential is unknown.
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International Journal of Systematic and Evolutionary Microbiology 64
Description of Vespertiliibacter pulmonis sp. nov.
Vespertiliibacter pulmonis gen. nov., sp. nov.
Table 2. Genome similarity of strain E127/08T to strains of the type species of the family Pasteurellaceae, and to Vespertiliibacter genomospecies 1 and 2, calculated based on recN gene sequences Strain(s) Vespertiliibacter pulmonis E146/08, E40/12 Vespertiliibacter genomospecies 1 E145/08 Vespertiliibacter genomospecies 2 E157/08 Nicoletella semolina CCUG 43639T Otariodibacter oris CCUG 59994T Bibersteinia trehalosi CCUG 27190T Actinobacillus lignieresii CCUG 41384T Phocoenobacter uteri CCUG 47322T Mannheimia haemolytica CCUG 12392T Haemophilus influenzae Rd KW20 Volucribacter psittacicida CCUG 47536T Pasteurella multocida CCUG 17976T Bisgaardia hudsonensis CCUG 43067T Basfia succiniciproducens CCUG 57335T Lonepinella koalarum ATCC 700131T Aggregatibacter actinomycetemcomitans D11S-1 Avibacterium gallinarum CCUG 12391T Histophilus somni CCUG 36157T Chelonobacter oris CCUG 55632T Gallibacterium anatis CCUG 15563T Necropsobacter rosorum CCUG 28028T
Similarity to strain 127/08T 0.93–0.94 0.78 0.77 0.39 0.39 0.34 0.32 0.29 0.28 0.09 0.08 0.07 0.07 0.04 0.04 0.03 0.03 0.03 0.00 0.00 0.00
Bisgaard, M., Nørskov-Lauritsen, N., de Wit, S. J., Hess, C. & Christensen, H. (2012). Multilocus sequence phylogenetic analysis of
Avibacterium. Microbiology 158, 993–1004. Blackall, P. J., Christensen, H., Beckenham, T., Blackall, L. L. & Bisgaard, M. (2005). Reclassification of Pasteurella gallinarum,
[Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium as Avibacterium gallinarum gen. nov., comb. nov., Avibacterium paragallinarum comb. nov., Avibacterium avium comb. nov. and Avibacterium volantium comb. nov. Int J Syst Evol Microbiol 55, 353–362.
Blackall, P. J., Bojesen, A. M., Christensen, H. & Bisgaard, M. (2007).
Reclassification of [Pasteurella] trehalosi as Bibersteinia trehalosi gen. nov., comb. nov. Int J Syst Evol Microbiol 57, 666–674. Christensen, H. & Bisgaard, M. (2006). The genus Pasteurella. In The
Prokaryotes, 3rd edn, vol. 6, pp. 1062–1090. Edited by M. Dworkin, S. Lyons, E. Rosenberg, K. H. Schleifer & E. Stackebrandt. New York: Springer. Christensen, H. & Bisgaard, M. (2004). Revised definition of
Actinobacillus sensu stricto isolated from animals. A review with special emphasis on diagnosis. Vet Microbiol 99, 13–30. Christensen, H., Bisgaard, M., Aalbaek, B. & Olsen, J. E. (2004).
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Acknowledgements The authors are grateful to Nicole Dinse for technical assistance and to Berliner Artenschutz Team – BAT-e.V., Elke Mu¨hlbach and Waltraud and Helmut Zoels for providing the bat carcasses. The study was supported by the Adolf and Hildegard Isler-Stiftung and the Klara Samariter-Stiftung.
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