IJSEM Papers in Press. Published September 1, 2014 as doi:10.1099/ijs.0.064626-0
1
Faecalicoccus acidiformans gen. nov., sp. nov. isolated from the chicken
2
caecum and reclassification of Streptococcus pleomorphus (Barnes et al.,
3
1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato
4
et al., 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis
5
gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov.,
6
respectively, within the family Erysipelotrichaceae
7
Celine De Maesschalck , Filip Van Immerseel , Venessa Eeckhaut , Siegrid De Baere , Margo
8
Cnockaert , Siska Croubels , Freddy Haesebrouck , Richard Ducatelle and Peter Vandamme
9
1
1
3
1
2
1
1
2
1
3
Department of Pathology, Bacteriology and Avian Diseases, Faculty of Veterinary Medicine, Ghent
10
University, Salisburylaan 133, B-9820 Merelbeke, Belgium
11
2
12
University, Salisburylaan 133, B-9820 Merelbeke, Belgium
13
3
14
Ghent, Belgium
15
Author for correspondence: Filip Van Immerseel,
[email protected], tel. +3292647447,
16
fax. +3292647789
17
Running title: Faecalicoccus acidiformans gen.nov., sp. nov.
18
Category: New Taxa – Firmicutes and Related Organisms
19
Footnotes: The GenBank accession numbers for the 16S rRNA sequences of strains LMG 27428
20
and LMG 27427 are HQ452864 and HQ452862. The GenBank accession numbers for the hsp60
21
sequence of strains LMG 27428 , Streptococcus pleomorphus LMG 17756 and LMG 27427 are
22
KJ512711, KJ512712 and KJ512713 respectively.
23
Non-standard abbreviations: SEM, scanning electron microscopy; hsp60, 60kDa heat-shock protein;
24
HPLC-UV, high pressure liquid chromatography-ultra violet; SCFA, short-chain fatty acid
25
Department of Pharmacology, Toxicology and Biochemistry, Faculty of Veterinary Medicine, Ghent
Laboratory of Microbiology, Faculty of Sciences, Ghent University, K.L. Ledeganckstraat 35, B-9000
T
T
T
26
Abstract
27
Strains LMG 27428T and LMG 27427 were isolated from the caecal content of a chicken and
28
produced butyric, lactic and formic acid as major metabolic end products. The genomic DNA
29
G+C content of strain LMG 27428T was 40.4 mol% and 38.8 mol% for LMG 27427. On the
30
basis of 16S rRNA gene sequence similarity, both strains were most closely related to the
31
generically misclassified Streptococcus pleomorphus ATCC 29734T. Strain LMG 27428T
32
could be distinguished from S. pleomorphus ATCC 29734T based on higher lactic acid and
33
less formic acid production in M2GSC medium, a higher DNA G+C content and absence of
34
acid phosphatase, leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine
35
arylamidase activity while strain LMG 27428 was biochemically indistinguishable from S.
36
pleomorphus. The novel genus Faecalicoccus within the family Erysipelotrichaceae is
37
proposed to accommodate strain LMG 27428T = (DSM 26963T) as Faecalicoccus
38
acidiformans sp. nov. and strain LMG 27427 (DSM 26962) as Faecalicoccus pleomorphus
39
comb. nov.. Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus
40
are the generically misclassified Eubacterium cylindroides DSM 3983T (94.4 % 16S rRNA
41
sequence similarity to the type strain) and Eubacterium biforme DSM 3989T (92.7 % 16S
42
rRNA sequence similarity to the type strain). We present genotypic and phenotypic data that
43
allow the differentiation of each of these taxa and formally propose to reclassify these
44
generically misnamed Eubacterium species as Faecalitalea cylindroides comb. nov. (DSM
45
3983T = ATCC 27803T = JCM 10261T) and Holdemanella biformis comb. nov. (DSM 3989T =
46
ATCC 27806T = CCUG 28091T), respectively.
47
Main text
48
The complex microbiota of the gastrointestinal tract is dominated by microorganisms
49
belonging to the phylum Firmicutes (Ley et al., 2008; Stanley et al., 2013). Eeckhaut et al.
50
(2011) investigated the diversity and phylogenetic relationships of butyrate-producing
51
bacteria isolated from the chicken caeca and observed that butyrate producers belonging to
52
Clostridium cluster XVI as determined by Collins et al. (1994) may play a more important role
53
in the chicken gut than in the human colon. Members of Clostridium cluster XVI or the
54
Erysipelotrichaceae family stain Gram-positive with incoherent cell morphology and include
55
the generically misclassified Streptococcus pleomorphus, Eubacterium cylindroides and
56
Eubacterium biforme (Collins et al., 1994). The recent isolation of butyrate-producing
57
bacteria from the caecal content of a 14-week old Isa Brown layer type pullet yielded strains
58
LMG 27428T and LMG 27427 (Eeckhaut et al., 2011). Based on their near-entire 16S rRNA
59
gene sequence, these strains appeared to be most closely related to the above mentioned
60
members of the Erysipelotrichaceae family. In the present study we describe the
61
morphological, biochemical and genotypic characterization of strains LMG 27428T and LMG
62
27427 and their nearest phylogenetic neighbours and propose a novel classification for each
63
of these taxa.
64
Strains LMG 27428T and LMG 27427 were isolated from chicken caecal content and grown
65
anaerobically for 48 h on solid M2GSC medium pH 6 at 38 °C (Eeckhaut et al., 2011). The
66
colonies of both strains were 0.5-1.5 mm in diameter and white in colour. The cell
67
morphology was investigated using Gram-staining and scanning electron microscopy (SEM).
68
Cells of both strains stained Gram-positive and were observed as cocci-bacilli-shaped pairs
69
measuring 1.1-1.2 µm and 0.9-1.0 µm for strains LMG 27428T and LMG 27427respectively
70
(Fig. 1). Spore formation was not detected.
71
DNA was extracted from strains LMG 27428T and LMG 27427 using an alkaline lysis
72
procedure. Universal bacterial primers fD1 and rD1 (Weisburg et al., 1991) and primers H279
73
and H280 (Goh et al., 1996) were used to amplify the 16S rRNA gene and part of the 60kDa
74
heat shock protein (hsp60) gene, respectively. After purification, the amplicons were
75
sequenced by GATC Biotech (GATC Biotech AG, European Genome and Diagnostics
76
Centre, Konstanz, Germany) using the same primers for hsp60 and primers pD, Gamma*, 3
77
and O* for 16S rRNA (Coenye et al., 1999). For the 16S rRNA sequence, the closest match
78
to the deduced sequences was found using the EzTaxon-e server (Kim et al., 2012), while
79
for the hsp60 gene sequence, an independent mapping against a reference cpn60 gene
80
database was used (Hill et al., 2004). The sequences were aligned with reference 16S rRNA
81
gene sequences and hsp60 sequences using the MUSCLE program (Edgar, 2004a, b).
82
Phylogenetic trees were constructed using MEGA 6 software (Tamura et al., 2013).
83
Clustering was determined with the maximum likelihood method based on the Tamura-Nei
84
model (Tamura & Nei, 1993) and bootstrap values were calculated based on 100 replications
85
(Fig. 2-3). Strain LMG 27428T showed moderate sequence similarity to Streptococcus
86
pleomorphus ATCC 29734T (96.0 %) and lower similarity to Eubacterium cylindroides DSM
87
3983T (94.4 %) and Eubacterium biforme DSM 3989T (92.7 %). The 16S rRNA gene
88
sequence of strain LMG 27427 was 99.6 % similar to that of S. pleomorphus ATCC 29734T.
89
Phylogenetic analysis of protein encoding genes such as the hsp60 gene is commonly used
90
as an alternative identification instrument to distinguish between closely related species. The
91
analysis shown in Figure 3 demonstrates that S. pleomorphus ATCC 29734T and strain LMG
92
27427 have highly similar (98.2 %) hsp60 gene sequences that can be used to differentiate
93
them from their nearest neighbour, i.e. strain LMG 27428T.
94
Genomic DNA of S. pleomorphus ATCC 29734T and strain LMG 27427 was prepared
95
according to Pitcher et al. (1989) and DNA-DNA hybridizations were performed as described
96
by Ezaki et al. (1989) with an adapted hybridization temperature of 35 °C. The level of DNA-
97
DNA hybridization between strain LMG 27427 and S. pleomorphus ATCC 29734T was 83 %
98
(the reciprocal hybridization values were 94 and 71 %), which demonstrated that they indeed
99
represent the same species. The mol% G+C content of strains LMG 27428T and LMG 27427
100
was determined using a Waters Breeze HPLC system and an XBridge Shield RP18 column
101
maintained at 37 °C (Mesbah & Whitman, 1989). The genomic DNA G+C content of strains
102
LMG 27428T and LMG 27427 was determined to be 40.4 and 38.8 mol% respectively, which
103
is similar to that of S. pleomorphus ATCC 29734T (39.4 mol%) (Barnes et al., 1977).
104
The whole cell fatty acid methyl ester (FAME) composition was determined for strains LMG
105
27428T, LMG 27427, ATCC 29734T, DSM 3983T and DSM 3989T using an Agilent
106
Technologies 6890N gas chromatograph (Santa Clara, CA, USA). Fatty acids extraction and
107
analysis of the fatty acid methyl esters were performed according to the recommendations of
108
the Microbial Identification System, Sherlock version 3.10 (MIDI, Hewlett Packard, Newark,
109
DE, USA). Fatty acids were extracted from cultures grown in M2GSC for 24 h at 38°C under
110
anaerobic conditions. The peaks of the profiles were identified using the TSBA50
111
identification library version 5.0 (MIDI, Hewlett Packard, Newark, DE, USA). The
112
predominant fatty acid for strain LMG 27428T was iso-C19:1 (17.6 %), while for the closest
113
phylogenetic neighbours it was C16:0 (13.5 % - 24.5 %) (Table 1). Also other fatty acids were
114
present in lower percentages (above 1%) for the 5 strains: C12:0 (3.4 %- 7.3 %), C14:0 (4.4 %-
115
9.0 %), C18:0 (4.6 %- 20.3%), C18:1 ω9c (8.0 %- 13.2%) and C18:1 ω7c (2.6 %- 6.8 %).
116
The fermentation pattern of the two strains and their closest phylogenetic neighbours was
117
analysed using HPLC-UV (De Baere et al., 2013). After 24 h growth in M2GSC broth, strains
118
LMG 27428T, LMG 27427 and S. pleomorphus ATCC 29734T produced 7 - 8.5 mM lactic
119
acid, 2.4 - 3.5 mM butyric acid and 1.5-6.5 mM formic acid (Table 1). Strains LMG 27428T
120
and LMG 27427 consumed 2.8-3.7 mM acetic acid and 0.8-1.1 mM propionic acid. Both
121
Eubacterium strains DSM 3983T and DSM 3989T produced acids in the range of 0.3-4.0 mM
122
except for lactic acid of which the concentration was much higher for Eubacterium
123
cylindroides DSM 3983T (13.6 mM). Substrate utilization properties of strains LMG 27428T
124
and LMG 27427 were compared to those of their nearest phylogenetic neighbour species
125
using the API 20 A, rapid ID 32A and API ZYM systems (bioMérieux) according to the
126
manufacturer’s instructions except that the incubation was performed anaerobically for API
127
ZYM. S. pleomorphus ATCC 29734T exhibited enzymatic activity for acid phosphatase and
128
different arylamidases like leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and
129
histidine and differed from strain LMG 27427 only in alanine arylamidase and in gelatin
130
hydrolysis. Strain LMG 27428T fermented D-glucose and D-mannose, but not D-mannitol, D-
131
lactose, D-saccharose, D-maltose, salicin, D-xylose, L-arabinose, glycerol, D-cellobiose, D-
132
melezitose, D-raffinose, D-sorbitol, L-rhamnose and D-trehalose. Hydrolysis of gelatin and
133
aesculin was not detected. Strain LMG 27428T did not exhibit arylamidase or acid
134
phosphatase activity and could thus easily be distinguished from S. pleomorphus ATCC
135
29734T. E. cylindroides DSM 3983T only fermented D-saccharose and D-raffinose, while E.
136
biforme DSM 3989T fermented D-raffinose, D-mannitol, salicin, D-xylose and L-arabinose.
137
Gelatin hydrolysis was observed for E. biforme DSM 3989T, while E. cylindroides DSM 3983T
138
hydrolysed aesculin and exhibited esterase, ester lipase and α-glucosidase activity, hence
139
allowing a straightforward differentiation of strain LMG 27428T and its nearest phylogenetic
140
neighbours (Table 2).
141
In conclusion, the high degree of phenotypic similarity together with the DNA-DNA
142
hybridisation value demonstrate that strain LMG 27427 should be classified within the
143
generically misclassified S. pleomorphus ATCC 29734T (Fig. 1-2). In addition, strain LMG
144
27428T is the nearest phylogenetic neighbour of the latter but can be distinguished from it by
145
a considerable 16S rRNA divergence (4.0 %), hsp60 sequence analysis, a higher lactic acid
146
production but lower formic acid production, a higher DNA G+C content and the absence of
147
acid phosphatase and various arylamidases activities. S. pleomorphus, strain LMG 27428T,
148
and the generically misclassified E. cylindroides and E. biforme all belong to a single line of
149
descent within the family Erysipelotrichaceae and can be distinguished by both genotypic
150
and phenotypic characteristics (Table 1). On the basis of these polyphasic taxonomic data
151
we propose to classify strain LMG 27428T into the new genus Faecalicoccus, as
152
Faecalicoccus acidiformans sp. nov., and to reclassify the generically misnamed S.
153
pleomorphus (Kawamura et al., 1995; Ludwig et al., 1988) into this novel genus as
154
Faecalicoccus pleomorphus comb. nov.. Furthermore, there is a growing consensus that the
155
genus Eubacterium sensu stricto should be restricted to the type species, Eubacterium
156
limosum ATCC 8486T, and its closest phylogenetic relatives, and that the majority of
157
Eubacterium species therefore needs reclassification (Kageyama et al., 1999; Moore et al.,
158
1976; Nakazawa & Hoshino, 1994; Willems & Collins, 1996). The considerable phylogenetic
159
divergence between E. biforme DSM 3989T, E. cylindroides DSM 3983T and their nearest
160
phylogenetic neighbours, and the difference in mol% G+C content, in lactic acid production
161
and in various other biochemical characteristics (Fig. 1-3 and Table 1), together warrant the
162
reclassification of E. biforme and E. cylindroides into two new genera, as Holdemanella
163
biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov.,
164
respectively.
165
DESCRIPTION OF FAECALICOCCUS GEN. NOV.
166
Faecalicoccus (Fa.e.ca.li.coc’cus. N.L. adj. faecalis (from L.n. faex faecis), pertaining to
167
feces; N.L. masc. n. coccus (from Gr. masc. n. kokkus, a grain, seed), a coccus; N.L. masc.
168
n. Faecalicoccus, coccoid bacteria that are isolated from faecal material.).
169
The cells are obligate anaerobic non-spore-forming cocci that stain Gram-positive. The
170
bacteria have lactic acid as major fermentation product from glucose and mannose, and
171
exhibit naphthol-A,S-BI-phosphohydrolase activity. The major fatty acid (> 10 % of total fatty
172
acids) is C16:0. The DNA G+C content is 39-41 mol%. Strains have been isolated from avian
173
caecum. The type species is Faecalicoccus acidiformans.
174
DESCRIPTION OF FAECALICOCCUS ACIDIFORMANS SP. NOV.
175
Faecalicoccus acidiformans (a.ci.di.for’mans. N.L. n. acidum (from L. adj. acidus, sour), an
176
acid; L. part. adj. formans, forming; N.L. part. adj. acidiformans, acid-forming bacteria.).
177
The cells are anaerobic, non-motile, Gram-positive non-sporulating cocci. Individual cells are
178
about 1.1-1.2 µm long and occur in pairs or short chains. On M2GSC agar plates, they form
179
minute colonies, white in colour and measure 1-1.5 mm in diameter after 48 h growth at 38
180
°C in an anaerobic workstation. The major fatty acids (> 10 % of total fatty acids) are C16:0,
181
C18:1 ω9c and iso-C19:1. Test for acid production using API 20 A shows positive reaction with
182
D-glucose and D-mannose, but not with D-mannitol, D-lactose, D-saccharose, D-maltose,
183
salicin, D-xylose, L-arabinose, D-cellobiose, D-melezitose, D-raffinose, D-sorbitol, L-
184
rhamnose and D-trehalose. There is no urease activity, no hydrolysis of aesculin and gelatin
185
observed in the API 20 A test. Test for enzyme activities by use of API ZYM shows positive
186
reaction with naphthol-AS-BI-phosphohydrolase, but not with acid phosphatase, alkaline
187
phosphatase, esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine
188
arylamidase,
189
galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase,
190
α-mannosidase and α-fucosidase. Test for enzyme activities by use of rapid ID 32 A shows
191
no positive reaction. High amounts of lactic acid are produced as main fermentation product
192
in M2GSC broth, in addition to moderate amounts of butyric acid and low amounts of formic
193
acid. The G+C content of the DNA is 40.4 mol%.
194
The type strain LMG 27428T (DSM 26963T) was isolated from the caecal content of a 14-
195
week old Isa Brown layer chicken in Ghent (Belgium) in 2006.
196
DESCRIPTION OF FAECALICOCCUS PLEOMORPHUS COMB. NOV.
197
Faecalicoccus pleomorphus (ple.o.mor’phus. N.L. masc. adj. pleomorphus (from Gr. adj.
198
pleos, full, and Gr. n. morphê, form, shape), pleomorphic, different forms for the bacteria.).
199
Basonym: Streptococcus pleomorphus Barnes et al., 1977 (Approved Lists 1980).
200
The description of Faecalicoccus pleomorphus is as given for Streptococcus pleomorphus by
201
Barnes et al. (1977), with the following additions. The major fatty acids (> 10 % of total fatty
202
acids) are C16:0 and C18:1 ω9c. Test for acid production by use of API 20 A shows positive
203
reaction with D-glucose and D-mannose, but not with D-mannitol, D-lactose, D-saccharose,
204
D-maltose, salicin, D-xylose, L-arabinose, D-cellobiose, D-melezitose, D-raffinose, D-sorbitol,
205
L-rhamnose and D-trehalose. There is no urease activity, no hydrolysis of aesculin while
cysteine
arylamidase,
trypsin,
α-chymotrypsin,
α-galactosidase,
β-
206
some strains shows the hydrolysis of gelatin by the use of API 20 A. Test for enzyme
207
activities by use of API ZYM shows positive reaction with acid phosphatase, leucine
208
arylamidase and naphthol-AS-BI-phosphohydrolase, but not with alkaline phosphatase,
209
esterase (C4), esterase lipase (C8), lipase (C14), valine arylamidase, cysteine arylamidase,
210
trypsin, α-chymotrypsin, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase,
211
β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Test for
212
enzyme activities by use of rapid ID 32 A shows positive reaction with leucine arylamidase,
213
arginine arylamidase, pyroglutamic acid arylamidase, glycine arylamidase, histidine
214
arylamidase and alanine arylamidase for some strains but not with arginine dihydrolase, α-
215
galactosidase,
216
glucosidase, α-arabinosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, glutamic acid
217
decarboxylase, α-fucosidase, alkaline phosphatase, proline arylamidase, phenylalanine
218
arylamidase, tyrosine arylamidase, serine arylamidase and glutamyl glutamic acid
219
arylamidase.. On M2GSC agar, the species grows in white colonies of 1.0-1.5 mm after 48 h
220
incubation in anaerobic conditions at 38 °C. Overnight growth in M2GSC broth results in
221
moderate amounts of butyric acid and formic acid.
222
The type strain is LMG 17756T (= ATCC 29734T, DSM 20574T).
223
DESCRIPTION OF HOLDEMANELLA GEN. NOV.
224
Holdemanella (Hol.de.man.el’la. N.L. fem. dim. n. Holdemanella, named in honor of Lillian V.
225
Holdeman Moore, a contemporary American microbiologist, for her outstanding contribution
226
to the bacteriology of anaerobes).
227
The members of the genus are strictly anaerobic. The cells stain Gram-positive, are coccus-
228
shaped and often form pairs or chains. They are non-motile and non-spore-forming. D-
229
glucose, D-mannitol, salicin, D-xylose, L-arabinose, D-mannose and D-raffinose are
230
fermented. The major fatty acids (> 10 % of total fatty acids) are C16:0 and C18:0. The DNA
β-galactosidase,
β-galactosidase
6
phophatase,
α-glucosidase,
β-
231
G+C content is 32-34 mol%. Strains have been isolated from human faeces. The type
232
species is Holdemanella biformis.
233
DESCRIPTION OF HOLDEMANELLA BIFORMIS COMB. NOV.
234
Holdemanella biformis (bi.for’mis L. fem. adj. biformis, two-shaped, two-formed (pertaining to
235
cellular morphology)).
236
Basonym: Eubacterium biforme (Eggerth, 1935) Prévot 1938 (Approved Lists 1980).
237
The description of Holdemanella biformis is as given for Eubacterium biforme by Moore and
238
Holdeman (1974), with the following additions. The major fatty acids (> 10 % of total fatty
239
acids) are C16:0 and C18:0. Test for acid production by use of API 20 A shows positive reaction
240
with D-glucose, D-mannitol, salicin, D-xylose, L-arabinose, D-raffinose and D-mannose, but
241
not with D-lactose, D-saccharose, D-maltose, D-cellobiose, D-melezitose, D-sorbitol, L-
242
rhamnose and D-trehalose. There is no urease activity, no hydrolysis of aesculin but gelatine
243
is hydrolyzed. Test for enzyme activities by use of API ZYM shows positive reaction with acid
244
phosphatase, alkaline phosphatase and naphthol-AS-BI-phosphohydrolase, but not with
245
esterase (C4), esterase lipase (C8), lipase (C14), leucine arylamidase, valine arylamidase
246
cysteine
247
glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase
248
and α-fucosidase. Test for enzyme activities by use of rapid ID 32 A shows no positive
249
reaction.
250
propionic acid are produced.
251
The type strain is DSM 3989T (= ATCC 27806T, CCUG 28091T), and has been isolated from
252
human faeces.
253
DESCRIPTION OF FAECALITALEA GEN. NOV.
254
Faecalitalea (Fa.e.ca.li.ta’le.a N.L. adj. faecalis (from L. n. faex faecis), pertaining to faeces;
255
L.fem. n. talea, a rod; N.L. fem. n. Faecalitalea, rods isolated from faeces.)
arylamidase,
trypsin,
α-chymotrypsin,
α-galactosidase,
β-galactosidase,
β-
In M2GSC broth, moderate amounts of butyric acid, acetic acid, lactic acid and
256
The members of the genus are strictly anaerobic. The cells stain Gram-positive and are
257
pleomorphic rods which occur single, in pairs or in short chains. Filamentous forms are often
258
seen. They are non-motile and non-spore-forming. D-glucose, D-sucrose, D-mannose and D-
259
raffinose are fermented. The major end products of metabolism are lactic and butyric acid.
260
The major fatty acids (> 10% of total fatty acids) are C16:0 and C18:1 ω9c. The DNA G+C
261
content is 26-35 mol%. Strains have been isolated from the human, chicken and pig gut. The
262
type species is Faecalitalea cylindroides.
263
DESCRIPTION OF FAECALITALEA CYLINDROIDES COMB. NOV.
264
Faecalitalea cylindroides (cy.lin.dro’i.des. Gr. n. kulindros, a cylinder; L. suff. –oides (from Gr.
265
suff. eides, from Gr. N. eidos, which is seen, form, shape, figure), resembling, similar; N.L.
266
fem. adj. cylindroides, cylinder-shaped.) .
267
Basonym: Eubacterium cylindroides (Rocchi 1908) Holdeman and Moore 1970 (Approved
268
Lists 1980).
269
The description of Faecalitalea cylindroides is as given for Eubacterium cylindroides by Cato
270
et al. (1974), with the following additions. The major fatty acids (> 10 % of total fatty acids)
271
are C16:0 and C18:1 ω9c. Test for acid production by use of API 20 A shows positive reaction
272
with D-glucose, D-saccharose, D-raffinose and D-mannose, but not with D-mannitol, D-
273
lactose, D-maltose, salicin, D-xylose, L-arabinose, D-cellobiose, D-melezitose, D-sorbitol, L-
274
rhamnose and D-trehalose. There is no urease activity, no hydrolysis of gelatin but aesculine
275
is hydrolyzed. Test for enzyme activities by use of API ZYM shows positive reaction with acid
276
phosphatase, alkaline phosphatase, esterase (C4), esterase lipase (C8), α-glucosidase and
277
naphthol-AS-BI-phosphohydrolase, but not with lipase (C14), leucine arylamidase, valine
278
arylamidase,
279
galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase
280
and α-fucosidase. Test for enzyme activities by use of rapid ID 32 A shows no positive
cysteine
arylamidase,
trypsin,
α-chymotrypsin,
α-galactosidase,
β-
281
reaction. High amounts of lactic acid are produced as main fermentation product in M2GSC
282
broth, in addition to moderate amounts of butyric acid.
283
The type strain is DSM 3983T (= ATCC 27803T, JCM 10261T) and has been isolated from
284
clinically normal human faeces.
285
Acknowledgements
286
We thank Wim Van Den Broeck, Jurgen De Craene and Bart De Pauw of the Department of
287
Morphology, Faculty of Veterinary Medicine, Ghent University, for scanning electron
288
microscopy. Dr. J.P. Euzéby is acknowledged for his expert nomenclatural advice.
289
References
290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339
Barnes, E. M., Impey, C. S., Stevens, B. J. & Peel, J. L. (1977). Streptococcus pleomorphus sp.nov.: an anaerobic streptococcus isolated mainly from the caeca of birds. J Gen Microbiol 102, 45-53. Cato, E. P., Salmon, C. W. & Holdeman, L. V. (1974). Eubacterium-Cylindroides (Rocchi) Holdeman and Moore - Emended Description and Designation of Neotype Strain. Int J Syst Bacteriol 24, 256-259. Coenye, T., Falsen, E., Vancanneyt, M., Hoste, B., Govan, J.R.W., Kersters, F & Vandamme, P. (1999). Classification of Alcaligenes faecalis-like isolates from the environment and human clinical samples as Ralstonia gilardii sp.nov. Int J Syst Bacteriol 49, 405-413 Collins, M. D., Lawson, P. A., Willems, A., Cordoba, J. J., Fernandez-Garayzabal, J., Garcia, P., Cai, J., Hippe, H. & Farrow, J. A. (1994). The phylogeny of the genus Clostridium: proposal of five new genera and eleven new species combinations. Int J Syst Bacteriol 44, 812-826. De Baere, S., Eeckhaut, V., Steppe, M., De Maesschalck, C., De Backer, P., Van Immerseel, F. & Croubels, S. (2013). Development of a HPLC-UV method for the quantitative determination of four short-chain fatty acids and lactic acid produced by intestinal bacteria during in vitro fermentation. J Pharm Biomed Anal 80, 107-115. Edgar, R. C. (2004a). MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113. Edgar, R. C. (2004b). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32, 1792-1797. Eeckhaut, V., Van Immerseel, F., Croubels, S., De Baere, S., Haesebrouck, F., Ducatelle, R., Louis, P. & Vandamme, P. (2011). Butyrate production in phylogenetically diverse Firmicutes isolated from the chicken caecum. Microb Biotechnol 4, 503-512. Eggerth, A. H. (1935). The Gram-positive Non-spore-bearing Anaerobic Bacilli of Human Feces. J Bacteriol 30, 277-299. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric Deoxyribonucleic Acid Deoxyribonucleic Acid Hybridization in Microdilution Wells as an Alternative to Membrane-Filter Hybridization in Which Radioisotopes Are Used to Determine Genetic Relatedness among Bacterial Strains. Int J Syst Bacteriol 39, 224-229. Goh, S. H., Potter, S., Wood, J. O., Hemmingsen, S. M., Reynolds, R. P. & Chow, A. W. (1996). HSP60 gene sequences as universal targets for microbial species identification: studies with coagulase-negative staphylococci. J Clin Microbiol 34, 818-823. Hill, J. E., Penny, S. L., Crowell, K. G., Goh, S. H. & Hemmingsen, S. M. (2004). cpnDB: a chaperonin sequence database. Genome research 14, 1669-1675. Holdeman, L. V. and Moore, W. E. C. (1970) Eubacterium. In Elizabeth P. Cato, C. S. Cummins, Lilian V. Holdeman, J. L. Johnoson, W. E. C. Moore, R. M. Smibert, and L. DS. Smith. Outline of clinical methods in anaerobic bacteriology, 2nd rev. Virginia Polytechnic Institute Anaerobe Laboratory, Blacksburg, Va. Kageyama, A., Benno, Y. & Nakase, T. (1999). Phylogenetic and phenotypic evidence for the transfer of Eubacterium aerofaciens to the genus Collinsella as Collinsella aerofaciens gen. nov., comb. nov. Int J Syst Bacteriol 49, 557-565. Kawamura, Y., Hou, X. G., Sultana, F., Miura, H. & Ezaki, T. (1995). Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int J Syst Bacteriol 45, 406408. Kim, O. S., Cho, Y. J., Lee, K., Yoon, S. H., Kim, M., Na, H., Park, S. C., Jeon, Y. S., Lee, J. H. & other authors (2012). Introducing EzTaxon-e: a prokaryotic 16S rRNA gene sequence database with phylotypes that represent uncultured species. Int J Syst Bacteriol 62, 716721.
340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375
Ley, R. E., Hamady, M., Lozupone, C., Turnbaugh, P. J., Ramey, R. R., Bircher, J. S., Schlegel, M. L., Tucker, T. A., Schrenzel, M. D. & other authors (2008). Evolution of mammals and their gut microbes. Science 320, 1647-1651. Ludwig, W., Weizenegger, M., Kilpperbalz, R. & Schleifer, K. H. (1988). PhylogeneticRelationships of Anaerobic Streptococci. Int J Syst Bacteriol y 38, 15-18. Mesbah, M. & Whitman, W. B. (1989). Measurement of deoxyguanosine/thymidine ratios in complex mixtures by high-performance liquid chromatography for determination of the mole percentage guanine + cytosine of DNA. Journal of chromatography 479, 297-306. Moore, W. E. & Holdeman, L. V. (1974). Human fecal flora: the normal flora of 20 JapaneseHawaiians. Appl Microbiol 27, 961-979. Moore, W. E. C., Johnson, J. L. & Holdeman, L. V. (1976). Emendation of Bacteroidaceae and Butyrivibrio and Descriptions of Desulfomonas Gen-Nov and 10 New Species in Genera Desulfomonas, Butyrivibrio, Eubacterium, Clostridium, and Ruminococcus. Int J Syst Bacteriol 26, 238-252. Nakazawa, F. & Hoshino, E. (1994). Genetic-Relationships among Eubacterium Species. Int J Syst Bacteriol 44, 787-790. Pitcher, D. G., Saunders, N. A. & Owen, R. J. (1989). Rapid Extraction of Bacterial Genomic DNA with Guanidium Thiocyanate. Letters in Applied Microbiology 8, 151-156. Prévot, A. R. (1938) études de systematique bactérienne. III. Invalidité du genre Bacteroides Castellani et Chalmers. Démembrement et réclassification. Ann. Inst. Pasteur 60: 292 Rocchi, G. (1908) Lo stato attuale delle nostre cognizioni sui germi anaerobi. Tossi-infezioni putirde e gangrenose. Capitolo II. Bull Sci. Med. 8, 457-479 Stanley, D., Geier, M. S., Denman, S. E., Haring, V. R., Crowley, T. M., Hughes, R. J. & Moore, R. J. (2013). Identification of chicken intestinal microbiota correlated with the efficiency of energy extraction from feed. Vet Microbiol 164, 85-92. Tamura, K. & Nei, M. (1993). Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molecular biology and evolution 10, 512-526. Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. (2013). MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular biology and evolution 30, 27252729. Weisburg, W. G., Barns, S. M., Pelletier, D. A. & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173, 697-703. Willems, A. & Collins, M. D. (1996). Phylogenetic relationships of the genera Acetobacterium and Eubacterium sensu stricto and reclassification of Eubacterium alactolyticum as Pseudoramibacter alactolyticus gen nov, comb nov. Int J Syst Bacteriol 46, 1083-1087.
376
Figures T
377
Fig. 1: Scanning electron micrograph of cells of Faecalicoccus acidiformans LMG 27428 . (scale bar = 1µm).
378
Fig. 2: Rooted tree showing the 16S rRNA phylogenetic relationship of Faecalicoccus pleomorphus gen. nov.,
379
comb. nov. and Faecalicoccus acidiformans sp. nov. with some other members of the Erysipelotrichaceae family.
380
The tree was constructed using the maximum likelihood method based on the Tamura-Nei model and based on a
381
comparison of approximately 1300 nucleotides. Percentage bootstrap values, based on 100 replications, are
382
shown at branch points. The accession numbers of reference organisms are included. Bar, 0.02 substitutions per
383
nucleotide position. Erysipelothrix rhusiopathiae ATCC19414 was used as outgroup. The evolutionary analyses
384
were conducted using MEGA 6 software.
385
Fig. 3: Phylogenetic tree based on the hsp60 (heat-shock protein 60kDa) gene sequences of the two unknown
386
and three reference strains. The hsp60 gene sequence of two reference strains DSM 3989 and DSM 3983 was
387
taken from the cpn60 database while for the two unknown strains and the reference strain LMG 17756 we
388
sequenced them in this study. The tree, constructed using the maximum likelihood method based on the Tamura-
389
Nei model, showed a comparison of two unknown and three reference strains of family Erysipelotrichaceae.
390
Numbers at the nodes indicate the percentages of bootstrap sampling, derived from 100 samples, supporting the
391
internal branches. The accession numbers of the peptide GenBank are included between brackets. Scale bar:
392
0.05 substitutions per nucleotide position. The evolutionary analyses were conducted using MEGA 6 software.
393
T
T
T
T
394
Tables
395
Table 1: Cellular fatty acids profiles of two strains LMG 27428 and LMG 27427 and their closer phylogenetic
396
neighbours of the family Erysipelotrichaceae (determined in this study). Strains: 1, Faecalicoccus acidiformans
397
LMG 27428 ; 2, F. pleomorphus LMG 27427; 3, F. pleomorphus ATCC 29734 ; 4, Faecalitalea cylindroides
398
ATCC 27803 ; 5, Holdemanella biformis DSM 3989 . Major differences are highlighted in bold. ND, not detected
T
T
T
T
Cellular fatty acid Saturated fatty acid C10:0 C11:0 C12:0 C13:0 C14:0 C16:0 C18:0 C19:0 Unsaturated fatty acids C15:1 ω6c C16:1 ω9c C17:1 ω8c C17:1 ω6c C18:1 ω9c C18:1 ω7c C18:1 ω6c C20:1 ω9c Branched fatty acids iso-C14:0 iso-C15:0 anteiso-C15:0 iso-C16:0 iso-C17:0 anteiso-C17:0 iso-C18:1 H iso-C19:1 I Hydroxyl fatty acids C16:0 3-OH Summed feature 1 Summed feature 2 Summed feature 3 Summed feature 4 Summed feature 5 Summed feature 6 Summed feature 7
T
1
2
3
4
5
0.9 0.8 5.3 1.0 7.5 12.6 4.6 ND
0.7 0.2 3.4 1.2 6.3 13.5 7.1 0.4
0.6 ND 4.0 1.7 9.0 15.5 10.6 ND
1.2 ND 4.5 1.7 9.0 16.1 7.0 ND
1.0 ND 7.3 ND 4.4 24.5 20.3 ND
ND 1.5 3.5 ND 10.7 5.3 ND 0.5
0.9 0.8 6.3 1.3 12.8 5.8 ND ND
1.4 1.0 4.1 0.8 13.2 6.8 ND ND
1.1 1.8 6.4 1.0 12. 3.7 ND ND
ND ND ND ND 8.0 2.6 3.5 ND
ND 0.5 1.0 ND ND ND 2.3 17.3
0.1 0.5 0.7 0.3 0.4 ND 3.2 10.7
ND ND 1.1 ND ND ND 1.6 8.7
ND ND 0.8 ND ND ND 1.7 6.5
ND 1.1 2.5 0.9 ND 1.3 ND 2.1
ND 0.4 ND 4.4 11.2 2.6 1.1 ND
0.4 0.3 0.7 5.4 9.6 2.3 1.9 0.2
0.6 ND ND 6.0 7.0 2.1 1.3 ND
ND 1.1 1.3 7.3 9.0 2.0 0.7 ND
1.4 ND ND 0.4 8.7 7.1 ND ND
399 400 401 402 403 404
T
Table 2: Comparison between the two strains LMG 27428 and LMG 27427 and their closest phylogenetic T
neighbours of the family Erysipelotrichaceae. Strains: 1, Faecalicoccus acidiformans LMG 27428 ; 2, F. T
T
Holdemanella biformis DSM 3989 . All data arefrom this study unless indicated otherwise. +, Positive; -, Negative; ®
®
ND, not detected (§ api 20A; ‖ api ZYM; ¶ rapid ID 32A)
Characteristic DNA G+C content (%mol) Fermentation acids (mM) Butyric acid Acetic acid Propionic acid Lactic acid Formic acid Acid production from (API system): § D-Mannitol § D-Saccharose § Salicin § D-Xylose § L-Arabinose § D-Raffinose Hydrolysis of: § Gelatin § Aesculin Production of (API system): ‖ Alkaline phosphatase ‖ Esterase (C4) ‖ Ester lipase (C8) ‖ Acid phosphatase ‖¶ α-Glucosidase ¶ Arginine arylamidase ¶ Leucyl glycine arylamidase ¶ Leucine arylamidase‖ ¶ Pyroglutamic acid arylamidase ¶ Alanine arylamidase ¶ Glycine arylamidase ¶ Histidine arylamidase
405 406 407
T
pleomorphus LMG 27427; 3, F. pleomorphus LMG 17756 ; 4, Faecalitalea cylindroides ATCC 27803 ; 5,
1
2
3
40.4
38.8
39.4
3.5 ± 0.2 -3.7 ± 0.6 -0.8 ± 0.1 8.5 ± 0.2 1.5 ± 0.1
2.4 ± 0.3 -2.8 ± 0.3 -1.1 ± 0.1 7.9 ± 0.2 3.8 ± 0.1
-
4 *
5 †
‡
31.0
33.8
3.2 ± 0.3 N.D N.D 7.0 ± 0.3 6.5 ± 0.3
2.6 ± 0.6 1.0 ± 0.3 1.3 ± 0.4 13.6 ± 2.1 0.7 ± 0.2
3.2 ± 0.8 2.4 ± 0.5 3.9 ± 0.2 2.1 ± 0.4 0.3 ± 0.1
-
-
+ +
+ + + + +
-
+ -
-
+
+ -
-
+ + + + + + + +
+ + + + + + +
+ + + + + -
+ + -
DNA G+C content data were taken from: *, (Barnes et al., 1977); †,(Cato et al., 1974); ‡, (Eggerth 1935)
408
409
410 411
412
