IJSEM Papers in Press. Published May 27, 2014 as doi:10.1099/ijs.0.056937-0

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Bifidobacterium aesculapii sp. nov., from the faeces of the baby common marmoset (Callithrix

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jacchus)

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Modesto M.1, Michelini S.1, Stefanini I. 1, Ferrara A.2, Tacconi S. 2, Biavati B. 1, Mattarelli P. 1*

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1

Department of Agricultural Sciences, University of Bologna Italy

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2

Aptuit srl, Verona, Italy

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*Corresponding author:

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Paola Mattarelli, 1Department of Agricultural Sciences, University of Bologna, Viale Fanin 42,

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40127 Bologna, Italy Phone+393497620217 Fax+390512096274

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[email protected]

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Key words: New species, Bifidobacterium, Bifidobacterium aesculapii, common marmoset, Callitrix

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jacchus

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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences and hsp60, rpoB

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and clpC partial gene sequences of B. aesculapii MRM 3/1T are KC807989, KC997237, KC 997239

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and KF 164211 and of B. aesculapii MRM4/2 are KC 807990, KC997238, KC997240 and

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KF164212.

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Gram-positive-staining, microaerophilic, non-spore-forming, fructose-6-phosphate phosphoketolase

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positive bacterial strains with a peculiar morphology were isolated from faecal samples of baby

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common marmoset (Callithrix jacchus). Cells of these strains showed a morphology never found in

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bifidobacterial species, which resembled a coiled snake, always coiled or ring shaped or forming a

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“Y” shape. MRM 3/1 and MRM 4/2 were chosen as representative strains and further characterized.

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The bacteria utilized a wide range of carbohydrates and produced urease. Glucose was fermented to

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acetate and lactate. The strain MRM 3/1 showed a peptidoglycan type unique among members of

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the genus Bifidobacterium. The DNA base composition was 64.7mol% G+C. Almost complete 16S

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rRNA, hsp60, clpC and rpoB gene sequences were obtained and phylogenetic relationships were

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determined. Comparative analysis of 16S rRNA gene sequences showed that strains MRM 3/1 and

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MRM 4/2 had the highest similarities to Bifidobacterium scardovii (DSM 13734T) (94.6%) and

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Bifidobacterium stellenboschense (DSMZ 23968T) (94.5%). Analysis of hsp60 showed that both

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strains were closely related to B. stellenboschense (97.5%), however, despite this high degree of

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similarity, our isolates could be distinguished from B. stellenboschense DSMZ 23968T also by low

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levels of DNA-DNA relatedness (30.4%). Therefore, strains MRM 3/1 and MRM 4/2 were located

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in an actinobacterial cluster and were more closely related to the genus Bifidobacterium than to

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other genera in Bifidobacteriaceae. On the basis of these results strains MRM 3/1T and MRM 4/2

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represent a novel species within the genus Bifidobacterium, for which the name Bifidobacterium

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aesculapii sp. nov. is proposed; the type strain is MRM 3/1T (=DSM 26737T;= JCM 18761T).

46 47

Bifidobacteria are Gram positive, anaerobic, non-motile and non-spore-forming bacteria and

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represent one of the great bacterial groups within the Actinobacteria. Bifidobacterium spp. are

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typically found in the gastrointestinal tracts (GIT) of humans and other mammals and hindgut of

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most social insects, such as honey bees, wasps, cockroaches and bumblebeesbees (Biavati &

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Mattarelli, 2012; Kopečný et al., 2010; Killer et al., 2009). They are generally host-animal-specific

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microorganisms and can be separated into “human” and “animal” group based on their distribution

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(Ventura et al., 2004). Bifidobacteria are well known to posses beneficial effects and to play an

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important role in maintaining the health of their host (Turroni et al., 2011). Hence, it is the

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importance of understanding diversity of bifidobacteria in GIT and faeces.

56

During the characterization of bifidobacterial distribution in primates, six bifidobacterial strains

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with similar morphology were isolated from fresh faecal samples of baby subjects of the common

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marmoset (Callithrix jacchus), which were individually collected from five animals kept in animal

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houses in Aptuit s.r.l. Verona, North Italy. The common marmoset is a small exudivore monkey

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from the new world, which has developed a large specialized caecum for the digestion of complex

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carbohydrates found in tree exudates (Caton et al., 1996; Bailey & Coe, 2002). As microbiota

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growth and composition are affected by GIT functioning, such as motility and nutrient availability

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in intestinal lumen, it is likely that this evolutionary adaptation may influence the concentrations

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and the types of bacteria that reside as part of the normal intestinal microbiota (Bailey & Coe,

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2002).

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Samples of fresh rectal swabs from common marmosets were serially diluted with Peptone Water

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(Merck) supplemented with cysteine hydrochloride (0.5 g/L); aliquots of each dilution were

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inoculated onto TPY agar supplemented with mupirocine (100 mg/L) (Applichem) which is a

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selective agent for bifidobacteria (Rada & Petr, 2000). In each subject we observed cells of a

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bacterium with a new and unusual morphology, resembling a coiled snake. A total of six isolates

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with this morphology were obtained from the 5 baby marmosets. They were namely MRM 3/1,

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MRM 4/2, MRM 5/13, MRM 8/7, MRM 4/6 and MRM 4/7. The isolates were subcultured on TPY,

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their cells were suspended in a 10 % (w/v) sterile skim milk solution, supplemented with lactose (3

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%) and yeast extract (0.3 %) and kept both freeze dried and frozen at – 120°C. For all experiments,

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the strains were cultivated under anaerobic conditions and maintained in TPY broth, pH 6.9 at 37

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°C, unless indicated otherwise.

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In the present study the morphological, biochemical and molecular characterizations of the isolates

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were carried out.

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Chromosomal DNA was obtained from isolates according to the procedure of Rossi et al. (2000),

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with slight modifications. Briefly, the cells of overnight cultures were pelletted and resuspended in

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1 ml of TE buffer (pH 7.6) containing 50 mg/ml lysozyme and then incubated overnight at 37°C.

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For discrimination of the isolates, molecular typing was performed using the enterobacterial

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repetitive intergenic consensus sequences (ERIC) PCR with the primer pairs ERIC1 (5’-

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ATGTAAGCTCCTGGGGATTCAC-3’) and ERIC2 (5’-AAGTAAGTGACTGGGGTGAGCG-3’)

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(Ventura et al., 2003). Each 20 µl reaction mixture contained 3.5 mM of MgCl2, 20 mM of Tris-

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HCl, 50 mM of KCl, 200 µM of each deoxynucleoside triphosphate (HotStarTaq plus DNA

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polymerase MasterMix Kit; Qiagen), 30 ng of DNA template and 2 µM of each primer.

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Amplifications were performed using an Applied Biosystem Veriti Thermal Cycler (Applied

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Biosystems, Foster City, CA) with the following temperature profiles: 1 cycle at 94 °C for 3 min;

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35 cycles at 94 °C for 30 s, at 48 °C for 30 s, and at 72 °C for 4 min; and 1 cycle at 72 °C for 6 min.

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Aliquots of each amplification reaction mixture (15 µl each) were separated by electrophoresis in 2

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% (w/v) agarose gels at a voltage of 7 V/cm. Gels were ethidium bromide stained (0.5 µg/ml) and

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photographed under 260-nm UV light.

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Given that isolates revealed two different ERIC-PCR profiles (see Supplementary fig. S1), strains

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MRM 3/1T and MRM 4/2 were selected as representatives and further characterized.

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The partial 16S rRNA genes of the strains MRM 3/1T and MRM 4/2 were amplified by PCR using

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the

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AAGGAGGTGATCCAGCCGCA-3’) (Kim et al., 2010). The partial hsp60, rpoB and clpC gene

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sequences were also obtained using the primer pairs HspF3 (5’-ATCGCCAAGGAGATCGAGCT-

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3’) and HspR4 (5’-AAGGTGCCGCGGATCTTGTT-3’), BifF (5’-TCGATCGGGCACATACGG-

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3’) and BifR2 (5’-CGACCACTTCGGCAACCG-3’) (Kim et al., 2010) and BClpC-F (5’–

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ATCGCSGARACBATYGAGA-3’)

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(Watanabe et al., 2009), respectively.

primer

pairs

Bif285

(5’-GAGGGTTCGATTCTGGCTCAG-3’)

and

BClpC-R

and

Bif261

(5’-

(5’-ATRATGCGCTTGTGCARYT-3’)

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Each PCR mixture (20 µl) contained 1.5 mM of MgCl2, 20 mM of Tris-HCl, 50 mM of KCl, 200

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µM of each deoxynucleoside triphosphate (HotStarTaq plus DNA polymerase MasterMix Kit;

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Qiagen), 0.1 µM of each primer and 30 ng or 200 ng of DNA template for 16S rRNA gene and for

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each housekeeping gene, respectively. Amplifications were performed using a TGradient thermal

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cycler (Biometra). A touch down PCR was used to amplify 16S rRNA gene and for all phylogenetic

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markers, and it was performed as follows: initial denaturation (95 °C, 5 min) for HotStarTaq plus

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activation; 4 cycles with denaturation at 94 °C for 60 s, annealing at 62 °C for 60 s, and extension at

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72 °C for 90 s; 21 cycles with denaturation at 94 °C for 60 s, annealing at 60°C for 60 s, and

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extension at 72 °C for 90 s; 15 cycles with denaturation at 94 °C for 60 s, annealing at 58°C for 60

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s, and extension at 72 °C for 90 s; the PCR was completed with a single elongation step (10 min at

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72 °C).

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All the resulting amplicons were separated on 2 % agarose gels, followed by ethidium bromide

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staining. PCR fragments were purified using the NucleoSpin extract II kit (Macherey-Nagel, Duren,

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Germany) following manufacturer’s instructions.

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The 16S rRNA gene sequences were directly sequenced whereas, for inferring a correct

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phylogenetic study hsp60, clpC and rpoB gene sequences were cloned using InsTAclone PCR

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Cloning Kit (Fermentas). All sequencing reactions were performed by Eurofins MWG Operon.

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Almost-complete 16S rRNA gene sequence assembly was performed using

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program (Huang, 1992), in BIOEDIT (Hall, 1999).

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After editing, the closest known relatives of the novel strains were determined by comparison with

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DataBase entries and sequences of the closely related species were retrieved from the EMBL and

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GenBank nucleotide databases. Pairwise nucleotide sequence similarity values were calculated

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using the EzTaxon server (www.eztaxon.org) which provides a web-based tool (Kim et al., 2012).

CAP,

contig assembly

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The 16S rRNA gene sequences (about 1421 bp) of strains MRM 3/1T and MRM 4/2 and of those of

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their closest relatives retrieved from the DDBJ/GenBank/EMBL databases were aligned by using

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the

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total of 43 partial 16S rRNA gene sequences including those of members of the genus

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Bifidobacterium and of related genera, was constructed with the neighbor-joining method (Saitou &

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Nei, 1987) and the evolutionary distances were computed using the Kimura 2-parameter method

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(Kimura, 1980) by using the

CLUSTAL_X2

program (version 1.82) (Thompson et al., 1997). A phylogenetic tree based on a

MEGA

5.05 program (Tamura et al., 2011). The tree was rooted using

T

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Micrococcus luteus DSM 20030 (Fig. 1). The statistical reliability of the tree was evaluated by

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bootstrap analysis of 1000 replicates (Felsenstein, 1985) and the tree topology was also confirmed

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with the maximum-likelihood (Cavalli-Sforza & Edwards, 1967) maximum-parsimony (Fitch,

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1971) and least squares (Fitch & Margoliash, 1967) methods, by using MEGA 5.05 program (Tamura

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et al., 2011). The 16S rRNA gene sequences similarity between the strains MRM 3/1T and MRM

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4/2 was about 99.6%. Furthermore they showed low sequence similarities to the known

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bifidobacteria and the highest values were found to Bifidobacterium scardovii and Bifidobacterium

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stellenboschence (94.6 and 94.5%, respectively) which are a recently new species described in red -

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handed tamarin (Saguinus midas) by Endo et al. (2012). Based on a neighbor joining analysis, the

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novel strains are phylogenetically related to B. scardovii (Fig. 1). Similar tree topologies were

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obtained by using the maximum-likelihood (see Supplementary Fig. S2), maximum-parsimony and

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least squares methods (data not shown).

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Multilocus sequence analysis is a reliable and robust technique for the identification and

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classification of bacterial isolates to the species level as alternatives to 16S rRNA gene sequence

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analysis (Martens et al., 2008). For this reason, the phylogenetic location of the novel strains was

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also verified by the analysis of three additional phylogenetic markers such as hsp60, clpC and rpoB

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genes, which have proven to be discriminative for classification of the genus Bifidobacterium (Kim

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et al., 2010; Ventura et al., 2006; Jian et al., 2001).

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For hsp60, clpC and rpoB genes, the sequences of strains MRM 3/1T and MRM 4/2 and of those of

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their closest relatives retrieved from the DDBJ/GenBank/EMBL databases were aligned by using

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MAFFT program, at

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Gblocks

168

(“http://molevol.cmima.csic.es/castresana/Gblocks_server.htm”) was then used to eliminate poorly

169

aligned positions and divergent regions of DNA alignments, so that they become more suitable for

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phylogenetic analysis (Talavera & Castresana 2007).

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For completing phylogenetic determination hsp60 partial gene sequence was amplified, purified,

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and directly sequenced from B. scardovii DSM 13734T as above described, whereas for B.

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stellenboschense DSM 23968T we used the partial gene sequence obtained by Stenico et al., (2014)

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and retrieved from the GenBank nucleotide databases (Accession number KF 294527).

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The GenBank/EMBL/DDBJ accession number for the hsp60 partial gene sequence of B. scardovii

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DSM 13734T is KJ689460.

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Three phylogenetic trees were then constructed using the neighbor joining method. Approximately

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645 bp of the hsp60 gene, 500 bp of the clpC and 524 bp of the rpoB gene sequences of the isolates

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and related species were used in the analyses.

CBRC (http://mafft.cbrc.jp/alignment/software/) (Katoh, 2013).

program

(version

0,91b)

as

server

tool

at

Castresana

Lab

180 181

The level of similarity for the partial hsp60 gene sequences of the strains MRM 3/1T and MRM 4/2

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was 99.5 % and in relation to their closest relatives the levels of similarity were about 97.5 % with

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B. stellenboschense, 96.2 % with Bifidobacterium saeculare, 96 % with Bifidobacterium pullorum

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and Bifidobacterium gallinarum, 94.4 % with Bifidobacterium biavatii and 94 % with

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Bifidobacterium callitrichos and 90.8 % with B. scardovii. Strains MRM 3/1T and MRM 4/2

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produced a subcluster in the B. pullorum group (Fig. 2).

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The sequences similarity between the clpC gene of strains MRM 3/1T and MRM 4/2 was 99.2 %.

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The highest sequence similarities were found to B. scardovii and Bifidobacterium bifidum (about

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86.7 % and 86.5 %, respectively). Strains MRM 3/1T and MRM 4/2 produced a subcluster in the B.

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scardovii group.

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The clpC phylogenetic tree is shown in Supplementary Fig. S3.

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The level of similarity for the partial rpoB gene sequence of the strains MRM 3/1T and MRM 4/2

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was 99.8 % and the levels of similarity in relation to their closest relatives were about 95.2 %, 95

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%,and 94 % to Bifidobacterium cuniculi, Bifidobacterium choerinum and B. pullorum, respectively.

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Based on the rpoB partial sequences, MRM 3/1T and MRM 4/2 are placed in a distinct cluster and

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were related to B. cuniculi. The rpoB phylogenetic tree is shown in Supplementary Fig. S4.

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This finding correlated with the results of Ventura et al. (2006) and Endo et al. (2012) and indicated

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that the phylogenetic position of Bifidobacterium spp. was highly influenced on the genes used for

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the analysis.

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The 16S rRNA gene sequence similarity values of strains MRM 3/1T and MRM 4/2 to known

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species were less than 97 % and it was lower than the recommended value for species

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differentiation (98.7- 99 %) (Tindall et al., 2010). However, analysis of hsp60 showed that both

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strains were closely related to B. stellenboschense DSM 23968T (97.5%)

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Due to this high level of similarity (cut-off value for bifidobacterial species differentiation of hsp60

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is 96%, Zhu et al., 2003) DNA-DNA hybridization between B. aesculapii and B. stellenboschense

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has been also performed. Estimation of level of homology among B. stellenboschense DSM 23968T

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and strain MRM 3/1T was determined by DSMZ, Braunschweig, Germany. Cells were disrupted by

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using a Constant Systems TS 0.75 KW (IUL Instruments, Germany). DNA in the crude lysate was

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purified by chromatography on hydroxyapatite as described by Cashion et al. (1977). DNA-DNA

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hybridization was carried out as described by De Ley et al. (1970) under consideration of the

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modifications described by Huss et al. (1983) using a model Cary 100 Bio UV/VIS-

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spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature

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controller with in situ temperature probe (Varian). Type strain MRM 3/1 shared 30.4 % DNA-DNA

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homology to B. stellenboschense DSMZ 23968T unequivocally supporting the description of B.

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aesculapii as new species.

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Estimation of the G+C content in bacterial chromosomal DNA of type strain MRM 3/1T was

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determined by DSMZ, Braunschweig, Germany. DNA was purified on hydroxyapatit according to

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the procedure of Cashion et al. (1977) and was enzymatically hydrolyzed by the method of Mesbah

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et al. (1989). The resulting deoxyribonucleosides were analyzed by HPLC as described by Tamaoka

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& Komagata (1984). Type strain MRM 3/1 had G+C content of 64.7 mol% G+C. This value was

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within the G+C content values for the genus of Bifidobacterium which ranged from 52-67 mol %

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(Biavati & Mattarelli, 2012; Killer et al., 2010) and in particular was very similar to that obtained

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from the species Bifidobacterium callitrichos recently described in marmoset by Endo et al. (2012).

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Morphological, cultural and biochemical characterization of the isolates according to standard

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techniques were performed at 37 °C unless otherwise stated.

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Morphology examined by phase-contrast microscopy is shown in Fig. 3(a), (b). Morphological

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characteristics determined using a Scanning Electron Microscope (SEM) are shown in Fig. 3(c). For

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SEM observation the strains were cultured in TPY agar at 37°C for 48 hours under anaerobic

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conditions. After culturing, a slice of agar was excised and dehydrated with a series of increasing

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ethanol concentrations (at 50, 70, 80, 90, 95 and 100% for 15 minutes each). The prepared cells

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were subsequently critical-point-dried in a critical point dryer apparatus (CPD Emitech K850) using

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liquid CO2 as a transitional fluid. Dried samples were mounted on aluminum stubs with silver glue,

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coated with gold palladium film, using an ion-sputtering unit (Emitech K500) and observed in a

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Philips 515 SEM at 7-10.0 Kv.

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The temperature growth range of the strains was tested using an anaerobic TPY broth at a

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temperature gradient of 20, 25, 30, 35, 37, 40, 42, 45 and 47 °C for 48 h. The sensitivity of the

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strains to low values of pH was determined at 37 °C in anaerobic TPY broth (pH gradient 3.5, 4.0,

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4.5, 5.0 and 5.5) for 48 h. The ability of the strains to grow under aerobic and microaerophilic

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conditions (CampyGen, Oxoid) was tested using TPY agar, TPY soft agar (0.6 %), TPY broth, skim

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milk and UHT whole milk at 37 °C for 48 h.

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Hemolytic activity was determined in Columbia Blood Agar (Biolife) at 37 °C under anaerobic

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conditions for 48 h (Pineiro & Stanton, 2007).

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Spore staining was performed using malachite green dye. Phase-contrast microscopy (ZEISS) was

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used to observe the morphology of individual cells as well as spore staining.

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Gram staining, catalase and oxidase activities were determined in cells grown on TPY agar at 37 °C

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for 48 h under anaerobic conditions using Gram staining individual reagents (Merck Millipore), a 3

254

% (v/v) hydrogen peroxide solution and cotton swabs impregnated with N,N,N’,N’- Tetramethyl-p-

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phenylendiamine dihydrochloride and dried (Oxibioswab, Biolife), respectively. The motility of

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strains was determined by stabbing into TPY medium containing 0.4 % agar, knowing that motile

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strains show a diffuse growth spreading from the line of inoculation. Fermentation products (short-

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chain fatty acids) were analyzed according to the method described by Holdeman et al. (1977).

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Briefly after growth in TPY broth with 1% glucose, volatile acids were extracted with diethyl ether.

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A Carlo Erba 5300 gas chromatograph, with a Nukol capillary column (30 cm) at 170 °C, flame-

261

ionization detector and hydrogen carrier gas, was used for the analysis. All strains tested fermented

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glucose to acetate and lactate in a variable ratio ranging from 2:1 to 1.5:1.

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Biochemical characterization was carried out by using the API20A, API20E and API 50CHL

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systems (BioMérieux) following the manufacturer’s instructions. The results are summarized in

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Table 1.

266

Bifidobacteria and related genera degrade hexoses via the fructose-6-phosphate phosphoketolase

267

(F6PPK) pathway. F6PPK is the key enzyme in this pathway and . is considered a taxonomic

268

marker for identification of Bifidobacterium spp. and related genera (Biavati & Mattarelli, 2012).

269

The detection of F6PPK activity was determined according to the method described by Scardovi

270

(1986) and modified by Orban & Patterson (2000). All the isolates possess F6PPK activity (Table

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1).

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The cell wall murein composition of strain MRM 3/1T was examined by DSMZ. Analysis of partial

273

acid hydrolysates revealed the presence of an A4alpha L-Lys-D-Ser-D-Asp. This murein type is

274

unique among members of the genus Bifidobacterium and related genera confirming the novelty of

275

this species.

276

According to the phylogenetic analyses based on 16S rRNA gene, on hsp60, clpc and rpoB partial

277

sequences, and to other data obtained, the strains MRM 3/1T, MRM 4/2, MRM 5/13, MRM 4/6,

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MRM 4/7 and MRM 8/7 are genetically and phenotypically distinguishable from the currently

279

recognized species of bifidobacteria and thus represent a novel species, for which we suggest the

280

name Bifidobacterium aesculapii sp. nov.

281

Description of Bifidobacterium aesculapii sp. nov.

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Bifidobacterium aesculapii (aes.cu.la'pi.i. L. gen. masc. n. aesculapii from the snake-like

283

appearance of the bacterium, resembling the serpent-entwined rod wielded by the Roman God

284

Aesculapius).

285

Cells grown in TPY broth are rods of various shapes, occasionally swollen, always coiled or ring

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shaped or forming a “Y” shape at both ends. They are Gram-positive-staining, non-motile,

287

asporogenous, non hemolytic, F6PPK-positive, catalase- and oxidase-negative, indole negative and

288

microaerophilic. There was no difference in the growth of the strains under both anaerobic and

289

microaerophilic conditions. The strains were also negative for Arginine DiHydrolase, Lysine

290

DeCarboxylase, Ornithine DeCarboxylase, negative for citrate utilization and H2S production.

291

They did not reduce nitrate as well as nitrite. The well isolated colonies on the surface of TPY agar

292

under anaerobic conditions are white, opaque, smooth and circular with entire edges while the

293

imbedded colonies are lens-shaped or elliptical. Colonies reach 1.7-2.5 mm in diameter after 3 days

294

of incubation. The temperature range is 25-42 °C; no growth occurs at 20 or 47°C. The optimum

295

temperature for growth is 35°-37 °C. They grow at pH 4.5 -7.0 with an optimum at pH 6.5-7.0. The

296

strains can grow in milk, in aerobiosis, microaerophilic and anaerobiosis conditions. Acid is

297

produced from D-glucose, D-lactose, maltose, salicin, D-xylose, L-arabinose, melezitose, D-sorbitol,

298

D-ribose, D-galactose,

299

production from D-mannitol D-sucrose, glycerol, cellobiose, D-mannose, raffinose, L-rhamnose, D-

300

trehalose, D-fructose, starch, D-inulin and glycogen is strain dependent. Acid is not produced from

301

xylitol, amygdalin, methyl-alpha-D-glucoside, N-acetylglucosamine and gluconate. Lactic and

302

acetic acid are produced as end product of glucose fermentation in a variable ratio ranging from 1:2

303

to 1:1.5. Aesculin is hydrolysed and urease is produced. The DNA G+C content of the type strain is

304

64.7 mol%. The peptidoglycan type is A4alpha L-Lys-D-Ser-D-Asp. Phylogenetic analysis of the

305

16S rRNA gene sequence places the species in the B. scardovii subgroup of the genus

306

Bifidobacterium.

307

The type strain MRM 3/1T (=JCM 18761T =DSM 26737T) and the reference strain MRM 4/2

308

(=JCM 18762 =DSM 26738) were isolated from fresh faecal samples of infant common marmoset

309

(Callithrix jacchus), which were individually collected from 5 animals kept in animal houses in

310

Aptuit s.r.l. Verona, North Italy in 2012.

311

Acknowledgements

312

We thank Dr. Pisi Annamaria and Dr. Filippini Gianfranco from Department of Agricultural

313

Sciences, University of Bologna, for their technical assistance with scanning electron microscope

314

observation.

315

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Cashion, P., Holder-Franklin, M. A., McCully, J. & Franklin, M. (1977). A rapid method for

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387

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388

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390

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411

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422

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423

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424 425

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426

Bifidobacterium mongoliense sp. nov., from airag, a traditional fermented mare’s milk product from

427

Mongolia. Int J Syst Evol Microbiol 59, 1535–1540.

428 429 430 431 432 433 434 435 436

437

Figure 1. Phylogenetic relationship of the two novel bifidobacteria to related species based on 16S

438

rRNA gene sequences. The tree was constructed by the neighbour-joining method and rooted with

439

Micrococcus luteus DSM 20030T. The percentage of replicate trees in which the associated taxa

440

clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Bootstrap

441

percentages above 70 are given at branching points.

442 443 444

Figure 2. Phylogenetic tree based on hsp60 gene sequences showing the relationship of the novel

445

strains isolated from baby marmoset with closely related species. The tree was constructed by the

446

neighbour-joining method on the basis of a comparison of approximately 559 positions, and the

447

sequence of Mycobacterium tuberculosis H37Rv was used as an outgroup. The percentage of

448

replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates)

449

are shown next to the branches. Bootstrap percentages above 70 are given at branching points.

450 451 452

Figure 3. Cellular morphology of cells grown in TPY. Phase-contrast photomicrographs: (a),

453

MRM 3/1T, (b), MRM 4/2. Scanning electron photomicrograph: (c) MRM 3/1T.

454 455 456 457 458 459

Table 1.

460

Differential characteristics between novel bifidobacteria and their closest phylogenetic relatives: B.

461

stellenboschense DSM 23968T, B. biavatii DSM 23967T, B. scardovii DSM 13734T and B. bifidum

462

DSM 20456T.

463 464

465

Table 1:

466

Differential characteristics between novel bifidobacteria and their closest phylogenetic relatives: B. stellenboschense (DSM 23968 T) and B. biavatii

467

(DSM 23967 T) B. scardovii (DSM 13734 T) and B. bifidum (DSM 20456 T).

468

MRM

MRM

MRM

MRM

MRM

B.

B.

MRM 3/1T

4/2

8/7

5/13

4/6

4/7

biavatii

bifidum

D_Mannitol

+

-

-

w

-

+

w

w

-

-

D _Saccharose

+

-

+

+

-

+

+

+

+

+

D_Maltose

+

+

+

w

+

+

+

+

+

-

Salicine

+

+

+

-

+

+

+

+

-

-

D_Xylose

+

+

+

+

+

+

+

+

-

-

L_Arabinose

+

+

+

+

+

+

w

+

-

-

Glycerol

+

-

-

-

-

+

+

w

-

+

D_Cellobiose

+

-

-

-

-

+

-

+

-

-

D_Mannose

+

-

-

-

-

+

-

w

-

-

D_Melezitose

+

w

w

w

w

+

w

+

-

-

D_Raffinose

+

w

+

+

-

+

+

+

+

-

D_Sorbitol

+

w

+

+

+

+

w

w

-

-

L_Rhamnose

+

-

-

-

-

+

-

w

-

-

D_Trehalose

+

-

-

-

-

+

-

+

-

-

D_Ribose

W

+

+

w

+

+

+

+

-

-

Characteristics

B. stellenboschense B. scardovii

Carbohydrates:

D_Galactose

W

+

+

+

+

+

+

+

-

+

D_Fructose

-

+

+

+

+

+

+

+

+

-

Starch

+

-

-

-

w

-

-

-

+

-

Gentiobiose

+

w

+

+

+

+

w

+

+

-

D_Turanose

W

+

+

+

+

+

+

+

w

-

Aburtine

+

+

+

+

+

+

+

+

-

-

D_Melibiose

W

w

+

+

+

w

+

+

w

-

Inulin

W

-

w

W

w

w

-

-

-

-

W

w

+

-

w

+

w

w

-

-

Glycogen

+

-

-

-

-

-

-

w

+

-

Xylitol

W

-

-

-

-

-

-

+

-

-

-

-

-

-

-

-

+

+

-

-

-

-

-

-

-

-

+

-

-

-

-

-

+

+

-

-

+

+

w

-

-

-

-

-

-

-

+

-

-

-

64.7

N.D.

N.D.

N.D.

N.D.

N.D.

66.3

60.1

63.1

58

+

+

+

+

+

-

-

-

-

Potassium Gluconate

Amiygdalin α-Methyl-DGlucopyranoside D_Turanose NAcetylglucosamine

DNA GC Content (Mol%) Enzymatic Activity Urease

+

469 470

Bifidobacterium aesculapii sp. nov., from the feaces of the baby common marmoset (Callithrix jacchus) Modesto M.1, Michelini S.1, Stefanini I. 1, Ferrara A.2, Tacconi S. 2, Biavati B. 1, Mattarelli P. 1* 1Department of Agricultural Sciences, University of Bologna Italy 2Aptuit srl, Verona, Italy

*Corresponding author: Paola Mattarelli, 1Department of Agricultural Sciences, University of Bologna, Viale Fanin 42, 40127 Bologna, Italy Phone+393497620217 Fax+390512096274 [email protected]

Figure 1. Phylogenetic relationship of the two novel bifidobacteria to related species based on 16S rRNA gene sequences. The tree was constructed by the neighbour-joining method and rooted with Micrococcus luteus DSM 20030T. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Bootstrap percentages above 70 are given at branching points.

B. choerinum ATCC 27686T (D86186)

82

B. animalis subsp. animalis JCM 1190T (D86185)

B. pseudolongum subsp. pseudolongum JCM 1205T (D86195) 81

B. cuniculi YIT 4093T (AB438223)

95

B. gallicum JCM 8224T (D86189)

B. magnum JCM 1218T (D86193)

B. coryneforme YIT 4092T (AB437358)

99 98

B. indicum JCM 1302T (D86188)

B. asteroides CCUG 24607T (EF187235)

B. tsurumiense OMB115T (AB241106)

B. thermophilum YIT 11868T (AB437364) 100

B. thermacidophilum subsp. thermacidophilum YIT 11849T (AB437362)

87

B. boum JCM 1211T (D86190)

B. reuteri DSM 23975T (AB613259) 100

B. pseudocatenulatum JCM 1200T (D86187)

B. catenulatum YIT 4016T (AB437357)

B. merycicum JCM 8219T (D86192)

B. angulatum ATCC 27535T (D86182)

99

B. callitrichos DSM 23973T (AB674319)

B. ruminantium JCM 8222T (D86197) 93 99

B. adolescentis YIT 4011T (AB437355)

B. stercoris Eg1T (FJ611793)

B. dentium ATCC 27534T (D86183) T 100 MRM 3/1 (KC807989)

MRM 4/2 (KC807990)

B. stellenboschense DSM 23968T (AB559505)

B. scardovii YIT 11867T (AB437363)

B. biavatii DSM 23969T (AB559506)

B. bifidum YIT 4039T (AB437356)

B. saguini DSM 23967T (AB559504)

75 81

B. longum subsp. longum YIT 4021T (AB437359)

B. breve ATCC 15700T (AB006658)

B. subtile DSM 20096T (D89378) T 99 B. gallinarum JCM 6291 (D86191)

B. saeculare DSM 6531T (D89328) 100

B. pullorum JCM 1214T (D86196)

B. psychraerophilum YIT 11814T (AB437351)

82

B. mongoliense DSM 21395T (AB433856)

B. minimum YIT 4097T (AB437350)

Gardnerella vaginalis ATCC 14018T (M58744)

Scardovia inopinata DSM 1010T (D89332) 100

Parascardovia denticolens DSM 10105T (D89331)

Micrococcus luteus DSM 20030T (AJ536198)

0.02

Bifidobacterium aesculapii sp. nov., from the feaces of the baby common marmoset (Callithrix jacchus) Modesto M.1, Michelini S.1, Stefanini I. 1, Ferrara A.2, Tacconi S. 2, Biavati B. 1, Mattarelli P. 1* 1Department of Agricultural Sciences, University of Bologna Italy 2Aptuit srl, Verona, Italy

*Corresponding author: Paola Mattarelli, 1Department of Agricultural Sciences, University of Bologna, Viale Fanin 42, 40127 Bologna, Italy Phone+393497620217 Fax+390512096274 [email protected]

Figure 2. Phylogenetic tree based on hsp60 gene sequences showing the relationship of the novel strains isolated from baby marmoset with closely related species. The tree was constructed by the neighbour-joining method on the basis of a comparison of approximately 559 positions, and the sequence of Mycobacterium tuberculosis H37Rv was used as an outgroup. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. Bootstrap percentages above 70 are given at branching points.

B. breve KCTC 3220T (GU361838)

85 93

B. longum subsp. longum KCTC 3128 T 8GU361846)

B. saguini DSM 23967T (AB674320)

97

B. reuteri DSM 23975T (AB674318)

B. scardovii DSM 13734T (KJ689469)

99

B. merycicum KCTC 3369T (GU361848)

B. ruminantium KCTC 3425T (GU361854)

83

B. dentium KCTC 3222T (GU361842)

76

B. catenulatum KCTC 3221T (GU361839)

90

B. pseudocatenulatum KCTC 3223T (GU361850)

96

B. bifidum KCTC 3202T (GU361836)

B. callitrichos DSM 239673T (AB674319) 100

MRM 3/1T (KC997237)

98

MRM 4/2 (KC997238)

B. stellenboschense DSM 23968T (KF294527)

B. biavatii DSM 23969T (AB674321)

B. pullorum KCTC 3274T (GU361853)

B. gallinarum KCTC 3235T (GU361844)

87 100

B. saeculare KCTC 3427T (GU361855)

B. boum KCTC 3227T (GU361837) 100

B. thermophilum KCTC 3225T (GU361857)

B. subtile KCTC 3272T (GU361856)

B. cuniculi KCTC 3276T (GU361841) 100

B. pseudolongum subsp. globosum KCTC 3234T (GU361851)

B. pseudolongum subsp. pseudolongum KCTC 3224T (GU361852)

B. choerinum KCTC 3275T (GU361840)

B. animalis subsp. animalis KCTC 3219T (GU361835)

B. gallicum KCTC 3277T (GU361843) 99

B. magnum KCTC 3228T (GU361847)

B. indicum KCTC 3230T 8GU361845)

B. minimum KCTC 3273T 8GU361849)

M. tubercolosis H37RvT (NC 000962) 0.05

Bifidobacterium aesculapii sp. nov., from the faeces of the baby common marmoset (Callithrix jacchus).

Six Gram-positive-staining, microaerophilic, non-spore-forming, fructose-6-phosphate phosphoketolase-positive bacterial strains with a peculiar morpho...
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