Genome sequence and description of Actinomyces polynesiensis str. MS2 sp. nov. isolated from the human gut.

Actinomyces polynesiensis strain MS2 gen. nov., sp. nov. is a newly proposed genus within the family Actinomycetaceae, isolated from the stools of a healthy individual in Raiatea Island (French Polynesia, South Pacific). Actinomyces massiliensis is an anaerobic, Gram-positive organism. Here we describe the features of this organism, together with the complete genome sequence and annotation-2 943 271 bp with a 70.80% G+C content, assembled into 15 scaffolds and containing 2080 genes.

Introduction

The genus Actinomyces belongs to the domain Bacteria, Phylum Actinobacteria, Class Actinobacteria, Order Actinomycetales and Family Actinomycetaceae. The genus consists of a heterogeneous group of Gram-positive bacteria that have a high G+C content [1].

The genus Actinomyces consists of a heterogeneous group of Gram-positive bacilli, mainly facultatively anaerobic or microaerophilic rods with various degrees of branching [2]. Actinomyces species mainly belong to the human commensal flora of the oropharynx, gastrointestinal tract, and urogenital tract [3].

Actinomycosis is a very rare disease usually caused by one of a group of opportunistic but otherwise harmless commensals and may be complicated by one or more of another group of co-pathogens [4]. Actinomycetes are prominent among the normal flora of the oral cavity but less prominent in the lower gastrointestinal tract and female genital tract. As these microorganisms are not virulent, they require a break in the integrity of the mucous membranes and the presence of devitalized tissue to invade deeper body structures and cause human illness [4]. Many studies based on phenotypic identification of members of the genus Actinomyces have been performed. Several flowcharts have been proposed to enable accurate differentiation. However, during the past few years, Actinomyces taxonomy has undergone much improvement, primarily driven by 16S rRNA gene sequence analysis [5]. The study of Actinomyces polynesiensis str. MS2 is part of a project called ‘Culturomics’, the comprehensive determination of the microbial composition of the gut microbiota and the relationships with health and disease, which are major challenges in the twenty-first century. Metagenomic analysis of the human gut microbiota detects mostly uncultured bacteria as reported here [6].

Materials and Methods

Growth conditions and identification

sp. nov., strain MS2 (=CSURP658= DSMZ 27066).

Actinomyces polynesiensis str. MS2 was isolated from the stools of a healthy individual in Raiatea Island (French Polynesia, South Pacific). Actinomyces polynesiensis MS2 was isolated for the first time using inoculation in a blood culture bottle with coconut milk and then 5% sheep blood agar. Growth was tested on Columbia agar supplemented with 5% sheep blood and chocolate agar + PolyViteX (bioMérieux, Marcy-l’Étoile, France) in aerobic and anaerobic condition using GasPak™ EZ Anaerobe Container System Sachets (BD, Franklin Lakes, NJ, USA) at 37°C and CO2 Gen (ThermoScientific, Waltham, MA, USA). The ability of the strain to grow at different temperatures (25°C, 37°C and 45°C) was investigated.

Gram staining and electron microscopy were performed with a TechnaiG2 Cryo (FEI Company, Hillsboro, OR, USA) at an operating voltage of 200 keV. Cells were grown on 5% sheep-blood agar for 24 h. A bacterial suspension was pre-fixed in 5% (volume/volume) glutaraldehyde in phosphate buffer (Gibco, Waltham, MA, USA) for at least 1 h at room temperature, washed in the same buffer and stained with 1% (weight/volume) ammonium molybdate 1%. Catalase activity, as determined by an ID Color catalase test kit (bioMérieux) and oxidase activity, assayed by applying the cells to moistened discs impregnated with dimethyl-p-phenylenediamine (bioMérieux). Biochemical tests were performed with the commercially available API ZYM and API 50 CH strips and were used to characterize the biochemical properties of the strain according to the manufacturer's instructions. The bacterium was identified by sequence analysis of the 16S rRNA. Phylogenetic relationship with closely related species were determined using MEGA version 5.1. The evolutionary history was inferred using the maximum likelihood method based on the JTT matrix-based model [7].

Antibiotic susceptibility testing

Antibiotic susceptibility testing was conducted using the disc diffusion method on Müller–Hinton agar supplemented with 5% sheep blood medium (bioMérieux) and the results were interpreted using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines [8]. The antibiotics used in this study were penicillin, oxacillin, vancomycin, teicoplanin, linezolid, gentamicin, ciprofloxacin, trimethroprim-sulphamethoxazole, fosfomycin, doxycycline, erythromycin, clindamycin, rifampicin and colistin.

Genome sequencing

DNA extracted through the BioRobot EZ 1 Advanced XL (Qiagen, Hilden, Germany) paired end library was pyrosequenced on the 454_Roche_Titanium. The global 297 979 passed filter sequences generated 85.84 Mb with a length average of 281 bp. These sequences were assembled on the gsAssembler from Roche with 90% identity and 40 bp as overlap (Roche, Basel, Switzerland). It leads to 15 scaffolds and 309 large contigs (>1500 bp), generating a genome size of 2.94 Mb, which corresponds to a coverage of 29.2 genome equivalent.

Genome annotation

The genome was annotated by Rapid Annotation using the Subsytem Technology (RAST) bioserver [9]. Blast2GO optimizes the function transfer from homologous sequences through an elaborate algorithm that considers the similarity, the extension of the homology, the database of choice, the GO hierarchy, and the quality of the original annotations [10]. The resistome was analysed with the ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) database [11] and BLASTp in GenBank. The prediction of rRNAs was carried out using the RNAmmer tools [12]. The exhaustive bacteriocin database available in our laboratories (Bacteriocins of the URMITE database; http://drissifatima.wix.com/bacteriocins) was performed by collecting all currently available sequences from the databases and from NCBI. Protein sequences from this database allowed putative bacteriocins from human gut microbiota to be identified using BLASTp methodology [13].

Results

Description of Actinomyces polynesiensis sp. nov., strain MS2

Phenotypic properties

Actinomyces polynesiensis (polinesiense masc. adj. of πολυνησίας, the ancient Greek name for the islands of French Polynesia, where the strain was isolated). Actinomyces polynesiensis grew slowly, with characteristics similar to those of the filamentous fungi; which have the ability to grow in branched filaments and to form settlers and the typical mycelium seen with other smaller beaded white colonies (Fig. 1). Actinomyces polynesiensis MS2 was grown anaerobically on 5% sheep-blood-enriched Columbia agar at 37°C. Optimal growth was achieved anaerobically using chocolate agar + PolyViteX with 5% CO2 at 37°C and no growth was observed under aerobic conditions. Catalase and oxidase activity were respectively positive and negative. A motility test was positive. Acid production was observed for the following carbohydrates, with API 50 CH negative for d-arabinose, l-xylose, d-adonitol, methyl-β-d-xylopyranoside, l-sorbose, l-rhamnose, dulcitol, inuline, d-tagatose, d-fucose, l-arabitol, potassium 2- and 5-gluconate; all other acid production was positive (Table 1). All activities were negative with API ZYM (Table 1).

Fig. 1

(a) Actinomyces polynesiensis str. MS2 isolated on Müller–Hinton agar supplemented with 5% sheep blood medium (bioMérieux); (b) Gram staining of Actinomyces polynesiensis str. MS2; (c) electron microscopy of Actinomyces polynesiensis str. MS2 performed with a TechnaiG2 Cryo (FEI Company) at an operating voltage of 200 keV.

Table 1

Acid production compared between four Actinomyces species

Test	1	2	3	4
Acid production
Glycerol	+	–	nd	nd
l-Arabinose	+	–	+	+
d-Ribose	+	–	nd	nd
d-Xylose	+	–	nd	nd
d-Galactose	+	+	nd	nd
d-Glucose	+	+	+	+
d-Fructose	+	+	nd	nd
d-Mannose	+	–	nd	–
Inositol	+	–	nd	nd
d-Mannitol	+	–	–	+
Methyl β-d-glucopyranoside	+	–	–	–
N-Acetylglucosamine	+	–	nd	nd
Amygdalin	+	–	nd	nd
Aesculin	+	–	+	+
Salicin	+	–	nd	nd
Cellobiose	+	–	nd	nd
Lactose	+	+	–	+
Melibiose	+	–	+	–
Raffinose	+	–	+	–
Gentiobose	+	–	nd	nd
Turanose	+	–	nd	nd
d-Lyxose	+	–	nd	nd
APi ZYM
Esterase	–	–	–	–
Valine arylamidase	–	+	–	–
α-Galactosidase	–	–	+	–
β-Galactosidase	–	+	+	+
α-Glucosidase	–	+	–	+
β-Glucosidase	–	–	+	+

1, Actinomyces polynesiensis str. MS2; 2, Actinomyces israelii CIP 103259; 3, Actinomyces suimastitidis DSM15538; 4, Actinomyces vaccimaxillae DSM 15804

Further identification was performed using a 16S rRNA nucleotide sequence (GenBank accession number HF952919) gene closely related (99.8%) to Actinomyces 152R-3 (GenBank accession number DQ278863.1) (Fig. 2). This value was lower than the 95% 16S rRNA gene sequence threshold recommended by Stackebrandt and Elbers to delineate a new genus without carrying out DNA-DNA hybridization [14]. The spectrum from MS2 was added to our MALDI-TOF mass spectra database.

Fig. 2

Molecular phylogenetic analysis by maximum likelihood method of representatives of genus Actinomyces inferred from 16SrRNA gene sequence.

Phenotypically, A. polynesiensis str. MS2 was resistant to oxacillin, second-generation (cefoxitine) and third-generation cephalosporins (ceftriaxone), trimethroprim-sulphamethoxazole, fosfomicin, erythromycin and clindamycin but was susceptible to vancomycin, teicoplanin, linezolid, gentamicin, ciprofloxacin, doxycycline, rifampicin and colistin (Fig. 2).

Genome features

The genome size of Actinomyces polynesiensis str. MS2 is 2 943 271 bp with a 70.80% G+C content assembled into 15 scaffolds (309 contig). A total of 2080 genes (91%) were assigned to putative functions (by clusters of orthologous groups) and 216 (9%) genes were identified as unknown function and 110 were RNAs, ten genes were 5S rRNA, nine genes were 16S rRNA and four genes were 23S rRNA. The distribution of genes into clusters of orthologous group functional categories is presented in Table 2 and the properties and statistics of the genome are summarized in Table 3.

Table 2

Number of genes associated with 25 general functional categories of clusters of orthologous groups (COGs)

COG class	Calue	Description
A	32	RNA processing and modification
C	126	Energy production and conversion
D	30	Cell cycle control, cell division, chromosome partitioning
E	204	Amino acid transport and metabolism
F	80	Nucleotide transport and metabolism
G	239	Carbohydrate transport and metabolism
H	70	Coenzyme transport and metabolism
I	61	Lipid transport and metabolism
J	156	Translation, ribosomal structure and biogenesis
K	183	Transcription
L	135	Replication, recombination and repair
M	120	Cell wall/membrane/envelope biogenesis
N	2	Cell motility
O	68	Post-translational modification, protein turnover, chaperones
P	132	Inorganic ion transport and metabolism
Q	24	Secondary metabolites biosynthesis, transport and catabolism
R	245	General function prediction only
S	216	Function unknown
T	87	Signal transduction mechanisms
U	37	Intracellular trafficking, secretion, and vesicular transport
V	49	Defence mechanisms

Table 3

Genome properties of Actinomyces polynesiensis str. MS2

	Size Mb	GC%	Genes	Protein
Actinomyces polynesiensis str. MS2	2.87	70.80	2514	2566
Actinomyces israelii CIP 103259	4.03	71	3262	3125
Actinomyces odontolyticus ATCC 17982	2.39	65.4	2054	1982
Actinomyces georgiaeDSM 6843	2.5	69.80	2102	2031
Actinomyces suimastitidis DSM15538	2.29	56.4	1960	1891
Actinomyces vaccimaxillae DSM 15804	2.34	57.6	2067	1991

Resistome

The resistome of this multidrug-resistant Actinomyces polynesiensis str. MS2 includes penicillin-binding protein (296 amino acids), metallo-hydrolase enzyme (239 amino acids) and the major facilitator superfamily (Table 4).

Table 4

List of genes associated with antibiotic resistant in Actinomyces polynesiensis str. MS2

Open reading frame	Gene name	GC%	Size aa	Function	Best blast hit in GenBank	% aa coverage	% aa identity
WP_043535219.1	MBL	74.4	239	metallo-hydrolase	Actinomyces SD1	100	65
WP052450410.1	MFS	71.3	510	MFS transporter	Leifsonia aquatica	96	58
WP_043535826.1	PBP	73	269	penicillin-binding protein	Actinomyces vaccimaxillae	99	58

aa, amino acids; MBL, metallo-hydrolase enzyme; MFS, the major facilitator superfamily; PBP, penicillin-binding protein.

Specific features

Toxin–antitoxin loci encode a stable toxin that is neutralized by a metabolically unstable antitoxin [15]. The analysis of the genome has not demonstrated the presence of bacteriocin and non-ribosomal polyketide synthase.

Conclusion

On the basis of the phylogenetic analysis, the novel species Actinomyces polynesiensis str. MS2 sp. nov. is proposed with the accession number CCXH01000000 in the GenBank database.

Genome Sequence Accession Number

The genome of Actinomyces polynesiensis str. MS2 has been submitted to the EBI database under bioproject ID: PRJEB1958 with accession number on GenBank database CCXH01000000 and 16S rRNA accession number HF952919.