Peptoniphilus lacydonensis sp. nov., a new human-associated species isolated from a patient with chronic refractory sinusitis.

Strain EL1T was isolated from a sinus sample of an 85-year-old man with chronic refractory sinusitis complicating ethmoidal adenocarcinoma. We studied its phenotypic and genomic characteristics. This is a Gram stain-positive, anaerobic and microaerophilic coccus. Cells are catalase negative, nonmotile and non-spore forming. The major fatty acids are saturated hexadecanoic acid (34%), unsaturated 9-octadecenoic acid (32%) and 9.12-octadecadienoic acid (21%). The 1.86 Mb long genome exhibits a 29.9% G+C content and contains 1750 protein-coding and 43 RNA genes. On the basis of these data, we propose the creation of the new human-associated bacterial species Peptoniphilus lacydonensis sp. nov.

Introduction

As of 31 August 2017, the genus Peptoniphilus contains 15 species with validly published names (http://www.bacterio.net/peptoniphilus.html). Bacteria from the genus Peptoniphilus are Gram-positive cocci that do not form spores and are strictly anaerobic and catalase negative [1]. All species have been isolated from human clinical material except three species that were isolated from animals. Peptoniphilus asaccharolyticus [1], P. olsenii [2], P. gorbachii [2], P. duerdenii [3], P. koenoeneniae [3], P. timonensis [4] and P. catoniae [5] were isolated from human clinical specimens. In addition, P. harei [1], P. ivorii [1], P. coxii [6] and P. tyrrelliae [6] were isolated from human clinical infections. Finally, P. indolicus [1], P. methioninivorax [7] and P. stercorisuis [8] were isolated from cattle, retail ground beef and a swine manure storage tank, respectively.

Recently, next-generation sequencing technology, which enables sequencing the whole genome of a prokaryote in a short time, and mass spectrometric analysis of bacteria have given easy access to genetic and proteomic information [9]. Therefore, we propose a polyphasic approach to describe new bacterial species that combines genomic properties with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) spectrum and phenotypic characteristics [10].

Herein we describe strain EL1T (= CSUR P2013 = DSM 100661), which was isolated from a sinus specimen of an 85-year-old patient with chronic refractory sinusitis. We present a summary classification and set of features for this strain, including the description of its complete genome sequencing and annotation that allow its description as a new Peptoniphilus species. These characteristics support the circumscription of the new species Peptoniphilus lacydonensis sp. nov.

Results

Strain identification by MALDI-TOF MS and 16S rRNA gene sequencing

No significant MALDI-TOF MS score was obtained for strain EL1T against the Bruker Daltonics (Leipzig, Germany) database, suggesting that our strain was not a member of any known species. The reference mass spectrum of strain EL1T was added to our database (Supplementary Fig. S1). A gel view comparing the spectrum of strain EL1T with those of other Peptoniphilus species is shown in Supplementary Fig. S2.

To identify strain EL1, the 16S rRNA gene was sequenced, and the obtained sequence (GenBank accession no. LN867000) showed a 96.76% similarity with P. tyrrelliae strain RMA 19911T (accession number GU938835), the phylogenetically closest bacterial species with a validly published name (Fig. 1). The general characteristics of strain EL1T are presented in Supplementary Table S1. According to the similarity level threshold defined by Kim et al. [11], we propose strain EL1T as type strain of a new species within the genus Peptoniphilus, for which we suggest the name Peptoniphilus lacydonensis sp. nov. (Supplementary Table S1).

Fig. 1

Phylogenetic tree obtained by comparison of 16S rRNA gene sequences showing position of Peptoniphilus lacydonensis sp. nov. strain EL1T relative to other type strains within genus Peptoniphilus. GenBank accession numbers are indicated in parentheses. Sequences were aligned using default parameters in MUSCLE; phylogenetic inferences were obtained using maximum-likelihood method with 1000 replicates within MEGA6 software. Scale bar represents 2% nucleotide sequence divergence.

Phenotypic description

Growth was obtained at 37°C but weakly at 29°C, with optimal growth at 37°C after 48 hours' incubation. The pH range for growth was 6 to 8.5, and growth occurred in the presence of 0 to 5 g/L NaCl. Strain growth was observed in both anaerobic and microaerophilic conditions, but not in aerobic atmosphere. Colonies were translucent and grey; they exhibited a diameter of 0.5 to 1 mm after 72 hours' incubation on 5% sheep's blood–enriched Columbia agar (bioMérieux, Marcy l’Etoile, France). Bacterial cells were Gram-positive cocci (Supplementary Fig. S3) with a mean diameter of 0.73 μm, and they were mostly grouped in pairs (Fig. 2). Cells were not motile and were non–spore forming.

Fig. 2

Transmission electron microscopy of Peptoniphilus lacydonensis strain EL1T using Tecnai G20 electron microscope (FEI Company). Scale bar = 200 nm.

Biochemical characterization and antibiotic susceptibility

Catalase and oxidase production reactions were negative for strain EL1T. Using an API 20A strip (bioMérieux), a positive reaction was observed for indole formation. No acid production was observed from d-glucose, d-mannitol, d-lactose, d-saccharose, d-maltose, salicin, d-xylose, l-arabinose, glycerol, d-cellobiose, d-mannose, d-melezitose, d-raffinose, d-sorbitol, l-rhamnose or d-trehalose. Urease, β-glucosidase and protease activities were absent.

Using an API ZYM strip (bioMérieux), a weak reaction was observed for naphthol-AS-BI-phosphohydrolase. Negative reactions were noted for alkaline phosphatase, esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, phosphatase acid, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, α-mannosidase, α-fucosidase and N-acetyl-β-glucosaminidase.

Using an API 50 CH strip (bioMérieux), negative reactions were observed for the fermentation of glycerol, erythritol, d-arabinose, l-arabinose, d-ribose, d-xylose, l-xylose, d-adonitol, methyl-β-d-xylopyranoside, d-galactose, d-glucose, d-fructose, d-mannose, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl-αd-xylopyranoside, methyl-αd-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, esculin, ferric citrate, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, d-saccharose, d-trehalose, inulin, d-melezitose, d-raffinose, amidone, glycogen, xylitol, gentiobiose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium-5-ketogluconate.

The major fatty acid was saturated hexadecanoic acid (34%), followed by unsaturated 9-octadecenoic acid (32%) and 9.12-octadecadienoic acid (21%). Both unsaturated and saturated fatty acids were detected (Supplementary Table S2).

Peptoniphilus lacydonensis strain EL1T was susceptible to amoxicillin (minimum inhibitory concentration 0.125 μg/mL), amoxicillin/clavulanic acid (0.125), ticarcillin (1), cefepime (1), imipenem (0.125), gentamicin (0.5), doxycycline (0.25), tigecycline (0.125), clindamycin (0.125), fosfomycin (0.125), rifampicin (0.06), ciprofloxacin (0.125), erythromycin (0.25) and vancomycin (0.5), but resistant to trimethoprim/sulfamethoxazole (>16). Table 1 lists the antibiotic susceptibility of closely related species.

Table 1

Differential phenotypic characteristics of Peptoniphilus lacydonensis strain EL1T and related type strains (Peptoniphilus lacrimalis, Peptoniphilus asaccharolyticus, Peptoniphilus indolicus, Peptoniphilus harei, Peptoniphilus ivorii and Peptoniphilus duerdenii[1], [6])

Property	P. lacydonensis	P. lacrimalis	P. asaccharolyticus	P. indolicus	P. harei	P. ivorii	P. duerdenii
Gram stain	Positive	Positive	Positive	Positive	Positive	Positive	Positive
Oxygen requirement	Anaerobic and microaerophilic	Obligately anaerobic	Obligately anaerobic	Obligately anaerobic	Obligately anaerobic	Obligately anaerobic	Obligately anaerobic
Motility	−	−	−	−	−	−	−
Spore formation	−	−	−	−	−	−	−
Production of:
Catalase	−	−	−	+	+	−	−
Indole	+	−	D	+	D	−	+
Urease	−	−	−	−	−	−	−
Alkaline phosphatase	−	−	−	+	−	−	−
Fermentation of:
Glucose	−	−	−	−	−	−	−
Lactose	−	−	−	−	−	−	−
Raffinose	−	−	−	−	−	−	−
Mannose	−	−	−	−	−	−	−
Activity of:
α-Galactosidase	−	−	−	−	−	−	−
β-Galactosidase	−	−	−	−	−	−	−
α-Glucosidase	−	−	−	−	−	−	−
β-Glucosidase	−	−	−	−	−	−	−
Leucine arylamidase	−	+	D	+	D	−	+
Habitat	Human	Human	Human	Animal	Human	Human	Human

+, positive result; −, negative result; D, strain dependent.

Genome properties

The final assembly was made of three contigs. The genome is 1 863 688 bp long (one chromosome, but no plasmid) with a 29.9% G+C content (Fig. 3, Supplementary Table S3). Of the 1793 predicted genes, 1750 were protein-coding genes and 43 were RNAs (two genes are 5S rRNA, one gene is 16S rRNA, one gene is 23S rRNA and 39 genes are tRNA genes). A total of 1321 genes (75.49%) were assigned a putative function. Twenty genes were identified as ORFans (1.14%). The remaining genes were annotated as hypothetical proteins (429 genes, 24.51%) (Supplementary Table S3).

Fig. 3

Graphical circular map of chromosome of Peptoniphilus lacydonensis sp. nov. strain EL1T. From outside to centre: genes on forward strand coloured by COGs category (only genes assigned to COGs), genes on reverse strand coloured by COGs category (only genes assigned to COGs), RNA genes (tRNAs green, rRNAs red), GC content and GC skew. COGs, Clusters of Orthologous Groups database.

Table 2 shows the distribution of genes into Clusters of Orthologous Groups (COGs) functional categories.

Table 2

Number of genes associated with 25 general COGs functional categories

Code	Value	%a	Description
J	138	7.89	Translation
A	0	0	RNA processing and modification
K	89	5.09	Transcription
L	123	7.03	Replication, recombination and repair
B	0	0	Chromatin structure and dynamics
D	17	0.97	Cell cycle control, mitosis and meiosis
Y	0	0	Nuclear structure
V	61	3.49	Defense mechanisms
T	42	2.4	Signal transduction mechanisms
M	59	3.37	Cell wall/membrane biogenesis
N	3	0.17	Cell motility
Z	0	0	Cytoskeleton
W	0	0	Extracellular structures
U	13	0.74	Intracellular trafficking and secretion
O	51	2.91	Posttranslational modification, protein turnover, chaperones
X	7	0.4	Mobilome: prophages, transposons
C	85	4.86	Energy production and conversion
G	36	2.06	Carbohydrate transport and metabolism
E	111	6.34	Amino acid transport and metabolism
F	52	2.97	Nucleotide transport and metabolism
H	52	2.97	Coenzyme transport and metabolism
I	41	2.34	Lipid transport and metabolism
P	75	4.29	Inorganic ion transport and metabolism
Q	9	0.51	Secondary metabolites biosynthesis, transport and catabolism
R	158	9.03	General function prediction only
S	111	6.34	Function unknown
—	417	23.83	Not in COGs

COGs, Clusters of Orthologous Groups database.

Total is based on total number of protein-coding genes in annotated genome.

The genome sequence has been deposited in GenBank under accession numbers FNWF01000001 to FNWF01000003.

Genome comparison

Phylogenomic analysis showed that the strain EL1T genome is closest to Peptoniphilus harei and Peptoniphilus timonensis (Fig. 4).

Fig. 4

Phylogenomic tree obtained by comparison of whole genome sequences showing position of Peptoniphilus lacydonensis sp. nov. strain EL1T relative to other type strains' genomes within genus Peptoniphilus. GenBank accession numbers are indicated in parentheses. Sequences were aligned using default parameters in Mauve alignment programme; phylogenetic inferences were obtained using maximum-likelihood method with 1000 replicates within FastTree software.

The draft genome of P. lacydonensis (1.86 Mb) has a smaller size than that of P. lacrimalis (1.88 Mb), P. indolicus (1.93 Mb), P. harei (1.87 Mb), and P. duerdenii (2.11 Mb) but is greater than those of P. timonensis (1.76 Mb) and P. coxii (1.84 Mb) (Table 3). The G+C content of P. lacydonensis (29.9%) was lower than that of P. timonensis (30.7%), P. lacrimalis (30.2%), P. indolicus (31.7%), P. harei (34.4%), P. duerdenii (34.2%) and P. coxii (44.6%) (Table 3).

Table 3

Genome comparison of closely related species to Peptoniphilus lacydonensis strain EL1

Organism name	GenBank Accession no.	Size (Mb)	G+C%	No. of proteins	Total genes
Peptoniphilus lacydonensis	FNWF00000000	1.86	29.9	1750	1793
Peptoniphilus coxii	LSDG00000000	1.84	44.6	1738	1775
Peptoniphilus duerdenii	AEEH00000000	2.08	34.2	1920	1947
Peptoniphilus harei	AENP00000000	1.84	34.4	1719	1754
Peptoniphilus indolicus	AGBB00000000	2.10	31.7	2205	2231
Peptoniphilus lacrimalis	ARKX00000000	1.85	30.2	1738	1780
Peptoniphilus timonensis	CAHE00000000	1.76	30.7	1914	1936

Similarly, the gene content of P. lacydonensis sp. nov. (1793) is greater than those of P. coxii, P. lacrimalis and P. harei (1775, 1780 and 1754, respectively) but is smaller than those of P. timonensis, P. indolicus and P. duerdenii (1936, 2231 and 1947, respectively) (Table 3). However, the distribution of genes into COGs categories was similar in all compared genomes (Supplementary Fig. S4).

Among Peptoniphilus species with standing in nomenclature, average genomic identity of orthologous gene sequences (AGIOS) values ranged from 61.20% between P. timonensis and P. coxii to 84.44% between P. timonensis and P. harei. When comparing P. lacydonensis sp. nov. to other species, AGIOS values were in the same range, from 62.54% with P. coxii to 80.54% with P. timonensis (Table 4). Among Peptoniphilus species with standing in nomenclature, digital DNA-DNA hybridization (dDDH) values ranged from 20.10% between P. indolicus and P. harei to 45.1% between P. indolicus and P. coxii. When comparing P. lacydonensis sp. nov. to other species, dDDH values were also in the same range, from 20.2% with P. indolicus to 39.5% with P. duerdenii (Supplementary Table S4). Among species with standing in nomenclature, average nucleotide identity (ANI) values ranged from 64.4% between P. timonensis and P. coxii to 82.8% between P. timonensis and P. harei. When comparing P. lacydonensis to other species, the ANI value ranged from 66.1% between P. lacydonensis and P. coxii to 79.7% between P. lacydonensis and P. timonensis (Supplementary Table S4).

Table 4

AGIOS values (lower left), numbers of orthologous proteins shared between genomes (upper right) and numbers of proteins per genome (bold numbers)

	Peptoniphilus lacydonensis	Peptoniphilus timonensis	Peptoniphilus duerdenii	Peptoniphilus harei	Peptoniphilus indolicus	Peptoniphilus lacrimalis	Peptoniphilus coxii
P. lacydonensis	1750	1008	943	1138	937	1001	877
P. timonensis	80.54%	1914	780	1029	826	900	743
P. duerdenii	69.20%	67.78%	1920	965	852	941	864
P. harei	78.78%	84.44%	69.48%	1719	964	1074	879
P. indolicus	68.96%	68.16%	67.21%	67.25%	2205	878	792
P. lacrimalis	72.20%	71.11%	69.52%	71.21%	68.54%	1738	835
P. coxii	62.54%	61.20%	63.71%	62.19%	61.45%	62.24%	1738

AGIOS, average genomic identity of orthologous gene sequences.

Discussion

Strain EL1T, which was isolated from a patient with chronic refractory ethmoidal sinusitis, exhibits a close phylogenetic relatedness to Peptoniphilus species but with specific features, notably its ability to grow in microaerophilic atmosphere and a 16S rRNA gene sequencing similarity of 96.76% with P. tyrrelliae [6], which is lower than the threshold recommended by Kim et al. [11] to delineate a new species. Phenotypic characterization, MALDI-TOF MS, 16S rRNA gene sequencing and comparative genomic analyses among close phylogenetic relatives enabled the identification of strain EL1T as a new species within the genus Peptoniphilus, with its closest relatives being P. tyrrelliae, P. harei, P. timonensis and P. gorbachii. ANI, AGIOS and dDDH values confirmed P. lacydonensis sp. nov. as a new species.

Taxonomic and nomenclatural proposals: description of Peptoniphilus lacydonensis sp. nov.

Peptoniphilus lacydonensis (la.cy.do.ne'n.sis, L. gen. masc. n., from lacydonensis, derived from Greek Λακυδω, the name of the ancient port of Marseille, where the patient from which strain EL1T was obtained, lived).

Colonies obtained after 72 hours' incubation on 5% sheep's blood–enriched Columbia agar exhibit a diameter of 0.5 to 1 mm. Bacterial cells are Gram-positive, non–spore-forming and nonmotile cocci. Strain EL1T optimally grows at 37°C after 48 hours in anaerobic atmosphere but is also able to grow in microaerophilic conditions. Catalase and oxidase production reactions are negative. The major fatty acid is the saturated hexadecanoic acid.

Using API 20A and API ZYM strips, positive reactions are observed for indole formation and naphthol-AS-BI-phosphohydrolase, respectively. Negative reactions are observed for alkaline phosphatase, esterase, esterase-lipase, lipase, leucine arylamidase, valine arylamidase, cystine arylamidase, trypsin, α-chymotrypsin, phosphatase acid, α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, α-mannosidase, α-fucosidase and N-acetyl-β-glucosaminidase using API ZYM strips. In addition, no acid production was observed from d-glucose, d-mannitol, d-lactose, d-saccharose, d-maltose, salicin, d-xylose, l-arabinose, glycerol, d-cellobiose, d-mannose, d-melezitose, d-raffinose, d-sorbitol, l-rhamnose and d-trehalose, and no activity was observed for urease, β-glucosidase or protease using API 20A strips. Negative reactions are observed for the fermentation of glycerol, erythritol, d-arabinose, l-arabinose, d-ribose, d-xylose, l-xylose, d-adonitol, methyl-β-d-xylopyranoside, d-galactose, d-glucose, d-fructose, d-mannose, l-sorbose, l-rhamnose, dulcitol, inositol, d-mannitol, d-sorbitol, methyl-αd-xylopyranoside, methyl-αd-glucopyranoside, N-acetylglucosamine, amygdalin, arbutin, esculin ferric citrate, salicin, d-cellobiose, d-maltose, d-lactose, d-melibiose, d-saccharose, d-trehalose, inulin, d-melezitose, d-raffinose, amidone, glycogen, xylitol, gentiobiose, d-turanose, d-lyxose, d-tagatose, d-fucose, l-fucose, d-arabitol, l-arabitol, potassium gluconate, potassium 2-ketogluconate and potassium-5-ketogluconate using API 50 CH strips.

The genome is 1 863 688 bp long with a 29.9% G+C content and is accessible under GenBank accession numbers FNWF01000001 to FNWF01000003. The 16S rRNA gene sequence is also accessible in GenBank under number LN867000. The type strain EL1T (= CSUR P2013 = DSM 100661) was isolated from the ethmoidal sinus of a patient with chronic refractory sinusitis complicating an adenocarcinoma in Marseille, France.

Material and methods

Sample information and EL1T strain isolation

In February 2015, a sinus sample from an 85-year-old man with chronic refractory sinusitis complicating an ethmoidal adenocarcinoma was collected in Marseille, France. The patient provided written informed consent, and the study was validated by the Sud Mediterannée 1 ethics committee under number 15-12. The study was also registered at ClinicalTrials.gov as NCT02407275. After 10 days' culture in an anaerobic blood culture vial that contained 5% sheep's blood–enriched and sterile-filtered sheep rumen, we grew a bacterial strain that we named EL1T (= CSUR P2013 = DSM 100661). The strain was subsequently subcultured on 5% sheep's blood–enriched Columbia agar (bioMérieux) in anaerobic atmosphere at 37°C.

Strain identification by MALDI-TOF MS

To identify the strain, MALDI-TOF MS protein analysis was performed as previously described using a Bruker Microflex spectrometer [12]. Twelve spectra obtained from 12 individual colonies were imported into MALDI BioTyper 2.0 software (Bruker) and analysed by standard pattern matching (with default parameter settings) against the main spectra of 8500 bacteria, including those of 15 Peptoniphilus species. Spectra were recorded in the positive linear mode for the mass range from 2000 to 20 000 Da (parameter settings: ion source 1 (ISI), 20 kV; IS2, 18.5 kV; lens, 7 kV). A spectrum was obtained after 675 shots with variable laser power. The time of acquisition was between 30 seconds and 1 minute per spot. From the resulting scores, the tested species may or may not be identified when compared to the instrument's database; a score ≥2 with a validly published species enables identification at the species level; a score ≥1.7 and <2 allows identification at the genus level; and a score <1.7 does not enable any identification.

Strain identification by 16S rRNA gene sequencing

The 16S rRNA gene nucleotide sequence was obtained by amplification and sequencing using the fD1 and rP2 primers as previously described [13]. Kim et al. [11] determined a 98.65% similarity level threshold to define a new species without performing DNA-DNA hybridization (DDH). A BLASTN was then systematically performed (http://blast.ncbi.nlm.nih.gov.gate1.inist.fr/Blast.cgi). Sequences were aligned and phylogenetic inferences obtained using MUSCLE [14] and MEGA (maximum-likelihood method) [15] software, respectively.

Growth conditions, phenotypic tests and biochemical characterization

The bacterium was subcultured on 5% sheep's blood–enriched Columbia agar (bioMérieux) and incubated for 24 hours at 37°C in anaerobic conditions. Growth was tested at different temperatures (29, 37 and 55°C). Strain growth was tested on 5% sheep's blood–enriched Columbia agar (bioMérieux) under anaerobic and microaerophilic conditions using the GENbag anaer and GENbag microaer systems, respectively (bioMérieux), and under aerobic atmosphere, with or without 5% CO2. Growth was tested for salt tolerance, with 0 to 5%, 50% and 100% (w/v) NaCl. The pH range for growth was tested at pH 6.5 and 8.5 using trypticase soy agar. API 20 A, API ZYM and API 50CH (bioMérieux) strips were used to perform biochemical assays. Oxidase (Becton Dickinson, Franklin Lakes, NJ, USA) and catalase (bioMérieux) assays were performed separately. Cellular fatty acid methyl ester analysis was performed by gas chromatography/mass spectrometry as previously described [16]. Antibiotic susceptibility was tested using the standard disc diffusion procedure on Mueller-Hinton agar with 5% blood using SirScan Discs antibiotics (i2a, Montpellier, France) as described by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) (http://www.eucast.org). The results were interpreted according to EUCAST breakpoints.

Electronic microscopy was performed with a formvar carbon 400-mesh nickel grid (Electron Microscopy Sciences, Hatfield, PA, USA) on which was deposited for 60 seconds a 3.5 μL drop of bacterial suspension. After drying on filter paper, bacteria were immediately stained with 1% ammonium molybdate (ACROS Organics, Illkirch, France) for 1 second. Electron micrographs were then acquired on a Tecnai G20 transmission electron microscope (FEI Company, Hillsboro, OR, USA) at an operating voltage of 200 kV.

Genomic DNA preparation

Strain EL1T was cultured on 5% sheep's blood–enriched Columbia agar (bioMérieux) at 37°C in an anaerobic atmosphere. Bacteria grown on three petri dishes were resuspended in 400 μL of Tris-EDTA (TE) buffer. Then 200 μL of this suspension was diluted in 1 mL TE buffer for lysis treatment, which included a 30-minute incubation with 2.5 μg/μL lysozyme at 37°C, followed by an overnight incubation with 20 μg/μL proteinase K at 37°C. Extracted DNA was then purified using three successive phenol/chloroform extractions and ethanol precipitations at −20°C overnight. After centrifugation, the DNA was resuspended in 160 μL TE buffer.

Genome sequencing and assembly

Genomic DNA (gDNA) of strain EL1T was sequenced using a MiSeq sequencer (Illumina, San Diego, CA, USA) with the mate-pair strategy. The gDNA was barcoded in order to be mixed with 11 other projects with the Nextera Mate Pair sample prep kit (Illumina).

The gDNA was quantified by a Qubit assay using the high-sensitivity kit (Life Technologies, Carlsbad, CA, USA) to 120.3 ng/μL. The mate-pair library was prepared with 1.5 μg of gDNA using the Nextera Mate Pair (Illumina) guide. The gDNA sample was simultaneously fragmented and tagged with a mate-pair junction adapter. The pattern of fragmentation was validated on an Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA, USA) with a DNA 7500 labchip. The DNA fragments ranged in size from 1.5 to 11 kb, with an optimal size at 5.316 kb. No size selection was performed, and 640.3 ng of tagged fragments was circularized. The circularized DNA was mechanically sheared to small fragments with an optimal size of 1550 bp on the Covaris device S2 in T6 tubes (Covaris, Woburn, MA, USA). The library profile was visualized on a High Sensitivity Bioanalyzer LabChip (Agilent), and the final concentration library was measured at 15.44 nmol/L. The libraries were normalized at 2 nM and pooled. After a denaturation step and dilution at 15 pM, the pool of libraries was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and sequencing run were performed in a single 2 × 301 bp run. Total information of 7.3 Gb was obtained from a 511K/mm2 cluster density, with clusters passing quality control filters of 97.0% (12 079 000 passing filter paired reads). Within this run, the index representation for strain EL1T was determined to be 7.71%. The 931 514 paired reads were trimmed, then assembled using SPAdes [17].

Genome annotation and comparison

Open reading frames (ORFs) were predicted using Prodigal [18] with default parameters. However, the predicted ORFs were excluded if they spanned a sequencing gap region. The predicted bacterial protein sequences were searched against the GenBank [19] and COGs databases using BLASTP. The tRNAs and rRNAs were predicted using the tRNAScan-SE [20] and RNAmmer [21] tools, respectively. Signal peptides and numbers of transmembrane helices were predicted using SignalP [22] and TMHMM [23], respectively. Mobile genetic elements were predicted using PHAST [24] and RAST [25]. The CRISPRFinder algorithm was used to identify putative CRISPR (clustered regularly interspaced short palindromic repeat) loci [26]. ORFans were identified if their BLASTP E value was lower than 1e-03 for an alignment length >80 amino acids. If alignment lengths were <80 aa, we used an E value of 1e-05. Such parameter thresholds have been used in previous work to define ORFans. Artemis [27] and DNAPlotter [28] were used for data management and visualization of genomic features, respectively.

A phylogenomic tree was constructed using an FastTree [29] based on Mauve programme genome alignment [30].

To evaluate the degree of genomic similarity at the nucleotide level among studied strains, we determined two parameters: dDDH and AGIOS. The former exhibits a high correlation with DDH, using Genome-to-Genome Distance Calculator (GGDC), formula 2, software [31]. The latter was designed to be independent from DDH. It combines Proteinortho [32] to detect orthologous proteins between genomes compared two by two, then retrieves the corresponding genes, and then determines the mean percentage of nucleotide sequence identity among orthologous ORFs using the Needleman-Wunsch global alignment algorithm [10]. ANI at the genome level between P. lacydonensis and the other species P. lacrimalis (ARKX01000001.1), P. indolicus (AGBB01000001), P. duerdenii (AEEH01000001), P. harei (AENP01000001.1), P. coxii (LSDG00000000) and P. timonensis (CAHE00000000) was estimated using the OrthoANI software [33].