Genome analysis of Lachnoclostridium phocaeense isolated from a patient after kidney transplantation in Marseille

Lachnoclostridium phocaeense is a new species in the genus Lachnoclostridium. Lachnoclostridium phocaeense is a Gram-positive anaerobic rod. This strain, Marseille-P3177T (CSUR = P3177) with the below described genome was isolated from the urine sample of a women after kidney transplantation. The strain genome is 3 500 754 bp long with 50.62% G + C content and consists of a single contig (GenBank accession number NZ_LT635479.1).


Introduction
Lachnoclostridium is a genus of Gram-positive, obligate anaerobic, spore-forming, motile bacteria. Organisms in this genus can grow in moderate 'mesophilic' as well as in extremely high 'thermophilic' temperatures, ranging from 20°C to 45°C and from 203°C to 633°C, respectively [1].
The Lachnoclostridium genus includes organisms from the Lachnospiraceae family and from several clostridial clusters such as Clostridium XIVa [1]. Clostridial cluster XIVa is known to make up a significant part of the human gut microflora [2]; it can exert anti-inflammatory effects and plays a role in homeostasis. In addition, via its components and metabolites, notably butyrate, clostridial cluster XIVa maintains intestinal health [3].
The human gut microbiota is a complex ecosystem that contains a variety of organisms including bacteria, fungi and viruses [4]. To explore this niche, bacterial cultures were used [5]; however, provided information on only the cultivable part of the humvan gut with a considerable fraction being uncultured. This is despite the advancement of molecular techniques such as metagenomics and 16S rRNA sequencing [6]. Recently, a new approach combining bacterial culturing under different conditions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and 16s rRNA sequencing, named culturomics, was implemented. Compared with metagenomics, this approach allows the cultivation of species corresponding to previously unassigned sequences [7].
Using a previously described taxonogenomic approach [8,9] combined with culturomics, we present here the phenotypic and genomic characteristics of a Lachnoclostridium novel species isolated from a patient admitted to the hospital in Marseille. This is part of the culturomics project, which aims to detect and isolate new bacterial species. The new species was deposited in the Collection de Souches de l'Unite des Rickettsies (CSUR, WDCM 875) under the number P3177 [10].

Phenotypic and biochemical characterization
Strain Marseille-P3177 appears as translucent and whitish circular colonies with a diameter of 0.7-1 mm on a 5% sheep blood Columbia agar medium (BioMérieux, Marcy-l'Étoile, France). This species developed under anaerobic conditions at 37°C and for a period of 5 days of incubation [10].
Biochemical characteristics of the isolated strain were determined using API ZYM and API 50CH (BioMérieux). Catalase assays (BioMérieux) and oxidase assays (Becton Dickinson, Le Pont de Claix, France) showed that this strain is oxidase and catalase negative. API ZYM revealed positive reactions for acid phosphatase, naphthol-AS-BIphosphohydrolase, α-galactosidase, β-galactosidase, α-glucosidase and β-glucosidase. On the other hand, using API 50CH, acid production was observed in the presence of starch (Table 1).
Antibiotic susceptibility testing was done using E-test (Bio-Mérieux) performed on Mueller-Hinton agar supplemented with 5% blood (BioMérieux). Interpretation of the results was done according to the European Committee on Antimicrobial susceptibility testing 2018 (EUCAST). The strain was susceptible to amoxicillin, cefotaxime, ertapenem, impipenem,  Phylogenetic tree analysis based on partial 16S ribosomal RNA sequences. Genbank accession numbers of partial 16S rRNA gene sequence are indicated in parenthesis. Sequences were aligned using CLUSTALW and the phylogenetic tree was obtained using the maximum likelihood bootstrap method and MEGA 7 software [20]. Numbers shown at the nodes are bootstrap percentages values obtained by 1000 times repetition analysis. meropenem, vancomycin, teicoplanin, metronidazole, trimethoprim-sulfamethoxazole, rifampicin and gentamicin; but resistant to ciprofloxacin, fosfomycin, colistin, doxycycline, oxofloxacin and erythromycin.

Genome sequencing
Extracted genomic DNA of L. phocaeense P3177 was sequenced using MiSeq (Illumina, San Diego, CA, USA) with the mate-pair strategy. Assembly and annotation were performed with a pipeline of different softwares (SPADES [13], VELVET [11], SOAP DENOVO [14], trimmed (TRIMMOMATIC), MISEQ [15] software or untrimmed data (only MISEQ software) and XEGEN (http:// www.xegen.fr/). To reduce assembly gaps, GAPCLOSER was used. Scaffolds with depth value < 25% of the mean depth and <800 bp were removed. Using different criteria (number of N, number of scaffolds and N50), the best assembly was selected. Genome coverage was 125×. The predicted bacterial protein sequences for L. phocaeense in addition to the five complete genomes of Lachnclostridium available on NCBI were searched against the Clusters of Orthologous Groups (COG) database and NR database using BLASTP [16].
The degree of genomic similarity of Marseille-P3177 with closely related species was estimated using the ORTHOANI software [17]. Values among closely related species (Fig. 4) ranged from 67.07% between Lachnoclostridium pacaense and Lachnoclostridium hylemonae to 76.60% between Lachnoclostridium bolteae and Lachnoclostridium pacaense. When strain Marseille-P3177 was compared with these closely related species, values ranged from 67.66% with L. saccharolyticum to 72.53% with L. scindens.

Genome description
Lachnoclostridium phocaeense strain Marseille P3177 genome (GenBank accession number NZ_LT635479.1) is 3 500 754 bp long with 50.62% G + C content ( Table 2). The genome coverage was 125×. Of the 3382 predicted genes, 3315 were protein-coding genes and 67 were RNAs (four genes were 5S rRNA, four genes were 16S rRNA, four genes were 23S rRNA, 55 genes were tRNA genes). A total of 2328 genes (70.23%) were assigned as putative function (by COGs or by NR blast). A total of 170 genes were identified as ORFans (5.13%). The remaining genes were annotated as hypothetical proteins (719 genes, 21.69%). Gene distribution into COG functional categories of L. phocaeense are presented in Table 3. The distribution of genes in COG categories was similar in all six species of Lachnoclostridium (Fig. 5).
Using the BIO-EDIT interface, a BLAST search was conducted against ARG-ANNOT, a database for acquired antibiotic   resistance genes (ARGs). The BLAST search was done under an e-value of 10 −5 , moderately stringent conditions for in silico ARG prediction [18]. ARG-ANNOT BLAST search revealed the presence of one resistance gene against tetracycline. This is in accordance with the antibiotic susceptibility testing performed, which showed that this strain was resistant to doxycycline. The bacteriocin database available in our research unit (Bacteriocins of the URMITE database BUR; available at http:// drissifatima.wix.com/bacteriocins) was set up through the collection of all available sequences from NCBI and databases. Protein sequences from the aforementioned database allow the identification of bacteriocins from the human gut microbiota using BLASTp methodology [19]. Resistome analysis via this database showed the presence of 25 bacteriocinassociated genes.
Lachnoclostridium phocaeense (pho.cae.en'se, L. neut. adj. phocaeense, referring to the town Phocaea, the Latin name of the city that was later named Marseille, in France, where the type strain was first isolated). Lachnoclostridium phocaeense strain Marseille-P3177 is a new species in the genus Lachnoclostridium that was isolated from a 51-year-old woman's urine sample after kidney transplantation in Marseille. The species' optimal growth conditions are 37°C for 5 days under anaerobic conditions. Colonies are 0.7-1 mm in diameter on blood-supplemented agar. Lachnoclostridium phocaeense is a strictly anaerobic Gram-positive rod. It is also catalase and oxidase negative.