Non-contiguous finished genome sequence and description of Kallipyga massiliensis gen. nov., sp. nov., a new member of the family Clostridiales Incertae Sedis XI

Kallipyga massiliensis strain ph2^T (CSUR=P241, DSM=26229) is the type strain of K. massiliensis gen. nov., sp. nov. This bacterium was isolated from the stool sample of an obese French patient as part of a study aiming at individually cultivating all species occurring within human feces [1-3]. It is a Gram-positive, anaerobic, indole-negative coccus. Defining the taxonomic status of bacterial isolates remains a challenging task. The taxonomic molecular tools currently available, including 16S rRNA sequence similarity, G + C content and DNA–DNA hybridization (DDH) [4,5], although considered as gold standards, have limitations [6,7]. The 16S rRNA sequence similarity and G+C content thresholds do not apply uniformly to all species or genera, and the DDH method lacks intra- and inter-laboratory reproducibility [5]. The advent of high-throughput genome sequencing and proteomic analysis [8] has granted unprecedented access to exhaustive genetic and protein information for bacterial isolates. We recently proposed a polyphasic approach to describe new bacterial species in which genome sequences and MALDI-TOF spectra are used along with phenotypic characteristics [9-30].

The family Clostridiales Incertae Sedis XI (Garrity and Holt 2001) was created in 2001 [31] and currently includes the 11 following genera: Anaerococcus (Ezaki et al. 2001) [32], Dethiosulfatibacter (Takii et al. 2007) [33], Finegoldia (Murdoch and Shah 2000) [34], Gallicola (Ezaki et al. 2001) [32], Helcococcus (Collins et al. 1993) [35], Parvimonas (Tindall and Euzéby 2006) [36], Peptoniphilus (Ezaki et al. 2001) [32], Sedimentibacter (Breitenstein et al. 2002) [37], Soehngenia (Parshina et al. 2003) [38], Sporanaerobacter (Hernandez-Eugenio et al. 2002) [39] and Tissierella (Collins and Shah 1986) [40]. Currently, 31 species with validly published names are reported in this family [41]. The species listed in the Clostridiales Incertae Sedis XI are mostly comprised of Gram-positive, obligate anaerobic cocci. Members belonging to this family were identified as pathogens in both humans and animals. In humans, they were often isolated from patients with septic arthritis, necrotizing pneumonia, prosthetic joint infection and other clinical conditions associated with vaginal discharges and ovarian, peritoneal and sacral abscesses [42-46].

Here we present a summary classification and a set of features for K. massiliensis gen. nov., sp. nov., strain ph2^T (CSUR=P241, DSM=26229) together with the description of the complete genomic sequencing and annotation. These characteristics support the circumscription of the genus Kallipyga and its type species, K. massiliensis within the Clostridiales Incertae Sedis XI family.

A stool sample was collected from a 26-year-old woman living in Marseille (France). She suffered from morbid obesity and had a body mass index of 48.2 (118.8 kg, 1.57 meter). At the time of stool sample collection she did not take any medication and was not on a diet. The patient gave an informed and signed consent, and the agreement of the ethics committee of the Institut Fédératif de Recherche (IFR48, Faculty of Medicine, Marseille, France) was obtained under reference 09-022. Another four new bacterial species, Alistipes obesi, Peptoniphilus grossensis, P. obesi and Enorma massiliensis [25-27,33], were also isolated from this specimen using various culture conditions. The fecal specimen was preserved at -80°C after collection. Strain ph2 ^T (Table 1) was isolated in 2011 by anaerobic culture on 5% sheep blood-enriched agar in anaerobic atmosphere at 37°C, following 26 days in a blood culture bottle with rumen and sheep blood. The 16S rRNA nucleotide sequence (GenBank accession number JN837487) of Kallipyga massiliensis strain ph2^T was 86.09% similar to Helcococcus sueciensis, the phylogenetically closest species (Figure 1). This value was lower than the 95.0% 16S rRNA gene sequence threshold recommended by Stackebrandt and Ebers (2006) to delineate a new genus without carrying out DNA-DNA hybridization [5].

By comparison to the Genbank database [57], strain ph2^T also exhibited a nucleotide sequence similarity greater than 98.7% with 16 sequences from uncultured bacteria from the human skin microbiome [58]. These bacteria are most likely classified within the same species as strain ph2^T.

Different growth temperatures (25, 30, 37, 45°C) were tested; no growth occurred at 25°C and 30°C, growth occurred between 37°C and 45°C, and optimal growth was observed at 37°C. Colonies were bright grey with a diameter of 1.0 mm on 5% blood-enriched Columbia agar. Growth of the strain was tested under anaerobic and microaerophilic conditions using GENbag anaer and GENbag microaer systems, respectively (BioMérieux), and in the presence of air, with or without 5% CO2. Optimal growth was obtained anaerobically. No growth was observed under aerobic and microaerophilic conditions. Gram staining showed Gram-positive cocci (Figure 2). A motility test was negative. Cells grown on agar are Gram-positive, have a diameter in electron microscopy ranging from 0.57µm to 0.78µm (mean, 0.67 µm, Figure 3) and are mostly grouped in pairs, short chains or small clumps.

Strain ph2^T exhibited neither catalase or oxidase activities. Using API 32A (BioMerieux), nitrate reduction, indole formation and urease production were negative. A positive reaction was obtained for α-galactosidase, arginine dihydrolase and arginine arylamidase, α-glucosidase and β-glucosidase. Strain ph2^T did not ferment mannose or raffinose. Negative reactions were observed for β-galactosidase, β-galactosidase-6-phosphate, α-arabinosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, glutamic acid decarboxylase, proline arylamidase, leucyl glycine arylamidase, phenylalanine arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase, alanine arylamidase, glycine arylamidase, histidine arylamidase, glutamyl glutamic acid arylamidase, and serine arylamidase. Using an API Zym (BioMerieux), positive reactions were observed for esterase lipase, leucine arylamidase, α-glucosidase, β-glucosidase and acid phosphatase. Negative reactions were obtained for esterase, lipase, valine and cysteine arylamidase, trypsine, α-chymotrypsine, naphthol-AS-BI-phosphohydrolase, α-galactosidase, β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. Using an API 50CH (BioMerieux), K. massiliensis weakly fermented D-ribose, D-glucose, D-fructose and aesculin. By comparison with its closest phylogenetic neighbors, K. massiliensis differed from Finegoldia magna in α-galactosidase and α-glucosidase production, D-ribose and D-fructose fermentation. It also differed from Helcococcus kunzii in oxygen requirement, α-galactosidase and leucine arylamidase production, D-ribose, D-glucose and esculin utilization. It differed from Parvimonas micra in alkaline phosphatase, glutamyl glutamic acid arylamidase, β-glucosidase, phenylalanine arylamidase and histidine arylamidase production and D-glucose fermentation. It differed from Peptoniphilus indolicus in α-galactosidase, indole, α-glucosidase, β-glucosidase, dihydrolase phenylalanine, phenylalanine arylamidase and histidine arylamidase production, and D-glucose fermentation (Table 2).

K. massiliensis is susceptible to amoxicillin, amoxicillin-clavulanic acid, gentamicin 500, penicillin, imipenem, vancomycin, rifampicin and nitrofurantoin, but resistant to ciprofloxacin, metronidazole, gentamicin 10, trimethoprim/sulfamethoxazole, ceftriaxon, erythromycin and doxycycline.

Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) MS protein analysis was carried out as previously described [59] using a Microflex spectrometer (Bruker Daltonics, Germany). Twelve distinct deposits were done for strain ph2^T from 12 isolated colonies. The twelve ph2^T spectra were imported into our database and compared to spectra from 3,769 bacteria using the MALDI BioTyper software (version 2.0, Bruker). A score enabled the presumptive identification and discrimination of the tested species from those in a database: a score > 2 with a validly published species enabled the identification at the species level; a score > 1.7 but < 2 enabled the identification at the genus level; and a score < 1.7 did not enable any identification. For strain ph2^T, no significant score was obtained, suggesting that our isolate was not a member of any known species or genus (Figures 4 and 5). A broader study incorporating MALDI-TOF and 16S rDNA and genomic DNA identity data may be conducted to define taxonomic criteria at the family level.

The genome is 1,770,679 bp long (one chromosome, no plasmid) with a G+C content of 51.40% (Figure 6 and Table 4). Of the 1,625 predicted chromosomal genes, 1,575 were protein-coding genes and 50 were RNAs. A total of 1,238 genes (76.18%) were assigned a putative function. Forty-two genes were identified as ORFans (2.66%) and the remaining genes were annotated as hypothetical proteins. The properties and statistics of the genome are summarized in Tables 4 and 5. The distribution of genes into COGs functional categories is presented in Table 5.

Currently, 35 genomes are available for members of the family Clostridiales Incertae Sedis XI. Here, we compared the genome sequence of K. massiliensis strain ph2^T with those of Finegoldia magna strain ATCC 29328, Helcococcus kunzii strain ATCC 51366, Peptoniphilus indolicus strain ATCC 29427 and Parvimonas micra strain ATCC 33270. The draft genome of K. massiliensis (1.77Mb) is smaller than all other genomes except P. micra (1.70Mb) and exhibits a higher G+C content (51.40) (Table 6A). The gene content of K. massiliensis is also lower than the other four genomes used for comparison (Table 6B). In addition, K. massiliensis shared 653, 549, 592 and 548 orthologous genes with F.magna, H. kunzii,P. indolicus and P.micra respectively. The average nucleotide sequence identity ranged from 58.61 to 69.17% among Clostridiales Incertae Sedis XI family species, and from 58.61 to 59.97% between K. massiliensis and other species, thus confirming its new genus status (Table 6B).