Standards in Genomic Sciences (2013) 8:336-351 DOI:10.4056/sigs.3567059 Non contiguous-finished genome sequence and description of Dielma fastidiosa gen. nov., sp. nov., a new member of the Family Erysipelotrichaceae Dhamodharan Ramasamy1, Jean-Christophe Lagier1, Thi Tien Nguyen1, Didier Raoult1 and Pierre-Edouard Fournier1* 1Aix-Marseille Université, URMITE, Faculté de médecine, Marseille, France *Corresponding author: Pierre-Edouard Fournier ([email protected]) Keywords: Dielma fastidiosa, Genome, Culturomics, Taxono-genomics Dielma fastidiosa strain JC13T gen. nov., sp. nov. is the type strain of D. fastidiosa gen. nov., sp. nov., the type species of a new genus within the family Erysipelotrichaceae. This strain, whose draft genome is described here, was isolated from the fecal flora of a healthy 16-year-old male Senegalese volunteer. D. fastidiosa is a Gram-negative anaerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 3,574,031 bp long genome comprises a 3,556,241-bp chromosome and a 17,790-bp plasmid. The chromosome contains 3,441 protein-coding and 50 RNA genes, including 3 rRNA genes, whereas the plasmid contains 17 protein-coding genes. Introduction Dielma fastidiosa strain JC13T (CSUR P149 / DSM and annotation. These characteristics support the 26099) is the type strain of D. fastidiosa gen. nov., circumscription of the genus Dielma and its type sp. nov., the type species of Dielma gen. nov. This species, D. fastidiosa within the family bacterium is a Gram-negative, anaerobic, catalase Erysipelotrichaceae. and indole-negative bacillus, isolated from the stool The family Erysipelotrichaceae was created in 2004 of a healthy Senegalese patient as part of a study [27] and includes the 10 following genera: aimed at cultivating individually all species within Allobaculum [28], Bulleidia [29], Catenibacterium human feces [1,2]. The conventional genotypic [30], Coprobacillus (Kageyama and Benno 2000) methods used in bacterial taxonomy include 16S [30], Eggerthia [31], Erysipelothrix [32], rRNA gene-based phylogeny and nucleotide simi- Holdemania [33], Kandleria [31], Solobacterium larity [3,4], determination of the G + C content and [30] and Turicibacter [34]. Currently, 12 species DNA–DNA hybridization (DDH) [5,6]. Although with validly published names are reported in this DDH and 16S rRNA gene similarity cutoffs are con- family [35]. The species listed in the sidered as gold standards in bacterial taxonomy, Erysipelotrichaceae are mostly comprised of Gram- they have some limitations as they do not apply positive, non-spore forming, rod-shaped, straight well to all species or genera [3]. Hence, there is a or slightly curved or irregularly shaped, need for alternative methods. The introduction of facultatively anaerobic or anaerobic, catalase nega- high-throughput genome sequencing and proteo- tive, chemoorganotrophic fermentative or respira- mic analyses [7] provided a source of comprehen- tory metabolism, acidifying glucose and other sug- sive information about studied bacterial isolates. ars [35]. Members of species within the radiation of Such data may now be included among the criteria Erysipelotrichaceae were identified as pathogens in used for taxonomic identification. We recently pro- both humans and animals. In humans, these bacte- posed to use a polyphasic approach to describe ria were isolated from patients with oral infection new bacterial taxa that is based on their genome and acute appendicitis [36-40]. sequence, MALDI-TOF spectrum and main pheno- typic characteristics [8-26]. Classification and features Here we present a summary classification and a set A stool sample was collected from a healthy 16- of features for D. fastidiosa gen. nov., sp. nov. strain year-old male Senegalese volunteer patient living JC13T (CSUR P149 / DSM 26099) together with the in Dielmo (rural village in the Guinean-Sudanian description of the complete genome sequencing zone in Senegal), who was included in a research The Genomic Standards Consortium Ramasamy et al. protocol. Written assent was obtained from this fermentation, glutamic acid decarboxylase, individual. For this study, no written consent was alkanine phospatase, arginine arylamidase, proline needed from his guardians because he was older arylamidase, leucyl glycine arylamidase, phenylala- than 15 years (in accordance with the previous nine aylamidase, leucine arylamidase, pyroglutamic project approved by the Ministry of Health of Sene- acid arylamidase, tyrosine arylamidase, alanine gal and the assembled village population and as arylamidase, glycine arylamidase, histidine published elsewhere [41].) Both this study and the arylamidase, glutamyl glutamic acid arylamidase, assent procedure were approved by the National and serine arylamidase. Using an API 20NE strip, a Ethics Committee of Senegal (CNERS) and the Eth- positive reaction was observed for esculine hydrol- ics Committee of the Institut Fédératif de Recher- ysis. No sugar fermentation was observed using che IFR48, Faculty of Medicine, Marseille, France API 50CH (Biomerieux). D. fastidiosa is susceptible (agreement numbers 09-022 and 11-017 Several to amoxicillin, imipenem, metronidazole and other new bacterial species were isolated from this ciprofloxacine, but resistant to trime- specimen using various culture conditions, includ- thoprim/sulfamethoxazole, rifampin, doxycycline, ing the recently described Alistipes senegalensis, and gentamicin. The differential phenotypic charac- Alistipes timonensis, Anaerococcus teristics with other species are summarized in Ta- senegalensis,Clostridium senegalense, Peptoniphilus ble 2. timonensis, Paenibacillus senegalensis, Different growth temperatures (25, 30, 37, 45°C) Herbaspirillum massiliense, Kurthia massiliensis, were tested; growth occurred between 25°C and Brevibacterium senegalense, Aeromicrobium 45°C and optimal growth was observed at 30°C. massilense, Cellulomonas massiliensis, Colonies were 0.5 to 1 mm in diameter on blood- Senegalemassilia anaerobia, Peptoniphilus enriched Columbia agar and BHI agar. Growth of senegalensis and Enterobacter massiliensis [9- the strain was tested under anaerobic and 20,23]. microaerophilic conditions using GENbag anaer The fecal specimen was preserved at -80°C after and GENbag microaer systems, respectively collection. Strain JC13 T (Table 1) was isolated in (BioMérieux) and in the presence of air with or January 2011 by cultivation on Brain Heart Infu- without 5% CO2. Growth was achieved only under sion agar (Becton Dickinson, Pont de Claix, France), anaerobic conditions. Gram staining showed a rod- after a 10 day preincubation in anaerobic blood shaped Gram-negative bacterium (Figure 2). The culture bottle. motility test was positive. Cells grown on agar have The 16S rRNA sequence (GenBank accession num- a mean diameter of 0.60 µm and a mean length of ber JF824807) of D. fastidiosa strain JC13T was 2.2 µm in electron microscopy (Figure 3). compared to sequences in GenBank using BLAST Matrix-assisted laser-desorption/ionization time- [50] and showed a highest similarity of 89.71% of-flight (MALDI-TOF) MS protein analysis was car- with Clostridium innocuum (Figure 1). By compari- ried out as previously described using a Microflex son with type species from genera within the family spectrometer (Bruker Daltonics, Leipzig, Germany) Erysipelotrichaceae, D. fastidiosa exhibited a 16S [7,51]. Briefly, a pipette tip was used to pick one rRNA sequence similarity ranging from 69.90 to isolated bacterial colony from a cultured agar plate, 89.71%. Since these values are lower than the 95% and spread it as a thin film on a MTP 384 MALDI- threshold recommended by Stackebrandt and TOF target plate (Bruker Daltonics). Twelve dis- Ebers to delineate new genera without performing tinct deposits were made for strain JC13 from DDH [3], we propose to classify strain JC13T within twelve isolated colonies. Each smear was overlaid a novel genus. with 2µL of matrix solution (saturated solution of Strain JC13T did not exhibit catalase or oxidase ac- alpha-cyano-4-hydroxycinnamic acid) in 50% ace- tivity. Using API Rapid ID 32A, positive reactions tonitrile, 2.5% tri-fluoracetic-acid, and allowed to were ob -fucosidase and pyroglutamic dry for five minutes. Measurements were per- acid arylamidase. Negative reactions were ob- formed with a Microflex spectrometer (Bruker). served fortained indole for αproduction, nitrate reduction, Spectra were recorded in the positive linear mode - - for the mass range of 2,000 to 20,000 Da (parame- - - ter settings: ion source 1 (IS1), 20 kV; IS2, 18.5 kV; urease, arginine-arabinosidase, dihydrolase, α-glucuronidase,galactosidase, Nβ- lens, 7 kV). A spectrum was obtained after 675 acetylgalactosidase- -glucosaminidase, 6 phosphate, man noseα glucosidase, and raffinose β shots at a variable laser power. The time of acquisi- glucosidase, α β http://standardsingenomics.orgβ 337 Dielma fastidiosa gen. nov., sp. nov. tion was between 30 seconds and 1 minute per species: a score > 2 with a validly published species spot. The twelve JC13 spectra were imported into enabled the identification at the species level, a the MALDI BioTyper software (version 2.0, Bruker) score > 1.7 but < 2 enabled the identification only at and analyzed by standard pattern matching (with the genus level; and a score < 1.7 did not enable any default parameter settings) against the reference identification. For strain JC13T, no significant score spectra from