NEW MICROBES IN HUMANS Noncontiguous finished genome E-mail: fl[email protected] The first two authors contributed equally to this article, and both sequence and description of should be considered first author. Weeksella massiliensis sp. nov. Introduction S. A. Sankar1,C.I.Lo1,2, B. Fall3, B. Sambe-Ba3, 1,2 3 1 4 3 O. Mediannikov , I. Diallo , N. Labas , N. Faye , B. Wade , The genus Weeksella (Holmes et al., 1986) was first described in 1,2,5 1 1,2 D. Raoult , P.-E. Fournier and F. Fenollar 1986 [1]. To date, this genus includes one species, Weeksella 1 )Aix-Marseille Université, URMITE, UM63, CNRS 7278, IRD 198, Inserm virosa, which has been isolated from human clinical specimens U1095, Faculté de médecine, Marseille, France, 2 )Campus International [1,2]. UCAD-IRD, 3 )Hôpital Principal, 4 )Université Cheikh Anta Diop de Dakar, The current classification of prokaryotes relies on a poly- Laboratoire de Parasitologie générale, Dakar, Senegal and 5 )Special phasic strategy combining phenotypic and genotypic charac- Infectious Agents Unit, King Fahd Medical Research Center, King Abdulaziz teristics [3,4]. These include 16S rRNA sequence similarity, University, Jeddah, Saudi Arabia G + C content and DNA-DNA hybridization (DDH). However, these tools have significant drawbacks, notably that the rec- ommended threshold values do not apply to all species or Abstract genera [5,6]. Thanks to the progress made in sequencing technologies and Strain FF8T (= CSUR P860 = DSM 28259) was isolated in Dakar, their lowering costs, almost 40 000 bacterial genome sequences Senegal, from the urine of a 65-year-old man with acute cystitis. are currently available, covering many phyla [7]. Recently we This strain shows a similarity of sequence of 16S rRNA of proposed to integrate phenotypic characteristics, notably the 98.38% with Weeksella virosa, and its GenBank accession numbers MALDI-TOF spectrum, and genomic analysis and comparison in are HG931340 and CCMH00000000. Matrix-assisted laser the taxonomic description of bacterial species [5,8,9].We desorption/ionization time-of-flight mass spectrometry analysis named this strategy taxonogenomics [5]. T had a poor score, ranging from 1.32 to 1.56, that did not allow Strain FF8 (= CSUR P860 = DSM 28259) was isolated from identification of the bacterium. Using a polyphasic study made of the urine of a 65-year-old man treated at the Hôpital Principal phenotypic and genomic analyses, strain FF8T was a Gram- de Dakar, Senegal. That is a Gram-negative bacterium, aerobic, negative, aerobic rod and a member of the family indole negative, nonmotile and rod shaped. This bacterium was Flavobacteriaceae. The sequenced genome is 2 562 781 bp with cultivated as part of the MALDI-TOF implementation at Hôpital one chromosome but no plasmid. It exhibits a G + C content of Principal de Dakar aiming to improve the routine laboratory 35.9% and contains 2390 protein-coding and 56 RNA genes, identification of microorganisms [10]. including a complete rRNA operon. On the basis of these data, Here we present a summary classification and a set of fea- we propose the creation of Weeksella massiliensis sp. nov. tures for Weeksella massiliensis sp. nov., together with the New Microbes and New Infections © 2015 The Authors. Published description of the complete genome sequencing and annota- by Elsevier Ltd on behalf of European Society of Clinical tion. These characteristics support the circumscription of the Microbiology and Infectious Diseases. species Weeksella massiliensis. Keywords: Culturomics, cystisis, genome, human, Organism Information taxonogenomics, urine, Weeksella massiliensis Original Submission: 29 May 2015; Revised Submission: 21 September 2015; Accepted: 23 September 2015 Classification and features Article published online: In July 2013, a urine sample was collected from a 65-year-old Senegalese man with acute cystitis. From this clinical sample strain FF8 (Table 1) was isolated by cultivation on 5% sheep’s Corresponding author: F. Fenollar, Aix-Marseille Université, – ’ URMITE, UM63, CNRS 7278, IRD 198, Inserm U1095, Faculté de blood enriched Columbia agar (bioMérieux, Marcy l Etoile, médecine, 27 Boulevard Jean Moulin, 13385 Marseille cedex 05, France). When analysed by MALDI-TOF, no identification was France obtained because the strain displayed low scores. New Microbe and New Infect 2015; 8: 89–98 New Microbes and New Infections © 2015 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) http://dx.doi.org/10.1016/j.nmni.2015.09.013 90 New Microbes and New Infections, Volume 8 Number C, November 2015 NMNI TABLE 1. Classification and general features of Weeksella Gram-negative, non-spore-forming rods of regular shape with massiliensis strain FF8T [19] rounded ends (Fig. 2) and have mean diameter of 0.3 μm (range, – μ μ – μ MIGS Evidence 0.2 0.5 m) and a mean length of 1.5 m (range, 0.8 2.1 m) ID Property Term codea (Fig. 3). Weeksella massiliensis strain FF8T does not grow on Classification Domain: Bacteria TAS [34] MacConkey agar [14]. Phylum: Bacteroidetes TAS [11,12] Class: Flavobacteriia TAS [35,36] Order: Flavobacteriales TAS [35–37] Chemotaxonomic information Family: Flavobacteriaceae TAS [38] Genus: Weeksella TAS [1] This bacterium possesses catalase and oxidase. Using an API Species: Weeksella IDA massiliensis ZYM strip (bioMérieux), positive reactions were observed for (Type) strain: FF8T IDA Gram stain Negative IDA alkaline phosphatase, esterase, esterase–lipase, leucine aryla- Cell shape Rod IDA Motility Not motile IDA midase, acid phosphatase and naphthol-AS-BI- Sporulation Non–spore forming NAS Temperature range Mesophile IDA phosphohydrolase. Negative reactions were noted for Optimum temperature 37°C IDA α-chymotrypsin, cystine arylamidase, valine arylamidase, Optimum pH range 7.2–7.4; 7.3 Carbon source Unknown trypsin, α-glucosidase, β-glucosidase, α-galactosidase, β-galac- MIGS-6 Habitat Human IDA MIGS-6.3 Salinity Unknown tosidase, β-glucuronidase, α-mannosidase, α-fucosidase and N- MIGS-22 Oxygen requirement Aerobic TAS T MIGS-15 Biotic relationship Free living TAS acetyl-β-glucosaminidase. Strain FF8 is susceptible to ceftri- MIGS-14 Pathogenicity Unknown MIGS-4 Geographic location Dakar TAS axone, amoxicillin/clavulanic acid, penicillin, imipenem, genta- MIGS-5 Sample collection November 28, 2013 TAS MIGS-4.1 Latitude 14.6937000 TAS micin and doxycycline but resistant to nitrofurantoin, MIGS-4.1 Longitude −17.4440600 TAS MIGS-4.4 Altitude 12 m above sea level TAS vancomycin, trimethoprim/sulfamethoxazole and metronida- MIGS, minimum information about a genome sequence. zole. The minimum inhibitory concentrations (MICs) for some aEvidence codes are as follows: IDA, inferred from direct assay; TAS, traceable antibiotics tested by Weeksella massiliensis strain FF8T sp. nov. author statement (i.e., direct report exists in the literature); NAS, nontraceable author statement (i.e., not directly observed for the living, isolated sample, but are listed in Table 2. A comparison of phenotypic characteris- based on a generally accepted property for the species or on anecdotal evidence). These evidence codes are from http://www.geneontology.org/GO.evidence.shtml tics with W. virosa, Bergeyella zoohelcum [15] and Moheibacter of the Gene Ontology project [39]. If the evidence is IDA, then the property was directly observed for a live isolate by one of the authors or an expert mentioned in sediminis [16] is presented in Table 3. the acknowledgements. Extended features descriptions MALDI-TOF protein analysis was performed with a Microflex LT (Bruker Daltonics, Leipzig, Germany), as previously re- Strain FF8 exhibited a 98.38% 16S rRNA sequence similarity ported [17,18]. The scores previously established by Bruker with Weeksella virosa strain DSM 16922T (GenBank accession allowing validating (or not) the identification of species number NR_074495), the phylogenetically closest bacterial compared to the database of the instrument were applied. species with standing in nomenclature. These values were Briefly, a score of 2.000 with a species with a validly pub- lower than the 98.7% 16S rRNA gene sequence threshold lished name provided allows the identification at the species recommended by Meier-Kolthoff et al., 2013, to delineate a new level; a score of 1.700 and < 2.000 allows the identification at species within phylum Bacteroidetes [11,12] without carrying the genus level; and a score of < 1.700 does not allow any out DDH [13]. A phylogenetic tree based on the 16S rRNA identification. We performed 12 distinct deposits from 12 sequence highlights the position of Weeksella massiliensis strain isolated colonies of strain FF8T. Two microliters of matrix so- FF8T among the family Flavobacteriaceae (Fig. 1). Different lution (saturated solution of α-cyano-4-hydroxycinnamic acid) growth temperatures (25, 30, 37, 45 and 56°C) were tested. in 50% acetonitrile and 2.5% trifluoroacetic acid were distrib- Growth was obtained between 25 and 37°C, with an optimal uted on each smear and submitted at air drying for 5 minutes. growth at 37°C. Growth of the strain was tested also under Then the spectra from the 12 different colonies were imported anaerobic and microaerophilic conditions using GENbag anaer into the MALDI BioTyper software (version 2.0, Bruker) and and GENbag microaer systems, respectively (bioMérieux), and analysed by standard pattern matching (with default parameter under aerobic conditions, with or without 5% CO . Thus the 2 settings) against the main spectra of 6252 bacteria. Scores optimal growth was observed under aerobic and micro- ranging from 1.32 to 1.56 were obtained for strain FF8T, sug- aerophilic conditions. No growth was observed under anaer- gesting that it was not a member of any known species. The obic conditions. The colonies were opaque, light yellow in reference mass spectrum from strain FF8T was incremented in color with a smooth surface, not haemolytic on 5% sheep’s our database (Fig.