G Model SYAPM-25874; No. of Pages 8 ARTICLE IN PRESS Systematic and Applied Microbiology xxx (2017) xxx–xxx Contents lists available at ScienceDirect Systematic and Applied Microbiology journal homepage: www.elsevier.de/syapm Vagococcus teuberi sp. nov., isolated from the Malian artisanal sour milk fènè a a a a Stephan Wullschleger , Christoph Jans , Clelia Seifert , Sarah Baumgartner , a b a a,∗ Christophe Lacroix , Bassirou Bonfoh , Marc J.A. Stevens , Leo Meile a Laboratory of Food Biotechnology, Institute of Food Science and Nutrition, ETH Zurich, Schmelzbergstrasse 7, CH-8092 Zurich, Switzerland b Centre Suisse de Recherches Scientifiques en Côte d’Ivoire (CSRS), KM 17 route de Dabou, Adiopodoumé Yopougon, Abidjan — 01 B.P. 1303, Abidjan, Cote d’Ivoire a r t i c l e i n f o a b s t r a c t Article history: Ten bacterial isolates belonging to the genus Vagococcus were obtained from Malian sour milk fènè Received 27 July 2017 produced from spontaneously fermented cow milk. However, these isolates could not be assigned to Received in revised form 3 November 2017 a species upon initial comparative 16S rRNA gene sequence analysis and were therefore further charac- Accepted 7 November 2017 T terized. Rep-PCR fingerprinting of the isolates yielded four strain clusters represented by strains CG-21 T T (=DSM 21459 ), 24CA, CM21 and 9H. Sequence identity of the 16S rRNA gene of DSM 21459 to its clos- Keywords: T est relative species Vagococcus penaei was 97.9%. Among the four rep strain clusters, DSM 21459 and Vagococcus teuberi sp. nov. 24CA shared highest 16S rRNA gene sequence identity of 99.6% while CM21 and 9H shared 98.6–98.8% Lactic acid bacteria T T T with DSM 21459 and V. penaei CD276 . DSM 21459 and 24CA were thus subjected to a polyphasic Spontaneous fermentation T typing approach. The genome of DSM 21459 featured a G + C content of 34.1 mol% for a 2.17-bp chro- Sour milk T Fènè mosome and a 15-kbp plasmid. Average nucleotide identity (ANI) of DSM 21459 to Vagococcus fluvialis T Mali bH819, V. penaei CD276 were 72.88%, 72.63%, respectively. DNA–DNA hybridization (DDH) similarities T of strain DSM 21459 to other Vagococcus species were <42.0%. ANI and DDH findings strongly supported T the 16S rRNA gene phylogenetic tree delineations. The fatty acid patterns of DSM 21459 was palmitic acid (C 16:0, 24.5%), oleic acid (C 18:1-␻9c, 32.8%), stearic acid (C 18:0, 18.9%). General physiological char- T acterization of DSM 21459 and 24CA were consistent with those of the genus Vagococcus. Strain DSM T 21459 and further strains are therefore considered to belong to a novel species, for which the nomen- T T clature Vagococcus teuberi sp. nov. is proposed. The type strain is named CG-21 (=DSM 21459 and LMG 24695T). © 2017 Elsevier GmbH. All rights reserved. Introduction isolated from other aquatic animals [20,30], Vagococcus carniphilus isolated from ground beef and dry sausages [39], Vagococcus elon- Vagococcus is a genus in the family Enterococcaceae in the phy- gatus isolated from a swine-manure storage pit [29], Vagococcus lum Firmicutes. The genus was created in 1990 from a group of penaei from contaminated cooked shrimp [22], Vagococcus acid- lactococci strains reacting with Lancefield group N antisera [6]. ifermentans from a food waste water bioreator [46], Vagococcus The type species Vagococcus fluvialis was originally isolated from entomophilus from a wasp digestive tract [25] and recently Vago- chicken feces and river water [19]. It was initially typed as a motile coccus humatus from soil [42]. All 10 species seem to be present Lactococcus-like strain with an unresolved phylogenetic position in different environments. Milk and milk products were so far not [38] and later associated with human or animal infections [1]. Nine described as primary habitat source of any Vagococcus species. other species of the genus Vagococcus have been described to date. In this report we describe a polyphasic taxonomic study on T These are the fish-pathogenic Vagococcus salmoninarum isolated strain DSM 21459 and genotypic and phenotypic inter-species dif- from rainbow trout [45], Vagococcus lutrae and Vagococcus fessus ferences of Vagococcus strains which were isolated from different spontaneously fermented milk (fènè) stocks at home- and small- scale production sites in Mali [48]. Based on the presented data, ∗ including whole genome sequences, we consider that these strains Corresponding author. belong to an undefined novel species of the genus Vagococcus. E-mail address: [email protected] (L. Meile). https://doi.org/10.1016/j.syapm.2017.11.003 0723-2020/© 2017 Elsevier GmbH. All rights reserved. Please cite this article in press as: S. Wullschleger, et al., Vagococcus teuberi sp. nov., isolated from the Malian artisanal sour milk fènè, Syst. Appl. Microbiol. (2017), https://doi.org/10.1016/j.syapm.2017.11.003 G Model SYAPM-25874; No. of Pages 8 ARTICLE IN PRESS 2 S. Wullschleger et al. / Systematic and Applied Microbiology xxx (2017) xxx–xxx Material and methods the universal 16S rRNA gene primer bak11 w [11] adapted to the ◦ higher annealing temperatures of 63 C of the V. teuberi-specific Microorganisms and culture conditions reverse primer va-r1 in order to amplify a 207-bp DNA fragment ◦ of the 16S rRNA gene. A total of 35 cycles of denaturation (95 C), ◦ ◦ All strains used during this study are summarized in Table S1. annealing (63 C) and replication (72 C) were executed for 30 s T ◦ Bacterial isolates CG-21 (subsequently deposited as Vagococcus each after an initial denaturation of 95 C for 2 min. For validation T teuberi DSM 21459 ), GG-1, 24CA, 24CB, 9H, CM21, CM22, CM11 of the assay, total DNA was extracted from single colonies or and CM12 were picked as white, round colonies from a KF Strep- liquid cultures grown in TSYE as described previously [12]. A tococcus agar plate (Labo-Life Sàrl, Pully, Switzerland) at up to the reference set comprised of Vagococcus spp., Enterococcus spp., −8 10 -dilution from fènè samples obtained from household produc- Lactobacillus spp., Lactococcus spp., Pediococcus spp., Weissella spp. tion in Kasséla, a village near Bamako, the capital city of Mali [48]. and Carnobacterium spp. was used for validation of the PCR assay Two so far unclassified isolates, 186B2.1R4 and 184A6.1, were orig- (Table S1). After validation, the assay was applied on 217 untyped inated from spontaneously fermented camel milk in Kenya [23]. isolates from fènè [48]. Isolates testing positive in this presumptive All these isolates were routinely cultured under aerobic conditions species-specific PCR assay were further subjected by rep-PCR using ◦ at 37 C in TSYE medium containing 30 g/l tryptic soy broth and the (GTG)5 primer [10] for strain differentiation. 3 g/l yeast extract. Vagococcus spp. type strains and other reference strains were provided by the German Collection of Microorganisms Antimicrobial resistance gene profile using DNA microarray and Cell Cultures (DSMZ, Braunschweig, Germany) and Culture T Collection University of Gothenburg (CCUG, Gothenburg, Sweden) Antimicrobial resistance (AMR) gene profiling of DSM 21459 (Table S1). was performed using a microarray containing probes for 90 antibiotic resistance genes of Gram-positive bacteria [35] using T Phenotypic and physiological characteristics of strain DSM 21459 Streptococcus pseudintermedius KM1381 [8] as positive AMR gene control. Hybridisation was performed using biotin labeling as T T Strains DSM 21459 (=CG-21 ) and 24CA were used as refer- described previously [35]. PCR primers targeting the entire dfr(G) ence pair for the species description and the following phenotypic gene were designed according to the dfr(G) sequence available and physiological characterization. Analysis of cell morphology under accession number FM204877. and motility was done by light microscopy and cell surface was inspected by electron microscopy. Gram-reaction of the strains Construction of phylogenetic trees based on 16S rRNA genes was carried out with KOH lysis [14]. Catalase activity was tested with 3% H2O2. Growth parameters, e.g. growth temperatures and 16S rRNA genes were amplified using the bak4 [15] and bak11w oxygen requirement, pH and salt tolerance (4% and 6.5% NaCl) as [11] primers and subsequently sequenced using Sanger methodol- well as other phenotypic assays were carried out in TSYE medium. ogy (Microsynth, Balgach, Switzerland). A phylogenetic tree was Gas production was measured with Durham tubes in MRS-glucose constructed based on these sequences and on sequences obtained broth (Labo-Life Sàrl). Hemolytic activity was tested by culturing from GenBank after filtering for type strains and sequence length of the strains on Columbia agar plates with 5% sheep blood. Tellurite at least 1200 nt and only minimal number of ambiguous basepairs. tolerance on TSYE agar plates was recorded with a 0.04% tellurite Sequences were aligned in BioEdit 7.0.9.0 using the ClustalW algo- concentration and serological reactions monitored with the Slidex rithm. Afterwards, sequences were trimmed to equal lengths to Strepto-Kit (bioMérieux, Geneva, Switzerland). Enzyme activities calculate the phylogenetic tree using Neighbor-joining algorithm and acid production from different carbon sources were both tested and 1000 bootstrap replications in MEGA7.0 [27]. The sequence with the API Rapid ID 32S system (bioMérieux). identity matrix was calculated in BioEdit 7.0.9.0 using the same set Proteolytic activity by precipitation of unhydrolyzed casein of aligned and trimmed 16S rRNA gene sequences. The chromo- T was assessed on CASO agar plates (VWR-International, Dietikon, some of V. teuberi DSM 21459 (NZ CP017267) harbored six copies Switzerland) containing 5% skim milk and incubated aerobically of the 16S rRNA gene, which were all included into this comparison ◦ at 37 C for 15 h [32].