Lactococcus petauri sp. nov., isolated from an abscess of a sugar glider

A strain of lactic acid bacteria, designated 159469T, isolated from a facial abscess in a sugar glider, was characterized genetically and phenotypically. Cells of the strain were Gram-stain-positive, coccoid and catalase-negative. Morphological, physiological and phylogenetic data indicated that the isolate belongs to the genus Lactococcus. Strain 159469T was closely related to Lactococcus garvieae ATCC 43921T, showing 95.86 and 98.08 % sequence similarity in 16S rRNA gene and rpoB gene sequences, respectively. Furthermore, a pairwise average nucleotide identity blast (ANIb) value of 93.54 % and in silico DNA–DNA hybridization value of 50.7  % were determined for the genome of strain 159469T, when compared with the genome of the type strain of Lactococcus garvieae. Based on the data presented here, the isolate represents a novel species of the genus Lactococcus, for which the name Lactococcus petauri sp. nov. is proposed. The type strain is 159469T (=LMG 30040T=DSM 104842T).

Sugar gliders (Petaurus breviceps) are small marsupials native to Australia and New Guinea and commonly kept as pets.Due to a specialized dental structure that is designed for peeling bark and not for eating soft diets, they are prone to oral cavity disease as companion animals; abscesses due to facial trauma are also common [1,2].Sugar gliders are susceptible to a number of bacterial and parasitic infections including Pasteurella multocida and Toxoplasma gondii [3], but are typically not associated with infections caused by members of the genus Lactococcus.
The genus Lactococcus is a member of the family Streptococcaceae composed of lactic acid fermenters.Members of this genus are commonly used in food production, especially in the dairy industry.Lactococcus garvieae, originally isolated from a mastitic cow udder [4], is a common pathogen of fish that is often isolated from environmental sources such as farm animal bedding.Lactococcosis is a serious concern for the global aquaculture industry.Although it rarely causes gastrointestinal disorders and infective endocarditis in humans [5,6], L. garvieae has been described as an emerging zoonotic pathogen [7,8].
Strain 159469 T was the predominant bacterial strain isolated from a facial abscess swab on a sugar glider submitted for routine clinical culture.The colonies had a distinctive bright orange pigment (Fig. S1, available in the online Supplementary Material).The strain was initially typed as a representative of L. garvieae based on biochemical profiles obtained from commercial diagnostic platforms and 16S rRNA gene Sanger sequencing.The pigmentation of the colonies, however, was inconsistent with this identification, and further characterization by whole-genome sequencing was performed.Phylogenetic and phenotypic analyses subsequently indicated that this strain represents a distinct species of the genus Lactococcus, sharing most of its sequence in common with L. garvieae and the pigmented phenotype of Lactococcus lactis.We propose to name 159469 T as the type strain of Lactococcus petauri sp.nov.
A 1537 bp 16S rRNA gene sequence was extracted from an assembled draft genome of isolate 159469 T using RNAmmer 1.2 [9] and checked for the presence of chimera using DECIPHER [10].NCBI BLAST identified the 16S rRNA gene sequence from L. garvieae strain M14 as the closest  ) in a maximumlikelihood tree reconstructed on the basis of 16S rRNA gene sequences in RAxML v. 8 using the general time-reversible (GTRGAMMAI) substitution model and 1000 bootstrap repetitions (Fig. 1; [11]).L. garvieae JCM 10343 T was confirmed as the closest relative of strain 159469 T based on the 95.86 % sequence similarity of aligned 16S rRNA gene sequences (MUSCLE; [12]).The 16S rRNA gene sequence identity of <97 % [13] suggested that isolate 159469 T represents a novel species.The same alignment was used to reconstruct the neighbour-joining (Fig. S2) and maximumparsimony trees in MEGA (Fig. S3) [12].All three tree reconstruction methods produced congruent clustering of L. petauri sp.nov.159469 T with L. garvieae JCM 10343 T , L. garvieae subsp.bovis BSN307 T and Lactococcus formosensis NBRC 109475 T .
The genome of the L. petauri sp.nov.159469 T was sequenced on an Illumina MiSeq platform with 2Â250 bp paired-end reads, which were assessed for quality with FastQC version 0.11.2 and assembled de novo with SPAdes version 3.6.2[14].Samtools version 1.3.1 [15] and QUAST version 3.2 [16] were used to confirm a high quality of the draft genome (e.g.319Â average coverage, 33 contigs >1 Kb, 2.4 Mb total length, N50 of 352908).The assembled genome of L. petauri sp.nov.159469 T and genomes of seven type strains of species and subspecies of the genus Lactococcus extracted from NCBI were analysed using kSNP version 2 with kmer size 19 [17] to identify core genome single-nucleotide polymorphisms (SNPs).Core genome SNPs (N=109) were used to reconstruct a maximum-likelihood phylogeny with 1000 bootstrap repetitions in RaxML (Fig. 2; [11]).
Based on the core genome SNPs, L. petauri sp.nov.159469 T clustered close to L. garvieae JCM 10343 T ; the divergence between these two strains was robust, as demonstrated by a high bootstrap value of 81.
The same eight genomes were used to compute pairwise average nucleotide identity BLAST (ANIb) (https://github.com/widdowquinn/scripts/blob/master/bioinformatics/cal-culate_ani.py).An ANIb pairwise similarity matrix was used to plot the dendrogram in R 3.3.2[18] using the 'hclust' method (Fig. 3).L. garvieae JCM 10343 T was shown to have the most similar genome (93.54 %) to L. petauri sp.nov.159469 T as suggested by ANIb.The pairwise ANIb values <95 % compared with representatives of other species in the genus Lactococcus confirmed strain 159469 T as a representative of a novel species [19].Interestingly, the pairwise ANIb value between L. petauri sp.nov.159469 T and another strain, currently classified as a representative of L. garvieae in the NCBI database (strain PAQ102015-99, BioSample accession number SAMN04958039) was 98.51 %.This indicates the existence of another strain of the novel species described here as L. petauri sp.nov.Strain PAQ102015-99 was described as a putative pathogen of salmonid fish used for vaccine development.
In silico DNA-DNA hybridization (DDH) analysis was performed on the aforementioned eight genomes using GGDC To confirm phylogenetic distinctiveness of L. petauri sp.nov.159469 T , the rpoB sequence was extracted from the whole-genome sequence.It was then aligned with rpoB sequences of 14 type strains of species and subspecies of the genus Lactococcus for which rpoB sequences of sufficient length were available in the NCBI database.This alignment was used to reconstruct a neighbour-joining tree with the Tamura 3-parameter substitution model and 1000 bootstrap repetitions in MEGA 6.0 (Fig. 4; [12]).Clustering of L. petauri sp.nov.159469 T based on rpoB phylogeny was consistent with that based on 16S rRNA gene phylogeny.
Strain 159469 T was isolated from a lesion on the chin of a 2-year-old female sugar glider at the North Dakota State University Veterinary Diagnostic Laboratory in 2016.The lesion was described as an abscess that was not responding to enrofloxacin.A beta-haemolytic, mucoid and pigmented member of the genus Lactococcus was isolated at 4+, which was interpreted as 'heavy growth'.Alpha-haemolytic streptococci and non-haemolytic staphylococci were also present in moderate numbers.A Gram stain was performed on the abscess swab, and low numbers of Gram-stain-negative rods and Gram-stain-positive cocci were present.This mixed growth was typical from an abscess, but the representative of the genus Lactococcus was the predominant colony type.Gram staining from those colonies revealed Gram-stainpositive cocci that were catalase-negative.No identification could be obtained using the biochemical profile on the Sensititre platform (Thermo Scientific).The Biolog and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) platforms both ranked L. garvieae as the closest match with scores of 0.734 and 2.1.The strain was propagated on trypticase soy agar (TSA) with 5 % Sheep Blood (BD) at 33-37 C and demonstrated growth typical of a facultative anaerobe.Production of acid from carbohydrates was determined by using API 50 CH kits (bioM erieux).The kits were used according to the manufacturer's instructions and incubated for 48 h at 30 C. Enzymic activity was assessed using the API ZYM kit (bioM erieux).The kit was used according to the manufacturer's instructions.Strains were initially grown aerobically at 30 C for 24 h on M17 agar.Once the strip was inoculated, incubation occurred for 4 to 4.5 h at 37 C.The results of the API tests can be seen in Tables 2, S1 and S2.
Analysis of fatty acid methyl esters was performed by Microbial ID according to the instructions of the Microbial Identification System.The organism was cultured on TSA for 24 h at 28 C before harvesting.The major fatty acids of L. petauri sp.nov.159469 T were C 16 : 0 (40.93 %) and C 14 : 0 (14.43 %).The complete fatty acid profile of the novel species is shown in Table 3. Comparisons with other species of the genus Lactococcus cannot be made due to the use of different incubation conditions by other authors for fatty acid analysis.Fatty acid profiles of other species in the genus can also be seen in Table 3 with the incubation conditions indicated.
DESCRIPTION OF LACTOCOCCUS PETAURI SP.NOV.
Cells are Gram-stain-positive cocci, catalase-negative, oxidasenegative, beta-haemolytic, mucoid and facultatively anaerobic.The type strain is orange-pigmented when grown aerobically on TSA with 5 % sheep blood or when grown anaerobically on M17 agar; cream coloured when grown aerobically on M17 agar.Lactococcus formosensis 516 T (AB775180.1) Lactococcus petauri 159469 T (MF141900) Fig. 4. Rooted rpoB neighbour-joining tree of Lactococcus petauri sp.nov.159469 T , 15 type strains of species and subspecies of the genus Lactococcus.The tree was reconstructed in MEGA version 6.0, using the
match to the 16S rRNA gene sequence of isolate 159469 T .Comparative phylogenetic analysis was carried out with 16S rRNA gene sequences of isolate 159469 T and type strains of 16 species and subspecies of the genus Lactococcus with validly publsihed names.Strain 159469 T clustered close to L. garvieae JCM 10343 T (=ATCC 43921 Downloaded from www.microbiologyresearch.orgby IP: 54.70.40.11On: Sun, 03 Feb 2019 21:49:47 Rooted 16S rRNA gene maximum-likelihood tree of Lactococcus petauri sp.nov.159469 T , 16 type strains of species and subspecies of the genus Lactococcus, and Escherichia coli K-12 as an outgroup.The tree was reconstructed in RAxML v. 8, using the GTRGAMMAI substitution model and 1000 bootstrap repetitions.Numbers at nodes indicate percentage bootstrap support.Bar, 0.2 substitutions per site.L. petauri sp.nov.159469 T (NCBI accession number KY548925) is presented in bold type.

Table 1 .
In silico computed DNA-DNA hybridization values for L. petauri sp.nov.159469T and seven species and subspecies of the genus Lactococcus Growth at various temperatures was determined by plating overnight cultures on M17 agar plates and incubating at 4, 6, 10 and 14 C for 21 days, 20 and 25 C for 14 days, 30, 35, 37 and 40 C for 7 days, and 45 and 55 C for 3 days.L. petauri sp.nov.159469T was able to grow at temperatures of between 6 and 40 C, but not at 45 C. Results of the temperature, pH and NaCl tests can be seen in Table2compared with those of other type strains of the genus Lactococcus.

Table 2 .
Tamura 3-parameter model and 1000 bootstrap repetitions.Bar, 0.05 substitutions per site.Lactococcus petauri sp.nov.159469 T (NCBI accession number MF141900) is presented in bold type.Phenotypic properties of L. petauri sp.nov.159469 T and type strains of species and subspecies of the genus Lactococcus with validly published names

Table 3 .
[24]ular fatty acid composition of L. petauri sp.nov.159469T and type strains of species and subspecies of the genus Lactococcus with validly published names L. lactis subsp.cremorisKCCM40699T; 10, L. lactis subsp.hordniaeKCTC3768T;11,L. lactis subsp.lactisKCTC3769T.Values represent the percentage of the total fatty acids as determined by the Microbial Identification System software.Data for strain 1 are from this study.Data for the rest of the strains are as indicated.Growth conditions for fatty acid analysis are as follows: this study, TSA agar, 24 h, 28 C; Chen et al.[22], MRS agar, 72 h, 37 C; Cai et al.[23], MRS agar, 48 h, undefined temperature; Cho et al.[24], TSA agar, 72 h, 30