Staphylococcus Debuckii Sp. Nov., a Coagulase-Negative Species from Bovine Milk

TAXONOMIC DESCRIPTION Naushad et al., Int J Syst Evol Microbiol DOI 10.1099/ijsem.0.003457

Staphylococcus debuckii sp. nov., a coagulase-negative species from bovine milk

Sohail Naushad,1,2,* Uliana Kanevets,1 Diego Nobrega,1,2 Domonique Carson,1,2 Simon Dufour,2,3 Jean-Philippe Roy,2,4 P. Jeffrey Lewis2,5 and Herman W. Barkema1,2

Abstract A novel type strain, designated SDB 2975T (=CECT 9737T=DSM 105892T), of the novel species Staphylococcus debuckii sp. nov. isolated from bovine milk is described. The novel species belongs to the genus Staphylococcus and showed resistance to tetracycline and was oxidase- and coagulase-negative, catalase-positive, and Gram-stain-positive. Phylogenetic relationships of Staphylococcus debuckii SDB 2975T to other staphylococcal species were inferred from 16S rRNA gene and whole- genome-based phylogenetic reconstruction. The 16S rRNA gene comparisons showed that the strain is closely related to Staphylococcus condimenti (99.73 %), Staphylococcus piscifermentans (99.66 %), Staphylococcus carnosus (99.59 %) and Staphylococcus simulans (98.03 %). Average nucleotide identity (ANI) values between S.taphylococcus debuckii SDB 2975T and its closely related Staphylococcus species were 83.96, 94.5, 84.03 and 78.09 %, respectively, and digital DNA–DNA hybridization (dDDH) values were 27.70, 58.02, 27.70 and 22.00 %, respectively. The genome of Staphylococcus debuckii SDB 2975T was sequenced with PacBio and Illumina technologies and is 2 691 850 bp long, has a G+C content of 36.6 mol% and contains 2678 genes and 80 RNAs, including six copies of each5S rRNA, 16S rRNA and 23S rRNA genes. Biochemical profiling and a newly developed PCR assay enabled differentiation of Staphylococcus debuckii SDB 2975T and three other SDB strains from its closest staphylococcal species. Differentiation was also achieved by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF). Genes unique to Staphylococcus debuckii were identified and a PCR- based assay was developed to differentiate Staphylococcus debuckii from other staphylococcal species. In conclusion, the results of phylogenetic analysis along with the ANI values <95 %, and dDDH values <70 % from closely related species along with the phenotypic and biochemical characteristics and specific MALDI-TOF profiles demonstrated that Staphylococcus debuckii SDB 2975T represents a novel species within the genus Staphylococcus, named Staphylococcus debuckii sp. nov. (SDB 2975T=CECT 9737T=DSM 105892T).

Non-aureus staphylococci (NAS), commonly known as genus Staphylococcus are Gram-positive cocci that occur coagulase-negative staphylococci (CNS), are the most fre- singly, in pairs, short chains and irregular grape-like clusters quently isolated organisms from bovine mastitis milk [9]. Since its description, classification of the genus Staphy- worldwide [1–4]. NAS belong to the genus Staphylococcus, lococcus has been under active revisions [10–12]. Many new first described in 1884 [5], and currently (as of September Staphylococcus species have been described in the last 2018) consists of 51 species and 28 subspecies (www.bac- decade [13–16]. However, in the past, description of novel terio.net/staphylococcus.html), out of which 25 NAS species staphylococcal species was solely based upon morphological are most commonly identified from bovine milk in Canada, and biochemical characteristics [9, 17]. However, these the phylogeny, distribution, virulence and antimicrobial methods do not always identify Staphylococcus species reli- resistance profiles of those 25 NAS species were described ably [15, 18, 19]. Many new rules of genome-based classifi- recently [2, 6–8]. Morphologically, the members of the cation and methods of identification have been published in

Author affiliations: 1Department of Production Animal Health, Faculty of Veterinary Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary AB, Canada; 2Canadian Bovine Mastitis and Milk Quality Research Network, St-Hyacinthe, QC, Canada; 3Department of Pathology and Microbiology, Faculty of Veterinary Medicine, University of Montreal, Saint-Hyacinthe QC, Canada; 4Department of Clinical Sciences, Faculte de medecine vet erinaire, Universite de Montreal, 3200 Rue Sicotte, St-Hyacinthe, Quebec, Canada; 5Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada. *Correspondence: Sohail Naushad, [email protected] Keywords: novel species; coagulase-negative staphylococci; bovine intramammary infection; whole-genome sequencing; virulence factors; antimicrobial resistance. Abbreviations: ANI, average nucleotide identity; ARG, antimicrobial resistant gene; CAS, coagulase-negative staphylococci; dDDH, digital DNA–DNA hybridization; GGDC, Genome-to-Genome Distance Calculator; ML, maximum-likelihood; MSA, multiple sequence alignment; NAS, non-aureus staphylococci; OGRI, overall genome related index; WGS, whole-genome sequencing. One supplementary table is available with the online version of this article.

003457 ã 2019 The Authors Downloaded from www.microbiologyresearch.org by This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. IP: 54.70.40.11 1 On: Sat, 22 Jun 2019 10:18:51 Naushad et al., Int J Syst Evol Microbiol the last decade [20–24]. With the advent of next-generation Each of them was originally assigned as Staphylococcus pisci- sequencing technologies, bacterial species are currently fermentans with scores of 1.757, 1.831 and 1.864, respec- being recognized based on whole-genome sequences, aug- tively. These scores are within the ‘probable genus mented with matrix-assisted laser desorption/ionization identification’ only, with insufficiently reliable species time-of-flight mass spectrometry (MALDI-TOF MS) demarcation. Following more analyses, we eventually and phenotypic and chemotypic characterization [23–26]. named them Staphylococcus debuckii SDB 3462 (MQM ID Two prokaryotic bacteria are typically regarded as different 3462) isolated from fourth quarter (right rear) of a cow 15 species if genomic DNA exhibits a DNA–DNA hybridiza- days after calving and Staphylococcus debuckii SDB 5244 tion (DDH) value <70 % [27, 28], with the DDH value (MQM ID 5244) and Staphylococcus debuckii SDB 5264 reflecting relatedness or similarity between two genomes. (MQM ID 5264), both isolated from the same second quar- However, after several studies proposed the replacement of ter (left rear) of a cow 5 and 13 days after calving, DDH with pairwise genome-sequence derived similarity respectively. indices, the overall genome related index (OGRI) has become an alternative (to DDH) accepted standard for pro- PHYLOGENY karyotic speciation [20, 23, 24]. Like DDH, OGRI can be Core genome (Fig. 1) and 16S rRNA gene (Fig. 2) trees of used to check if a strain belongs to a known species by cal- T culating the relatedness between genome sequences of novel staphylococcal species showed that strain SDB 2975 was strains and type strain of a species [24]. Recently, average most closely related to Staphylococcus condimenti (99.73 %), nucleotide identity (ANI) and digital DDH (dDDH) have S. piscifermentans (99.66 %), S. carnosus (99.59 %) and S. been most widely used for bacterial species demarcation simulans (98.03 %), with percentage similarity (16S – rRNA gene) for each species shown in parentheses. How- [13, 26, 29 31]. The proposed and generally accepted spe- T cies boundary for ANI and dDDH values are 95~96 and ever, apart from distinct branching of strain SDB 2975 in 70 %, respectively [24]. the phylogenetic trees, the 16S rRNA gene sequences similarities were not distinct enough for determining the species. Following recommended guidelines [17, 32] and the new Traditionally, newly discovered bacterial strains in most rules of classification [20, 24], herein we describe strain SDB T T T cases have been defined as members of the same species if 2975 (=CECT 9737 =DSM 105892 ) as the type strain of they share 97 % or greater 16S rRNA gene sequence similar- the novel species Staphylococcus debuckii sp. nov. We also ity [23, 34, 35]. However, many studies have pointed out report the phenotypic and chemotypic characterization of S. limitations in 16S rRNA gene-based classification of prokar- debuckii, along with the whole-genome sequencing (WGS), yotes, and in many cases bacteria have been misclassified as genome annotation and characterization and phylogenetic members of incorrect taxonomic groups [36–39]. The avail- characteristics. We also generated MALDI-TOF profiles to ability of the whole genomes has enabled resolving many distinguish Staphylococcus debuckii from other closely incorrect taxonomical relationships [40, 41]. In order to related staphylococcal species. compute the core genome phylogeny of Staphylococcus debuckii, complete genomes of S. carnosus LTH 3730 ISOLATION AND HOME HABITAT (CP016760.1), S. condimenti DSM 11674T (NZ_CP015114), Strain SDB 2975T was isolated from the milk of the right S. simulans FDAARGOS_124 (NZ_CP014016.1) and S. pis- fore quarter of an eighth lactation cow at 35 days-in-milk cifermentans NCTC13836 (NZ_LT906447.1) were down- housed in a tie-stall herd in Quebec, Canada. The cow pro- loaded from the NCBI. Genome sequences of 25 other bovine duced on average 29 kg of milk per day on a preceding dairy NAS species were used from BioProject PRJNA342349 [7]. herd improvement test. The herd average bulk tank somatic Core genome tree reconstruction was performed as cell count was 253 000 cells mlÀ1. The sample was collected described previously [7, 42]. Briefly, the core set of protein on 13 May 2007 and was stored in the Mastitis Pathogen families (60 % sequence identify and 80 % sequence length), Culture Collection of the Canadian Bovine Mastitis and present in 90 % of the input genomes were identified Milk Quality Research Network (CBMQRN) under the bar- by using the CD-HIT program [43, 44]. Protein families code 31302975 [33]. The sample showed mix growth of which contained potential paralogous sequences (duplicated Staphylococcus aureus and Staphylococcus debuckii at first sequence in same genome) were excluded from further anal- inoculation. The subsequent subcultures separated the two ysis. Multiple sequence alignment (MSA) of each protein cultures. We obtained pure culture of Staphylococcus family was performed with the MAFFT 7 [45] algorithm. debuckii and it was stored under ID 31 302 975–263. Three Aligned amino acid positions which contained gaps in more other related Staphylococcus debuckii strains, collected in than 50 % of genomes were excluded from further analysis. January to April 2016 from milk of two different cows from Remaining amino acid positions were concatenated to cre- two different herds in Quebec, Canada, were stored in ate a combined dataset consisting of 889 core proteins. the Maritime Quality Milk (MQM) collection (University of Poorly aligned regions from this concatenated alignment Prince Edward Island). They were identified as CNS based were removed using Gblocks 0.92 [46]. This combined data- on their coagulase (negative) results and in MALDI-TOF set was further trimmed with TrimAl to contain 165 454 MS based BioTyper classifications performed in April phylogenetically informative positions [47]. The maximum- (MQM ID 3462) and June (MQM IDs 5244 and 5264) 2016. likelihood (ML) tree based on this alignment was

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Staphylococcus cohnii SNUC 969 Staphylococcus nepalensis SNUC 4337 Staphylococcus equorum SNUC 116 Staphylococcus xylosus SNUC 237 Staphylococcus saprophyticus SNUC 2042 Staphylococcus succinus SNUC 1184 Staphylococcus gallinarum SNUC 741 Staphylococcus kloosii SNUC 4696 Staphylococcus arlettae SNUC 1330 Staphylococcus auricularis SNUC 993 Staphylococcus hominis SNUC 4474 Staphylococcus devriesei SNUC 760 Staphylococcus haemolyticus SNUC 192 Staphylococcus aureus 9 Staphylococcus pasteuri SNUC 5329 Staphylococcus warneri SNUC 1678 Staphylococcus epidermidis SNUC 29 Staphylococcus caprae SNUC 4023 Staphylococcus capitis SNUC 2159 Staphylococcus simulans SNUC 824 Staphylococcus debuckii SDB 2975 Staphylococcus piscifermentans NCTC1383 Staphylococcus condimenti DSM 11674 Staphylococcus carnosus LTH 3730 Staphylococcus chromogenes SNUC 104 Staphylococcus hyicus SNUC 5959 Staphylococcus agnetis SNUC 719 Staphylococcus sciuri SNUC 209 Staphylococcus ﬂeurettii SNUC 182 Staphylococcus vitulinus SNUC 2204 Macrococcus caseolyticus ATCC 13548

Fig. 1. Core genome tree of bovine and other closely related staphylococcal species, showing the phylogenetic placement of Staphylo- coccus debuckii SDB 2975T. The tree was reconstructed using the maximum-likelihood method based on the Whelan and Goldman substitution model. The percentage of trees in which the associated taxa clustered together is shown next to the branches. The tree was drawn to scale and was rooted with Macrococcus caseolyticus ATCC 13548. reconstructed using FastTree 2.1 [48], using the Whelan The resulting alignment, consisting of 1369 bp, was used to and Goldman substitution model [49]. For the 16S rRNA reconstruct an ML tree, with 100 bootstrap replicates, using gene tree the 16S rRNA gene sequence of strain SDB 2975T the general time reversible model [53] in MEGA 6.0 [54]. was extracted from the complete genome using Barrnap version 0.8 [50]. The 16S rRNA gene sequences for other GENOMIC CHARACTERIZATION type strains of Staphylococcus species were downloaded The complete genome of strain SDB 2975T was 2 691 850 bp from Ribosomal Database Project (RDP) release 11 [51]. long and had 340Â (average) depth of sequencing coverage MSA of 16S rRNA gene sequences was created using (PacBio). The complete genome had 36.6 mol% G+C con- MUSCLE version 3.8.31 [52]. Poorly aligned regions in the tent and contained a total of 2678 genes, composed of 2535 beginning and end of the MSA were removed manually. CDS, 18 rRNA genes (six copies of each 5S rRNA, 16S

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Staphylococcus lutrae DSM 10244 (X84731) Staphylococcus lugdunensis ATCC 43809 (AB009941) Staphylococcus devriesei KS-SP 60 (FJ389206) Staphylococcus haemolyticus CCM2737 (X66100) Staphylococcus hominis DSM 20328 (X66101) Staphylococcus petrasii MCC10046 (AY953148) Staphylococcus warneri ATCC 27836 (L37603) Staphylococcus simiae CCM 7213 (AY727530) Staphylococcus schweitzeri NCTC13712 (LR134304.1) Staphylococcus aureus ATCC 35844 (D83355) Staphylococcus argenteus MSHR1132 (FR821777) Staphylococcus saccharolyticus ATCC 14953 (L37602) Staphylococcus caprae ATCC 35538 (AB009935) Staphylococcus capitis ATCC 49326(AB009937) Staphylococcus epidermidis ATCC 14990(D83363) Staphylococcus pasteuri ATCC 51129 Staphylococcus warneri ATCC 27836 (L37603) Staphylococcus kloosii ATCC 43959 (AB009940) Staphylococcus gallinarum ATCC 35539 (D83366) Staphylococcus arlettae ATCC 43957 (AB009933) Staphylococcus cohnii ATCC 29974 (D83361) Staphylococcus nepalensis CW1 (AJ517414) Staphylococcus equorum ATCC 43958 (AB009939) Staphylococcus succinus AMG-D1 (AF004220) Staphylococcus saprophyticus GTC 843 (AB233327) Staphylococcus edaphicus CCM 8730 (KY315825.1) Staphylococcus xylosus ATCC 29971 (D83374) Staphylococcus auricularis ATCC 33753 (D83358) Staphylococcus pettenkoferi B3117 (AF322002) Staphylococcus argensis M4S-6 (KP716705.1) Staphylococcus simulans ATCC 27848 (D83373) Staphylococcus condimenti DSM 11674 (Y15750) Staphylococcus piscifermentans SK03 (Y15754) Staphylococcus debuckii SDB 2975 (MK121623) Staphylococcus carnosus ATCC 51365 (AB009934) Staphylococcus massiliensis CCUG 55927 (EU707796) Staphylococcus schleiferi DSM 4807 (S83568) Staphylococcus delphini ATCC 49171 (AB009938) Staphylococcus intermedius ATCC 29663 (D83369) Staphylococcus pseudintermedius LMG 22219 (AJ780976) Staphylococcus agnetis 6-4 (HM484980) Staphylococcus hyicus ATCC 11249 (D83368) Staphylococcus chromogenes ATCC 43764 (D83360) Staphylococcus felis ATCC 49168 (D83364) Staphylococcus microti CCM 4903 (EU888120) Staphylococcus muscae DSM7068 (FR733703) Staphylococcus rostri ARI 262 (FM242137) Staphylococcus sciuri DSM 20345 (AJ421446) Staphylococcus vitulinus ATCC 51145 (AB009946) Staphylococcus lentus ATCC 29070 (D83370) Staphylococcus ﬂeurettii GTC 1999 (AB233330) Staphylococcus stepanovicii 196 (GQ222244) Macrococcuscaseolyticus ATCC 13548(D83359)

Fig. 2. Phylogenetic tree based on alignment of 16S rRNA gene sequence of strain SDB 2975T with all (type) species of the genus Staphylococcus. The tree was reconstructed using the maximum-likelihood method employing the general time reversible model. Num- bers at nodes indicate the percentage of bootstrap support based on 100 replications. The tree was drawn to scale and was rooted with Macrococcus caseolyticus ATCC 13548. The numbers in the parentheses represent GenBank identifiers.

rRNA and 23S rRNA), 1 tmRNA, 62 tRNAs and 62 ncRNAs strain SDB 2975T was sequenced using Illumina Miseq and and does not contain CRISPR repeats. The whole genome of PacBio sequencing technologies. For Miseq sequencing,

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dDDH ANI

S. carnosus LTH 3730 45.0 91.6 100 27.7 84.03 90 S. condimenti DSM 11674 80 27.9 27.7 83.96 84.1 70 60 21.7 27.8 S. debuckii SDB 2975 84.0 78.6 50 40 21.8 58.02 94.5 78.2 30 S. piscifermentans NCTC 13836 20 22.0 78.09 10 21.8 78.0 0 S. simulans FDAARGOS_124

Fig. 3. Pairwise comparisons of average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) values between strain SDB 2975T and Staphylococcus piscifermentans, Staphylococcus carnosus, Staphylococcus condimenti and Staphylococcus simulans. ANI values and the corresponding heatmap was generated with OrthoANI software. Whereas the dDDH values were obtained using the Genome-to-Genome Distance Calculator (GGDC 2.0), and the corresponding heatmap was generated manually in PowerPoint (Microsoft). The proposed and generally accepted species boundary for ANI and DDH values are 95~96 and 70 %, respectively.

DNA was extracted with the DNeasy Blood and Tissue Kit pairwise ANI (and OrthoANI) values between strain SDB (Qiagen), following the recommended protocol for Gram- 2975T and S. piscifermentans NCTC 13836, S. carnosus LTH positive bacteria, and DNA sequencing and assembly was 3730, S. condimenti DSM 11674T and S. simulans FDAAR- performed as described previously [7]. For PacBio sequenc- GOS_124 were calculated using the ANI calculator (from ing, high molecular weight DNA, in the recommended con- EzBiocloud.net) as described by [60]. In silico dDDH values centration (~5 ug), was extracted separately with phenol were calculated with the Genome-to-Genome Distance Cal- chloroform extraction after employing modifications spe- culator (GGDC 2.0), using the recommended cific to S. aureus genomic DNA extraction [55]. Quality and BLAST+ method [61]. The dDDH results were based on rec- quantity of DNA was checked using a NanoVue plus spec- ommended formula 2 (identities/HSP length), which is trophotometer (GE Healthcare Life Sciences) and the Qubit independent of genome length and thus can serve as robust 2.0 fluorometer (Invitrogen). Purified DNA was sent to the indicator for incomplete draft genomes. OrthoANI values Genetic Resources Core Facility (GRCF) of John Hopkins between strain SDB 2975T and S. piscifermentans, S. carno- School of Medicine, Baltimore, MD, USA, where sequencing sus, S. condimenti and S. simulans were 94.5 84.03, 83.96 was performed with the P6-C4 version 2 kit (Pacific Bio- and 78.09 %, respectively (Fig. 3), which were below the rec- sciences) on the PacBio RS II sequencing system. The ommended cut-off (<95–96 % ANI) threshold for species genome assembly was performed with the PacBio HGAP3 delineation [24]. Similarly, in silico dDDH values (Fig. 3) SMRT analysis pipeline [56]. The assembled genome was between strain SDB 2975T and S. piscifermentans, S. carno- polished with Unicycler-Polish pipeline, which utilizes Pilon sus, S. condimenti and S. simulans were 58.02 % (with prob- [57] and ALE (github.com/sc932/ALE) for single nucleoti- ability that two strains represent the same species: 46.25 %), des and InDels corrections. The polished genome was sub- 27.70 % (0.04 %), 27.70 % (0.04 %) and 22.00 % (0 %), mitted to NCBI for gene predictions and annotations with the respectively. According to Thompson et al. [62], strains Prokaryotic Genome Annotation Pipeline (PGAP) [58]. from the same species share 70 % or higher in silico dDDH The Completed PacBio genome (accession no. CP033460), values. The dDDH difference of strain SDB 2975T from raw PacBio reads (SRR8464298) and Illumina short reads other closely related Staphylococcus species, consistent with (SRR8146325) were submitted to NCBI under bioproject their orthoANI an ANI values, confirm the designation of PRJNA406952. The complete genome of strain SDB 2975T Staphylococcus debuckii SDB 2975T as a distinct novel was analysed on the RAST server version 2.0 [59] for G+C species. content and functional categorization of genes. To further characterize the genome of strain SDB 2975T With the availability of cost-effective genome sequencing identification of virulence and antimicrobial resistant technologies, new standards and guidelines for the descrip- genes (ARGs) was performed [6, 8]. Briefly, the complete tion of prokaryotic species have become available [20, 23, genome of strain SDB 2975T was screened against 24]. To comply with the new standards and guidelines, a previously reconstructed database of 191 virulence genes

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[8] and the ARG database [6], using BLAST+ 2.5.0 [63]. À Homology between query proteins sequences and BLAST hits was determined by calculating H (homology) scores + + + + + + + + [6, 64]. The H scores between protein sequences, labelled as Ha (where ‘a’ represents amino acid) were calculated + + + ++ + + + + + + using formula Ha=Qid x Lm/Lq [6, 8], with Qid representing the identities between query sequence and identified protein sequence (ranging from 0 to 1), Lm representing À À À the length (excluding gaps) of the matching sequence from the hit, whereas Lq denotes the length of the query + + + sequence. A cut-off of 60 % sequence similarity and 60 % query length coverage was used for initial searches [6]. All genomic hits that met the minimal cut-off for each indi- -arginine; URE, urea. Columns in blue colour

L vidual query were selected at this stage. A final BLAST hit table containing all possible hits for all query sequences ÀÀÀÀÀ ÀÀÀÀÀ ÀÀ ÀÀÀÀÀÀÀÀÀÀÀ ÀÀÀÀÀ ÀÀ from strain SDB 2975T genomes was imported into R ver-

+ sion 3.4.2 [65]. Hits from each query sequence were then arranged according to Ha score, using dplyr version 0.7.6 in R [66]. From this list, only the hits with highest Ha À score (highest sequence similarity and query length coverage) were selected as potential virulence and ARG. The + + + + + + + + + + + + ++ + + + + + + + +

acetyl-glucosamine; ADH, identification of known and new mutations in ARGs asso- -mannitol; XLT, xylitol; MEL, melibiose; NIT, potassium nitrate; PAL, alkaline N- D ciated with AMR were identified as described [6]. The identification of bacteriocin clusters was performed as described [67]. The screening of AMR genes showed no ÀÀ ÀÀ ÀÀÀÀ ÀÀ ÀÀ ARGs detected in the SDB 2975T complete genome (Pac- Bio). However, in antimicrobial susceptibility T ÀÀÀ ÀÀÀ testing, strain SDB 2975 appeared to be resistant to tetracycline. The MIC values observed were 0.12 µg mlÀ1 for

-glucopyranoside; NAG, À1 À1

D –

- ampicillin (0.12 8 µg ml ), 4 µg ml for chlorampheni- + + + + + + + + a col, 0.5 µg mlÀ1 for ceftiofur, 2 µg mlÀ1 for cephalotin, 1 µg mlÀ1 for ciprofloxacin, 0.5 µg mlÀ1 for clindamy- + + ++ + + + + + + cin, 0.5 µg mlÀ1 for daptomycin, 0.25 µg mlÀ1 for erythromycin, 2 µg mlÀ1 for gentamicin, 0.25 µg mlÀ1 À1 À1 À À for levofloxacin, 2 µg ml for linezolid, 0.25 µg ml for À1

from other closely relates staphylococcal species. moxifloxacin, 32 µg ml for nitrofurantoin, 0.25 µg T À1 À1 ÀÀÀ ÀÀÀ ÀÀ ÀÀÀ ÀÀÀ ÀÀ ml for oxacillin +2 % NaCl, 0.06 µg ml for penicillin, 1/2 µg mlÀ1 for the combination of penicillin and novo- À1 À1 and other closely related staphylococcal species biocin, 0.5 µg ml for pirlamycin, 0.5 µg ml for the SDB 2975 combination of quinupristin and dalfopristin, 0.5 µg mlÀ1

-mannose; MAL, maltose; LAC, lactose; TRE, trehalose; MAN, À1 D for rifampin, 32 µg ml for sulphadimethoxine, 16 µg + + + + + + + + + + ++ + + + + + + mlÀ1 for tetracycline, 0.12 µg mlÀ1 for tigecycline, 0.05/ 9.5 µg mlÀ1 for the combination of trimethoprim and sul- À À À À À À À À À 0 GLU FRU MNE MAL LAC TRE MAN XLT MEL NIT PAL VP RAF XYL SACfamethoxazole, MDG NAG ADH URE and 0.5 µg ml 1 for vancomycin. Accord- ing to CLSI breakpoints [68], this isolate was considered -fructose; MNE, T D resistant only to tetracycline. Previous studies showed that Staphylococcus debuckii Staphylococcus debuckii T the tetracycline-resistance gene in staphylococcal species T 20309886

T was mostly found on plasmids [69]. This was confirmed

DSM 7373 on screening of the Illumina sequenced genome of SDB T DSM 11674 2975 as it contained tetracycline resistance gene tet(k), -glucose; FRU, Proskauer; RAF, raffinose; XYL, xylose; SAC, sucrose; MDG, methyl CBMQRN D DSM 20501 SDB 5264 SDB 5244 SDB 2975 SDB 3462 – which is possibly located on a plasmid. The likely explana- tion for tet(k) to be missing from the PacBio sequences is that for PacBio sequencing higher molecular DNA was isolated and plasmid DNA was likely eliminated during this procedure. In genome screening for bacteriocins, no Biochemical characteristics of clusters of bacteriocins were detected in strain SDB 2975T. The identification of unique genes was carried out as Staphylococcus piscifermentans Staphylococcus condimenti Staphylococcus debuckii Staphylococcus simulans Staphylococcus debuckii Strains Staphylococcus debuckii Staphylococcus debuckii Staphylococcus carnosus 0, Negative control; GLU, phosphatase; VP, Voges Table 1. represent differentiating characters of described previously [70]. Briefly, BLASTn searches were

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Staphylococcus simulans DSM 20723 Staphylococcus simulans DSM 20037 Staphylococcus simulans DSM 20324 Staphylococcus simulans DSM 20323 Staphylococcus simulans DSM 20322T Staphylococcus simulans CCUG 58188 Staphylococcus simulans CCUG 46177 Staphylococcus piscifermentans 900400383 LBK Staphylococcus piscifermentans DSM 7373T Staphylococcus debuckii sp . nov. strain SDB 2975 T Staphylococcus condimenti DSM 11675 Staphylococcus condimenti DSM 11674T Staphylococcus carnosus sspcarnosus DSM 20501T Staphylococcus carnosus ssputilis DSM 11677 Staphylococcus carnosus ssputilis DSM 11676T 10 90 80 70 60 50 40 30 20 10 00 0 0 0 0 0 0 0 0 0 Distance Level

Fig. 4. Main spectrum (MSP) dendrogram (unrooted) displaying the taxonomic placement of the custom Staphylococcus debuckii sp. nov. SDB 2975T MSP within the Staphylococcus carnosus, Staphylococcus condimenti, Staphylococcus piscifermentans and Staphylococ- cus simulans species group. MSPs for S. simulans strains CCM 2724 (different MSP creation method) and 10w374209 RLH (outlier strain) from MALDI-Biotyper database 7311 were excluded.

conducted on all ORFs from the strain SDB 2975T against staphylococcal species (Table S1), including most closely T the NCBI nr database. The ORFs for which no hits (default related S. condimenti DSM 11674 , S. piscifermentans megablast alignment parameters with at least 50 % align- DSM 7373T, S. carnosus DSM 20501T and S. simulans ment length and at least 30 % sequence similarity) were (CBMQRN 22205803 and 20309886). These SDB-specific found in the nr database were considered unique genes. PCR assays provide useful tools for quick and accurate Primers sets were designed against two different unique identification and detection of Staphylococcus debuckii, genes designated SDB242F/R (SDB2424F: TTGCGGA especially in those laboratory settings which do not have TTTATTGGACACA and SDB2424R: TACAAAACGGG- access/resources to WGS or MALDI-TOF technologies. CAACATTGA) and SDB654F/R (SDB654F: GGGACGA- GAACGATATACCAC) and SDB654R: GGTGCTCG PHENOTYPIC AND CHEMOTAXONOMIC TCGATTTGAATTA) with Primer3 (using default settings). Specificity of the primers was tested against CHARACTERIZATION the sequenced SDB 2975T strain and three other SDB Colony morphology of all Staphylococcus debuckii SDB strains (for which WGS was not performed). The cross- strains was observed after 24 h growth on 5 % defibrinated reactivity of the primers was tested against DNA from 26 sheep blood agar plates (BD Diagnostics). The growth and Staphylococcus species (Table S1, available in the online other physiological and biochemical characteristics of version of this article). The DNA of these 26 staphylococ- Staphylococcus debuckii were determined following recom- cal species was extracted as described previously [7]. The mended minimal standards for description of new staphylo- PCR reactions were performed using the TopTaq DNA coccal species [17]. Colonies of all SDB strains were opaque, Polymerase kit (Qiagen). PCR reactions were performed in white, circular, slightly raised and convex with entire mar- a total volume of 25 ul, containing 20 ng genomic DNA, gins, and had smooth and shiny surfaces. The SDB 2975T 1Âfinal buffer concentration, Q buffer, coral red, 1.5 mM and SDB 3462 colonies were 1.5–2.0 mm in diameter, MgCl2, and 200 uM of each dNTP, 0.4 uM of each primer whereas SDB 5244 and 5264 showed slightly smaller diame- and 0.2 U Taq DNA polymerase. The PCR was performed ter range (1.0–1.5 mm) for same age, same media lot and in a programmable thermal cycler (Bio-Rad), starting with same culture conditions. All SDB strains were haemolytic, 3 min denaturation at 94 C, followed by 35 cycles at 94 C Gram-positive, non-spore-forming cocci, which occurred for 30 s, annealing at 58 C for 30 s, and extension at 72 C singly, in pairs and in small clusters. Cells were non-motile for 60 s, with the final extension step of 10 min at 72 C. when observed with phase-contrast microscopy. All Staphy- PCR products were analysed by electrophoresis in 2 % (w/ lococcus debuckii strains were catalase-positive, coagulase- v) agarose gel in 1ÂTris-EDTA buffer. The gels were visu- negative (after 1, 2, 4 and 24 h), oxidase-negative and did alized in a Bio-Rad gel documentation system. The unique not produce DNase. Coagulase activity was not visible after gene primers SDB242F/R and SDB654F/R amplified frag- 1, 2, 4 and 24 h. Staphylococcus debuckii strains were incapa- ments of the expected sizes, 347 bp and 346 bp, from all ble of growth at 45 C and on 15 % sodium chloride agar, Staphylococcus debuckii strains, but not from 26 other and sensitive to Novobiocin (1.6 µg mlÀ1). The growth of

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Table 2. Classification summary for Staphylococcus debuckii strains SDB 2975T, 3462, 5244 and SDB 5264 using MALDI-BioTyper MBT database 7311 and custom MSP for Staphylococcus debuckii sp. nov. SDB 2975T.

Classification results

Top choice as Staphylococcus debuckii sp. nov. Second choice as Staphylococcus T Analyte* SDB 2975 piscifermentans†‡ Classification scores Classification scores Strains

Type No. % Mean±SEM Min Max % Mean±SEM Min Max Staphylococcus debuckii sp. nov. SDB 2975 T§ CE 24 100 2.804±0.004 2.756 2.837 100 1.992±0.008 1.875 2.064 Staphylococcus debuckii sp. nov. SDB 2975T DS 46 100 2.390±0.014 2.164 2.518 97.8 1.861±0.014 1.596 1.992 Staphylococcus debuckii SDB 3462 DS 40 100 2.325±0.014 2.054 2.466 100.0 1.849±0.015 1.632 2.008 Staphylococcus debuckii SDB 5244 DS 40 100 2.344±0.015 2.130 2.474 100.0 1.856±0.013 1.614 2.012 Staphylococcus debuckii SDB 5264 DS 40 100 2.355±0.012 2.162 2.476 100.0 1.862±0.018 1.534 2.026 Staphylococcus debuckii strains (total) DS 166 100 2.355±0.007 2.054 2.518 99.4 1.857±0.008 1.534 2.026

*Colony extracted (CE) and direct transfer colony smear (DS) represent custom MSP creation (24) and classification spectra (166), respectively. †1 of 46 S. debuckii SDB 2975T. Direct transfer colony Smears (DS) identified S. condimenti DSM 11674T as second choice (score 1.492). ‡S. piscifermentans MSP strain preference was for DSM 7373T (164 of 166 colony smear analytes). §Colony extracted spectra used to create custom Staphylococcus debuckii sp. nov. SDB 2975T MSP.

Staphylococcus debuckii was checked at different tempera- (CBMQRN 22205803 and 20309886). From the details of tures (15, 20, 25, 30, 37 and 45 C), under anaerobic condi- individual reactions it is evident that Staphylococcus tions at 37 C, under different pH values (pH 6, 6.5, 7 and debuckii can be differentiated from its closest neighbour S. 8.5) and in different NaCl concentrations (0, 5, 7.5, 10 and piscifermentans based on the negative acidification reaction 15 %). Oxidase activity was investigated using Oxidase (of LAC, SAC and NAG) and positive acidification of MAN Strips (Hardy Diagnostics). Catalase activity was deter- and positive reaction for PAL (Table 1). Differential charac- mined by the slide catalase test using Catalase Test Reagent teristics of Staphylococcus debuckii from other closely (PML Microbiologicals). DNase activity was tested on related staphylococcal species S. condimenti, S. carnosus and DNase test agar with Methyl Green (BD Difco). Phenotypic S. simulans (CBMQRN 22205803) can be derived from characteristics of the novel species were compared with Table 1. The identification and differentiation of strain SDB those of S. condimenti DSM 11674T, S. piscifermentans 2975T from closely related staphlococcal species was also DSM 7373T, S. carnosus DSM 20501T and S. simulans achieved through MALDI-TOF MS analysis. The MALDI- (CBMQRN 22205803 and 20309886). These strains were all TOF MS identification was performed using colony extrac- Gram-positive cocci, occurring in singles, pairs and clusters. tion and direct colony transfer methods. Samples were pre- All were catalase-positive, coagulase-negative and DNase- pared using standard liquid phase formic acid extraction, as negative. S. piscifermentans and S. simulans were haemolytic well direct colony smears. The custom main spectra profile on BAP after 24 h growth. Staphylococcus. condimenti and (MSP), named Staphylococcus debuckii sp. nov SDB 2975T, S. carnosus were non-haemolytic. S. simulans showed some was created in BioTyper OC 3.1 (version 3.1, build 66) using growth at 45 C, while there was no growth observed for the MSP Dendrogram Creation Standard Method (version other strains at 45 C. All strains grew in 0, 5, 7.5 and 10 % 1.4) with default parameters. Classification results for both NaCl concentrations, but none grew in 15 % NaCl concen- methods are interpreted by the manufacturer (MALDI Bio- tration. The Staphylococcus debuckii strains had positive Typer 3.1 User Manual, section 2.18.5), in part, using the reactions (Table 1) for the following tests on the API Staph following scoring parameters: values between 2.300–3.000 strip (bioMerieux): acidification by fermentation of D-glu- are considered secure genus and species identification, val- cose (GLU), D-fructose (FRU), trehalose (TRE) and D-man- ues between 2.000–2.299 are considered secure genus and nitol (MAN); reduction of nitrates to nitrites (NIT); probable species identification, values between 1.700–1.999 presence of alkaline phosphatase (PAL); acetyl-methyl-car- are probable genus identification and scores less than 1.699 binol production (VP); presence of arginine dihydrolase are considered unreliable identifications. Custom Staphylo- (ADH) and urease (URE). The following tests resulted in a coccus debuckii sp. nov. SDB 2975T MSP self-classified cor- negative reaction (Table 1): acidification by fermentation of rectly with a score of 3.000. MALDI-BioTyper (MBT 7311) T D-mannose (MNE), maltose (MAL), lactose (LAC), xylitol database entries for S. piscifermentans strains DSM 7373 (XLT), melibiose (MEL), raffinose (RAF), D-xylose (XYL), (score 2.063) and 900400383 LBK1 (score 1.701) were sec- sucrose (SAC), methyl a-D-glucopyranoside (MDG) and N- ond and third choices, respectively. S. condimenti DSM acetyl-glucosamine (NAG). The API Staph tests were also 11674T (score 1.669) and S. carnosus ssp. utilis DSM 11677 performed for S. condimenti DSM 11674T, S. piscifermen- (score 1.479) were fourth and fifth choices, respectively. All tans DSM TT, S. carnosus DSM 20501T and S. simulans 24 colony extracted spectra utilized to create S. debuckii sp.

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T nov. SDB 2975 custom MSP classified correctly and scores melibiose, raffinose, D-xylose and sucrose. Reduction of exceeded manufacturer recommended (see Bruker Dalton- nitrates and utilization of urea, L-arginine (ADH), alkaline ics Custom MSP and Library Creation, revision I, 2015) phosphatase (PAL) and sodium pyruvate (VP) is positive, minimum classification score guideline of >2.700 (see but methyl a-D-glucopyranoside (MDG) and N-acetyl-glu- Table 2, top row). A dendrogram (Fig. 4) supports the cus- cosamine (NAG) are not utilized. tom MSP classification results placing S. debuckii sp. nov. The type strain, SDB 2975T (=CECT 9737T=DSM 105892T), SDB 2975T MSP in the S. carnosus ssp., S. condimenti, S. pis- was isolated from the milk of the right fore quarter of an cifermentans and S. simulans taxonomic group. Table 2 eighth lactation cow at 35 days-in-milk housed in a tie-stall summarizes classification results for a total of 166 colony herd in Quebec, Canada. The genome of the type strain is extracted and direct transfer colony smears (analytes) repre- characterized by a size of 2 691 850 bp and the G+C content senting Staphylococcus debuckii SDB 2975T, SDB 3462, SDB is 36.6 mol%. 5244 and SDB 5264. Correct classification as S. debuckii sp. nov. SDB 2975T occurred in all cases with 127 of 166 analytes showing scores>2.300 (secure genus and species identifi- Funding information cation) and the remainder scoring between 2.000 and 2.299 We acknowledge financial support from ‘Eyes High Postdoc award’ (secure genus and probable species identification) (data not from University of Calgary for S. N. This work was partially funded by NSERC CREATE in Milk Quality grant and NSERC Industrial Research shown). Typically, the second-choice classification was S. Chair in Infectious Diseases of Dairy Cattle (Grant 463100–13). piscifermentans (165 of 166 analytes) and rarely S. condimenti (see Table 2). Despite the close taxonomic relation- Acknowledgements ship between Staphylococcus debuckii strains and S. The authors thank Natasha Robinson and Dr Greg Keefe from the T Atlantic Veterinary College, University of Prince Edward Island, for piscifermentans DSM 7373 (see Fig. 4), creation of a cus- help and assistance with logistics and sample handling. They are also tom S. debuckii sp. nov. strain SDB 2975T MSP allows quick grateful for technical assistance from Sandi Nishikawa and Ben Cad- and reproducible resolution of these species. dey from the University of Calgary. Conflicts of interest PROPOSAL OF STAPHYLOCOCCUS DEBUCKII The authors declare that there are no conflicts of interest

SP. NOV. References Staphylococcus debuckii SDB 2975T represents the type 1. De Visscher A, Piepers S, Haesebrouck F, de Vliegher S. Intra- mammary infection with coagulase-negative staphylococci at par- strain of the novel species Staphylococcus debuckii within turition: species-specific prevalence, risk factors, and effect on the genus Staphylococcus that can be distinguished from its udder health. J Dairy Sci 2016;99:6457–6469. nearest neighbours by genotypic (Fig. 3; ANI value <95 %, 2. Condas LAZ, De Buck J, Nobrega DB, Carson DA, Naushad S et al. and dDDH values<70 %) and phenotypic characteristics Prevalence of non-aureus staphylococci species causing intra- (Table 1; no acid production from lactose and no utilization mammary infections in Canadian dairy herds. J Dairy Sci 2017; 100:5592–5612. of N-acetyl-glucosamine), by sequence-specific PCR 3. Vanderhaeghen W, Piepers S, Leroy F, van Coillie E, (Table S1), and by a distinct MALDI-TOF MS profile Haesebrouck F et al. Invited review: effect, persistence, and viru- (Fig. 4). The genotypic and phenotypic difference of Staphy- lence of coagulase-negative Staphylococcus species associated lococcus debuckii SDB 2975T from other closely related with ruminant udder health. J Dairy Sci 2014;97:5275–5293. Staphylococcus species confirm the designation of Staphylo- 4. De Vliegher S, Fox LK, Piepers S, McDougall S, Barkema HW. coccus debuckii SDB 2975T as a distinct novel species. Invited review: mastitis in dairy heifers: nature of the disease, potential impact, prevention, and control. J Dairy Sci 2012;95: 1025–1040. DESCRIPTION OF STAPHYLOCOCCUS 5. Rosenbach FJ. Microorganismen bei den wund-infections-krank- DEBUCKII SP. NOV. heiten des menschen. J.F. Bergmann, Wiesbaden 1884;122:1. 6. Nobrega DB, Naushad S, Naqvi SA, Condas LAZ, Saini V et al. Staphylococcus debuckii (de.buck’i.i. N.L. gen. n. debuckii of Prevalence and genetic basis of antimicrobial resistance in Non- De Buck, named after prominent bovine bacteriologist Prof. aureus Staphylococci isolated from Canadian dairy herds. Front Dr. Jeroen De Buck). Microbiol 2018;9:256. 7. Naushad S, Barkema HW, Luby C, Condas LA, Nobrega DB et al. The description of the novel species is based on characteris- Comprehensive phylogenetic analysis of bovine non-aureus Staph- tics of four isolates originating from bovine milk. All strains ylococci species based on whole-genome sequencing. Front are Gram-positive, appear as cocci mostly in pairs and small Microbiol 2016;7:7. clusters. All strains are non-motile and non-spore-forming. 8. Naushad S, Naqvi SA, Nobrega D, Luby C, Kastelic JP et al. Com- prehensive virulence gene profiling of bovine non-aureus Staphy- After 24 h growth, colonies are 1.0–2.0 mm in diameter, lococci based on whole-genome sequencing data. mSystems smooth, shiny, circular and haemolytic. All strains are cata- 2019;4:e00098–18. lase-positive, coagulase-negative, oxidase-negative and do 9. Kloos WE. Natural populations of the genus Staphylococcus. Annu not produce DNase. All strains are incapable to growth at Rev Microbiol 1980;34:559–592. 45 C, sensitive to novobiocin (1.6 µg mlÀ1) and sensitive to 10. Kloos WE, Ballard DN, George CG, Webster JA, Hubner RJ et al. 15 % NaCl salt concentration. All strains produce acid from Delimiting the genus Staphylococcus through description of Macro- coccus caseolyticus gen. nov., comb. nov. and Macrococcus equiper- of D-glucose, D-fructose, trehalose and D-mannitol. No acid cicus sp. nov., and Macrococcus bovicus sp. no. and Macrococcus is produced from D-mannose, maltose, lactose, xylitol, carouselicus sp. nov. Int J Syst Bacteriol 1998;48 Pt 3:859–877.

Downloaded from www.microbiologyresearch.org by IP: 54.70.40.119 On: Sat, 22 Jun 2019 10:18:51 Naushad et al., Int J Syst Evol Microbiol

11. Kloos WE, George CG, Olgiate JS, van Pelt L, Mckinnon ML et al. 28. Rosselló-Mora R. DNA-DNA reassociation methods applied to Staphylococcus hominis subsp. novobiosepticus subsp. nov., a novel microbial taxonomy and their critical evaluation. In: Stackebrandt trehalose- and N-acetyl-D-glucosamine-negative, novobiocin- and E (editor). Molecular Identification, Systematics, and Population multiple-antibiotic-resistant subspecies isolated from human Structure of Prokaryotes Springer; 2006. pp. 23–50. – blood cultures. Int J Syst Bacteriol 1998;48 Pt 3:799 812. 29. Peeters C, Meier-Kolthoff JP, Verheyde B, de Brandt E, Cooper 12. Bannerman TL, Kloos WE. Staphylococcus capitis subsp. ureolyti- VS et al. Phylogenomic study of Burkholderia glathei-like organ- cus subsp. nov. from human skin. Int J Syst Bacteriol 1991;41: isms, proposal of 13 novel Burkholderia species and emended 144–147. descriptions of Burkholderia sordidicola, Burkholderia 13. Pantu cek R, Sedlacek I, Indrakov a A, Vrbovska V, Mašlaňova I zhejiangensis and Burkholderia grimmiae. Front Microbiol 2016;7: et al. Staphylococcus edaphicus sp. nov., isolated in Antarctica, 877. Harbors the mecC gene and genomic Islands with a suspected 30. Huang CH, Liou JS, Lee AY, Tseng M, Miyashita M et al. Polypha- role in adaptation to extreme environments. Appl Environ Microbiol sic characterization of a novel species in the Lactobacillus casei 2018;84:e01746–17. group from cow manure of Taiwan: Description of L. chiayiensis – 14. Novakov a D, Pantu cek R, Hubalek Z, Falsen E, Busse HJ et al. sp. nov. Syst Appl Microbiol 2018;41:270 278. Staphylococcus microti sp. nov., isolated from the common vole 31. Lugli GA, Mangifesta M, Duranti S, Anzalone R, Milani C et al. Phy- (Microtus arvalis). Int J Syst Evol Microbiol 2010;60:566–573. logenetic classification of six novel species belonging to the genus 15. Tong SY, Schaumburg F, Ellington MJ, Corander J, Pichon B et al. Bifidobacterium comprising Bifidobacterium anseris sp. nov., Bifido- Novel staphylococcal species that form part of a Staphylococcus bacterium criceti sp. nov., Bifidobacterium imperatoris sp. nov., Bifi- aureus-related complex: the non-pigmented Staphylococcus argen- dobacterium italicum sp. nov., Bifidobacterium margollesii sp. nov. and Bifidobacterium parmae sp. nov. Syst Appl Microbiol 2018;41: teus sp. nov. and the non-human primate-associated Staphylococ- – cus schweitzeri sp. nov. Int J Syst Evol Microbiol 2015;65:15–22. 173 183. € 16. Åvall-Ja€askel€ ainen€ S, Taponen S, Kant R, Paulin L, Blom J et al. 32. Tindall BJ, Rosselló-Móra R, Busse HJ, Ludwig W, Kampfer P. Comparative genome analysis of 24 bovine-associated Staphylo- Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 2010;60:249–266. coccus isolates with special focus on the putative virulence genes. PeerJ 2018;6:e4560. 33. Reyher KK, Dufour S, Barkema HW, des Coteaux^ L, Devries TJ et al. The National Cohort of Dairy Farms-a data collection plat- 17. Freney J, Kloos WE, Hajek V, Webster JA, Bes M et al. Recom- form for mastitis research in Canada. J Dairy Sci 2011;94:1616– mended minimal standards for description of new staphylococcal 1626. species. Subcommittee on the taxonomy of staphylococci and streptococci of the International Committee on Systematic Bacte- 34. Oren A. The phyla of prokaryotes - cultured and uncultured. In: riology. Int J Syst Bacteriol 1999;49 Pt 2:489–502. Oren A and Papke RT (editors). Molecular Phylogeny of Microorganisms. Norfolk, UK: Caister Academic Press; 2010. pp. 18. Svec P, Vancanneyt M, Sedlacek I, Engelbeen K, Stetina V et al. 85–107. Reclassification of Staphylococcus pulvereri Zakrzewska-Czerwin- ska et al. 1995 as a later synonym of Staphylococcus vitulinus 35. Tindall BJ, Kampfer€ P, Euzeby JP, Oren A. Valid publication of Webster et al. 1994. Int J Syst Evol Microbiol 2004;54:2213–2215. names of prokaryotes according to the rules of nomenclature: past history and current practice. Int J Syst Evol Microbiol 2006;56: 19. Švec P, Petraš P, Pantucek R, Doškar J, Sedlacek I. High intra- 2715–2720. species heterogeneity within Staphylococcus sciuri and rejection of its classification into S. sciuri subsp. sciuri, S. sciuri subsp. carnati- 36. Ramasamy D, Mishra AK, Lagier JC, Padhmanabhan R, Rossi M cus and S. sciuri subsp. rodentium. Int J Syst Evol Microbiol 2016; et al. A polyphasic strategy incorporating genomic data for the 66:5181–5186. taxonomic description of novel bacterial species. Int J Syst Evol Microbiol 2014;64:384–391. 20. Chun J, Rainey FA. Integrating genomics into the taxonomy and systematics of the Bacteria and Archaea. Int J Syst Evol Microbiol 37. Yarza P, Yilmaz P, Pruesse E, Glöckner FO, Ludwig W et al. Unit- 2014;64:316–324. ing the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol 2014; 21. Varghese NJ, Mukherjee S, Ivanova N, Konstantinidis KT, 12:635–645. Mavrommatis K et al. Microbial species delineation using whole genome sequences. Nucleic Acids Res 2015;43:6761–6771. 38. Oren A, Garrity GM. Then and now: a systematic review of the systematics of prokaryotes in the last 80 years. Antonie van 22. Konstantinidis KT, Stackebrandt E. Defining taxonomic ranks. The Leeuwenhoek 2014;106:43–56. Prokaryotes, vol. 1 Springer; 2013. pp. 229–254. 39. Janda JM, Abbott SL. 16S rRNA gene sequencing for bacterial 23. Kim M, Oh HS, Park SC, Chun J. Towards a taxonomic coherence identification in the diagnostic laboratory: pluses, perils, and pit- between average nucleotide identity and 16S rRNA gene sequence falls. J Clin Microbiol 2007;45:2761–2764. similarity for species demarcation of prokaryotes. Int J Syst Evol Microbiol 2014;64:346–351. 40. Naushad S, Adeolu M, Wong S, Sohail M, Schellhorn HE et al. A phylogenomic and molecular marker based taxonomic framework 24. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR et al. Pro- for the order Xanthomonadales: proposal to transfer the families posed minimal standards for the use of genome data for the tax- – Algiphilaceae and Solimonadaceae to the order Nevskiales ord. nov. onomy of prokaryotes. Int J Syst Evol Microbiol 2018;68:461 466. and to create a new family within the order Xanthomonadales, the 25. Singhal N, Kumar M, Kanaujia PK, Virdi JS. MALDI-TOF mass family Rhodanobacteraceae fam. nov., containing the genus Rhoda- spectrometry: an emerging technology for microbial identification nobacter and its closest relatives. Antonie van Leeuwenhoek 2015; and diagnosis. Front Microbiol 2015;6:791. 107:467–485. 26. Nicholson AC, Gulvik CA, Whitney AM, Humrighouse BW, 41. Naushad S, Adeolu M, Goel N, Khadka B, Al-Dahwi A et al. Phylo- Graziano J et al. Revisiting the taxonomy of the genus Elizabeth- genomic and molecular demarcation of the core members of the kingia using whole-genome sequencing, optical mapping, and polyphyletic pasteurellaceae genera actinobacillus, haemophilus, MALDI-TOF, along with proposal of three novel Elizabethkingia and pasteurella. Int J Genomics 2015;2015:1–15. species: Elizabethkingia bruuniana sp. nov., Elizabethkingia ursingii 42. Adeolu M, Alnajar S, Naushad S, S Gupta R. Genome-based phy- sp. nov., and Elizabethkingia occulta sp. nov. Antonie van logeny and taxonomy of the ’Enterobacteriales’: proposal for – Leeuwenhoek 2018;111:55 72. Enterobacterales ord. nov. divided into the families Enterobacteria- 27. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, ceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersinia- Vandamme P et al. DNA-DNA hybridization values and their rela- ceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., tionship to whole-genome sequence similarities. Int J Syst Evol and Budviciaceae fam. nov. Int J Syst Evol Microbiol 2016;66:5575– Microbiol 2007;57:81–91. 5599.

Downloaded from www.microbiologyresearch.org by IP: 54.70.40.1110 On: Sat, 22 Jun 2019 10:18:51 Naushad et al., Int J Syst Evol Microbiol

43. Li W, Godzik A. Cd-hit: a fast program for clustering and compar- 58. Tatusova T, Dicuccio M, Badretdin A, Chetvernin V, Nawrocki EP ing large sets of protein or nucleotide sequences. Bioinformatics et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids 2006;22:1658–1659. Res 2016;44:6614–6624. 44. Fu L, Niu B, Zhu Z, Wu S, Li W. CD-HIT: accelerated for clustering 59. Aziz RK, Bartels D, Best AA, Dejongh M, Disz T et al. The RAST the next-generation sequencing data. Bioinformatics 2012;28: Server: rapid annotations using subsystems technology. BMC 3150–3152. Genomics 2008;9:75. 45. Katoh K, Standley DM. MAFFT multiple sequence alignment soft- 60. Lee I, Ouk Kim Y, Park SC, Chun J. OrthoANI: an improved algo- ware version 7: improvements in performance and usability. Mol rithm and software for calculating average nucleotide identity. Int – Biol Evol 2013;30:772–780. J Syst Evol Microbiol 2016;66:1100 1103. 46. Castresana J. Selection of conserved blocks from multiple align- 61. Meier-Kolthoff JP, Auch AF, Klenk HP, Göker M. Genome ments for their use in phylogenetic analysis. Mol Biol Evol 2000; sequence-based species delimitation with confidence intervals 17:540–552. and improved distance functions. BMC Bioinformatics 2013;14: 60. 47. Capella-Gutierrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic 62. Thompson CC, Chimetto L, Edwards RA, Swings J, Stackebrandt E et al. Microbial genomic taxonomy. BMC analyses. Bioinformatics 2009;25:1972–1973. Genomics 2013;14:913. 48. Price MN, Dehal PS, Arkin AP. FastTree 2-approximately maxi- 63. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J et al. mum-likelihood trees for large alignments. PLoS One 2010;5: BLAST+: architecture and applications. BMC Bioinformatics 2009; e9490. 10:421. 49. Whelan S, Goldman N. A general empirical model of protein evo- 64. Li J, Tai C, Deng Z, Zhong W, He Y et al. VRprofile: gene-cluster- lution derived from multiple protein families using a maximum- detection-based profiling of virulence and antibiotic resistance – likelihood approach. Mol Biol Evol 2001;18:691 699. traits encoded within genome sequences of pathogenic bacteria. 50. Seemann T. Prokka: rapid prokaryotic genome annotation. Brief Bioinform 2018;19:566–574. – Bioinformatics 2014;30:2068 2069. 65. Core Team R. R: A Language and environment for statistical com- 51. Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM et al. Ribosomal puting. R Foundation for Statistical Computing, Vienna, Austria. database project: data and tools for high throughput rRNA analy- 2016 http://www.R-project.org/. sis. Nucleic Acids Res 2014;42:D633–D642. 66. Wickham H, Francois R, Henry L, Müller K. dplyr: A grammar of 52. Edgar RC. MUSCLE: multiple sequence alignment with high accu- data manipulation. R package version (0.7.6.); 2018. https://CRAN. racy and high throughput. Nucleic Acids Res 2004;32:1792–1797. R-project.org/package=dplyr. 53. Nei M, Kumar S. Molecular Evolution and Phylogenetics. Oxford Uni- 67. Carson DA, Barkema HW, Naushad S, De Buck J. Bacteriocins of versity Press; 2000. non-aureus Staphylococci isolated from bovine milk. Appl Environ Microbiol 2017;83:e01015–01017. 54. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: 68. Clinical and Laboratory Standards Institute (CLSI). Performance molecular evolutionary genetics analysis version 6.0. Mol Biol Evol Standards for Antimicrobial Susceptibility Testing. Pennsylvania – 2013;30:2725 2729. USA: Wayne; 2016. 55. Chapaval L, Moon DH, Gomes JE, Duarte FR, Tsai SM. An alterna- 69. Chopra I, Roberts M. Tetracycline antibiotics: mode of action, tive method for Staphylococcus aureus DNA isolation. Arquivo applications, molecular biology, and epidemiology of bacterial Brasileiro de Medicina Veterinaria e Zootecnia 2008;60:299–306. resistance. Microbiol Mol Biol Rev 2001;65:232–260. 56. Chin CS, Alexander DH, Marks P, Klammer AA, Drake J et al. 70. Naushad HS, Lee B, Gupta RS. Conserved signature indels and Nonhybrid, finished microbial genome assemblies from long-read signature proteins as novel tools for understanding microbial phy- SMRT sequencing data. Nat Methods 2013;10:563–569. logeny and systematics: identification of molecular signatures 57. Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A et al. Pilon: an that are specific for the phytopathogenic genera Dickeya, Pecto- integrated tool for comprehensive microbial variant detection and bacterium and Brenneria. Int J Syst Evol Microbiol 2014;64:366– genome assembly improvement. PLoS One 2014;9:e112963. 383.

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