Staphylococcus pseudoxylosus sp. nov., isolated from bovine mastitis

MacFadyen AC1, Leroy S2, Harrison EM3, Parkhill J3, Holmes MA4 and Paterson

GK1*

1Royal (Dick) School of Veterinary Studies and The Roslin Institute, University of

Edinburgh, Easter Bush, Campus, Midlothian, EH25 9RG, UK.

2Université Clermont Auvergne-INRA, MEDIS, 63000 Clermont-Ferrand, France.

3The Wellcome Sanger Institute, Wellcome Trust, Genome Campus, Hinxton, CB10

1SA, UK.

4Department of Veterinary Medicine, University of Cambridge, Madingley Road,

Cambridge CB3 0ES, UK.

*gavin.paterson@ed.ac.uk

Non-standard abbreviations

ANI: average nucleotide identity

dDDH: digital DNA-DNA hybridisation

OGRI: overall genome related index

GGDC: genome-to-genome distance calculator

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The 16S rDNA sequence of Staphylococcus pseudoxylosus S04009T is available

under accession MH643903.

The genome sequence data from Staphylococcus pseudoxylosus S04009T is

available under these accessions: Sequence Read Archive accession number;

ERR163436 and assembly accession number; RCVN00000000.

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Abstract

Strain S04009T, a Gram-stain-positive, coagulase-negative staphylococcus was

isolated from bovine mastitis in France. 16S rDNA analysis revealed it to be closely

related to the coagulase-negative species; Staphylococcus xylosus, Staphylococcus

saprophyticus, Staphylococcus caeli and Staphylococcus edaphicus. At the whole

genome level, S04009T had an average nucleotide identity of < 95% and an inferred

DNA–DNA hybridization of < 70 % when compared to these species. Furthermore,

phenotypic characteristics distinguished S04009T from those species. From these

related species only S04009T and S. xylosus are able to ferment xylose and these

two can be distinguished by the inability of S04009T to express urease activity.

Based on the genotypic and phenotypic results, it is proposed that this isolate is a

novel species, with the name Staphylococcus pseudoxylosus sp. nov. The type

strain is S04009T (=DSM 107950T= CCUG 72763T=NCTC 14184T).

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The genus Staphylococcus consists of Gram-stain-positive, cocci, being either

coagulase positive or negative. Coagulase-negative staphylococci are a large

heterogeneous group, made up of numerous species, isolated from diverse sources,

such as animals, birds, humans and food (1). They can cause a range of infections

including mastitis in dairy cattle (2). Isolate S04009T, a Staphylococcus isolated from

bovine mastitis, has been characterised as Staphylococcus pseudoxylosus sp. nov.,

as described here.

Isolate S04009T was isolated in 2002 from a case of bovine mastitis in France and

initially identified as being Staphylococcus xylosus, with suppressive and subtractive

hybridization being consistent with that identification (3). The genome of S04009T

was subsequently sequenced and found to encode mecC1 (4), a novel allotype of

the mecC methicillin resistance determinant (5). An allotype being a mec gene

homologue sharing ≥70% to <95% nucleotide identity to the corresponding

archetypal mec gene (6). Although mecC1 in S04009T carries a nonsense mutation

and the isolate is methicillin susceptible (4), a finding which to the best of our

knowledge has not been reported in other mecC positive staphylococcal isolates.

Initial analysis of the genome focused on the mecC1 region and showed that this

was distinct from the corresponding region seen in mecC-positive methicillin-

resistant Staphylococcus aureus (4). Here we present further analysis of the

S04009T isolate and its genome sequence to demonstrate that it does not belong to

S. xylosus but instead to a closely-related taxon Staphylococcus pseudoxylosus sp.

nov.

Whole genome sequencing was performed using Illumina Hi-Seq technology with

library preparation carried out as described (7) and sequencing performed following

the manufacturer’s standard protocols (Illumina, Inc., UK). Assembly was done using

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Velvet (8) and the resultant sequencing reads and draft genome (total length

3045959 bases, 124 contigs, N50 = 211400, L50 = 6, coverage c. 106-fold) were

deposited in GenBank with the Sequence Read Archive accession number

ERR163436 and assembly accession RCVN00000000. Sequence analysis of the

16S rDNA gene of S04009T revealed the isolate to be most closely related to

Staphylococcus saprophyticus subsp. saprophyticus ATCC 15305T and

Staphylococcus edaphicus CCM 8730T (Table 1 and Figure 1). However, further

analysis by comparison of the genes hsp60, sodA, dnaJ, rpoB, tuf and gap, often

used to distinguish similar staphylococcal species (9, 10), fails to discriminate

S04009T from S. xylosus in each case (Table 1). In addition, S04009T cannot be

distinguished from S. saprophyticus subsp. saprophyticus based on rpoB sequence

alone or from S. edaphicus based on dnaJ sequence alone (Table 1) (9, 10). To

resolve the identity of S04009T the overall genome related index (OGRI) methods of

average nucleotide identity (ANI), calculated by OrthoANI (11) and digital DNA-DNA

hybridisation (dDDH), calculated by the genome-to-genome distance calculator

(GGDC) version 2.1 (12) were used. Both methods demonstrate that S04009T is a

novel species, with the resultant ANI and dDDH values lower than those considered

to represent the same species (13, 14) (Table 1). Production of a maximum-

likelihood phylogenetic tree based on the alignment of 107 single-copy core genes,

utilised by bcgTree (15), with bootstrap support values from 500 replicates, further

supports the designation of S04009T as a novel species (Figure 2). The phylogeny

differentiates S04009T from S. xylosus, demonstrating a greater taxonomic resolution

between S. xylosus and S04009T than seen between isolate pairings belonging to

different subspecies ie. the subspecies of S. saprophyticus, Staphylooccus succinus

and Staphylococcus equorum (Figure 2).

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To compare S04009T with the population diversity of S. xylosus, a further bcgTree

based on the same 107 core genes was generated with S04009T and twenty-two

assembled S. xylosus genomes (Figure 3). This phylogeny places S04009T as an

outlier, distinct from the S. xylosus population and further supports the designation of

S04009T as a novel species, separate to S. xylosus. Among this collection of

genome-sequenced S. xylosus (Figure 3) only isolate 47-83 encodes mecC. 47-83

carries the archetypal mecC allele within a SCCmecXI element highly conserved

with that seen in S. aureus (16) and distinct from that described in S04009T (4).

Phenotypic analysis of S04009T revealed it to be coagulase-negative using rabbit

plasma in the tube test and DNAse-negative using DNAse agar with methyl green

(Oxoid, Basingstoke, UK). Coagulase production in the slide agglutination assay

could not be assessed due autoagglutination of S04009T in both water and 0.85%

saline. The Vitek2 GP card (bioMérieux, Basingstoke, UK) was used to perform

biochemical profiling of S04009T and related Staphylococcus type strains following

the manufacturer’s instructions (Table 2). S04009T can be distinguished

phenotypically from S. saprophyticus, S. edaphicus, S. caeli type strains based on

several biochemical reactions, and in agreement with the core gene phylogenetic

tree S04009T was most similar phenotypically to S. xylosus NCTC 11043T (Table 2).

However, S04009T was positive for sorbitol fermentation and negative for urease

production while S. xylosus NCTC 11043T was negative and positive respectively for

these properties (Table 1). Both phenotypic differences were confirmed by an

independent method. S04009T produced a weakly positive reaction in phenol red

broth or andrade peptone water supplemented with 1% (w/v) sorbitol while S.

xylosus NCTC 11043T gave a negative result. In contrast, S. xylosus NCTC 11043T

produced a positive reaction for urease using urea agar slopes (Oxoid, Basingstoke,

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UK) while S04009T was negative. Furthermore, S. xylosus NCTC 11043T did not

display autoagglutination in water or 0.85% saline as seen with S04009T. Finally,

S04009T colonies on columbia horse blood agar (37oC 18 hr in atmospheric

conditions) displayed an irregular edge and contoured surface distinct from the

smooth, complete-edged colonies of S. xylosus NCTC 11043T, although a variety of

colony morphologies have been described for S. xylosus (17). Using the API® Staph

system following the manufacturer’s instructions (bioMérieux, Basingstoke, UK),

S04009T is positive for acid production from; D-glucose, D-fructose, D-mannose, D-

maltose, D-lactose, D-trehalose, D-mannitol, D-xylitol, D-xylose, D-sucrose and N-

acetyl-ᴅ-glucosamine but not from D-melobiose and raffinose. Likewise, S4009 T is

positive for β-naphthyl phosphate, potassium nitrate, methyl-αD-glucopyranoside,

sodium pyruvate (Voges Proskauer) and alkaline phosphatase activity and negative

for arginine dihydrolase and urease activity. API and Vitek2 results were consistent

in five replicates and tests shared between API and Vitek2 produced the same result

although it is worth highlighting the case of arginine dihydrolase activity. S04009T is

negative for arginine dihydrolase activity assessed by API and Vitek2 well ADH2s

(containing 0.27 mg arginine) but positive according to Vitek2 well ADH1 (0.111 mg

arginine). S. xylosus NCTC 11043T produced the same arginine dihydrolase activity

results as described for S04009T.

Antimicrobial sensitivity testing was performed using the AST-P634 card on Vitek2

following the manufacturer’s instructions and interpreted according to European

Committee on Antimicrobial Susceptibility Testing Version 9 2019 criteria for

coagulase-negative staphylococci. S04009T is susceptible to oxacillin, gentamicin,

ciprofloxacin, linezolid, daptomycin, teicoplanin, vancomycin, chloramphenicol,

rifampicin and trimethoprim. S4009T is negative in the cefoxitin screen and shows

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intermediate resistance to nitrofurantoin. It is resistant to benzylpenicillin,

clindamycin, tetracycline and fusidic acid.

Description of Staphylococcus pseudoxylosus sp. nov.

Staphylococcus pseudoxylosus (pseu.do.xy.lo'sus. Gr. adj. pseudes false; N.L.

masc. adj. xylosus pertaining to xylose, and a specific epithet; N.L. masc. adj.

pseudoxylosus a false (Staphylococcus) xylosus, due to the similarity to S. xylosus

and initial identification as such.

The Gram-positive, non-spore forming, facultative anaerobe, coagulase-negative S.

pseudoxylosus forms non-pigmented colonies approximately 2 mm in diameter with

an irregular edge and a contoured surface, after 18hr of growth on Columbia horse

blood agar at 37oC in atmospheric conditions. S. pseudoxylosus is negative for

DNase, and coagulase. Using the on Vitek2 system S. pseudoxylosus S04099T is

able to produce acid from xylose, lactose, ᴅ-mannitol, ᴅ-sorbitol,

saccharose/sucrose, ᴅ-maltose, ᴅ-trehalose, ᴅ-mannose, N-acetyl-ᴅ-glucosamine,

and methyl-β-ᴅ-glucopyranoside but is unable to do so from ᴅ-amygdalin, ᴅ-ribose,

ᴅ-raffinose, pullulan, cyclodextran, ᴅ-galactose and salicin. S. pseudoxylosus

S04009T is positive for catalase, β-galactosidase, β-glucuronidase, phosphatase, L-

pyrrolydonyl-arylamidase but negative for phosphatidylinositol phospholipase C,

urease, alanine-phenylalanine-proline-arylamidase, leucine-arylamidase, L-

aspartate-arylamidase, L-proline-arylamidase, tyrosine-arylamidase, alanine-

arylamidase, β-galactopyranosidase, L-lactate alkalinisation, α-galactosidase, α-

glucosidase and α-mannosidase activity. S. pseudoxylosus S04009T is able to grow

in 6.5% NaCl and is resistant to novobiocin and optochin but susceptible to polymixin

B and bacitracin.

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The type strain S04009T (=DSM 107950T=CCUG 72763T=NCTC 14184T) was

isolated in 2002 from a case of bovine mastitis in France. The genome assembly of

S04009T (accession RCVN00000000) is 3 056 842 bp, with a DNA G+C content of

32.86 mol%.

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Author Statements

Funding Information

Work was supported by internal funding at the University of Edinburgh, Medical Research

Council Partnership Grant (G1001787/1) and the Wellcome Trust (Grant 098051). EMH is

supported by a UK Research and Innovation (UKRI) Fellowship: MR/S00291X/1.

Acknowledgements

The help of the core sequencing and informatics teams, and the Pathogen Informatics team

at the Wellcome Trust Sanger Institute is gratefully acknowledged. The technical assistance

and expertise of Jennifer Harris and Sarah Goodbrand in the R(D)SVS microbiology

laboratory is also gratefully acknowledged.

Conflicts of Interest

The authors declare no conflicts of interest.

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References

1. Becker K, Heilmann C, Peters G. Coagulase-negative staphylococci. Clin Microbiol Rev 2014;27(4):870-926.2. Naushad S, Barkema HW, Luby C, Condas LA et al. Comprehensive phylogenetic analysis of bovine non-aureus Staphylococci species based on whole-genome sequencing. Front Microbiol 2016;7:1990.3. Dordet-Frisoni E, Dorchies G, De Araujo C, Talon R, Leroy S. Genomic diversity in Staphylococcus xylosus. Appl Environ Microbiol 2007;73(22):7199-209.4. Harrison EM, Paterson GK, Holden MTG, Morgan FJE, Larsen AR et al. A Staphylococcus xylosus Isolate with a new mecC allotype. Antimicrob Agents Chemother 2013;57(3):1524-8.5. Paterson GK, Harrison EM, Holmes MA. The emergence of mecC methicillin-resistant Staphylococcus aureus. Trends Microbiol 2014;22(1):42-7.6. Ito T, Hiramatsu K, Tomasz A, de Lencastre H, Perreten V et al. Guidelines for reporting novel mecA gene homologues. Antimicrob Agents Chemother 2012;56(10):4997-9.7. Quail MA, Kozarewa I, Smith F, Scally A, Stephens PJ et al. A large genome center's improvements to the Illumina sequencing system. Nat Methods 2008;5:1005.8. Zerbino DR, Birney E. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 2008;18(5):821-9.9. Ghebremedhin B, Layer F, Konig W, Konig B. Genetic classification and distinguishing of Staphylococcus species based on different partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf gene sequences. J Clin Microbiol 2008;46(3):1019-25.10. Shah MM, Iihara H, Noda M, Song SX, Nhung PH et al. dnaJ gene sequence-based assay for species identification and phylogenetic grouping in the genus Staphylococcus. I Int J Syst Evol Microbiol 2007;57:25-30.11. Lee I, Ouk Kim Y, Park SC, Chun J. OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 2016;66(2):1100-3.12. Meier-Kolthoff JP, Auch AF, Klenk HP, Goker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics 2013;14:60.13. Chun J, Oren A, Ventosa A, Christensen H, Arahal DR et al. Proposed minimal standards for the use of genome data for the taxonomy of prokaryotes. Int J Syst Evol Microbiol 2018;68(1):461-6.14. Ciufo S, Kannan S, Sharma S, Badretdin A, Clark K et al. Using average nucleotide identity to improve taxonomic assignments in prokaryotic genomes at the NCBI. Int J Syst Evol Microbiol 2018;68(7):2386-92.15. Ankenbrand MJ, Keller A. bcgTree: automatized phylogenetic tree building from bacterial core genomes. Genome 2016;59(10):783-91.16. MacFadyen AC, Harrison EM, Ellington MJ, Parkhill J, Holmes MA et al. A highly conserved mecC-encoding SCCmec type XI in a bovine isolate of methicillin-resistant Staphylococcus xylosus. J Antimicrob Chemother 2018;73(12):3516-8.17. Schleifer KH, Kloos WE. Isolation and characterization of Staphylococci from human skin I. Amended descriptions of Staphylococcus epidermidis and Staphylococcus saprophyticus and descriptions of three new species: Staphylococcus cohnii, Staphylococcus haemolyticus, and Staphylococcus xylosus. Int J Syst Evol Microbiol 1975;25(1):50-61.18. Sivadon V, Rottman M, Quincampoix JC, Avettand V, Chaverot S et al. Use of sodA sequencing for the identification of clinical isolates of coagulase-negative staphylococci. Clin Microbiol Infect 2004;10(10):939-42.19. Fairbrother RW. Coagulase production as a criterion for the classification of the staphylococci. J Pathol Bacteriol 1940;50(1):83-8.20. Shaw C, Stitt JM, Cowan ST. Staphylococci and their classification. Microbiol 1951;5(5):1010-23.

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21. Hajek V, Meugnier H, Bes M, Brun Y, Fiedler F et al. Staphylococcus saprophyticus subsp. bovis subsp. nov., isolated from bovine nostrils. Int J Syst Evol Microbiol 1996;46(3):792-6.22. Pantucek R, Sedlacek I, Indrakova A, Vrbovska V, Maslanova I et al. Staphylococcus edaphicus sp. nov., isolated in Antarctica, harbours mecC gene and genomic islands with suspected role in adaptation to extreme environment. Appl Environ Microbiol 2017.23. MacFadyen AC, Drigo I, Harrison EM, Parkhill J, Holmes MA et al. Staphylococcus caeli sp. nov., isolated from air sampling in an industrial rabbit holding. Int J Syst Evol Microbiol 2019;69(1):82-6.24. Bergey's Manual of Systematic Bacteriology Second ed: Springer; 2009.25. Manual of Clinical Microbiology. 11th Edition ed: ASM Press; 2015.

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Table 1. Results for Species delineation methods/gene comparisons.

ANI was calculated by OrthoANI (11), with dDDH calculated by the genome-to-genome distance calculator (GGDC) version 2.1

(12). Species cut-off values for individual genes/methods are shown in brackets.

Strain (reference) Gene/Method

16S rRNA

(98.7%) (13)

tuf

(98%) (9)

rpoB

(93.6%)

(9)

dnaJ

(88.8%)

(10)

hsp60

(93%) (9)

sodA

(97%) (18)

gap

(96%) (9)

ANI

(95-96%)

(13)

dDDH

(70%) (13)

S. xylosus NCTC 11043T (17) 99.8% 98.5% 98% 98.4% 96.9% 98.6% 98.4% 93.30% 51%

S. saprophyticus subsp. saprophyticus ATCC 15305T (19, 20) 99.9% 96.8% 93.7% 87.2% 88.2% 94.3% 93.2% 80.64% 24.7%

S. saprophyticus subsp. bovis CCUG 38042T (21) 99.7% 96.9% 93.6% 87.1% 88.2% 94.3% 93.1% 80.86% 25.0%

S. edaphicus CCM 8730T (22) 99.9% 96.5% 93.6% 89.5% 87.6% 93.5% 95.1% 80.50% 23.9%

S. caeli NCTC 14063T (23) 99.8% 96.1% 92.1% 86.8% 88.3% 93.8% 94.4% 79.95% 24.1%

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Table 2. Phenotypic characterisation of S. pseudoxylosus sp. nov strain S04009T and closely

related staphylococcal species.

Phenotypic data from Vitek 2 performed in this study on Strains: 1, S. pseudoxylosus

S04009T; 2, S. xylosus NCTC 11043T; 3, S. saprophyticus subsp. saprophyticus ATCC

15305T; 4, S. saprophyticus subsp. bovis DSM 18869T; 5, S. edaphicus DSM 104441T; 6, S.

caeli NCTC 14063T. +, positive; -, negative. Vitek 2 experiments with S04009T and NCTC

11043T were performed five times and twice for other isolates with consistent results

throughout. Results presented in [ ] represent those of the species as a whole, where such

data are available, with symbols indicating: +, 90% or more of strains are positive; -, 90% or

more of strains are negative; d, 11 to 89% of strains are positive; ND, not determined. In the

case of species phenotypic data these are taken from (24, 25), except for sorbitol for S.

xylosus taken from (17).

Biochemical Test1 2 3 4 5 6

D-xylose + +[+] -[-] -[-] - -

β-galactosidase + +[+] +[d] -[d] - +

Phosphatase + +[d] -[-] -[d] - -

β-glucuronidase + +[d] -[-] -[d] + +

D-Galactose - -[d] +[-] -[d] + -

D-ribose - -[d] -[-] +[+] - -

Lactose + +[d] +[d] +[d] - +

N-acetyl-d-glucosamine + +[+] -[d] -[+] - -

D-mannitol + +[d] +[d] +[+] + +

D-mannose + +[+] -[-] -[d] + +

Salicin - -[d] -[-] -[ND] - -

D-sorbitol + -[d] -[-] -[ND] - -

Urease - +[+] +[+] -[+] + +

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Figure 1. Phylogenetic relationship of Staphylococcus type strains based on 16S rDNA

sequence. Arrow indicates the position of S. pseudoxylosus S04009T. The phylogenetic tree

was generated from a MUSCLE alignment of the 16S rDNA gene sequences of

Staphylococcus type strains and the type strain of Macrococcus caseolyticus. The

neighbour-joining method was used and bootstrapping the analysis based on 1500

replicates was performed using MEGA10 software. The optimal tree, with a branch length

sum of 0.38740990, is shown. Evolutionary distances were calculated using the Maximum

Composite Likelihood method, the scale bar represents number of base substitutions per

site. Gamma distribution was used to model the rate of variation. The units used for

evolutionary distances to infer phylogeny are the same as those used for branch lengths,

with the tree drawn to scale. rDNA sequences were obtained from the SILVA Living Tree

Project [18], except for S. argensis (partial sequence of 1080 bp), S. caeli and S.

cornubiensis, with sequence accession numbers for each 16S rDNA sequence shown after

the species name.

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Figure 2. Phylogenetic relationship of Staphylococcus type strains most closely related to

S04009T. Arrow indicates the position of S. pseudoxylosus S04009T. Alignment of 107

single-copy core genes utilised by bcgTree, was used to reconstruct a maximum-likelihood

phylogenetic tree, with bootstrap support values from 500 replicates. The scale bar

represents the number of amino acid substitutions per site. M. caseolyticus DSM 20597 has

been used as an outlier.

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Figure 3. Phylogenetic relationship of S04009T compared to the S. xylosus population. S.

xylosus type strain denoted by a superscript T. A maximum-likelihood phylogenetic tree was

generated from the alignment of 107 single-copy core genes utilised by bcgTree, with

bootstrap support values from 500 replicates. The scale bar represents the number of

amino acid substitutions per site. S. caeli 82BT has been used as an outlier.

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