Novel staphylococcal species that form part of a Staphylococcus aureus-related complex: the non-pigmented Staphylococcus argenteus sp. nov. and the non-human primate-associated Staphylococcus schweitzeri sp. nov.

We define two novel species of the genus Staphylococcusthat are phenotypically similar to and have near identical 16S rRNA gene sequences to Staphylococcus aureus. However, compared to S. aureus and each other, the two species, Staphylococcus argenteus sp. nov. (type strain MSHR1132T = DSM 28299T = SSI 89.005T) and Staphylococcus schweitzeri sp. nov. (type strain FSA084T = DSM 28300T = SSI 89.004T), demonstrate: 1) at a whole-genome level considerable phylogenetic distance, lack of admixture, average nucleotide identity <95 %, and inferred DNA–DNA hybridization <70 %; 2) different profiles as determined by MALDI-TOF MS; 3) a non-pigmented phenotype for S. argenteus sp. nov.; 4) S. schweitzeri sp. nov. is not detected by standard nucA PCR; 5) distinct peptidoglycan types compared to S. aureus; 6) a separate ecological niche for S. schweitzeri sp. nov.; and 7) a distinct clinical disease profile for S. argenteus sp. nov. compared to S. aureus.

been identified as S. aureus according to phenotype, but on the basis of multi-locus sequence typing (MLST) and a single genome sequence, the lineages are allied to but significantly diverged from S. aureus. We describe here investigations including the use of whole-genome sequence analysis to justify classification as three separate species, S. aureus, and two novel species of the genus Staphylococcus.
Strain MSHR1132 T was isolated from blood cultures of an Indigenous patient from Darwin, Australia (Holt et al., 2011); and strain FSA084 T was isolated from the nares of a red-tailed monkey (Cercopithecus ascanius) from Gabon, Africa (Schaumburg et al., 2012). Both strains grew on tryptone soy agar (TSA) at 37 u C with large, round, smooth colonies similar to typical S. aureus. Colonies of FSA084 T have a yellowish-pigmented appearance while those of MSHR1132 T are non-pigmented, displaying a creamy white appearance. The difference in pigmentation between typical S. aureus and strain MSHR1132 T is particularly evident after growing on chocolate agar (Oxoid) for 48 h at 37 u C (Holt et al., 2011). Strains MSHR1132 T and FSA084 T are both catalase-positive, coagulase-positive by tube coagulase test and colonies demonstrate b-haemolysis on blood agar. Gram staining tests revealed Gram-stainpositive cocci in clusters for both strains. MLST revealed MSHR1132 T as ST1850 and FSA084 T as ST2022. We also selected for further investigation five strains that clustered according to MLST with MSHR1132 T (LBSA043, JABA32044, M260, M051, H115100079) and five that clustered with FSA084 T (FSCB1B, FSCB5, FSA096, FSA090, FSA037). The MSHR1132 T lineage strains were all recovered from human hosts from northern Australia (Brennan et al., 2013;McDonald et al., 2006), Fiji (Jenney et al., 2014) and the UK. The FSA084 T lineage strains were recovered from non-human primates in Gabon and Cô te d'Ivoire, Africa (Schaumburg et al., 2012). These additional strains demonstrated the same cell and colonial morphology as MSHR1132 T and FSA084 T respectively. All MSHR1132 T lineage strains appeared non-pigmented.
Whole-genome sequencing using the Illumina HiSeq platform of the 12 strains, followed by core-genome single nucleotide polymorphism (SNP)-based maximum-likelihood trees demonstrated that MSHR1132 T lineage strains, FSA084 T lineage strains, and reference S. aureus genomes, form three distinct clusters with 100 % bootstrap support (Fig. 1). The details and GenBank accession numbers for these strains are provided in Table 1. Compared to S. aureus, the 16S rRNA gene sequence (1474 nt) is identical in strain MSHR1132 T and differs at one position in strain FSA084 T . However, pairwise average nucleotide identity (ANI), as calculated using JSpecies (Richter & Rosselló -Mó ra, 2009), across the genomes within and between these  (Table 3). An analysis of orthologous core genes shared by all three groups using Bayesian Analysis of Population Structure (BAPS) software ) demonstrated three BAPS clusters and an absence of admixture between the groups (Fig. 1). All MSHR1132 T lineage strains lacked the carotenoid pigment operon.
PCR amplification of the nucA gene that is used as a standard confirmatory marker for S. aureus is positive in strain MSHR1132 T but negative in strain FSA084 T . An examination of the nucA gene and in particular the primer sites for nucA (Brakstad et al., 1992) reveal one and two mismatches for the forward primer, and five and five mismatches for the reverse primer, for MSHR1132 T and FSA084 T , respectively (Fig. 2). The presence of mismatches at the 39 end of primers for strain FSA084 T most likely contributes to the lack of amplification of product for strain FSA084 T . There were two in-frame deletions of 9 and 12 bp and one in-frame insertion of 3 bp in both MSHR1132 T and FSA084 T nucA sequences compared to S. aureus.
Biochemical profiling was performed with the Vitek2 GP card platform (bioMérieux) according to the manufacturer's instructions. We tested in triplicate each of the 12 strains together with 18 strains of S. aureus from the ATCC collection (ATCC 12600 T , 13565, 13709, 14458, 19095, 19636, 23235, 25904, 25923, 27664, 29213, 29247, 33591, 33592, 43300, 49230, 49775, 51811) ( Two novel staphylococcal species related to S. aureus respectively. The MSHR1132 T lineage strains were positive for urease in 56 % of tests compared to 0 % for both S. aureus and FSA084 T lineage strains. S. aureus was positive for N-acetyl-D-glucosamine in 98 % of tests compared to 33 % and 28 % for FSA084 T lineage strains and MSHR1132 T lineage strains, respectively. We attempted to discriminate 12 MSHR1132 T lineage strains (an additional six strains to those already described), 12 FSA084 T lineage strains (an additional six strains to those already described), and 22 consecutive standard clinical strains of S. aureus by using matrixassisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (Microflex LT MALDI-TOF instrument; Bruker Daltonik) ( Table 4). We prepared samples using liquid phase formic acid extraction, according to the manufacturer's recommendations, and compared the spectral profiles gained to the existing Bruker standard clinical database of profiles using MALDI Biotyper 2.1 software (Bruker Daltonik) with default settings. Strains of S. aureus were confidently identified. The MSHR1132 T lineage strains and FSA084 T lineage strains profiles were most similar to the S. aureus profile, but identity scores were much lower than for the strains of S. aureus (P,0.0001 for both compared to S. aureus) and fell below the manufacturer's recommended threshold for a species level identification. We generated new reference profiles with three MSHR1132 T lineage strains and three FSA084 T lineage strains and repeated the analysis of all 46 strains. All strains were then confidently identified into their different groups. These findings are consistent with the three groups being separate species based on cell proteomic analysis.
Analyses of fatty acids, respiratory quinones and peptidoglycans were carried out by the Identification Service of the Leibniz-Institut DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen), Braunschweig, Germany. S.
aureus ATCC 29213, MSHR1132 T and FSA084 T were cultured and tested for fatty acid composition under identical conditions. The fatty acid profiles of strains MSHR1132 T and FSA084 T were similar and dominated by anteiso-C 15 : 0 and anteiso-C 17 : 0 and corresponded in their composition to S. aureus ATCC 29213 (Table 5). Strains MSHR1132 T and FSA084 T contained the menaquinones MK-7, MK-8 and MK-9 at ratios of 11 : 70 : 11 plus a nonidentified peak (MSHR1132 T ) and 7 : 80 : 13 (FSA084 T ).
In Conclusion, although MSHR1132 T lineage strains and FSA084 T lineage strains share identical or near identical 16S rRNA gene sequences, have similar fatty acid and menaquinone compositions to S. aureus, and are phylogenetically the closest known relatives of S. aureus, there are strong justifications for assigning these lineages to two novel species of the genus Staphylococcus, for which the names Staphylococcus argenteus sp. nov. (type strain MSHR1132 T ) and Staphylococcus schweitzeri sp. nov. (type strain FSA084 T ) are proposed. These justifications are: 1) phylogenetic distance, lack of admixture, ANI ,95 %, and inferred DDH ,70 %; 2) different profiles as determined by MALDI-TOF MS; 3) non-pigmented phenotype of S. argenteus sp. nov.; 4) S. schweitzeri sp. nov. cannot be detected by standard nucA PCR; 5) distinct peptidoglycan types compared to S. aureus; 6) a separate ecological niche for S. schweitzeri sp. nov., which has only once been Fig. 2. Sequence alignment of representative nucA gene sequences. The standard primer sites (Brakstad et al., 1992) are indicated in bold underline (S. aureus), light grey shading (S. argenteus sp. nov.) or dark grey shading (S. schweitzeri sp. nov.). MSSA476 represents a reference S. aureus sequence, MSHR1132 T and LBSA043 represent S. argenteus sp. nov., and FSA084 T and FSA090 represent S. schweitzeri sp. nov. Table 4. Comparison of MALDI-TOF MS identity scores using the standard clinical database and an amended database with reference profiles from S. argenteus sp. nov. and S. schweitzeri sp. nov. groups Identity score values are graded as highly probable species identification (score value 2.300-3.000), secure genus and probable species identification (2.000-2.299) and probable genus identification (1.700-1.999). Values are mean (standard deviation) of identity scores.  (Schaumburg et al., 2012); 7) distinct clinical disease profile for S. argenteus sp. nov. compared to S. aureus .

Standard database
Description of Staphylococccus argenteus sp. nov.
The type strain FSA084 T (5DSM 28300 T 5SSI 89.004 T ) was isolated from the nares of a non-human primate (Cercopithecus ascanius) from Gabon, Africa within 12 h after the death of the animal in 2010. The type strain has also been deposited in the Robert Koch Institute (Germany) and the National Collection of type Cultures, Public Health England (UK).

Acknowledgements
The work described here was funded by the Wellcome Trust through core funding for the Sanger Institute Pathogen Variation Group and by the Deutsche Forschungsgemeinschaft (DFG, EI 247/8-1). *Differentiation between these two fatty acids was not possible.
Two novel staphylococcal species related to S. aureus