Description of Staphylococcal Strains from Straw-Coloured Fruit Bat (Eidolon helvum) and Diamond Firetail (Stagonopleura guttata) and a Review of their Phylogenetic Relationships to Other Staphylococci

Abstract

The phylogenetic tree of the Staphylococcus aureus complex consists of several distinct clades and the majority of human and veterinary S. aureus isolates form one large clade. In addition, two divergent clades have recently been described as separate species. One was named Staphylococcus argenteus, due to the lack of the "golden" pigment staphyloxanthin. The second one is S. schweitzeri, found in humans and animals from Central and West Africa. In late 2021, two additional species, S. roterodami and S. singaporensis, have been described from clinical samples from Southeast Asia. In the present study, isolates and their genome sequences from wild Straw-coloured fruit bats (Eidolon helvum) and a Diamond firetail (Stagonopleura guttata, an estrildid finch) kept in a German aviary are described. The isolates possessed staphyloxanthin genes and were closer related to S. argenteus and S. schweitzeri than to S. aureus. Phylogenetic analysis revealed that they were nearly identical to both, S. roterodami and S. singaporensis. We propose considering the study isolates, the recently described S. roterodami and S. singaporensis as well as some Chinese strains with MLST profiles stored in the PubMLST database as different clonal complexes within one new species. According to the principle of priority we propose it should be named S. roterodami. This species is more widespread than previously believed, being observed in West Africa, Southeast Asia and Southern China. It has a zoonotic connection to bats and has been shown to be capable of causing skin and soft tissue infections in humans. It is positive for staphyloxanthin, and it could be mis-identified as S. aureus (or S. argenteus) using routine procedures. However, it can be identified based on distinct MLST alleles, and "S. aureus" sequence types ST2470, ST3135, ST3952, ST3960, ST3961, ST3963, ST3965, ST3980, ST4014, ST4075, ST4076, ST4185, ST4326, ST4569, ST6105, ST6106, ST6107, ST6108, ST6109, ST6999 and ST7342 belong to this species.

Keywords: DNA microarray; Staphylococcus argenteus; Staphylococcus aureus; Staphylococcus roterodami; Staphylococcus schweitzeri; Staphylococcus singaporensis; whole genome sequencing (WGS).

Figure 1

SplitsTree analysis for 154 core genomic markers of the study isolates, “S. roterodami” EMCR19 as well as “S. singaporensis” SS60 and SS90 compared to diverse S. aureus, S. argenteus and S. schweitzeri CCs. Note, for the genes that were found inverted in the Zoo-28 genome, reverse complement sequences were used for analysis.

Figure 2

(A) SNP analysis comparing 2292 non-motile genes of BDS-53E with the Zoo-28 (green), “S. roterodami” EMCR19 (blue) as well as with “S. singaporensis” SS60 and SS90 (red and yellow). For each single gene, the number of differences ( Supplemental File 7B ) to the comparator strains was counted and expressed in percentage. For genes that were present in one, but absent in the other isolate, this percentage was set as 100%. Note, for the genes that were found inverted in the Zoo-28 genome, reverse complement sequences were used for analysis. (B) SNP analysis comparing 2292 non-motile genes of BDS-53E with S. schweitzeri (NCTC13712, LR134304.1, brown), S. argenteus (MSHR1132, FR821777.2, grey), S. aureus CC1 (MW2, BA000033.2, yellow) and S. aureus CC152 (SA17_S6, CP010941.1, red). The genes around pos. 1550 represent a genomic island that is absent in the study isolates (protease genes splF, splE, splD2, splC, splB, splA, leukocidin lukD/E, lantibiotic epidermin gene cluster). The highly conserved genes around pos. 1850 are those encoding ribosomal proteins.

Figure 3

Matrix of differences between BDS-53E, Zoo-28, “S. roterodami” (EMCR19, CAJGUT01), “S. singaporensis” (SS60 and SS90, NZ_JABWHF and NZ_JABWHD), S. schweitzeri, (NCTC13712, LR134304.1), S. argenteus (MSHR1132, FR821777.2) and three S. aureus lineages, CC1 (MW2, BA000033.2), CC93 (JKD6159, CP002114.2) and CC152 (SA17_S6, CP010941.1). The percentages were calculated as explained for Figure 2 and the image shows the median values over all these 2292 genes for each genome sequence compered to all others.

Figure 4

The left diagram shows a simplified version of the blot from Figure 2 , comparing BDS-53E to the reference sequences of S. schweitzeri NCTC13712 (red) and the S. aureus CC1 strain MW2 (green). Differences are evenly distributed all across the genome, suggesting a continuous evolution and an accumulation of more mutations compared to S. aureus than to S. schweitzeri corresponding supposedly to a longer time of separate evolution. The right diagram shows the same analysis for a chimeric strain in which a fragment of “red” origin spanning from approximately position 1750 over oriC (pos. 0) to pos. 200 is integrated into a backbone genome of “green” provenance. These are actually S. aureus CC140 and CC8, in a ST6610 strain, see (Burgold-Voigt et al., 2021). For the Y-axes, different scales were used because differences between the different species are larger than those between different CCs of S. aureus.