Genomic Stability of Composite SCC mec ACME and COMER-Like Genetic Elements in Staphylococcus epidermidis Correlates With Rate of Excision

Abstract

The epidemiological success of methicillin-resistant Staphylococcus aureus USA300 has been associated with the presence of two mobile elements, the arginine catabolic mobile element (ACME) and the copper and mercury resistance (COMER) element. These two mobile elements are associated with resistance to copper, which has been related to host fitness and survival within macrophages. Several studies found that ACME is more prevalent, and exhibits greater diversity, in Staphylococcus epidermidis while COMER has not been identified in S. epidermidis or any other staphylococcal species. We aimed in this study to evaluate the presence and diversity of ACME and COMER-like elements in our S. epidermidis clinical isolates. The genomes of 58 S. epidermidis clinical isolates, collected between 2009 and 2018 in a Scottish hospital, were sequenced. A core-genome phylogenetic tree and genome based MLST typing showed that more than half of the isolates belong to the clinically predominant sequence type2 (ST2) and these isolates have been found to split into two lineages within the phylogenetic tree. Analysis showed the presence of SCCmec in the majority of isolates. Comparative analysis identified a cluster of ACME-positive isolates with most of them belonging to ST48. ACME showed high variation even between isolates of the same ACME type and ST. COMER-like elements have been identified in one of the two major hospital adapted drug resistant ST2 lineages; and showed high stability. This difference in stability at the genomic level correlates well with the up to one hundred times higher excision frequency found for the SCCmec elements in ACME-containing isolates compared to COMER-like element containing isolates. ACME/COMER-like element positive isolates did not show a significant phenotype of decreased copper susceptibility, while resistance to mercury was over-represented in COMER-like element positive isolates. To the best of our knowledge, this is the first molecular characterization of COMER-like elements in S. epidermidis isolates. The presence of the COMER-like elements is the most prominent accessory genome feature of these successful lineages suggesting that this chromosomal island contributes to the success and wide clinical distribution of ST2 S. epidermidis.

Keywords: ACME; COMER; SCCmec; Staphylococcus epidermidis; mobile genetic element.

FIGURE 1

Maximum likelihood phylogenetic tree constructed using core genome alignment from the 58 S. epidermidis clinical isolates. The phylogenetic tree is annotated with the isolate’s sequence type ST. Colored boxes to the right of each strain name illustrated the distribution of the SCCmec (blue), cop operon (yellow), ACME (red), and COMER-like element (green) characteristic genes among the S. epidermidis isolates. S. epidermidis BPH0662 used as reference strain in this phylogenetic tree.

FIGURE 2

Schematic map of the COMER-like elements in S. epidermidis. The COMER element previously described in MRSA USA300 strain CA12 (GenBank accession number CP007672) (A) and the SCCmec-III element previously described in strain 85/2082 (GenBank accession number AB037671) (C) are included for comparison. Characteristic genes of the COMER-like element are colored including the mer operon, the abi genes, and the restriction modification system (green), the cop operon (yellow-brown) and ars operon (purple). The SCCmec element genes colored include the mecA, ccrAB3 genes (blue), and the cad operon (light green). An additional module harboring a metal transporter, ccrAB4 (bright green) and speG genes (light purple) is found upstream SCCmec-III in our S. epidermidis strains (B). Homologous regions between the different elements are shown by shading. The direct repeats (DR) are indicated.

FIGURE 3

Schematic map of the ACME-I in S. epidermidis. comparative organization of ACME-I composite island in the seven ST48, ST54, ST35 S. epidermidis isolates. The size of each ACME composite island is indicated after the strain names. Four distinct ACME-I composite island were defined according to the additional modules in these elements (A–D). Each gene or group of genes of interest is shaded in color; arc and opp3 operons (red), speG (light purple), copB (brown), mco (light yellow), copL (ornage), ars operon (dark purple), mecA, mecR1, IS431, IS1272, and ccrAB2 (dark blue). The direct repeat (DR) sequences DRA, DR-B, DR-C, and the new DR are indicated.

FIGURE 4

Core genome phylogenetic tree of a world-wide collection of S. epidermidis genomes. Maximum likelihood tree of the 58 Scottish isolates (Table 1) (isolates in blue) and the dataset from a world-wide collection of 225 isolates (isolates in gray) (NCBI BioProject numbers: PRJEB12090, PRJNA470534, and PRJNA470752) (Lee et al., 2018). As in Figure 1, the tree is annotated with the isolate’s sequence type (ST) and displays the colored boxes of the heatmap illustrating the distribution of the characteristic genes of the SCCmec (blue), cop operon (brown/yellow), ACME (red) and COMER-like element (green) among the S. epidermidis isolates. Isolate BPH0662 (isolate in black) is used as reference strain in this phylogenetic tree.

FIGURE 5

Susceptibility profiles of S. epidermidis to different metals. Disc diffusion inhibition zones were defined for the 58 S. epidermidis isolates by exposing bacteria to discs containing 10 μl of 1M metal stocks (0.2 M for mercury and arsenic) [(A) copper, (B) cadmium, (C) mercury, (D) manganese, (E) zinc, (F) nickel, (G) cobalt, (H) iron, and (I) arsenic]. Metals tested included copper sulfate (CuSO₄), nickel sulfate (Niso₄), iron chloride (FeCl₃), manganese sulfate (MnSO₄), zinc sulfate (ZnSO₄), cobalt bromide (CoBr₂), cadmium chloride (CdCL₂), mercuric chloride (HgCl₂), and sodium arsenate (AsNA₂). In the bar charts ACME containing isolates are shown in red, COMER-like element containing isolates in green and all other isolates in gray. ECOFFs are shown in arrows. Statistical analysis of the association of isolates carrying COMER-like elements with cadmium and mercury susceptibility is shown (Fisher’s exact test).

FIGURE 6

Excision rates of composite SCCmec elements. The rate of excision was measured using real-time qPCR by comparing the excised circularized element and the reconstituted chromosomal attachment site to the integrated element using this formula: 2^{–Δ CT}. The columns represent the rate of excised element, SCCmec alone (gray) and the composite SCCmec/ACME (white), in isolates STAPH79, STAPH3, and STAPH16. Error bars represent the mean SD of the excised element with and without mitomycin C, asterisk (*) indicates p-value smaller than 0.05 (p < 0.05). (A) Induction with mitomycin C did not affect the excision ratio of the SCCmec (gray) nor the composite SCCmec/ACME (white) in isolate STAPH79 comparing to the control (Ctrl) (B). In STAPH60, like in STAPH79, the mitomycin C induction had no effect on excision frequency with respect to control (Ctrl) (C). The schematic representation of the excision of SCCmec and ACME/COMER-like elements is shown in (D–H) including the chromosome containing the integrated SCCmec (blue) with the ACME/COMER elements (green) (D), the circular form of the SCCmec (E) and circular form SCCmec with ACME/COMER-like element (F), the chromosome with the ACME/COMER element after excision of SCCmec only (G) and the reconstituted chromosome after excision of the whole composite element (H). The att sites are shown in red and the primers used for qPCR are shown as blue arrows and TaqMan probes as purple arrows. The maps are not drawn to scale.