DNA extraction

Total bacterial DNA was isolated from an overnight culture of HL1 (ST72) using lysostaphin digestion and the method of Marmur [16]. The pellet of a 1-ml overnight culture was resuspended with 400 µl buffer P1 (Qiagen), to which 20 µl lysostaphin were added. The sample was incubated at 37°C for 15 min. Then 20 µl of a saturated sodium dodecylsulfate solution (in 45% ethanol) was added, the sample was vortexed, and incubated at 37°C for 5 min. Then 130 µl of a 5 M sodium perchlorate solution were added, and the sample was vortexed again. Six hundred µl of phenol/chloroform/isoamylalcohol (25:24:1 by volume) were added, the sample was vortexed, and the bottom phase transferred to a new tube. This extraction was repeated twice, then 800 µl ice-cold ethanol was added to precipitate the DNA, which was harvested by centrifugation for 15 min at 13,000 rpm in a tabletop centrifuge. The DNA was washed twice with 70% ethanol and resuspended in water.

Genome sequencing, annotation and comparative analysis

DNA sequencing was performed using fragment and paired-end libraries on a Roche 454 FLX genome sequencer using Titanium chemistry (454 Life Sciences [a Roche company], Branford, CT), Coverage of 40-60× or higher was obtained according to the manufacturer’s recommendations. Reads were assembled using the GS Assembler Version 2.5 software program. 454 reads were re-aligned to the contigs to check for assembly accuracy, and misassembled portions were corrected All gaps between contigs were closed by oligonucleotide primer design, PCR fragment generation, and Sanger sequencing of the PCR products on an Applied Biosystems 3730XL DNA sequencer (Applied Biosystems, Foster City, CA), Primer walking of large gaps or correction of ambiguous base calls was performed by PCR and Sanger sequencing. Open reading frame (ORF) calling was performed using public and proprietary algorithms, with a minimum length cutoff of 40 amino acids, as previously described [17,18]. The genome sequence and annotation of HL1 (ST72) are deposited in DDBJ/EMBL/GenBank under the NCBI accession numbers CP003979 (chromosome), CP003980 (pHL1), CP003981 (pHL2). ORFs displaying evidence of frameshifts or mutations leading to premature stop codons were identified by proprietary algorithms and were manually verified. Genome comparisons were performed using ClustalW alignments.

Genome analysis

To compare genome contents, we used a proprietary algorithm (Integrated Genomics) and compared every ORF from the HL1 genome with the ORFs from the two selected S. aureus genomes (FPR3757, MW2). Each ORF was compared using three metrics: similarity score (1e-10), functional annotation, and protein length. For ORFs to be considered for inclusion the following criteria had to be satisfied. The considered ORFs must have a P-Score similarity to an ORF from the HL1 genome of 1e-10 or less. In addition, the ORF in consideration should either have same functional annotation as of the HL1 genome’s ORF or have at least 80% matching protein length with that of the corresponding ORF in the HL1 genome. Further genome analyses were performed using ERGO (Integrated Genomics).

Overview

The CA-MRSA isolate HL1, previously also termed CN1 [13], was obtained from the pus of a necrotizing fasciitis infection in an 80-year old patient in the Seoul area of South Korea [15]. The isolate was then determined to be PVL-negative, resistant to clindamycin and erythromycin, belong to ST72 and spa type t324, and harbor SCCmec type IVa. Furthermore, we previously showed that it has high virulence in a rabbit model of skin infection and promotes neutrophil lysis to an extent almost as pronounced as seen with strain USA300 and within the range of other global CA-MRSA isolates [13].

The isolate has a genome of 2,757,070 base pairs (bp), with 2,726 assigned ORFs, of which 1970 have assigned functions, 53 tRNAs, and 9 rRNAs. The overall GC content is 32.79%. The HL1 genome is thus a little shorter than those of the prominent CA-MRSA strains USA300 (strain FPR3757, 2,917,469 bp, 2672 ORFs) and USA400 (strain MW2, 2,820,462 bp, 2644 ORFs) [4,8], which will serve as comparison in this study. HL1 also harbors two plasmids, which we named pHL1 and pHL2, of 3332 and 2472 bp, respectively.

Virulence factors and pathogenicity islands

HL1 harbors the vSaα, vSaβ, and vSaγ genomic islands and the ΦSa3 prophage (Tab. 1). The vSaα, vSaβ, and vSaγ genomic islands occur in most S. aureus strains and harbor a series of virulence factors. For example, vSaα encodes a series of exotoxins and a restriction/modification system. vSaβ encodes another restriction/modification system, four serine proteases, and the bicomponent leukocidin LukDE. vSaγ encodes exotoxins, fibrinogen-binding proteins, a formyl peptide receptor 1 inhibitory protein, α-toxin, and the phenol-soluble modulins (PSMs) PSMβ1 and PSMβ2. The ΦSa3 prophage is not always present in S. aureus; for example, it is absent from the HA-MRSA strain COL, representing the archaic MRSA lineage. It contains genes encoding immune evasion factors, namely the chemotaxis inhibitory protein CHIPS, the complement inhibitor SCIN, and staphylokinase. Of note, ΦSa3 splits the gene encoding the sphingomyelinase β-toxin into two non-functional parts.

These islands and the ΦSa3 prophage are also present in the USA300 and USA400 CA-MRSA strains. Although the overall genetic composition differs, there are no considerable differences in known virulence determinants (Figs. 1, 2). As a notable exception, HL1 vSaβ does not contain the bsa operon coding for the biosynthesis of the epidermin-like lantibiotic aureodermin. It has been shown that this operon encodes a functional lantibiotic [19]. However, production levels are very low under all conditions tested so far, and the role of aureodermin in S. aureus physiology is unclear [20]. Importantly, HL1 does not contain virulence factors encoded on genomic islands or prophages that are absent from USA300 and USA400. Vice versa, both USA300 and USA400 contain the ΦSa2 (ΦSLT) prophage harboring the lukSF genes coding for PVL. USA300 and USA400 also contain the vSa3, and USA400 the vSa4 pathogenicity islands, which are absent from HL1. vSa3 contains two enterotoxin genes with unknown function in virulence, and vSa4 does not comprise known virulence factors. Finally, USA300 contains ACME, harboring the recently described virulence and colonization factor speG [21].

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Figure 1. Genomic islands in ST72 HL1 compared to USA300 and USA400.

A, vSaα genomic island. B, vSaβ genomic island. C, vSaγ genomic island. Broken lines, missing ORFs; red, virulence determinants; green, type I restriction-modification system specificity subunit; yellow, lipoproteins; blue, bacteriocin-related genes; yellow, determinants; grey= ORF not identical to other strains.

https://doi.org/10.1371/journal.pone.0072803.g001

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Figure 2. ΦSa3 prophage in ST72 HL1 compared to USA300 and USA400.

Broken lines, missing ORFs; red, virulence determinants; blue, phage gene or phage-related gene; grey, ORF not identical to other strains.

https://doi.org/10.1371/journal.pone.0072803.g002

A genome-wide analysis on 103 virulence genes to determine whether virulence factors present in HL1 are also present in USA300 and USA400 further confirmed that the overall composition of virulence genes in HL1, USA300, and USA400 is almost identical (Tab. 2). USA300 is missing two enterotoxin genes on vSaβ present in USA400 and HL1 (Fig. 1); and USA400 lacks the chemotaxis-inhibiting protein (CHIPs, gene chs), present in HL1 and USA300 on the ΦSa3 prophage (Fig. 2). The gene coding for the staphylococcal complement inhibitor SCIN is truncated in HL1, but not in USA300 or USA400. The gene crtN coding for the biosynthesis of the carotenoid staphyloxanthin immune evasion factor is annotated as truncated in HL1, because it appears to be split in a very short gene and a larger gene. However, the larger gene likely codes for the functional, previously described CrtN protein [22], while the shorter ORF may be a pseudo-gene. Similarly, HL1 and USA400 have a split coagulase gene, whereas it is not split in USA300. Finally, 28 surface proteins, identified by the sortase substrate LPXTG motif [23], did not show differences in composition between the three analyzed strains (Tab. 3).

PSMs, short, amphipathic, α-helical peptides have recently been recognized as key determinants of CA-MRSA virulence [24]. They are produced by all S. aureus strains, except in naturally occurring Agr-defective mutants, in which no PSMs are detectable. HL1, USA300, and USA400 produce comparably high amounts of PSMs, while HA-MRSA often lack pronounced production of PSMs owing to low activity or mutation of the Agr system [13]. Accordingly, all three strains harbor the genetic loci encoding the PSMα peptides PSMα1 through PSMα4, PSMβ1 and PSMβ2, and the δ-toxin, which is encoded within RNAIII of the Agr quorum-sensing virulence regulator. Notably, the psmα genes are often not annotated in S. aureus genomes, due to their short length, but we annotated them in the HL1 genome and ascertained presence in USA300 and USA400.

Altogether, these findings are in good accordance with the comparable virulence of HL1 and USA400 in the rabbit model of skin infection that we performed previously [13]. The slightly higher virulence of USA300 in that model may be due to ACME-encoded speG, which a recent report indicates promotes virulence during skin infection [7]. Furthermore, they are in line with previous epidemiological studies and infection models performed in the PVL-sensitive rabbit indicating that PVL, as the only major virulence determinant that is absent from HL1 and present in USA300 and USA400, does not have a significant impact on virulence during CA-MRSA skin infection [12,13,25].

SCCmec

The comparison of the HL1 genome with those of USA300 and USA400 only revealed pronounced differences, encompassing a series of genes, in a very limited number of locations. The strongest difference was seen in and surrounding the SCCmec IVa element (Fig. 3). As previously described by Park et al., the class B mec cassette of ST72 SCCmec IVa element contains a tnp20 IS element and a pUB110 region in addition to the SCCmec IVa element of USA300 and USA400 [26]. The tnp20 IS element is also found at another location in the genome of USA300, but not USA400. The pUB110 region comprises genes involved in kanamycin and bleomycin resistance, in addition to plasmid replication and recombination enzymes. In the left extremity (L-C) region of HL1 SCCmec, there are four genes with high similarity to genes found in the S. epidermidis ATCC12228 genome, indicating that they may have been acquired from S. epidermidis – similar to SCCmec IV in general, which is believed to have originated from S. epidermidis at an earlier time [27]. One of the genes in this region has a homologue at another location in the USA400 genome, but not in USA300, while the others lack homologues in those strains. The tnp20 IS element and the USA400 homologue are in vicinity of the nifR3 gene, encoding a putative nitrogen regulatory protein, in the respective USA300 and USA400 genomes, while the nifR3 gene is found close to the J1 region of HL1 SCCmec, indicating that recombination between these two genomic sites occurred.

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Figure 3. SCCmec IVa of ST72 HL1.

The HL1 SCCmec IVa element and the adjacent unique downstream region are compared to the SCCmec IVa elements of USA300 and USA400. SCCmec regions are shaded in grey boxes. Colored internal boxes in the case of HL1 show differing regions. Regions present in USA300 and USA400, but absent from HL1 are in dark grey, internal boxes. Solid grey bar, class B mec region; dotted grey bar, left extremity (L–C) region. Rep, replication protein; Mob, plasmid mobilization protein; Bleo-Resis, bleomycin resistance protein; Kan Nucl-T, kanamycin nucleotidyltransferase; (R), (R)-specific enoyl-CoA-hydratase; HMGCoA, hydroxymethylglutaryl CoA reductase; ACME, arginine catabolic mobile element (downstream of SCCmec in USA300).

https://doi.org/10.1371/journal.pone.0072803.g003

Plasmids

Plasmid pHL1 (3332 bp) contains four ORFs, encoding a plasmid replication protein, a hypothetical protein, and two cadmium resistance proteins (Figure 4). Plasmid pHL2 (2472 bp) encodes a plasmid replication protein, a hypothetical protein, and an rRNA (adenine-N6-) methyltransferase (Figure 4). No virulence factors are encoded on the HL1 plasmids.

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Figure 4. Plasmids in ST72 HL1.

https://doi.org/10.1371/journal.pone.0072803.g004

Concluding remarks

Our analysis of a CA-MRSA genome of a previously not analyzed ST advances our understanding of the CA-MRSA pandemic, especially as it represents the first sequenced genome of a PVL-negative CA-MRSA strain. It further strengthens the hypothesis that the success of CA-MRSA as pathogens is multi-factorial rather than dependent on the acquisition of specific, CA-MRSA-characteristic virulence determinants. In particular, we did not find virulence determinants in ST72 with an apparent role of substituting for the absence of PVL, such as, especially, other leukotoxins. While further detailed functional and gene expression analyses will be necessary, these findings suggest that the virulence of ST72 CA-MRSA is mainly dependent, as previously suggested for CA-MRSA in general [5,14], on gene regulatory adaptations enhancing the expression of core genome-encoded virulence determinants.