Ethical Statement

Isolates were obtained as part of routine diagnostic testing and were analyzed anonymously and the isolates, not humans, were studied. All data was collected in accordance with the European Parliament and Council decision for the epidemiological surveillance and control of communicable disease in the European community [16,17]. Ethical approval and informed consent were not required.

Bacterial isolates

Bacterial isolates were identified as part of the FP7-HEALTH program CONCORD (Control of Community-acquired MRSA: Rationale and Development of Counteractions) [18]. The collection was not part of a structured survey with pre-defined criteria of isolate collection. It was a convenience sample composed of 568 S. aureus isolates obtained from infection (74%) and colonization (17%) of patients attending health-care centers and hospitals, collected within 48 hours of hospitalization. Among this set of isolates three patients were identified with pairs of isolates for which the first was an isolate from colonization and the second from an infection site. All isolates were obtained in 2005. Isolates S1800 and S1805 were obtained 11 days apart, isolates S2395 and S2396 29 days, and S2397 and S2398 17 days apart. The infection for the first set was cellulitis, whereas the latter two sets both caused a furuncle.

In addition, a set of livestock-associated methicillin-resistant (LA-MRSA) isolates were identified at the same time on a single farm. One isolate was from the nose of a colonized pig (S0462) the other from the nose of the colonized pig farmer (S0460).

Whole sequencing and analysis

Whole genome sequencing and contig assembly for the three patients pairs were performed by BaseClear (Leiden, the Netherlands) using Illumina GA IIx technology. The LA-MRSA isolates were sequenced by KeyGene (Wageningen, The Netherlands) using 454 GS FLEX technology. The sequences have the following EMBL accession nrs: S0460: CAVH010000001-CAVH010000167; S0462: CAVX010000001-CAVX010000134; S1800: CAWB010000001-CAWB010000401; S1805: CAVY010000001-CAVY010000125; S2395: CAVU010000001-CAVU010000120; S2396: CAWA010000001-CAWA010000096; S2397: CAVV010000001-CAVV010000075; S2398: CAVW010000001-CAVW010000078.

The contigs were mapped against strain Newman (GenBank accession number AP009351)for S1800 and S1805; MSSA476 (GenBank accession number BX571857) for S2395 and S2396; and WKZ-1 (GenBank project ID 40253) for S2397 and S2398, as these strains appeared to most closely resemble the sequences of the isolates. The LA-MRSA isolates were mapped against S0385, the first sequenced LA-MRSA (GenBank accession number AM990992) [19]. Novel sequences were based on BLAST against GenBank databases. Annotations was performed using Kodon (Applied Maths, Sint-Martens-Latem, Belgium). The isolates were then compared pairwise by alignment for differences in gene content and SNPs using Kodon. Differences in gene content were confirmed by PCR.

Sequencing coverage was 9, 10.5, 96, 118, 275, 114, 306, and 178 times for S0460, S0462, S01800, S1805, S2395, S2396, S2397, and S2398, respectively. SNPs in the three pairs of human derived isolates were excluded when 1) they were present within 50 bp of the end of a contig; 2) were within 10 bp of a GGC motif [20]; 3) were present in multiple sequences >50 bp with >98% similarity; remaining potential SNPs were validated by mapping the reads. Regions smaller than 1 kb with more than 3 mutations were sequenced by conventional sequencing to determine SNP status.

SNPs in S. aureus isolates S0460, S4062 were identified slightly differently, because the genome was sequenced using 454 technology. SNPs were identified by mapping the 454 sequencing reads against the reference genome using GS reference mapper software (Roche) and by sequence alignment of the contigs against the reference genome using MUMmer [21]. SNPs located in polyA/T tracts were excluded. The remaining SNPs were categorized as true, possible or probable depending the following criteria: 1) supported by at least 5 reads; 2) at least 90% SNP agreement between reads; 3) SNP position in the contig is at least 50 nucleotides away from the border of the contig; 4) quality score assigned by 454 sequencing. Only if all criteria were met the SNP was deemed true, otherwise they were reported as possible or probable when one or two criteria were not met, respectively. SNPs located in mobile genetic elements not conserved among the isolates, repeat regions or paralogous genes were excluded from further analysis. We selected 20 SNPs for PCR amplification and traditional capillary sequencing in all four isolates which confirmed the criteria used to select true SNPs.

S1800 and S1805

Both S1800 and S1805 belong to sequence type 80 (ST80) and spa-type t-044 indicating that both isolates belong to the European clone of CA-MRSA. Isolate S1800 was a carrier isolate whereas isolate S1805 caused an infection.

A Staphylococcal Cassette Chromosome mec (SCCmec) type IV was present in both S1800 and S1805. The region of the mec element also contains a Fok type II restriction methylase and restriction endonuclease, which is also present in the sequence of the first published ST80 strain [22].

Both isolates also contained a plasmid. A part of the plasmid encoding 7 proteins is present in Streptococccus mitis strain B6 (GenBank Acc no. FN568063.1) (Table 1). Compared to S. mitis B6 a number of mutations occur and some nucleotides are missing in a scattered pattern between bp 28,227-28,555 and a stretch of nucleotides lacks between bp 30,426 and 30,448 in the S. mitis isolate. The missing stretch is in the intergenic region between a transcriptional regulator and the aminoglycoside 3'-phosphotransferase and the scattered changes are in the streptomycin aminoglycoside 6-adenyltransferase. This may represent a novel variant of this gene.

Comparison with the first completely sequenced ST80 (GenBank accession number CP003194) showed that it carried an additional prophage compared to S1805. This prophage was partially present in S1800. The other main difference is that the SCCmec of the first completely sequenced ST80 contained additional sequences (approximately 23 kb) between the ccr genes and mecA.

When S1800 and S1805 were compared for gene content major differences were observed. S1800 contained an additional prophage without any striking proteins. Both isolates contained the genes for staphylokinase and the staphylococcal complement inhibitor which are located on a prophage. The isolates also contained the genes for Panton-Valentine toxin. In addition, then gene encoding SplF protease and a stretch encoding 3 hypothetical proteins were additionally present in S1800. The S1805 contained an integrative conjugative element similar to one reported for MRSA ST398 strain S0385 [19]. In S1805 genes encoding a LPTXG-motif cell wall anchor protein, exotoxin 9, a putative lipoprotein and a hypothetical protein were present that were not found in S1805.

Initial sequence comparison between S1800 and S1805 showed the presence of 188 potential SNPs. A total of 31 potential SNPs were analyzed by conventional sequencing because they were present close to each other at seven different loci. Four of these potential SNPs could be confirmed. They were present in the ATB-binding subunit of a multidrug resistance ABC-transporter, an auto-inducer sensor protein, the sensor-histidine kinase KdpD, protein A, and the GTP-sensing transcriptional pleiotropic repressor CodY. Remarkably, three of these confirmed mutations were found in proteins involved directly or indirectly in gene regulation. However, the majority of the false-positive SNPs were present in genes with similar sequences elsewhere or within the same gene, e.g., the repeats of the protein A gene. Of the remaining 157 SNPs 69 were considered sequence artifacts, present in sequences that occurred multiple times in the genomes, or were phage encoded, leaving 88 potential SNPs. Mapping of the reads showed that 99-100% all reads for each SNP matched for these 88 potential SNPs. So, in total at least 92 true SNPs were present between S1800 and S1805. Seventeen SNPs were located at intergenic sequences, 20 were synonymous mutations and the remaining 55 SNPs were non-synonymous. These SNPs were present in a variety of proteins (Table S1). The majority of these would be involved in house-keeping, but some more interesting genes might be a type I restriction modification system protein and MutS involved in mutator phenotypes.

From these data we conclude that despite the fact that both isolates have the same ST, spa-type and the strongest epidemiological link that can be found (isolates from the same patient), the isolates are not clonal.

S2395 and S2396

The sequenced isolates (S2395 and S2396) belong to ST1 and have spa-type t-590. S2396 was an isolate from colonization obtained before the isolate from infection, S2395. When the isolates were compared for gene content only one major difference was noted. S2395 contained a bacteriophage that was absent in the other isolate. Both isolates contained the genes staphylokinase, staphylococcal enterotoxin A, and staphylococcal complement inhibitor which are located on a prophage. When compared to MSSA476, a completely sequenced ST1 MSSA, several significant differences were present. MSSA476 contained a SCC element encoding amongst others fusidic acid resistance [23], and two sequences both encoding two hypothetical proteins. Isolates S2395 and S2396 additionally contained a 13.5 kb sequence with partial homology to the staphylococcal pathogenicity island (SaPi) of strain RN3984 (GenBank accession number EF010993.1). Eight of the 14 genes showed more than 90% similarity with SaPibov (GenBank accession number AF217235). One gene encoded a PemK-like growth inhibitor associated with a SaPi in strain JKD6008, a ST239 member (Genbank accession nr CP002120.1). Five genes encoded hypothetical proteins without a homologue in the GenBank database. Furthermore, a second 15 kb SaPi is present. Highest similarity was found with the bovine SaPi and the SaPi of strain T0131. No remarkable genes could be identified, but three genes encoded hypothetical proteins not related to pathogenicity islands. A transposon, Tn916-like, encoding tetracycline was also present in both isolates but not in MSSA476. This 17.6 kb transposon was with 99% similarity also present in the first sequenced ST398 strain (S0385). The isolates and MSSA476 differed in a prophage. Finally, genes encoding an enoyl-(acyl carrier protein) reductase and two hypothetical proteins were present in the set of isolates and absent in MSSA476.

After initial data analysis 114 potential SNPs were found between S2395 and S2396. A total of 59 potential SNPs were analyzed by conventional sequencing because they were present close to each other at 11 different loci. None of these potential SNPs could be confirmed. Of the remaining SNPs 49 were considered sequence artifacts, present in sequences that occurred multiple times in the genomes, or were phage encoded. Four of the remaining 6 potential SNPs could not be confirmed by mapping of the reads. This was most likely also due to multiple copies of the sequences since approximately half of the reads showed one nucleotide and the other half another nucleotide. The remaining 2 SNPs were confirmed by mapping the reads. One was an A to G change in an intergenic region in front of a putative peptidase in S2395 and the second was a non-synonymous G to T mutation in a gene encoding a putative membrane protein resulting in a valine to fenylalanine amino acid change.

The presence of a low number of potential SNPs and the fact that the 59 resequenced potential SNPs confirmed their absence suggest that the isolates are closely related despite a difference in prophage content.

S2397 and S2398

The two sequenced isolates (S2397 and S2398) belong to ST30 and have spa-type t-433. S2398 was an isolate from colonization obtained before the isolate from infection, S2397. Community-associated MRSA with this sequence type are considered to belong to the South-West Pacific clone. The isolates do not differ in gene content. The genomes do not contain any novel sequences.

The differences between this pair of isolates and a representative of ST30 we sequenced before, MSSA WKZ-1 [24] were considerable. The most important differences were that WKZ-1 possessed SaPi2 encoding, amongst others, toxic shock syndrome toxin, a second SaPi structure encoding at least two putative enterotoxins, an integrative conjugative element, transposon Tn552 encoding an ß-lactamase, an unnamed plasmid-like structure encoding amongst others arsenic resistance that is also found in MRSA252. Furthermore, two prophages differed between the pair and WKZ-1, but no virulence factors could be identified on these. Both isolates contained the prophage encoded immune evasion cluster encoding staphylokinase, staphylococcal enterotoxin A, staphylococcal complement inhibitor, chemotaxis inhibitory protein of S. aureus. Isolate S2397 and S2398 contained 4 additional genes on 2.8 kb DNA, but no function could be assigned to these genes. This sequence has also been observed in a number of other S. aureus such strains TW20 and TCH60 (GenBank accession numbers CP002120 and CP002110, respectively).

After initial data analysis 105 potential SNPs were found between S2397 and S2398. A total of 39 potential SNPs were analyzed by conventional sequencing because they were present close to each other at six different loci. None of these potential SNPs could be confirmed. Of the remaining potential SNPs 65 were considered sequence artifacts. One SNP was identified, a synonymous mutation (a to g in S2398) in a putative lipoprotein.

These data are in agreement with our hypothesis that the isolates form a pair, because only one of the potential SNPs was confirmed. In this case most likely no adaptation occurred when the strain went from carrier status to infection.

S0460 and S0462

S0460 and S0462 belong to ST398, have spa-type t-011, and contain a SCCmec type IV. .There were no differences in gene content between the bacterial chromosomes of the two farm derived S. aureus ST398 isolates. Many of their characteristics are shared with the reference genome S. aureus ST398 isolate S0385. All isolates possessed the allelic variants of νSaα and νSaß islands described previously for ST398 and which seem to unique for this sequence type. νSaß is most notable of these, because it lacks the genes encoding type I restriction-modification system, enterotoxins, serine proteases, lantibiotic biosynthesis proteins and LukE/D hemolysins commonly found in the other S. aureus genomes. SaPi-S0385 encodes two excreted proteins with homology to the staphylococcal complement inhibitor (SCIN) and von Willebrand factor binding protein (Wbp) [19]. Like in isolate S0385, resistance to penicillin and tetracycline is conferred by Tn552 and Tn916, encoded by bla and tet(M), respectively. A third transposon (Tn7-like) with unknown function(s) was present in both isolates. Furthermore, a prophage very similar to φSa6S0385 is inserted; we could not identify any known or hypothetical virulence genes on this prophage.

Comparison with the reference strain S0385 also showed divergence between the genomes, most of which can be explained by the presence or absence of complete mobile genetic elements (MGE). The farm derived isolates all possess SCCmec type IV while the reference has SCCmec type V integrated into orfX. A second prophage is present in the farm isolates, but no genes with homology to known virulence factors were found. Integrated into the gene encoding for the DNA repair protein RadC is a Tn554-like element encoding a dihydrofolate reductase enzyme dfr in accordance with the trimethoprim resistance observed in the farm derived isolates.

The two isolates were very similar and did show only 12 mutations (Table 2). Remarkably, the majority of these were non-synonymous SNP or insertions or deletions (indels) and resulted in the truncation of two genes. The genes belong to a number of different functional categories (Table 2).