Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus

Abstract

Background: Community acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) increasingly causes disease worldwide. USA300 has emerged as the predominant clone causing superficial and invasive infections in children and adults in the USA. Epidemiological studies suggest that USA300 is more virulent than other CA-MRSA. The genetic determinants that render virulence and dominance to USA300 remain unclear.

Results: We sequenced the genomes of two pediatric USA300 isolates: one CA-MRSA and one CA-methicillin susceptible (MSSA), isolated at Texas Children's Hospital in Houston. DNA sequencing was performed by Sanger dideoxy whole genome shotgun (WGS) and 454 Life Sciences pyrosequencing strategies. The sequence of the USA300 MRSA strain was rigorously annotated. In USA300-MRSA 2658 chromosomal open reading frames were predicted and 3.1 and 27 kilobase (kb) plasmids were identified. USA300-MSSA contained a 20 kb plasmid with some homology to the 27 kb plasmid found in USA300-MRSA. Two regions found in US300-MRSA were absent in USA300-MSSA. One of these carried the arginine deiminase operon that appears to have been acquired from S. epidermidis. The USA300 sequence was aligned with other sequenced S. aureus genomes and regions unique to USA300 MRSA were identified.

Conclusion: USA300-MRSA is highly similar to other MRSA strains based on whole genome alignments and gene content, indicating that the differences in pathogenesis are due to subtle changes rather than to large-scale acquisition of virulence factor genes. The USA300 Houston isolate differs from another sequenced USA300 strain isolate, derived from a patient in San Francisco, in plasmid content and a number of sequence polymorphisms. Such differences will provide new insights into the evolution of pathogens.

Figure 1

Histogram of the prevalence of community-acquired MR and MS strains of S. aureus at TCH over a four-year study. All cases are shown in red, MR cases in green and MS cases in purple.

Figure 2

PFGE of Sma I-digested USA300-HOU-MR and USA300-HOU-MS. Replicates of MR (left) and MS (right) profiles are shown. Molecular weight markers are shown in kilobases.

Figure 3

Chromosomal map of USA300-HOU-MR with landmarks indicating the SCCmec-ACME region (outer circle, green), pathogenicity islands (outer circle, light blue), prophage (outer circle, lavender), the 13 kb insertion sequence (outer circle, orange), and two regions of sequence not found in USA300-HOU-MS (outer circle, yellow). ORFs on both strands are represented by the second circle and are colored according to functional categories as follows and as shown in the key: cell processes, tan; cell structure, violet; DNA replication and recombination, red; general metabolism, blue; regulation, green; transcription, yellow; translation, orange; transport, cyan; virulence, fuchsia; unknown, black. The third circle shows RNAs: rRNAs, blue; tRNAs, red; ncRNAs, green. The inner circle shows the location of SNPs between USA300-HOU-MR and FPR3757.

Figure 4

Linear representation of BLASTN comparison of S. aureus strains to USA300-HOU-MR. Strains are listed on the y-axis and the x-axis shows the USA300-HOU-MR coordinates. Bars represent regions of at least 50 bp in length present in USA300-HOU-MR but absent in the other strains.

Figure 5

Boot-strapped phylogenetic trees of the prophages found in USA300-HOU-MR: a) phiSLT phage and b) phiβC family phage. The numbers on the branches indicate the number of times the sets co-partitioned after 100 iterations.

Figure 6

Linear representations of circular plasmids observed in USA300-HOU-MR (a, b) and USA300-MS (c). Replication and regulatory genes are colored red, recombination/transposition genes are green, antibiotic/heavy metal/bacteriocin resistance genes are blue, other genes are white and hypothetical, conserved hypothetical (CHP) and staphylococcal conserved hypotheticals (SCHP) are colored grey. Horizontal bars indicate the regions of pUSA300-HOU-MR and pUSA300-HOU-MS having homology with other sequenced staphylococcal plasmids.

Figure 7

DNA sequence of the sarU promoters in USA300-HOU-MR and FPR3757 showing -10 and -35 sequences (red) and SarA consensus binding sites (boxed).

Figure 8

Dendrogram of seventeen fully sequenced genomes generated by Mauve [35] and drawn using MEGA [58]. RP62A and ATCC12228 are S. epidermidis strains, JCSC1435 is an S. haemolyticus strain, and ATCC15305 is an S. saprophyticus strain. All others are S. aureus strains. The bar represents coalescent units.