Purpose: The purpose of this study was to sequence the genome and define the basal transcriptome of a CA-MRSA USA300 strain, an emerging foodborne pathogen, using next generation sequencing (NGS) as a preliminary step toward improved control and therapeutic strategies.
Methods: Genomic DNA was extracted from MRSA SF8300, a representative CA-MRSA USA300 strain; libraries were sequenced on the Illumina MiSeq platform (Illumina TruSeq Sample Preparation Kit; > 700,000 reads; 250bp paired-end reads). RNA was extracted from early log phase (OD600 = 0.4) and stationary phase (OD600 = 1.0 + 3hr) from MRSA SF8300 cells; libraries were quantified on the Illumina HiSeq 2500 platform in triplicate (Epicentre ScriptSeq Library Preparation Kit; > 60 million reads each; 100 bp paired-end reads). RNA-Seq reads were mapped to the MRSA TCH1516 genome sequence and normalized to fragments per kilobase per million reads (FPKM) values using the Tophat and Cufflinks packages; MULTTEST model with p-value adjustments based on False Discovery Rate (FDR) (SAS v9.3) was performed for statistical significance.
Results: Genomic DNA for strain SF8300 was assembled into 33 scaffolds with 63X coverage. Actively transcribed genes were defined as (FPKMlog/FPKMstat > 2; adj. P-value < 0.05) with 109/2,578 (4.2%) and 162/2,578 (6.2%) genes exhibiting increased transcriptional activity in log and stationary phase, respectively, over the total number of genes exhibiting activity. Major virulence gene regulator, agr, exhibited higher transcriptional activity in stationary phase cultures.
Significance: This study defines the log and stationary phase transcriptomes of CA-MRSA as well as the genome sequence of a typical USA300 MRSA strain. These data provide preliminary insight into methods for strain differentiation, interventions and improved therapeutics.