Comparison of Clinical Staphylococcus aureus Strains Demonstrating Differing Susceptibilities to the Biocide Ethanol

Ethanol is utilized widely in medicine for both its antiseptic and disinfectant capabilities. In an effort to understand the effects of ethanol on a very common human pathogen, we have determined the ethanol-stimulon of two well characterized unrelated clinical S. aureus methicillin resistant strains LP9 and MM66 with ethanol minimum inhibitory concentrations (MIC) of 10 and 7% respectively. Using S. aureus microarrays and array data analysis protocols (NIAID's Pathogen Functional Genomics Resource Center) we have characterized the transcriptomes of LP9 and MM66 following 15 min exposure to 10% ethanol. Induction with 10 % ethanol led to the altered regulation of 797 genes in LP9 and 899 genes in MM66. There was an overlap of 600 commonly altered genes in both strains and of these, SAMMD identified 50 genes that are uniquely altered only in the ethanol stimulon compared to the other 93 transcriptomes used for comparison. SAMMD analysis further revealed that 259 of the 600 (43%) commonly Eth-altered genes, overlapped with the stringent response induced with the antibiotic mupirocin. Interestingly the gene SACOL2518 up-regulated 20.4 and 23 fold in LP9 and MM66 respectively has a RelA- and SpoT- like domain. We have also found the MICs and MBCs for both LP9 and MM66 to be affected by the addition of certain amino acids. Genes involved with the heat shock response were also among some of the highest similarly up-regulated genes. Overall, SAMMD found 30% (43 genes) in common between the heat shock and ethanol transcriptomes of LP9 and MM66. clpB encoding the clpB chaperone was the highest induced gene in both strains. groES, groEL, hrcA, grpE and dnaK were also some of the highest up-regulated genes in both strains. S. aureus pangenome microarrays were utilized to determine transcriptome alterations occurring in clinical strains LP9 and MM66 which have dissimilar levels of ethanol susceptibility. Overnight grown S. aureus LP9 and MM66 were inoculated in MHB medium (20 ml) in a 50 ml Erlenmeyer flask and incubated at 37°C, with shaking at 200 rpm. Growth was measured at regular intervals at 625 nm until OD= ~0.5. Based on previous minimum inhibitory concentration studies a 10% final ethanol concentration was added for 15 min of challenge. Total bacterial RNA was isolated as previously described from S. aureus strains LP9 and MM66 grown in Mueller Hinton broth to an OD625nm of 0.5 (37°C, 200 rpm). The RNA samples were then converted to fluorescently-labeled cDNA and hybridized to S. aureus microarrays version 6 (NIAID's Pathogen Functional Genomics Resource Center). Contents of raw data files: Channel A = Cy3 dye, Channel B = Cy5 dye. File names ending with S1,S3 = Cy3 untreated, Cy5 treated. File names ending with S2,S4 = Cy5 untreated, Cy3 treated: a.txt,b.txt is S2 file--cy5 for treated, cy3 untreated c.txt,d.txt is S4 file--cy5 for treated, cy3 untreated e.txt,f.txt is s1 file---cy3 for untreated, cy5 treated g.txt, h.txt is s3 file---cy3 for untreated, cy5 treated 2.txt, 4.txt is s5 file---cy3 for LP9, cy5 for MM66. No treatment for both. 1.txt, 3.txt is s6 file---cy5 for LP9, Cy3 for MM66. No treatment for both. The combined replicate results (of Samples GSM434525-GSM434530) representing the final processed data are linked below as supplementary files. Real-time PCR and physiological experiments were performed to validate the microarray data.