Antisense <i>yycG</i> Regulation of Antibiotic Sensitivity of Methicillin-Resistant <i>Staphylococcus aureus</i> in Chronic Osteomyelitis

Abstract

<b><i>Background:</i></b> Methicillin-resistant <i>Staphylococcus aureus</i> (MRSA) is an urgent medical problem in osteomyelitis. The YycFG two-component regulatory system (TCS) allows bacteria to adapt rapidly to physical, chemical, and biological stresses. The recombinant plasmid shuttle vector was used to overexpress an antisense RNA (asRNA) to inhibit target gene expression by sequence-specific double-stranded RNA complex degradation. In the current study, antisense <i>yycG</i> RNA (AS<i>yycG</i>)<b>-</b>overexpression MRSA clinical isolates were constructed. <b><i>Methods:</i></b> Bacterial growth was monitored, and biofilm biomass was determined by crystal violet microtiter assay. Quantitative reverse transcription polymerase chain reaction analysis was used to identify expression of <i>yycF/G/H</i> and <i>icaA/D</i> in MRSA and AS<i>yycG</i> strains. The expression of YycG protein was quantified by Western blot assays. The antibiotic resistance of AS<i>yycG</i> strains was compared with that of the MRSA strains. <b><i>Results:</i></b> The AS<i>yycG</i> strains showed a decrease in growth rate compared with the MRSA strains. Of note, overexpression of AS<i>yycG</i> led to a reduction in biofilm formation and adhesion force. AS<i>yycG</i> strains had decreased expressions of the <i>yycF/G/H</i> and <i>icaA/D</i>. Furthermore, Western blot data showed that expression of the YycG protein decreased by 40% in AS<i>yycG</i> strains compared with MRSA strains. In addition, the effect of <i>yycG</i> asRNA improved the susceptibility of AS<i>yycG</i> strains to cefoxitin. <b><i>Conclusions:</i></b> The AS<i>yycG</i> strains inhibited biofilm organization and increased antibiotic sensitivity, which may be attributed to altered intracellular polysaccharide construction.