Abstract
ObjectivesMethicillin-resistant Staphylococcus aureus (MRSA) strains present an urgent medical problem in osteomyelitis cases. Our previous study indicated that the YycFG two-component regulatory pathway is associated with the bacterial biofilm organization of MRSA strains. The aim of this study was to investigate the regulatory roles of ASyycG in the bacterial biofilm formation and the pathogenicity of MRSA strains using an antisense RNA strategy.MethodsAn ASyycG-overexpressing MRSA clinical isolate was constructed. The bacterial growth was monitored, and the biofilm biomass on bone specimens was examined using scanning electron microscopy and confocal laser scanning microscopy. Furthermore, quantitative RT-PCR (QRT-PCR) analysis was used to measure the expression of yycF/G/H and icaA/D in the MRSA and ASyycG strains. The expression of the YycG protein was quantified by Western blot assays. We validated the role of ASyycG in the invasive ability and pathogenicity of the strains in vivo using histology and peptide nucleic acid fluorescent in situ hybridization.ResultsThe results showed that overexpression of ASyycG lead to a reduction in biofilm formation and exopolysaccharide (EPS) synthesis compared to the control MRSA strains. The ASyycG strains exhibited decreased expression of the yycF/G/H and icaA/D genes. Furthermore, Western blot data showed that the production of the YycG protein was inhibited in the ASyycG strains. In addition, we demonstrated that ASyycG suppressed the invasive ability and pathogenicity of the strain in vivo using an SPF (specific pathogen free) rat model.ConclusionIn summary, the overexpression of ASyycG leads to a reduction in biofilm formation and bacterial pathogenicity in vivo, which provides a potential target for the management of MRSA-induced osteomyelitis.
Highlights
Osteomyelitis is one type of severe bone infection, and open fractures of long bones are the most frequent etiology [1]
Antisense yycG downregulated the expression of biofilm formation genes and negatively affected the production of the YycG protein in biofilms in vitro The growth curves of the S. aureus strains were compared in three independent experiments (Fig. 1a)
The antisense yycG (ASyycG) strains showed decreased expression of the genes associated with biofilm formation when compared to Methicillin-resistant Staphylococcus aureus (MRSA) strains during biofilm growth
Summary
Osteomyelitis is one type of severe bone infection, and open fractures of long bones are the most frequent etiology [1]. Among the various types of open long bones fractures, tibia fractures have the highest rate of infection due to the lack of soft tissue. In cases of very serious infection, the surgical consequences include radical debridement, resulting in bone or soft-tissue defects and amputation or establishment of a continuous fistula, which may be the only treatment alternative [7]. An extended hospital stay and excessive antibiotic therapy are required in patients with a definite diagnosis of MRSA infection; these patients have 2-fold higher mortality rates than noninfected patients [9]. In the USA, the annual cost of treating MRSA infections was documented to be between $3.2 billion and $4.2 billion [10]
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