Abstract
Staphylococcus aureus is an opportunistic pathogen that colonizes human hosts and causes a wide variety of diseases. Two interacting regulatory systems called agr (accessory gene regulator) and sar (staphylococcal accessory regulator) are involved in the regulation of virulence factors. The aim of this study was to evaluate the effect of vancomycin on hld and spa gene expression during the exponential and post-exponential growth phases in multidrug-resistant (MDR) S. aureus.Methods: Antibiotic susceptibility was evaluated by the standard microdilution method. The phylogenetic profile was obtained by pulsed-field gel electrophoresis (PFGE). Polymorphisms of agr and SCCmec (staphylococcal cassette chromosome mec) were analyzed by multiplex polymerase chain reaction (PCR). The expression levels of hld and spa were analyzed by reverse transcription-PCR. An enzyme-linked immunosorbent assay (ELISA) was performed to detect protein A, and biofilm formation was analyzed via crystal violet staining.Results: In total, 60.60% (20/33) of S. aureus clinical isolates were MDR. Half (10/20) of the MDR S. aureus isolates were distributed in subcluster 10, with >90% similarity among them. In the isolates of this subcluster, a high prevalence (100%) for the agrII and the cassette SCCmec II polymorphisms was found. Our data showed significant increases in hld expression during the post-exponential phase in the presence and absence of vancomycin. Significant increases in spa expression, protein A production and biofilm formation were observed during the post-exponential phase when the MDR S. aureus isolates were challenged with vancomycin.Conclusion: The polymorphism agrII, which is associated with nosocomial isolates, was the most prevalent polymorphism in MDR S. aureus. Additionally, under our study conditions, vancomycin modified hld and spa expression in these clinical isolates. Therefore, vancomycin may regulate alternative systems that jointly participate in the regulation of these virulence factors.
Highlights
Staphylococcus aureus is an opportunistic pathogen capable of causing a wide variety of diseases in humans, ranging from localized infections of the skin and soft tissues to life-threatening systemic infections (Archer, 1998; Shopsin and Kreiswirth, 2001; David and Daum, 2010; Sowash and Uhlemann, 2014)
The aim of this study was to assess agr system expression by quantifying hld and spa expression in multidrug-resistant (MDR) S. aureus clinical isolates cultured from the exponential to postexponential growth phases in the presence of vancomycin
The purified RNA of all S. aureus clinical isolates was employed for reverse transcription (RT)-Polymerase Chain Reaction (PCR) assays with the GeneAmp RNA PCR Kit (Applied Biosystems, Foster City, CA, USA), using specific primers for the spa and hld genes (Table 2)
Summary
Staphylococcus aureus is an opportunistic pathogen capable of causing a wide variety of diseases in humans, ranging from localized infections of the skin and soft tissues to life-threatening systemic infections (Archer, 1998; Shopsin and Kreiswirth, 2001; David and Daum, 2010; Sowash and Uhlemann, 2014). S. aureus can produce a remarkable array of wall surface and secreted virulence factors that contribute to the establishment and maintenance of infection (Novick et al, 1993). These cell surface virulence factors include microbial surface components that recognize extracellular matrix proteins, such as fibrinogen, laminin, plasminogen, vitronectin, fibronectin, thrombospondin, and bone sialoprotein (Falord et al, 2011; Yamamoto et al, 2013). The expression of virulence genes in S. aureus is regulated under the partial control of the two-component quorum-sensing system encoded by genes at the agr locus (Bronner et al, 2004). An increase in protein A during the post-exponential phase has been observed in agr-deleted S. aureus strains (Novick, 2003)
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