Vancomycin modifies the expression of the agr system in multidrug-resistant Staphylococcus aureus clinical isolates

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.

Características moleculares de Staphylococcus aureus suscetível à vancomicina poderia ajudar a prever falhas no tratamento devido à reduzida suscetibilidade à vancomicina

“…7,11,[20][21][22] Regarding agr typing, group II was more frequent among MRSA isolates, as already seen in other analysis. 8,13 Its was also high among VT-MRSA and hVISA, being associated with reduced vancomycin susceptibility. 12,23,24 Many VISA are highly enriched for the agr group II.…”

“…7 Additionally, this reduced vancomycin suscepti-bility is associated with dysfunction in the accessory gene regulator (agr) locus, a quorum sensing system that controls the expression of genes encoding virulence factors. 12,13 In this context, to understand the epidemiology and some molecular characteristics of MRSA with reduced vancomycin susceptibility is required to assist in the antimicrobial therapy and control the spread of these multiresistant microorganism. This study was designed to determine the prevalence of SCCmec and agr types among MRSA isolates, and to evaluate the association of these molecular characteristics with reduced susceptibility to vancomycin.…”

Options and Limitations in Clinical Investigation of Bacterial Biofilms

“…Early microarray analysis revealed that during the formation of E. coli biofilms and subsequent colonization, 38% of genes were modified, resulting in the altered expression of 600 genes (176). Similar to microarray analyses, quantitative real-time PCR (qPCR) examines a subset of genes from biofilm-isolated cells, while multiplex PCR detects multiple target sequences and is a useful tool for genetic analysis of polymicrobial/multispecies communities or gene polymorphisms (177)(178)(179) (Table 3). PCR product detection and quantification are based on probe chemistry.…”

The Role of Antibiotics in Modulating Virulence in Staphylococcus aureus

“…Subsequent to RNA extraction and purification, two assays are used to measure specific mRNA levels: Northern blotting and quantitative reverse transcription-PCR (qRT-PCR). Virulence determinants whose expression was explored by specific mRNA quantitation were hla (4,13,29,34,35,39,42), pvl (4,5,12,13,26), and spa (13,16,38,39,43); mRNA levels were normalized with respect to the expression of housekeeping genes, mostly the 16S rRNA gene or gyrB. Variation in mRNA levels may occur either by transcriptional modulation or by posttranscriptional mechanisms involving mRNA turnover.…”