Molecular analysis of accessory gene regulator functionality and virulence genes in Staphylococcus aureus derived from pediatric wound infections

Highlights

• Agr functionality in S. aureus done by qRT-PCR.

• The Agr Group III was dominate in S. aureus wound isolates.

• MRSA isolates were significantly associated with group agr III.

• There is no significant correlation between agr types and S. aureus wound isolates.

Abstract

Staphylococcus aureus is a major human pathogen causing infections with high morbidity and mortality in both healthcare and community settings. The accessory gene regulator (Agr) is a key genetic element controlling the expression of numerous virulence factors in S. aureus. The significance of a functional Agr system in clinical S. aureus isolates derived from pediatric wound infections is still unclear. Therefore, the present study was conducted to identify virulence genes and determine Agr functionality from this cohort of patients. A total of 48 S. aureus wound isolates were collected from patients referred to Tehran Children's Medical Center Hospital from April 2017 to April 2018. In addition, in vitro antimicrobial susceptibility of the isolates was assessed using the disk diffusion and E-test methods. Conventional PCR was performed for the detection of toxins (tsst-1, hla, hlb, hld, eta, etb, etd, edin-A, edin-B, edin-C) and Agr typing (agrI, agrII, agrIII, agrIV). Agr functionality was assessed by quantitative reverse transcriptase real-time PCR (qRT-PCR). All S. aureus isolates were found to be susceptible to linezolid and vancomycin. The most frequently detected toxin gene was eta (100%), and the most prevalent Agr type was agrIII (56.3%). Importantly, qRT-PCR revealed that Agr was functional in 28 (58%) of wound isolates. Consequently, our data suggests that a functional Agr system may not be required for the development of S. aureus wound infections.

Introduction

Staphylococcus aureus is an ever-present opportunistic pathogen that can cause a variety of diseases. The severity of S. aureus-associated infections ranges from benign localized skin abscesses to life-threatening diseases, such as arthritis, osteomyelitis, and endocarditis (Francois et al., 2006; von Eiff et al., 2004). In recent decades, methicillin-resistant S. aureus (MRSA) strains have emerged as a predominant cause of invasive diseases, namely skin and soft tissues, as well as musculoskeletal infections in children (Kaushik and Kest, 2018). This bacterium is one of the most dominant commensals on human skin and nasal mucosa and can express a multitude of virulence factors, such as surface adhesins, enterotoxins and hemolysins which are central in the development of disease. (Kassam et al., 2017; Stevens et al., 2017). The synchronized expression of these virulence determinants is tightly controlled by the cumulative action of several regulatory elements, such as the accessory gene regulator (agr), staphylococcal accessory regulator A (sarA), and the alternative sigma factor B (σB) (Manna and Cheung, 2001).

The Agr system plays a central role in the growth-phase dependent modulation of virulence gene expression (Bronner et al., 2004; Sakoulas et al., 2003a). The agr operon is an autocatalytic system controlled in a cell density-dependent fashion through the production and sensing of auto-inducing peptides (AIP). At high cell density, the Agr system increases the production of many secreted virulence factors, including Toxic shock syndrome toxin −1 (TSST-1), delta-hemolysin and exfoliative toxins A and B (ETA and ETB). In contrast, Agr decreases the expression of several colonization factors such as fibronectin binding proteins, important in adhesion and biofilm formation (Li et al., 2018). The agr locus consists of two distinct transcripts, RNAII and RNAIII, which are transcribed by two promoters, P2 and P3 respectively. The activation of P2 induces the expression of the components involved in cell-to-cell quorum-sensing communication (AgrBDCA) (Bibalan et al., 2014). Both AgrB and AgrD function to process and secrete the auto-inducing peptide (AIP), which acts as the chemical messenger critical for Agr activity (Wang et al., 2014). Upon reaching a critical density, AIPs interact with the sensor kinase, AgrC which promotes phosphorylation of the DNA binding response regulator AgrA. Phosphorylated AgrA undergoes a conformational change permitting interaction and binding to the intergenic region between P2 and P3 facilitating their expression. P3 activation leads to the expression of RNAIII, the effector of target gene regulation (Novick and Geisinger, 2008).

Several studies have demonstrated a correlation between agr types and particular diseases. For example phylogenetic group AF1 (agr group IV) strains are closely related to generalized exfoliative syndromes and bullous impetigo whereas endocarditis is mainly caused by phylogenetic group AF2 (agr groups II and I) strains (Jarraud et al., 2002). In addition, it has been suggested that agr group III and IV strains are associated with toxic shock syndrome (Gomes et al., 2005). To the best of our knowledge, there is no published study evaluating Agr functionality among Iranian S. aureus isolates. The present study was conducted to determine dominant Agr types, Agr activity and presence of specific virulence genes in S. aureus isolates derived from pediatric wound infections.