Methodology for the Study of Horizontal Gene Transfer in Staphylococcus aureus

Summary

We describe here three different protocols for the in vitro investigation of conjugation, transduction, and natural transformation in Staphylococcus aureus.

Abstract

One important feature of the major opportunistic human pathogen Staphylococcus aureus is its extraordinary ability to rapidly acquire resistance to antibiotics. Genomic studies reveal that S. aureus carries many virulence and resistance genes located in mobile genetic elements, suggesting that horizontal gene transfer (HGT) plays a critical role in S. aureus evolution. However, a full and detailed description of the methodology used to study HGT in S. aureus is still lacking, especially regarding natural transformation, which has been recently reported in this bacterium. This work describes three protocols that are useful for the in vitro investigation of HGT in S. aureus: conjugation, phage transduction, and natural transformation. To this aim, the cfr gene (chloramphenicol/florfenicol resistance), which confers the Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A (PhLOPSA)-resistance phenotype, was used. Understanding the mechanisms through which S. aureus transfers genetic materials to other strains is essential to comprehending the rapid acquisition of resistance and helps to clarify the modes of dissemination reported in surveillance programs or to further predict the spreading mode in the future.

Introduction

Staphylococcus aureus is a commensal Gram-positive bacterium that naturally inhabits the skin and nasal cavity of human beings and animals. This bacterial species is the leading cause of nosocomial infections in hospitals and healthcare settings. Moreover, its ability to develop resistance to different antimicrobial compounds has made the management of the infections caused by this bacterium into a global concern.

Two main pathways involved in the spreading of resistance phenotypes are known: the clonal dissemination of resistant genotypes and the dissemination of genetic determinants among the bacterial pool. In the case of S.....

Protocol

NOTE: The strains and materials used in this work are listed in Table 1 and the Table of Materials, respectively. In the transmission experiments, N315 and COL cfr-positive derivatives were used as donors of the cfr gene (N315-45 and COL-45). These strains were previously obtained by conjugation, using as the donor a clinical cfr-positive Staphyloccocus epidermidis strain (ST2), following the standard conjugation protocol (see below). This strain harbored the cfr gene on a pSCFS7-like plasmid⁷.

1. Conjugation Using the Filter-mating Method

Results

The results represented here have been previously published (adapted from reference⁷ with the publisher's permission). We studied the potential transmission pathways of the cfr gene, which causes low-level linezolid resistance and the expression of the PhLOPSA-resistance phenotype^14,15 in S. aureus strains, by investigating three mechanisms of HGT.

Discussion

This work describes the three major methods to study the HGT of genetic determinants in S. aureus. Although transduction and conjugation have been studied for decades, the existence of natural transformation was only recently recognized². Thus, S. aureus is equipped with all of the three major modes of HGT, and testing all of them is required to clarify the possible dissemination pathways of genetic determinants. The aim of this work is to compile complete protocols and to provid.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tryptic Soy Broth (TSB)	Becton Dickinson	211825
Brain Heart Infusion (BHI)	Becton Dickinson	211059
Nutrient Broth No. 2	Oxoid	CM0067
Sheep blood agar	Eiken Chemical Co.,Ltd.	E-MR96	Tryptic soy agar added with 5% (v/v) sheep blood according to the manufacturer.
Agar powder	Wako Pure Chemical Industries	010-08725
Sodium citrate (Trisodium citrate dihydrate)	Wako Pure Chemical Industries	191-01785
Cellulose Ester Gridded 0.45 μL HAWG filter	Merck Milipore	HAWG 02500
QIAfilter Plasmid Midi kit	QIAGEN	12243