Genotyping of Staphylococcus aureus by Ribosomal Spacer PCR (RS-PCR)

Summary

Here, ribosomal spacer PCR (RS-PCR) is used together with a miniaturized electrophoresis system as a fast and high resolution method for genotyping S. aureus at moderate costs allowing a high throughput.

Abstract

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, therefore, suitable to be used for routine purposes. For best resolution, data evaluation and data management, a miniaturized electrophoresis system is required. Together with such an electrophoresis system and the in-house developed software (freely available here) assignment of the pattern of bands to a genotype is standardized and straight forward. DNA extraction is simple (boiling prep), setting-up of the reactions is easy and they can be run on any standard PCR machine. PCR cycling is common except prolonged ramping and elongation times. Compared to spa typing and Multi Locus Sequence Typing (MLST), RS-PCR does not require DNA sequencing what simplifies the analysis considerably and allows a high throughput. Furthermore, the resolution for bovine strains of S. aureus is at least as good as spa typing and better than MLST or pulsed-field gel electrophoresis (PFGE). The RS-PCR data base includes presently a total of 141 genotypes and variants. The method is highly associated with the virulence gene pattern, contagiosity and pathogenicity of S. aureus strains involved in bovine mastitis. S. aureus genotype B (GTB) is contagious and causes herds problems causing large costs in the Switzerland and other European countries. All the other genotypes observed in Switzerland infect individual cows and quarters. Genotyping by RS-PCR allows the reliable prediction of the epidemiological and the pathogenic potential of S. aureus involved in bovine intramammary infection (IMI), two key factors for clinical veterinary medicine. Because of these beneficial properties together with moderate costs and a high sample throughput the goal of this publication is to give a detailed, step-by-step protocol for easily establishing and running RS-PCR for genotyping S. aureus in other laboratories.

Introduction

Staphylococcus (S. aureus) is known as the most important pathogen worldwide responsible for intramammary infection (IMI) in cattle ¹. The study by Fournier et al. ² demonstrated in Swiss cows and the studies by Cosandey et al. ³ and Boss et al. ⁴ in cows of 12 European countries that S. aureus isolated from bovine IMI is a genetically heterogeneous group. By PCR amplification of the 16S-23S rRNA intergenic spacer region (RS-PCR), a total of 17 genotypes were initially detected in 101 epidemiologically independent isolates ². Genotype B and genotype C (GTC) wer....

Protocol

1. Staphylococcus aureus Isolates

1. Spread 10 µl of S. aureus stock culture or one colony grown on a selective medium on Columbia agar plates containing 5% sheep blood (BA).
2. Incubate aerobically at 37 °C for 18 hr to 24 hr.

2. DNA Extraction

1. Prepare TEL buffer containing 10 mM Tris-HCl and 10 mM Na₂EDTA, pH 8.5. Prepare aliquots and autoclave at 121 °C for 15 min.
2. Collect 1 colony from BA using a sterile plastic loop or a sterile wooden tooth pick and resuspend in 100 µl TEL. Use 1.5 ml tubes with screw caps.
3. Incubate at 95 &....

Results

Definition of a genotype and its variants

An electrophoretic profile differing in more than one band from all identified genotypes is considered as a new genotype. New genotypes are named and extended according to Fournier et al. ⁵ leading to the genotypes GTA to GTZ, followed by the genotypes GTAA to GTAZ, and GTBA to GTBZ, and GTCA to GTCC at present. Variation in just one band is regarded as a genotypic va.......

Discussion

The most critical step of the whole procedure is when there is an excess of DNA in RS-PCR (>30 ng pure DNA/assay) ². In general, resolution of a profile is optimal if all of its peaks start and end at the baseline. If two peaks are too close together, resolution is incomplete. Under these conditions, the bioanalyzer software may lead to inappropriate peak identification so that manual identification is required.

All the visibly separated and relevant peaks expressed as bp or FU .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tris(hydroxymethyl)-aminomethan	Merck	1.08382.0500	Mw = 121.14 g/mol
Titriplex III	Merck	1.08418.0250	Mw = 372.24 g/mol; Substance is equivalent to Na2EDTA·2H2O
Hydrochloric acid 5 mol/L	Merck	1.09911.0001
Columbia agar+5% sheep blood	bioMérieux	43049
Agilent DNA7500 Kit	Agilent Technologies	5067-1504
Agilent 2100 Bioanalyzer	Agilent Technologies	G2940CA
Agilent 2100 expert sofware	Agilent Technologies
HotStarTaq master mix	Qiagen	203445
Primer L1	Mycrosinth		5'CAA GGC ATC CAC CGT3'
Primer G1	Mycrosinth		5'GAA GTC GTA ACA AGG3'