A Novel Approach Based on Real-Time PCR with High-Resolution Melting Analysis for the Simultaneous Identification of Staphylococcus aureus and Staphylococcus argenteus

Abstract

Staphylococcus (S.) aureus is a pathogenic bacterium able to cause several diseases in humans and animals as well as foodborne intoxications. S. argenteus, being phenotypically and genotypically related to S. aureus, is part of the so-called S. aureus complex and recently recognized as an emerging pathogen able to cause, like S. aureus, several diseases both in humans and animals, and foodborne poisoning outbreaks. However, it has been reported that the widely used conventional PCR of Brakstad et al. [Journal of Clinical Microbiology, 30(7), 1654-1660, (1992)] targeting the thermostable nuclease gene may provide false-positive S. aureus, as it is able to amplify also S. argenteus. Here, we developed a novel two-step approach that, following the PCR of Brakstad et al. (1992), discriminates S. aureus from S. argenteus by a real-time PCR with high-resolution melting analysis (rt-PCR-HRM). In particular, targeting a polymorphic 137 bp region of the sodA gene, our developed rt-PCR-HRM method clearly discriminated S. aureus from S. argenteus, showing a remarkable difference in their amplification product melting temperatures (approximately 1.3 °C) as well as distinct melting curve shapes. The good sensitivity, reproducibility, user friendliness, and cost effectiveness of the developed method are advantageous attributes that will allow not only its easy employment to correctly identify misidentified isolates present in various collections of S. aureus, but also expand the still lacking knowledge on the prevalence and distribution of S. argenteus.

Keywords: HRM; Staphylococcus argenteus; Staphylococcus aureus; Staphylococcus aureus complex; high-resolution melting; real-time PCR; sodA gene; species-specific PCR; superoxide dismutase.

Figure 1

Alignment of the sodA genes of S. aureus DSM 20231^T and S. argenteus NCTC 13711^T, obtained by Clustal Omega [34] and visualized in Jalview [35]. Highlighted in yellow are the annealing sites of the forward primer sodAaur-argF (5′-TTTGGTTCAGGTTGGGCTTG-3′) and the reverse primer sodAaur-argR (5′-AGGTAATAAGCGTGTTCCCAT-3′).

Figure 2

Derivative melt curves generated by HRM analysis of the amplification products of 19 representative isolates tested in triplicate within one intra-assay reproducibility test. In red are shown the derivative melt curves of strains identified as S. aureus and the reference strain S. aureus DSM 20231^T; in green are shown the derivative melt curves of the reference strain S. argenteus DSM 28299^T (ISPA-CNR).

Figure 3

Normalized melt curves generated by HRM analysis of the amplification products of 19 representative isolates tested in triplicate within one intra-assay reproducibility test. In red are shown the normalized melt curves of strains identified as S. aureus and the reference strain S. aureus DSM 20231^T; in green are shown the normalized melt curves of the reference strain S. argenteus DSM 28299^T (ISPA-CNR).

Figure 4

Difference plot generated by HRM analysis of the amplification products of 19 representative isolates tested in triplicate within one intra-assay reproducibility test. In red are shown the melt curves of strains identified as S. aureus and the reference strain S. aureus DSM 20231^T; in green are shown the melt curves of the reference strain S. argenteus DSM 28299^T (ISPA-CNR).

Figure 5

Derivative melt curves generated by HRM analysis of the amplification products of 11 representative Staphylococcus spp. isolates tested in triplicate within one intra-assay reproducibility test. The red line shows the average melting temperature (Tm) of strains identified as S. aureus and CoNSA; in blue are the average melting temperature (Tm) values of the reference strain S. argenteus DSM 28299^T and internal control S. argenteus S1 2021 (MMARLab).

Figure 6

Derivative melt curves generated by HRM analysis of the amplification products of 22 S. aureus and 2 S. argenteus isolates tested in triplicate within one intra-assay reproducibility test. The red/pink lines show the average melting temperatures (Tm) of strains identified as S. aureus; the blue line shows the average melting temperature (Tm) of the reference strain S. argenteus DSM 28299^T and internal control S. argenteus S1 2021; the yellow line shows the Tm of the S1 2019 strain previously misidentified as S. aureus (MMARLab).