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Open AccessArticle

Amplicon-Based High-Throughput Sequencing Method Capable of Species-Level Identification of Coagulase-Negative Staphylococci in Diverse Communities

Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of Sciences and Bio-engineering Sciences, Vrije Universiteit Brussel, B-1050 Brussels, Belgium
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Microorganisms 2020, 8(6), 897; https://doi.org/10.3390/microorganisms8060897
Received: 6 May 2020 / Revised: 6 June 2020 / Accepted: 11 June 2020 / Published: 14 June 2020
(This article belongs to the Section Food Microbiology)
Coagulase-negative staphylococci (CNS) make up a diverse bacterial group, appearing in a myriad of ecosystems. To unravel the composition of staphylococcal communities in these microbial ecosystems, a reliable species-level identification is crucial. The present study aimed to design a primer set for high-throughput amplicon sequencing, amplifying a region of the tuf gene with enough discriminatory power to distinguish different CNS species. Based on 2566 tuf gene sequences present in the public European Nucleotide Archive database and saved as a custom tuf gene database in-house, three different primer sets were designed, which were able to amplify a specific region of the tuf gene for 36 strains of 18 different CNS species. In silico analysis revealed that species-level identification of closely related species was only reliable if a 100% identity cut-off was applied for matches between the amplicon sequence variants and the custom tuf gene database. From the three primer sets designed, one set (Tuf387/765) outperformed the two other primer sets for studying Staphylococcus-rich microbial communities using amplicon sequencing, as it resulted in no false positives and precise species-level identification. The method developed offers interesting potential for a rapid and robust analysis of complex staphylococcal communities in a variety of microbial ecosystems. View Full-Text
Keywords: coagulase-negative staphylococci; high-throughput sequencing; metagenetics coagulase-negative staphylococci; high-throughput sequencing; metagenetics
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Van Reckem, E.; De Vuyst, L.; Leroy, F.; Weckx, S. Amplicon-Based High-Throughput Sequencing Method Capable of Species-Level Identification of Coagulase-Negative Staphylococci in Diverse Communities. Microorganisms 2020, 8, 897.

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