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

Abstract

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.

Comparison of three amplicon sequencing approaches to determine staphylococcal populations on human skin

“…Besides the three amplicon NGS schemes analysed here, other studies have used different tuf gene fragments for the analysis of staphylococcal populations [43][44][45]. For example, the tuf gene fragment used by McMurray et al [43] was predicted to distinguish fourteen out of eighteen strains of mock community M2 (data not shown).…”

“…For example, the tuf gene fragment used by McMurray et al [43] was predicted to distinguish fourteen out of eighteen strains of mock community M2 (data not shown). Two additional tuf schemes were developed in the last year [44,45]; these schemes were predicted to distinguish both 13 strains of mock community M2 (data not shown). Thus, these additional three schemes based on tuf gene fragments were predicted to distinguish fewer strains of the mock community M2 than the tuf2 scheme.…”

“…One of the most established gene targets is tuf, which codes for elongation factor Tu (EF-Tu) [41]. The tuf gene, or rather fragments thereof, is also used as a target for analysing mixed staphylococcal communities with NGS methods, hereafter referred to as amplicon NGS [42][43][44][45]. Furthermore, the staphylococcal rpsK gene that encodes the 30S ribosomal protein S11 was recently proposed for amplicon NGS [46].…”

Application of a High-Throughput Amplicon Sequencing Method to Chart the Bacterial Communities that Are Associated with European Fermented Meats from Different Origins

“…To unravel the staphylococcal communities in more detail, a 379-bp region of the tuf gene with sufficient discriminatory power to distinguish different CNS species was amplified using primer set Tuf387/765 ( Table 1 ). This primer set has been designed previously, based on a custom database containing 2556 tuf gene sequences, representing 48 staphylococcal species selected from the European Nucleotide Archive of the European Bioinformatics Institute (ENA/EBI), and allows distinguishing staphylococcal communities to species level [ 33 ]. The PCR assay conditions comprised an initial denaturation step at 94 °C for 2 min, 25 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 60 s, and extension at 72 °C for 3 min, and a final extension at 72 °C for 7 min.…”

“…The objective of this study was to assess if a previously developed amplicon-based HTS method targeting the 16S rRNA and tuf genes [ 33 ] can be successfully applied to chart the bacterial communities in European fermented meats. A second goal was to assess if this also allows one to reveal a relationship between those communities and the country of origin and the applied processing parameters of each product (i.e., pH and salt concentration).…”