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

Emiel Van Reckem,Luc De Vuyst,Frédéric Leroy,Stefan Weckx

doi:10.3390/microorganisms8060897

Emiel Van Reckem, Luc De Vuyst + Show 2 more

Open Access

https://doi.org/10.3390/microorganisms8060897

Copy DOI

Journal: Microorganisms	Publication Date: Jun 14, 2020
Citations: 10	License type: CC BY 4.0

Affiliation: Vrije Universiteit Brussel

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.

Highlights

Staphylococcus is a genus of Gram-positive, catalase-positive, facultative anaerobic bacteria that belongs to the family Staphylococcaceae, order Bacillales [1]
To construct mock communities containing staphylococcal DNA to evaluate the culture-independent identification method developed in the current study, 36 bacterial strains were used, belonging to 18 different coagulase-negative staphylococci (CNS) species, namely Staphylococcus auricularis, Staphylococcus arlettae, Staphylococcus carnosus, S. chromogenes, Staphylococcus cohnii, S. epidermidis, S. equorum, Staphylococcus fleuretti, S. hominis, S. haemolyticus, Staphylococcus lugdunensis, Staphylococcus pasteuri, S. saprophyticus, Staphylococcus sciuri, S. simulans, Staphylococcus succinus, Staphylococcus warneri, and S. xylosus (Table 1)
The mannitol salt phenol-red agar (MSA) counts of S. carnosus IMDO-S15, S. epidermidis IMDO-S30, S. equorum IMDO-S35, S. saprophyticus IMDO-S59, and S. xylosus IMDO-S81 amounted to 5.7, 5.3, 6.1, 6.9, and 6.2 log (CFU/mL) after 4 h of growth and to 8.2, 8.1, 8.1, 8.7, and 8.5 log (CFU/mL) after 12 h of growth, respectively

Summary

Introduction

Staphylococcus is a genus of Gram-positive, catalase-positive, facultative anaerobic bacteria that belongs to the family Staphylococcaceae, order Bacillales [1]. CNS or NAS form a very large and genetically diverse phylogenetic group, consisting of five distinct clades based on whole genome sequencing analysis, spanning a wide variety of microbial ecosystems [5]. As such, they are known as opportunistic pathogens in humans and animals, for example as the main cause of intramammary infections, and as valuable microorganisms for the production of fermented foods, such as fermented sausages [5,6,7]. Staphylococcus equorum, Staphylococcus saprophyticus, and Staphylococcus xylosus are most prominent CNS species in food fermentations, whereas species such as S. chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, S. saprophyticus, and Staphylococcus simulans are most frequently encountered as pathogens in both humans and animals [8,9,10,11]

Objectives

Methods

Results

Conclusion