Full-Length SSU rRNA Gene Sequencing Allows Species-Level Detection of Bacteria, Archaea, and Yeasts Present in Milk.

Isabel Abellan-Schneyder,Katharina Hofmann,Mareike Wenning,Annemarie Siebert,Klaus Neuhaus

doi:10.3390/microorganisms9061251

Abstract

Full-length SSU rRNA gene sequencing allows species-level identification of the microorganisms present in milk samples. Here, we used bulk-tank raw milk samples of two German dairies and detected, using this method, a great diversity of bacteria, archaea, and yeasts within the samples. Moreover, the species-level classification was improved in comparison to short amplicon sequencing. Therefore, we anticipate that this approach might be useful for the detection of possible mastitis-causing species, as well as for the control of spoilage-associated microorganisms. In a proof of concept, we showed that we were able to identify several putative mastitis-causing or mastitis-associated species such as Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, Escherichia coli and Staphylococcus aureus, as well as several Candida species. Overall, the presented full-length approach for the sequencing of SSU rRNA is easy to conduct, able to be standardized, and allows the screening of microorganisms in labs with Illumina sequencing machines.

Highlights

Sequencing has become a reliable and fast method over the years, allowing a timeefficient long-term screening perspective of bacterial communities from many different habitats, allowing the detection of potential pathogens
The most frequently used method to study the microbiota of a given sample is short amplicon 16S rRNA gene sequencing, where one, two, or three adjacent variable regions of the 16S rRNA gene are sequenced on a short-read sequencer, e.g., Illumina’s MiSeq
The number of eukaryotic and archaeal reads varied from sample to sample, accounting for up to over 50% of all of the reads

Summary

Introduction

Sequencing has become a reliable and fast method over the years, allowing a timeefficient long-term screening perspective of bacterial communities from many different habitats, allowing the detection of potential pathogens. The 16S rRNA gene comprises nine variable regions (V-regions, V1–V9) that are separated by constant regions [1]. The more stable evolutionary constant regions are used for primer binding. The variable regions, evolved under varying evolutionary processes, are enclosed within a PCR product and are used for taxonomic classification and differentiation [2]. The drawbacks, on the other hand, are the read-length limitations of 600 bp maximum (due to the short-read sequencers) and the comparability issues of taxonomic profiles when using different short amplicon sequencing protocols or processing pipelines after the sequencing

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