Accurate and Strict Identification of Probiotic Species Based on Coverage of Whole-Metagenome Shotgun Sequencing Data

Abstract

Identifying the microbes present in probiotic products is an important issue in product quality control and public health. The most common methods used to identify genera containing species that produce lactic acid are matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA sequence analysis. However, the high cost of operation, difficulty in distinguishing between similar species, and limitations of the current sequencing technologies have made it difficult to obtain accurate results using these tools. To overcome these problems, a whole-genome shotgun sequencing approach has been developed along with various metagenomic classification tools. Widely used tools include the marker gene and k-mer methods, but their inevitable false-positives (FPs) hampered an accurate analysis. We therefore, designed a coverage-based pipeline to reduce the FP problem and to achieve a more reliable identification of species. The coverage-based pipeline described here not only shows higher accuracy for the detection of species and proportion analysis, based on mapping depth, but can be applied regardless of the sequencing platform. We believe that the coverage-based pipeline described in this study can provide appropriate support for probiotic quality control, addressing current labeling issues.

Shotgun metagenomic analysis of kombucha mutualistic community exposed to Mars‐like environment outside the International Space Station

“…Shotgun metagenomics allows to overcome the challenges that affect both assembly based and mapping‐based metagenomic profiling (Laudadio et al ., 2019), particularly, of high‐complexity samples (Walsh et al ., 2016; Seol et al ., 2019; Sirén et al ., 2019), including KMC (Arıkan et al ., 2020; Villarreal‐Soto et al ., 2020). Hence, we have implemented a shotgun metagenomic approach to gather an unbiased snapshot of the community structure and potential functionality in KMC that were previously exposed to space and different levels of Mars‐like conditions, simulated on low Earth orbit outside the International Space Station (ISS), within the BIOMEX project (de Vera et al ., 2019; Podolich et al ., 2019).…”

Evaluation of Dietary Supplements Containing Viable Bacteria by Cultivation/MALDI-TOF Mass Spectrometry and PCR Identification

“…2016; Chen et al, 2017;Ansari et al, 2019;Di Pierro et al, 2019;Korona-Glowniak et al, 2019;Seol et al, 2019;Aldawsari et al, 2020;Dioso et al, 2020;Kesavelu et al, 2020;Shehata and Newmaster, 2020;Vermeulen et al, 2020).…”

“…The need for stricter control of orally administered probiotic formulations, which should contain the specified number of viable bacteria and be free of contaminating microorganisms, was also recently highlighted by the European Society for Paediatric Gastroenterology Hepatology and Nutrition Working Group for Probiotics and Prebiotics ( Kolacek et al, 2017 ). Although significant improvements in the quality of these products have been observed in other markets in recent years, deficiencies in the content and viability of the declared microorganisms are still regularly reported ( Lewis et al, 2016 ; Chen et al, 2017 ; Ansari et al, 2019 ; Di Pierro et al, 2019 ; Korona-Glowniak et al, 2019 ; Seol et al, 2019 ; Aldawsari et al, 2020 ; Dioso et al, 2020 ; Kesavelu et al, 2020 ; Shehata and Newmaster, 2020 ; Vermeulen et al, 2020 ).…”

“…Products containing multiple strains present a particular challenge, as not all strains need to be present in the same quantity at the time of production and can vary greatly in viability during storage. In addition to conventional cultivation-based and PCR-based methods for viability assessment and identification, more advanced methods are increasingly being used, such as next-generation sequencing, terminal restriction fragment length polymorphism, and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) ( Angelakis et al, 2011 ; Lewis et al, 2016 ; Morovic et al, 2016 ; Patro et al, 2016 ; Čanžek Majhenič et al, 2018 ; Huang and Huang, 2018 ; Vecchione et al, 2018 ; Celandroni et al, 2019 ; Seol et al, 2019 ; Dioso et al, 2020 ; Vermeulen et al, 2020 ). Although some of them, such as viability PCR methods and advanced flow cytometry methods, are rapid and provide high-quality results, they may not be as useful for routine quality control because they require expensive equipment and chemicals as well as well-trained personnel.…”

The food-gut axis: lactic acid bacteria and their link to food, the gut microbiome and human health

“…This is the case of the methodology developed in (Seol et al . 2019 ) and aimed at reducing errors in terms of false positive rate for the specific identification of LAB and probiotic species.…”