High-throughput qPCR and 16S rRNA gene amplicon sequencing as complementary methods for the investigation of the cheese microbiota

Abstract

Background Next-generation sequencing (NGS) methods and especially 16S rRNA gene amplicon sequencing have become indispensable tools in microbial ecology. While they have opened up new possibilities for studying microbial communities, they also have one drawback, namely providing only relative abundances and thus compositional data. Quantitative PCR (qPCR) has been used for years for the quantification of bacteria. However, this method requires the development of specific primers and has a low throughput. The constraint of low throughput has recently been overcome by the development of high-throughput qPCR (HT-qPCR), which allows for the simultaneous detection of the most prevalent bacteria in moderately complex systems, such as cheese and other fermented dairy foods. In the present study, the performance of the two approaches, NGS and HT-qPCR, was compared by analyzing the same DNA samples from 21 Raclette du Valais protected designation of origin (PDO) cheeses. Based on the results obtained, the differences, accuracy, and usefulness of the two approaches were studied in detail. Results The results obtained using NGS (non-targeted) and HT-qPCR (targeted) show considerable agreement in determining the microbial composition of the cheese DNA samples studied, albeit the fundamentally different nature of these two approaches. A few inconsistencies in species detection were observed, particularly for less abundant ones. The detailed comparison of the results for 15 bacterial species/groups measured by both methods revealed a considerable bias for certain bacterial species in the measurements of the amplicon sequencing approach. We identified as probable origin to this PCR bias due to primer mismatches, variations in the number of copies for the 16S rRNA gene, and bias introduced in the bioinformatics analysis. Conclusion As the normalized microbial composition results of NGS and HT-qPCR agreed for most of the 21 cheese samples analyzed, both methods can be considered as complementary and reliable for studying the microbial composition of cheese. Their combined application proved to be very helpful in identifying potential biases and overcoming methodological limitations in the quantitative analysis of the cheese microbiota.

Lactic acid bacteria in cow raw milk for cheese production: Which and how many?

“…Different studies are instead available in the literature about the characterization of raw milk microbiota by amplicon-based high-throughput sequencing (HTS) analysis. This method is successful to describe changes in microbiota related to seasonality, geographical origin, and the microbiota evolution at different steps of cheese making, but it suffers from bias because results are usually reported as relative abundance of taxa which are not converted to quantitative values ( Skeie et al, 2019 ), and identification of bacteria beyond the genus level is often not possible ( Claesson et al, 2010 ; Dreier et al, 2022 ). On the other hand, a fundamental advantage of these studies is the possibility to use the generated raw sequences to perform meta-analysis.…”

A proof of concept infant-microbiota associated rat model for studying the role of gut microbiota and alleviation potential of Cutibacterium avidum in infant colic

“…The fecal microbiota profiles of C1 rats identified by 16S rRNA gene amplicon sequencing were discordant with qPCR findings for Bifidobacterium and Bacteroides . The bias can be partly explained by the occurrence of species with different numbers of 16S rRNA gene copies within the targeted taxa, and differences in the efficiency of primers to amplify different members within a taxonomic group ( 43 ). The metabolic profiles identified in the ceca of rats corresponded with the metabolic capacity of the most abundant fecal colonizers identified.…”

Significant antimicrobial-producing vegetation uniquely shapes the stormwater biofilter microbiome with implications for enhanced faecal pathogen inactivation

“…level) based on the limited discriminatory power of V3-4 region of the 16S rRNA gene [59,64]. Consequently, taxonomic analyses in the present study should be interpreted as preliminary only and further investigation, such as through directed qPCR analysis [65], is required for validation. The authors recommend employing a mock community standard which more closely resembles the microbiota inhabiting biofilters in future investigations, so as to obtain a more tangible evaluation of the efficiency of DNA extraction and accuracy of taxonomic classification [59,61].…”