A Comprehensive Evaluation of Enterobacteriaceae Primer Sets for Analysis of Host-Associated Microbiota.

Carolina N Resendiz-Nava,Hilda V Silva-Rojas,Edmundo M Mercado-Silva,Angel Rebollar-Alviter,Dulce M Rivera-Pastrana,Gerardo M Nava

doi:10.3390/pathogens11010017

Carolina N Resendiz-Nava, Hilda V Silva-Rojas + Show 4 more

Open Access

https://doi.org/10.3390/pathogens11010017

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Abstract

Enterobacteriaceae is one of the most important bacterial groups within the Proteobacteria phylum. This bacterial group includes pathogens, commensal and beneficial populations. Numerous 16S rRNA gene PCR-based assays have been designed to analyze Enterobacteriaceae diversity and relative abundance, and, to the best of our knowledge, 16 primer pairs have been validated, published and used since 2003. Nonetheless, a comprehensive performance analysis of these primer sets has not yet been carried out. This information is of particular importance due to the recent taxonomic restructuration of Enterobacteriaceae into seven bacterial families. To overcome this lack of information, the identified collection of primer pairs (n = 16) was subjected to primer performance analysis using multiple bioinformatics tools. Herein it was revealed that, based on specificity and coverage of the 16S rRNA gene, these 16 primer sets could be divided into different categories: Enterobacterales-, multi-family-, multi-genus- and Enterobacteriaceae-specific primers. These results highlight the impact of taxonomy changes on performance of molecular assays and data interpretation. Moreover, they underline the urgent need to revise and update the molecular tools used for molecular microbial analyses.

Highlights

Enterobacteriaceae is an important member of the Proteobacteria phylum; this bacterial group comprises numerous genera known to colonize the small and large intestine of mammals, including humans [1]
The present analysis revealed that selected primer pairs varied drastically in the number of 16S rRNA gene sequences targeted
After the most recent taxonomic restructuration of Enterobacteriaceae (December 2016, [28]), databases such as LPSN, NCBI, Ribosomal Database Project (RDP) and SILVA are still considering different genera assigned to Enterobacteriaceae (37, 29, 33 and 23 genera, respectively, as of October, 2021)

Summary

Introduction

Enterobacteriaceae is an important member of the Proteobacteria phylum; this bacterial group comprises numerous genera known to colonize the small and large intestine of mammals, including humans [1]. Enterobacteriaceae includes numerous recognized pathogens and opportunistic bacteria associated with the occurrence of enteric illnesses, urinary tract infections, sepsis and meningitis in humans [2,3,4,5]. Microbial molecular analyses have linked Enterobacteriaceae abundance to intestinal disorders such as ulcerative colitis, irritable bowel syndrome, diverticulitis, and Crohn’s disease [6,7,8,9,10]. Due to its biological importance, PCR-based approaches and massive 16S rRNA gene sequencing have been designed and implemented for analyses of diversity and abundance of the Enterobacteriaceae family in different environmental samples [14,15,16]. A comprehensive evaluation of their performance, specificity and coverage has not yet been

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Conclusion