AUTHOR=Pereira Rui P. A. , Peplies Jörg , Mushi Douglas , Brettar Ingrid , Höfle Manfred G. 

TITLE=Pseudomonas-Specific NGS Assay Provides Insight Into Abundance and Dynamics of Pseudomonas Species Including P. aeruginosa in a Cooling Tower

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.01958

DOI=10.3389/fmicb.2018.01958

ISSN=1664-302X

ABSTRACT=<p><italic>Pseudomonas</italic> species are frequent inhabitants of freshwater environments and colonizers of water supply networks via bioadhesion and biofilm formation. <italic>P. aeruginosa</italic> is the species most commonly associated with human disease, causing a wide variety of infections with links to its presence in freshwater systems. Though several other <italic>Pseudomonas</italic> species are of ecological and public health importance, little knowledge exists regarding environmental abundances of these species. In the present study, an Illumina-based next-generation sequencing (NGS) approach using <italic>Pseudomonas</italic>-specific primers targeting the 16S rRNA gene was evaluated and applied to a set of freshwater samples from different environments including a cooling tower sampled monthly during 2 years. Our approach showed high <italic>in situ</italic> specificity and accuracy. NGS read counts revealed a precise quantification of <italic>P. aeruginosa</italic> and a good correlation with the absolute number of <italic>Pseudomonas</italic> genome copies in a validated genus-specific qPCR assay, demonstrating the ability of the NGS approach to determine both relative and absolute abundances of <italic>Pseudomonas</italic> species and <italic>P. aeruginosa</italic>. The characterization of <italic>Pseudomonas</italic> communities in cooling tower water allowed us to identify 43 phylotypes, with <italic>P. aeruginosa</italic> being the most abundant. A shift existed within each year from a community dominated by phylotypes belonging to <italic>P. fluorescens</italic> and <italic>P. oleovorans</italic> phylogenetic groups to a community where <italic>P. aeruginosa</italic> was highly abundant. Co-occurrence was observed between <italic>P. aeruginosa</italic> and other phylotypes of <italic>P. aeruginosa</italic> group as well as the potentially pathogenic species <italic>P. stutzeri</italic>, but not with phylotypes of the <italic>P. fluorescens</italic> group, indicating the need to further investigate the metabolic networks and ecological traits of <italic>Pseudomonas</italic> species. This study demonstrates the potential of deep sequencing as a valuable tool in environmental diagnostics and surveillance of health-related pathogens in freshwater environments.</p>