Investigating reagent contamination and its influence on drinking wáter microbiome analysis

RAJAL, VERONICA; WOO, YISSUE; CRUZ, MERCEDES CECILIA; WUERTZ, STEFAN

Congreso; Congreso Argentino de Microbiologia; 2019

Drinking water (DW) is not sterile; it contains many microorganisms that can become primary colonizers, forming biofilms on
the pipes. This may eventually degrade water quality and result in a public
health risk. All microorganisms in the system form the so-called DW microbiome. Utilities need to understand the bioprocesses that
allow biofilms to form and their potential to host microorganisms, so strategies can be developed to
optimize operational activities. Thus, microbial community analysis using 16S rRNA gene amplicon sequencing has been widely adopted for studying DW microbiomes. However, this methodology has a limitation: since DW microbiomes have very low biomass, reagent DNA contaminants can impact our interpretation of these communities. Here, we aim to investigate the potential influence of DNA extraction methods and reagent contaminants on the determination of DW microbial community.16S rRNA amplicon sequencing of a pure culture of Enterococcus faecalis was used to demonstrate the presence of contaminating DNA. We performed four rounds of serial ten-fold dilutions (108 cells in the undiluted sample, to 104 cells in the last dilution). Nucleic acids from culture, dilutions, elution buffers, and dilution water used were extracted using two commercial kits: FASTDNA kit (MP Biomedicals, FS) and BioFlux (BF). Two batches of the former kit were compared (FS1 and FS2), all kits were run by duplicates. The total extracted gDNA was then used for amplicon sequencing. The 16S rRNA genes were PCR-amplified using a primer set targeting the V4-V5 hypervariable region. The PCR amplicons were sequenced using the Illumina MiSeq platform, 300PE reads. The raw reads were quality trimmed and adapters were removed using cutadapt-1.2.1. Next, they were processed with the DADA2 pipeline, using chimera removal and Silva database for taxonomy assignment.In total 305 OTUs were detected. The most diluted samples (104 x) showed more OTUs, from 64 in the BF kit to 49 in the FS kit, suggesting contamination had occurred. E. faecalis was detected in all spiked samples. For the two kits, the undiluted and first dilution samples showed >99.3% of reads assigned to E. faecalis. However, in 104 x dilutions, the assignment dropped to 31% and 64% with BF and FS kits, respectively. Ralstonia was the main contaminant genus (up to 54% abundance) in the FS kit. Meanwhile, Comamonas was the most abundant contaminant (35.3%) in samples extracted with the BF kit, followed by Ralstonia (11.4%) and Herbaspirillum (10.34%). Interesting, high abundances of Brochothrix (29.7%) and Aquabacterium (12.9%) were found only with the FS1 batch.This study provides evidence of DNA contamination in extraction kits. It varies greatly in composition among different kits and kit batches; thus, it could critically influence results obtained from samples with low microbial biomass. Many contaminant OTUs are also known to be present in DW samples, and we strongly advise to perform concurrent sequencing of negative control samples.