Abundance and environmental host range of the SXT/R391 ICEs in aquatic environmental communities

Abstract

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EpicPCR 2.0: Technical and Methodological Improvement of a Cutting-Edge Single-Cell Genomic Approach

“…Using these primers allows to get robust qPCR amplifications (efficiency: 97.3%; R 2 : 0.999; Quantification limit = Detection limit: 3 copies). Moreover, the sequencing of amplicons obtained with these primers while using environment genomic DNA as template confirmed they only amplified a sequence specific to SXT/R391 ICEs [9]. Therefore, we considered their design to be very good and very likely suitable for being used in epicPCR amplifications.…”

“…These choices have been made as the vast majority of SXT/R391-carrying bacteria have been isolated from water or fecal samples and that the sampling point is precisely located in a river downstream the release point of a large wastewater treatment plant (500,000 equivalent inhabitants). Moreover, although the conductivity of river waters usually ranges from 50 to 1500 µS/cm [13], that of the Meurthe River water at the sampling point reaches 4500 µS/cm [9], that may favor the survival/growth of some already known SXT/R391 hosting bacteria as Vibrio sp. [14].…”

“…The primers used to target SXT/R391 ICEs have been recently designed to target a conserved gene of these elements, traB, by quantitative PCR [9]. Using these primers allows to get robust qPCR amplifications (efficiency: 97.3%; R 2 : 0.999; Quantification limit = Detection limit: 3 copies).…”

“…The fusion PCR and the subsequent nested PCR amplifications were then performed using the conditions depicted in Spencer et al (2016), i.e., using the Phusion DNA Polymerase GC buffer, but no PCR product resulting from the fusion of SXT/R391 ICE and 16S rDNA gene fragments could be observed in agarose gel electrophoresis, unless BP were omitted (Figure 1, wells #1 and 2 vs. 4 and 5). This difficult amplification in epicPCR was surprising in regards of the amplification robustness that these primers allow to reach in qPCR [9]. We therefore rather considered replacing the GC buffer by the standard Phusion DNA Polymerase HF buffer because its use allows a lower error rate while incorporating nucleotides.…”

“…So far, epicPCR has only been reported for the taxonomic assignment of some ARGs and class 1 integrons that appeared as relatively frequent marker in bacterial Microorganisms 2021, 9, 1649 2 of 15 communities [8]. In a twin work very recently published elsewhere [9], we explored in depth the environmental host range of another ARG carrying-MGE, namely the integrative and conjugative elements (ICEs) of the SXT/R391 family [10], using different culture-based and molecular-based approaches. Here, we report in detail on significant improvements of the epicPCR workflow made from the original protocol described by Spencer et al (2016) and Hultman et al (2018) that allow for pushing forward the limits of the epicPCR.…”