Development of a genus-specific next generation sequencing approach for sensitive and quantitative determination of the Legionella microbiome in freshwater systems

Abstract

BackgroundNext Generation Sequencing (NGS) has revolutionized the analysis of natural and man-made microbial communities by using universal primers for bacteria in a PCR based approach targeting the 16S rRNA gene. In our study we narrowed primer specificity to a single, monophyletic genus because for many questions in microbiology only a specific part of the whole microbiome is of interest. We have chosen the genus Legionella, comprising more than 20 pathogenic species, due to its high relevance for water-based respiratory infections.MethodsA new NGS-based approach was designed by sequencing 16S rRNA gene amplicons specific for the genus Legionella using the Illumina MiSeq technology. This approach was validated and applied to a set of representative freshwater samples.ResultsOur results revealed that the generated libraries presented a low average raw error rate per base (<0.5%); and substantiated the use of high-fidelity enzymes, such as KAPA HiFi, for increased sequence accuracy and quality. The approach also showed high in situ specificity (>95%) and very good repeatability. Only in samples in which the gammabacterial clade SAR86 was present more than 1% non-Legionella sequences were observed. Next-generation sequencing read counts did not reveal considerable amplification/sequencing biases and showed a sensitive as well as precise quantification of L. pneumophila along a dilution range using a spiked-in, certified genome standard. The genome standard and a mock community consisting of six different Legionella species demonstrated that the developed NGS approach was quantitative and specific at the level of individual species, including L. pneumophila. The sensitivity of our genus-specific approach was at least one order of magnitude higher compared to the universal NGS approach. Comparison of quantification by real-time PCR showed consistency with the NGS data. Overall, our NGS approach can determine the quantitative abundances of Legionella species, i. e. the complete Legionella microbiome, without the need for species-specific primers.ConclusionsThe developed NGS approach provides a new molecular surveillance tool to monitor all Legionella species in qualitative and quantitative terms if a spiked-in genome standard is used to calibrate the method. Overall, the genus-specific NGS approach opens up a new avenue to massive parallel diagnostics in a quantitative, specific and sensitive way.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-017-0987-5) contains supplementary material, which is available to authorized users.

Environmental DNA detection of rare and invasive fish species in two Great Lakes tributaries

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The occurrence of antibiotic resistance genes in tap water – a review

“…This hindrance may be overcome with the application of newly designed technologies for high-throughput sequencing and bioinformatics investigations of obtained sequences. The increasingly used in environmental microbiology studies Next Generation Sequencing (NGS) approach becomes more available and allows to gain seemingly more information of tested sample [66][67][68]. However, as indicated in this study, the successful implementation of NGS in tap water samples research requires proven, reliable and reproductive procedures of highefficiency DNA extraction from this medium.…”

Detection of pathogenic bacteria in hot tap water using the qPCR method: preliminary research

“…WHO guidelines briefly mention the issue of hot tap water quality, indicating that hot tap water systems should be designed to minimise the proliferation of Legionella [5]. To date, several studies considered the bacterial biodiversity and microbial contamination in hot tap water samples [13][14][15][16]. The majority of them focus on the presence of genus Legionella.…”