Since the beginning of the COVID-19 outbreak, SARS-CoV-2 whole-genome sequencing (WGS) has been performed at unprecedented rate worldwide with the use of very diverse Next-Generation Sequencing (NGS) methods. Herein, we compare the performance of four NGS-based approaches for SARS-CoV-2 WGS. Twenty-four clinical respiratory samples with a large scale of Ct values (from 10.7 to 33.9) were sequenced with four methods. Three used Illumina sequencing: an in-house metagenomic NGS (mNGS) protocol and two newly commercialised kits including a hybridisation capture method developed by Illumina (DNA Prep with Enrichment kit and Respiratory Virus Oligo Panel, RVOP), and an amplicon sequencing method developed by Paragon Genomics (CleanPlex SARS-CoV-2 kit). We also evaluated the widely used amplicon sequencing protocol developed by ARTIC Network and combined with Oxford Nanopore Technologies (ONT) sequencing. All four methods yielded near-complete genomes (>99%) for high viral loads samples (n = 8), with mNGS and RVOP producing the most complete genomes. For mid viral loads (Ct 20–25), amplicon-based enrichment methods led to genome coverage >99 per cent for all samples while 1/8 sample sequenced with RVOP and 2/8 samples sequenced with mNGS had a genome coverage below 99 per cent. For low viral loads (Ct ≥25), amplicon-based enrichment methods were the most sensitive techniques. All methods were highly concordant in terms of identity in complete consensus sequence. Just one mismatch in three samples was observed in CleanPlex vs the other methods, due to the dedicated bioinformatics pipeline setting a high threshold to call SNP compared to reference sequence. Importantly, all methods correctly identified a newly observed 34nt-deletion in ORF6 but required specific bioinformatic validation for RVOP. Finally, as a major warning for targeted techniques, a loss of coverage in any given region of the genome should alert to a potential rearrangement or a SNP in primer-annealing or probe-hybridizing regions and would require further validation using unbiased metagenomic sequencing.

Legionella pneumophilais an environmental bacterium and the leading cause of Legionnaires’ disease. Just five sequence types (ST), from more than 2000 currently described, cause nearly half of disease cases in northwest Europe. Here, we report the sequence and analyses of 364L. pneumophilagenomes, including 337 from the five disease-associated STs and 27 representative of the species diversity. Phylogenetic analyses revealed that the five STs have independent origins within a highly diverse species. The number of de novo mutations is extremely low with maximum pairwise single-nucleotide polymorphisms (SNPs) ranging from 19 (ST47) to 127 (ST1), which suggests emergences within the last century. Isolates sampled geographically far apart differ by only a few SNPs, demonstrating rapid dissemination. These five STs have been recombining recently, leading to a shared pool of allelic variants potentially contributing to their increased disease propensity. The oldest clone, ST1, has spread globally; between 1940 and 2000, four new clones have emerged in Europe, which show long-distance, rapid dispersal. That a large proportion of clinical cases is caused by recently emerged and internationally dispersed clones, linked by convergent evolution, is surprising for an environmental bacterium traditionally considered to be an opportunistic pathogen. To simultaneously explain recent emergence, rapid spread and increased disease association, we hypothesize that these STs have adapted to new man-made environmental niches, which may be linked by human infection and transmission.

Legionella pneumophila is the causative agent of Legionnaires' disease. This bacterium is ubiquitous in aqueous environments and uses amoebae as an intracellular replicative niche. Real-time PCR has been developed for rapid detection of Legionella DNA in water samples. In addition to culturable bacteria, this method may also detect dead and viable but noncultivable (VBNC) legionellae. In order to understand the significance of positive PCR results in this setting, we prepared water samples containing known concentrations of L. pneumophila and analyzed them comparatively by means of conventional culture, real-time PCR, viability labeling, and immunodetection (solid-phase cytometry). We also examined the influence of chlorination on the results of the four methods. The different techniques yielded similar results for nonchlorinated water samples but not for chlorinated samples. After treatment for 24 h with 0.5 and 1 ppm chlorine, all cultures were negative, PCR and immunodetection showed about 10 ⁶ genome units and bacteria/ml, and total-viable-count (TVC) labeling detected 10 ⁵ and 10 ² metabolically active bacteria/ml, respectively. Thus, PCR also detected bacteria that were VBNC. The recoverability of VBNC forms was confirmed by 5 days of coculture with Acanthamoeba polyphaga . Therefore, some TVC-positive bacteria were potentially infective. These data show that L. pneumophila PCR detects not only culturable bacteria but also VBNC forms and dead bacterial DNA at low chlorine concentrations.

Natural transformation (i.e., the uptake of DNA and its stable integration in the chromosome) is a major mechanism of horizontal gene transfer in bacteria. Although the vast majority of bacterial genomes carry the genes involved in natural transformation, close relatives of naturally transformable species often appear not competent for natural transformation. In addition, unexplained extensive variations in the natural transformation phenotype have been reported in several species. Here, we addressed this phenomenon by conducting a genome-wide association study (GWAS) on a panel of isolates of the opportunistic pathogen Legionella pneumophila . GWAS revealed that the absence of the transformation phenotype is associated with the conjugative plasmid pLPL. The plasmid inhibits transformation by simultaneously silencing the genes required for DNA uptake and recombination. We identified a small RNA (sRNA), RocRp, as the sole plasmid-encoded factor responsible for the silencing of natural transformation. RocRp is homologous to the highly conserved and chromosome-encoded sRNA RocR which controls the transient expression of the DNA uptake system. Assisted by the ProQ/FinO-domain RNA chaperone RocC, RocRp acts as a substitute of RocR, ensuring that the bacterial host of the conjugative plasmid does not become naturally transformable. Distinct homologs of this plasmid-encoded sRNA are found in diverse conjugative elements in other Legionella species. Their low to high prevalence may result in the lack of transformability of some isolates up to the apparent absence of natural transformation in the species. Generally, our work suggests that conjugative elements obscure the widespread occurrence of natural transformability in bacteria.