One is not enough: On the effects of reference genome for the mapping and subsequent analyses of short-reads.
Mapping of high-throughput sequencing (HTS) reads to a single arbitrary reference genome is a frequently used approach in microbial genomics. However, the choice of a reference may represent a source of errors that may affect subsequent analyses such as the detection of single nucleotide polymorphisms (SNPs) and phylogenetic inference. In this work, we evaluated the effect of reference choice on short-read sequence data from five clinically and epidemiologically relevant bacteria (Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Pseudomonas aeruginosa and Serratia marcescens). Publicly available whole-genome assemblies encompassing the genomic diversity of these species…
The power and limitations of genomic surveillance of bacteria.
Hepatitis C virus early kinetics and resistance-associated substitution dynamics during antiviral therapy with direct-acting antivirals
The emergence of resistance-associated substitutions (RASs) can compromise the high efficacy of direct-acting antivirals (DAAs). Little is known about RASs selection at very early time points during DAA treatment. Therefore, we analyzed the potential emergence of RASs immediately after therapy initiation. Samples of 71 patients treated with different DAAs were collected at baseline, during therapy (hours 4 and 8; days 1-7; weeks 2-4) or until target not detected. HCV-RNA levels were determined by qPCR, and RASs were detected by deep sequencing. Sixty-three (89%) patients achieved a sustained virological response (SVR), 7 (10%) relapsed, and 1 (1%) experienced a breakthrough. Almost all non-…
Hepatitis C virus intrinsic molecular determinants may contribute to the development of cholestatic hepatitis after liver transplantation
Cholestatic hepatitis C (CHC) is a severe form of hepatitis C virus (HCV) infection recurrence that leads to high graft loss rates early after liver transplantation (LT). To investigate the pathogenic mechanisms of CHC, we analysed HCV quasispecies in CHC patients compared to a control group (mild hepatitis C recurrence) by deep pyrosequencing. At the time of LT, NS5B quasispecies complexity was similar between the two groups but, after LT, it decreased more sharply in CHC patients than in the control group. Interestingly, the major variant before LT propagated efficiently and remained as the dominant sequence after LT in 62 % of CHC patients versus 11 % of controls (P=0.031). Sequence anal…
Molecular epidemiology and drug-resistance mechanisms in carbapenem-resistant Klebsiella pneumoniae isolated in patients from a tertiary hospital in Valencia, Spain
Abstract Objectives The aim of this study has been to characterize carbapenem-resistant Klebsiella pneumoniae isolates and to determine the resistance mechanisms involved, the clonal relationship between strains and clinical and demographical data of the infected patients. Methods Clinical and demographical data from patients were collected and statistically analysed. Antimicrobial susceptibility testing was performed and resistance genes were detected both phenotypically and genotypically. Conjugation assays were performed to show horizontal transferability of resistance genes. Clonal relationship was also studied. Next-generation sequencing (NGS) was performed to obtain information regard…
Use of next generation sequencing technologies for the diagnosis and epidemiology of infectious diseases
[ES]: Por primera vez, la tecnología de secuenciación masiva permite acceder a la información genómica a un precio y a una escala tales, que se está implementado en la práctica clínica y epidemiológica rutinaria. Los obstáculos para dicha implementación son todavía muchos. Sin embargo, ya existen muchos ejemplos de las grandes ventajas que supone en comparación con métodos anteriores. Esto es, sobre todo, porque con una sola determinación podemos obtener simultáneamente información epidemiológica del microorganismo causante, así como de su perfil de resistencias, si bien estas ventajas están más o menos desarrolladas según el patógeno considerado. En esta revisión se repasan varios ejemplos…
High-throughput sequencing (HTS) for the analysis of viral populations
The development of High-Throughput Sequencing (HTS) technologies is having a major impact on the genomic analysis of viral populations. Current HTS platforms can capture nucleic acid variation across millions of genes for both selected amplicons and full viral genomes. HTS has already facilitated the discovery of new viruses, hinted new taxonomic classifications and provided a deeper and broader understanding of their diversity, population and genetic structure. Hence, HTS has already replaced standard Sanger sequencing in basic and applied research fields, but the next step is its implementation as a routine technology for the analysis of viruses in clinical settings. The most likely appli…
Genomic Analysis of Bacterial Outbreaks
The study of outbreaks of infectious diseases has been revolutionized by the current availability of fast and efficient, high-throughput methods capable of yielding the nucleotide sequence of complete genomes of viruses and bacteria within a few days, or even hours. These methods are replacing previous molecular techniques which have been used for the past 30 years, although many of them are still the usual approach for many such investigations. Here we review the major technologies currently in use for high-throughput sequencing of bacterial genomes emphasizing their advantages and drawbacks for the analysis of outbreaks. The use of more efficient methods does not necessarily mean that all…
Draft genome sequences of Vibrio vulnificus strains recovered from moribund tilapia
Potentially zoonotic Vibrio vulnificus strains were isolated from vibriosis outbreaks occurring on eastern Mediterranean tilapia farms between 2016 and 2019. In this work, the draft genome sequences of three representative isolates are presented.
Phylogenetic analysis of an epidemic outbreak of acute hepatitis C in HIV-infected patients by ultra-deep pyrosequencing.
Abstract Background The incidence of acute hepatitis C (AHC) among HIV-infected men who have sex with men (MSM) has increased significantly in the last 10 years. Several studies point to a social and sexual network of HIV-positive MSM that extends internationally. Objectives The aim of our study was to investigate the dynamics of HCV transmission in an outbreak of AHC in HIV-infected MSM in Barcelona by ultra-deep pyrosequencing. Study design Between 2008 and 2013, 113 cases of AHC in HIV-infected MSM were diagnosed in the Infectious Diseases Unit, Hospital Clinic, Barcelona. Massive sequencing was performed using the Roche 454 GS Junior platform. To define possible transmission networks, m…
Complete analysis of the epidemiological scenario around a SARS-CoV-2 reinfection: previous infection events and subsequent transmission
9 páginas, 3 figuras. The data that support the findings of this study (Fastq files) are publicly available. Fastq files above the GISAID thresholds were deposited at GISAID (hCoV-19/Spain/MD-IBV-99007733/2020, hCoV-19/Spain/MD-IBV-99007151/2020, hCoV-19/Spain/MD-IBV-99007734/2020, and hCoV-19/Spain/MDIBV-99007170/2020). All sequences were also deposited at the ENA (European Nucleotide Archive; https:// www.ebi.ac.uk/ena/browser/home) (ERR5698024, ERR5697187, ERR6459974, ERR5698025, and ERR5697254).
The first wave of the Spanish COVID-19 epidemic was associated with early introductions and fast spread of a dominating genetic variant
The COVID-19 pandemic has shaken the world since the beginning of 2020. Spain is among the European countries with the highest incidence of the disease during the first pandemic wave. We established a multidisciplinar consortium to monitor and study the evolution of the epidemic, with the aim of contributing to decision making and stopping rapid spreading across the country. We present the results for 2170 sequences from the first wave of the SARS-Cov-2 epidemic in Spain and representing 12% of diagnosed cases until 14th March. This effort allows us to document at least 500 initial introductions, between early February-March from multiple international sources. Importantly, we document the …
The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms
Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, ge…
The widespread presence of a family of fish virulence plasmids in Vibrio vulnificus stresses its relevance as a zoonotic pathogen linked to fish farms
Vibrio vulnificus is a pathogen of public health concern that causes either primary septicemia after ingestion of raw shellfish or secondary septicemia after wound exposure to seawater. In consequence, shellfish and seawater are considered its main reservoirs. However, there is one aspect of its biology that is systematically overlooked: its association with fish in its natural environment. This association led in 1975 to the emergence of a zoonotic clade within phylogenetic lineage 2 following successive outbreaks of vibriosis in farmed eels. Although this clade is now worldwide distributed, no new zoonotic clades were subsequently reported. In this work, we have performed phylogenetic, ge…