Real-Time Genomic Surveillance for SARS-CoV-2 Variants of Concern, Uruguay.

We developed a genomic surveillance program for real-time monitoring of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) in Uruguay. We report on a PCR method for SARS-CoV-2 VOCs, the surveillance workflow, and multiple independent introductions and community transmission of the SARS-CoV-2 P.1 VOC in Uruguay.

simurg: simulate bacterial pangenomes in R.

MOTIVATION: The pangenome concept describes genetic variability as the union of genes shared in a set of genomes and constitutes the current paradigm for comparative analysis of bacterial populations. However, there is a lack of tools to simulate pangenome variability and structure using defined evolutionary models. RESULTS: We developed simurg, an R package that allows to simulate bacterial pangenomes using different combinations of evolutionary constraints such as gene gain, gene loss and mutation rates. Our tool allows the straightforward and reproducible simulation of bacterial pangenomes using real sequence data, providing a valuable tool for benchmarking of pangenome software or comparing evolutionary hypotheses. AVAILABILITY AND IMPLEMENTATION: The simurg package is released under the GPL-3 license, and is freely available for download from GitHub (https://github.com/iferres/simurg). SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Leptospira venezuelensis sp. nov., a new member of the intermediate group isolated from rodents, cattle and humans.

Three strains, CLM-U50T, CLM-R50 and IVIC-Bov1, belonging to the genus Leptospira, were isolated in Venezuela from a patient with leptospirosis, a domestic rat (Rattus norvegicus) and a cow (Bos taurus), respectively. The initial characterisation of these strains based on the rrs gene (16S rRNA) suggested their designation as a novel species within the 'intermediates' group of the genus Leptospira. Further phylogenomic characterisation based on single copy core genes was consistent with their separation into a novel species. The average nucleotide identity between these three strains was >99 %, but below 89 % with respect to any previously described leptospiral species, also supporting their designation as a novel species. Given this evidence, these three isolates were considered to represent a novel species, for which the name Leptospiravenezuelensis sp. nov. is proposed, with CLM-U50T (=CIP 111407T=DSM 105752T) as the type strain.

Genomic and clinical evidence uncovers the enterohepatic species Helicobacter valdiviensis as a potential human intestinal pathogen.

BACKGROUND: Helicobacter valdiviensis is a recently described enterohepatic species isolated from wild bird's fecal samples. Currently, its pathogenic potential and clinical significance are unknown mainly due to the lack of whole-genome sequences to compare with other helicobacters and the absence of specific screenings to determine its prevalence in humans. MATERIALS AND METHODS: The species type strain (WBE14T ) was whole-genome-sequenced, and comparative analyses were carried out including the genomes from other Helicobacter species to determine the exact phylogenetic position of H. valdiviensis and to study the presence and evolution of virulence determinants. In parallel, stools from diarrheic patients and healthy individuals were screened by PCR to assess the clinical incidence of H. valdiviensis. RESULTS: Helicobacter valdiviensis belongs to a monophyletic clade conformed by H. canadensis, H. pullorum, H. winghamensis, H. rodentium, and H. apodemus. Its predicted genome size is 2 176 246 bp., with 30% of G+C content and 2064 annotated protein-coding genes. The patterns of virulence factors in H. valdiviensis were similar to other enterohepatic species, but evidence of horizontal gene transfer from Campylobacter species was detected for key genes like those coding for the CDT subunits. Positive PCR results confirmed the presence of H. valdiviensis in 2 of 254 (0.78%) stools of patients with acute diarrhea while not a single sample was positive in healthy individuals. CONCLUSIONS: Horizontal gene transfer has contributed to shape the gene repertory of H. valdiviensis, which codes for virulence factors conserved in other pathogens that are well-known human pathogens. Additionally, the detection of H. valdiviensisDNA in diarrheic patients supports its role as a potential emergent intestinal pathogen. Further, sampling efforts are needed to uncover the clinical relevance of this species, which should be accomplished by the isolation of H. valdiviensis from ill humans and the obtention of whole genomes from clinical isolates.

Fast and cost-effective SARS-CoV-2 variant detection using Oxford Nanopore full-length spike gene sequencing.

Most biologically relevant and diagnostic mutations in the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) genome have been identified in the S gene through global genomic surveillance efforts. However, large-scale whole-genome sequencing (WGS) is still challenging in developing countries due to higher costs, reagent delays and limited infrastructure. Consequently, only a small fraction of SARS-CoV-2 samples are characterized through WGS in these regions. Here, we present a complete workflow consisting of a fast library preparation protocol based on tiled amplification of the S gene, followed by a PCR barcoding step and sequencing using Nanopore platforms. This protocol facilitates fast and cost-effective identification of main variants of concern and mutational surveillance of the S gene. By applying this protocol, report time and overall costs for SARS-CoV-2 variant detection could be reduced, contributing to improved genomic surveillance programmes, particularly in low-income regions.

Protocol for post-processing of bacterial pangenome data using Pagoo pipeline.

Multiple downstream analyses are necessary to interpret the output of bacterial pangenome reconstruction software. This requires integrating diverse kinds of genetic and phenotypic data, which to date are left to each user's criterion. To fill this gap, we created Pagoo, a pangenome post-processing tool that leverages a standardized but flexible and extensible framework for data integration, analysis, and storage. Here, we provide the protocol for running Pagoo and performing from simple to more complex comparative analyses on bacterial pangenome data. For complete details on the use and execution of this protocol, please refer to Ferrés and Iraola (2021).

An object-oriented framework for evolutionary pangenome analysis.

Pangenome analysis is fundamental to explore molecular evolution occurring in bacterial populations. Here, we introduce Pagoo, an R framework that enables straightforward handling of pangenome data. The encapsulated nature of Pagoo allows the storage of complex molecular and phenotypic information using an object-oriented approach. This facilitates to go back and forward to the data using a single programming environment and saving any stage of analysis (including the raw data) in a single file, making it sharable and reproducible. Pagoo provides tools to query, subset, compare, visualize, and perform statistical analyses, in concert with other microbial genomics packages available in the R ecosystem. As working examples, we used 1,000 Escherichia coli genomes to show that Pagoo is scalable, and a global dataset of Campylobacter fetus genomes to identify evolutionary patterns and genomic markers of host-adaptation in this pathogen.

Pangenome analysis reveals genetic isolation in Campylobacter hyointestinalis subspecies adapted to different mammalian hosts.

Campylobacter hyointestinalis is an emerging pathogen currently divided in two subspecies: C. hyointestinalis subsp. lawsonii which is predominantly recovered from pigs, and C. hyointestinalis subsp. hyointestinalis which can be found in a much wider range of mammalian hosts. Despite C. hyointestinalis being reported as an emerging pathogen, its evolutionary and host-associated diversification patterns are still vastly unexplored. For this reason, we generated whole-genome sequences of 13 C. hyointestinalis subsp. hyointestinalis strains and performed a comprehensive comparative analysis including publicly available C. hyointestinalis subsp. hyointestinalis and C. hyointestinalis subsp. lawsonii genomes, to gain insight into the genomic variation of these differentially-adapted subspecies. Both subspecies are distinct phylogenetic lineages which present an apparent barrier to homologous recombination, suggesting genetic isolation. This is further supported by accessory gene patterns that recapitulate the core genome phylogeny. Additionally, C. hyointestinalis subsp. hyointestinalis presents a bigger and more diverse accessory genome, which probably reflects its capacity to colonize different mammalian hosts unlike C. hyointestinalis subsp. lawsonii that is presumably host-restricted. This greater plasticity in the accessory genome of C. hyointestinalis subsp. hyointestinalis correlates to a higher incidence of genome-wide recombination events, that may be the underlying mechanism driving its diversification. Concordantly, both subspecies present distinct patterns of gene families involved in genome plasticity and DNA repair like CRISPR-associated proteins and restriction-modification systems. Together, our results provide an overview of the genetic mechanisms shaping the genomes of C. hyointestinalis subspecies, contributing to understand the biology of Campylobacter species that are increasingly recognized as emerging pathogens.

Recurrent Dissemination of SARS-CoV-2 Through the Uruguayan-Brazilian Border.

Uruguay is one of the few countries in the Americas that successfully contained the coronavirus disease 19 (COVID-19) epidemic during the first half of 2020. Nevertheless, the intensive human mobility across the dry border with Brazil is a major challenge for public health authorities. We aimed to investigate the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains detected in Uruguayan localities bordering Brazil as well as to measure the viral flux across this ∼1,100 km uninterrupted dry frontier. Using complete SARS-CoV-2 genomes from the Uruguayan-Brazilian bordering region and phylogeographic analyses, we inferred the virus dissemination frequency between Brazil and Uruguay and characterized local outbreak dynamics during the first months (May-July) of the pandemic. Phylogenetic analyses revealed multiple introductions of SARS-CoV-2 Brazilian lineages B.1.1.28 and B.1.1.33 into Uruguayan localities at the bordering region. The most probable sources of viral strains introduced to Uruguay were the Southeast Brazilian region and the state of Rio Grande do Sul. Some of the viral strains introduced in Uruguayan border localities between early May and mid-July were able to locally spread and originated the first outbreaks detected outside the metropolitan region. The viral lineages responsible for Uruguayan urban outbreaks were defined by a set of between four and 11 mutations (synonymous and non-synonymous) with respect to the ancestral B.1.1.28 and B.1.1.33 viruses that arose in Brazil, supporting the notion of a rapid genetic differentiation between SARS-CoV-2 subpopulations spreading in South America. Although Uruguayan borders have remained essentially closed to non-Uruguayan citizens, the inevitable flow of people across the dry border with Brazil allowed the repeated entry of the virus into Uruguay and the subsequent emergence of local outbreaks in Uruguayan border localities. Implementation of coordinated bi-national surveillance systems is crucial to achieve an efficient control of the SARS-CoV-2 spread across this kind of highly permeable borderland regions around the world.

Emergence and Spread of a B.1.1.28-Derived P.6 Lineage with Q675H and Q677H Spike Mutations in Uruguay.

Uruguay controlled the viral dissemination during the first nine months of the SARS-CoV-2 pandemic. Unfortunately, towards the end of 2020, the number of daily new cases exponentially increased. Herein, we analyzed the country-wide genetic diversity of SARS-CoV-2 between November 2020 and April 2021. We identified that the most prevalent viral variant during the first epidemic wave in Uruguay (December 2020-February 2021) was a B.1.1.28 sublineage carrying Spike mutations Q675H + Q677H, now designated as P.6, followed by lineages P.2 and P.7. P.6 probably arose around November 2020, in Montevideo, Uruguay's capital department, and rapidly spread to other departments, with evidence of further local transmission clusters; it also spread sporadically to the USA and Spain. The more efficient dissemination of lineage P.6 with respect to P.2 and P.7 and the presence of mutations (Q675H and Q677H) in the proximity of the key cleavage site at the S1/S2 boundary suggest that P.6 may be more transmissible than other lineages co-circulating in Uruguay. Although P.6 was replaced by the variant of concern (VOC) P.1 as the predominant lineage in Uruguay since April 2021, the monitoring of the concurrent emergence of Q675H + Q677H in VOCs should be of worldwide interest.

Draft Genome Sequences of 40 Pathogenic Leptospira Strains Isolated from Cattle in Uruguay.

Pathogenic Leptospira species represent a major concern for livestock but also for human health, as they cause zoonotic infections. Forty strains representing L. interrogans, L. borgpetersenii, and L. noguchii were isolated from naturally infected cattle in Uruguay. Here, we report the whole-genome sequences for these strains.

MLSTar: automatic multilocus sequence typing of bacterial genomes in R.

Multilocus sequence typing (MLST) is a standard tool in population genetics and bacterial epidemiology that assesses the genetic variation present in a reduced number of housekeeping genes (typically seven) along the genome. This methodology assigns arbitrary integer identifiers to genetic variations at these loci which allows us to efficiently compare bacterial isolates using allele-based methods. Now, the increasing availability of whole-genome sequences for hundreds to thousands of strains from the same bacterial species has allowed us to apply and extend MLST schemes by automatic extraction of allele information from the genomes. The PubMLST database is the most comprehensive resource of described schemes available for a wide variety of species. Here we present MLSTar as the first R package that allows us to (i) connect with the PubMLST database to select a target scheme, (ii) screen a desired set of genomes to assign alleles and sequence types, and (iii) interact with other widely used R packages to analyze and produce graphical representations of the data. We applied MLSTar to analyze more than 2,500 bacterial genomes from different species, showing great accuracy, and comparable performance with previously published command-line tools. MLSTar can be freely downloaded from http://github.com/iferres/MLSTar.

Deciphering the unexplored Leptospira diversity from soils uncovers genomic evolution to virulence.

Despite recent advances in our understanding of the genomics of members of the genus Leptospira, little is known on how virulence has emerged in this heterogeneous bacterial genus as well as on the lifestyle of pathogenic members of the genus Leptospira outside animal hosts. Here, we isolated 12 novel species of the genus Leptospira from tropical soils, significantly increasing the number of known species to 35 and finding evidence of highly unexplored biodiversity in the genus. Extended comparative phylogenomics and pan-genome analyses at the genus level by incorporating 26 novel genomes, revealed that, the traditional leptospiral 'pathogens' cluster, as defined by their phylogenetic position, can be split in two groups with distinct virulence potential and accessory gene patterns. These genomic distinctions are strongly linked to the ability to cause or not severe infections in animal models and humans. Our results not only provide new insights into virulence evolution in the members of the genus Leptospira, but also lay the foundations for refining the classification of the pathogenic species.