The Catalan initiative for the Earth BioGenome Project: contributing local data to global biodiversity genomics.

The Catalan Initiative for the Earth BioGenome Project (CBP) is an EBP-affiliated project network aimed at sequencing the genome of the >40 000 eukaryotic species estimated to live in the Catalan-speaking territories (Catalan Linguistic Area, CLA). These territories represent a biodiversity hotspot. While covering less than 1% of Europe, they are home to about one fourth of all known European eukaryotic species. These include a high proportion of endemisms, many of which are threatened. This trend is likely to get worse as the effects of global change are expected to be particularly severe across the Mediterranean Basin, particularly in freshwater ecosystems and mountain areas. Following the EBP model, the CBP is a networked organization that has been able to engage many scientific and non-scientific partners. In the pilot phase, the genomes of 52 species are being sequenced. As a case study in biodiversity conservation, we highlight the genome of the Balearic shearwater Puffinus mauretanicus, sequenced under the CBP umbrella.

The Effect of Dietary Nitrate on the Oral Microbiome and Salivary Biomarkers in Individuals with High Blood Pressure.

BACKGROUND: Green leafy vegetables (GLV) contain inorganic nitrate, an anion with potential prebiotic effects on the oral microbiome. However, it remains unclear whether GLV and pharmacological supplementation [potassium nitrate (PN)] with a nitrate salt induce similar effects on the oral microbiome. OBJECTIVES: This study aimed to compare the effect of GLV with PN supplementation on the oral microbiome composition and salivary biomarkers in individuals with high blood pressure. METHODS: Seventy individuals were randomly allocated to 3 different groups to follow a 5-wk dietary intervention. Group 1 consumed 300 mg/d of nitrate in form of GLV. Group 2 consumed pills with 300 mg/d of PN and low-nitrate vegetables. Group 3 consumed pills with potassium chloride (placebo: PLAC) and low-nitrate vegetables. The oral microbiome composition and salivary biomarkers of oral health were analyzed before and after the dietary intervention. RESULTS: The GLV and PN groups showed similar microbial changes, probably nitrate-dependent, including an increase in the abundance of Neisseria, Capnocytophaga, Campylobacter species, and a decrease in Veillonella, Megasphaera, Actinomyces, and Eubacterium species after the treatment. Increased abundance of Rothia species, and reduced abundance of Streptococcus, Prevotella, Actinomyces, and Mogibacterium species were observed in the GLV group, which could be nitrate-independent. GLV and PN treatments increased salivary pH, but only GLV treatment showed an increase in the salivary buffering capacity and a reduction of lactate. CONCLUSION: The combination of nitrate-dependent and nitrate-independent microbial changes in the GLV group has a stronger effect to potentially improve oral health biomarkers compared with PN.

Long-term stability of acquired drug resistance and resistance associated mutations in the fungal pathogen Nakaseomyces glabratus (Candida glabrata).

The limited number of available antifungal drugs and the increasing number of fungal isolates that show drug or multidrug resistance pose a serious medical threat. Several yeast pathogens, such as Nakaseomyces glabratus (Candida glabrata), show a remarkable ability to develop drug resistance during treatment through the acquisition of genetic mutations. However, how stable this resistance and the underlying mutations are in non-selective conditions remains poorly characterized. The stability of acquired drug resistance has fundamental implications for our understanding of the appearance and spread of drug-resistant outbreaks and for defining efficient strategies to combat them. Here, we used an in vitro evolution approach to assess the stability under optimal growth conditions of resistance phenotypes and resistance-associated mutations that were previously acquired under exposure to antifungals. Our results reveal a remarkable stability of the resistant phenotype and the underlying mutations in a significant number of evolved populations, which conserved their phenotype for at least two months in the absence of drug-selective pressure. We observed a higher stability of anidulafungin resistance over fluconazole resistance, and of resistance-conferring point mutations as compared with aneuploidies. In addition, we detected accumulation of novel mutations in previously altered resistance-associated genes in non-selective conditions, which suggest a possible compensatory role. We conclude that acquired resistance, particularly to anidulafungin, is a long-lasting phenotype, which has important implications for the persistence and propagation of drug-resistant clinical outbreaks.

Comparison between 16S rRNA and shotgun sequencing in colorectal cancer, advanced colorectal lesions, and healthy human gut microbiota.

BACKGROUND: Gut dysbiosis has been associated with colorectal cancer (CRC), the third most prevalent cancer in the world. This study compares microbiota taxonomic and abundance results obtained by 16S rRNA gene sequencing (16S) and whole shotgun metagenomic sequencing to investigate their reliability for bacteria profiling. The experimental design included 156 human stool samples from healthy controls, advanced (high-risk) colorectal lesion patients (HRL), and CRC cases, with each sample sequenced using both 16S and shotgun methods. We thoroughly compared both sequencing technologies at the species, genus, and family annotation levels, the abundance differences in these taxa, sparsity, alpha and beta diversities, ability to train prediction models, and the similarity of the microbial signature derived from these models. RESULTS: As expected, the results showed that 16S detects only part of the gut microbiota community revealed by shotgun, although some genera were only profiled by 16S. The 16S abundance data was sparser and exhibited lower alpha diversity. In lower taxonomic ranks, shotgun and 16S highly differed, partially due to a disagreement in reference databases. When considering only shared taxa, the abundance was positively correlated between the two strategies. We also found a moderate correlation between the shotgun and 16S alpha-diversity measures, as well as their PCoAs. Regarding the machine learning models, only some of the shotgun models showed some degree of predictive power in an independent test set, but we could not demonstrate a clear superiority of one technology over the other. Microbial signatures from both sequencing techniques revealed taxa previously associated with CRC development, e.g., Parvimonas micra. CONCLUSIONS: Shotgun and 16S sequencing provide two different lenses to examine microbial communities. While we have demonstrated that they can unravel common patterns (including microbial signatures), shotgun often gives a more detailed snapshot than 16S, both in depth and breadth. Instead, 16S will tend to show only part of the picture, giving greater weight to dominant bacteria in a sample. Therefore, we recommend choosing one or another sequencing technique before launching a study. Specifically, shotgun sequencing is preferred for stool microbiome samples and in-depth analyses, while 16S is more suitable for tissue samples and studies with targeted aims.

Symmetric and asymmetric DNA N6-adenine methylation regulates different biological responses in Mucorales.

DNA N6-adenine methylation (6mA) has recently gained importance as an epigenetic modification in eukaryotes. Its function in lineages with high levels, such as early-diverging fungi (EDF), is of particular interest. Here, we investigated the biological significance and evolutionary implications of 6mA in EDF, which exhibit divergent evolutionary patterns in 6mA usage. The analysis of two Mucorales species displaying extreme 6mA usage reveals that species with high 6mA levels show symmetric methylation enriched in highly expressed genes. In contrast, species with low 6mA levels show mostly asymmetric 6mA. Interestingly, transcriptomic regulation throughout development and in response to environmental cues is associated with changes in the 6mA landscape. Furthermore, we identify an EDF-specific methyltransferase, likely originated from endosymbiotic bacteria, as responsible for asymmetric methylation, while an MTA-70 methylation complex performs symmetric methylation. The distinct phenotypes observed in the corresponding mutants reinforced the critical role of both types of 6mA in EDF.

Several secondary metabolite gene clusters in the genomes of ten Penicillium spp. raise the risk of multiple mycotoxin occurrence in chestnuts.

Penicillium spp. produce a great variety of secondary metabolites, including several mycotoxins, on food substrates. Chestnuts represent a favorable substrate for Penicillium spp. development. In this study, the genomes of ten Penicillium species, virulent on chestnuts, were sequenced and annotated: P. bialowiezense. P. pancosmium, P. manginii, P. discolor, P. crustosum, P. palitans, P. viridicatum, P. glandicola, P. taurinense and P. terrarumae. Assembly size ranges from 27.5 to 36.8 Mb and the number of encoded genes ranges from 9,867 to 12,520. The total number of predicted biosynthetic gene clusters (BGCs) in the ten species is 551. The most represented families of BGCs are non ribosomal peptide synthase (191) and polyketide synthase (175), followed by terpene synthases (87). Genome-wide collections of gene phylogenies (phylomes) were reconstructed for each of the newly sequenced Penicillium species allowing for the prediction of orthologous relationships among our species, as well as other 20 annotated Penicillium species available in the public domain. We investigated in silico the presence of BGCs for 10 secondary metabolites, including 5 mycotoxins, whose production was validated in vivo through chemical analyses. Among the clusters present in this set of species we found andrastin A and its related cluster atlantinone A, mycophenolic acid, patulin, penitrem A and the cluster responsible for the synthesis of roquefortine C/glandicoline A/glandicoline B/meleagrin. We confirmed the presence of these clusters in several of the Penicillium species conforming our dataset and verified their capacity to synthesize them in a chestnut-based medium with chemical analysis. Interestingly, we identified mycotoxin clusters in some species for the first time, such as the andrastin A cluster in P. flavigenum and P. taurinense, and the roquefortine C cluster in P. nalgiovense and P. taurinense. Chestnuts proved to be an optimal substrate for species of Penicillium with different mycotoxigenic potential, opening the door to risks related to the occurrence of multiple mycotoxins in the same food matrix.

Proof of Concept Study: Comparability of Microbiome Diversity in Self- and Physician-Collected HPV-Positive and HPV-Negative Cervicovaginal Samples.

Recent studies have revealed the impact of human papillomavirus (HPV) infections on the cervicovaginal microbiome; however, few have explored the utility of self-collected specimens (SCS) for microbiome detection, obtained using standardised methods for HPV testing. Here, we present a proof-of-concept analysis utilising Oxford Nanopore sequencing of the 16S rRNA gene in paired samples collected either by the patient using an Evalyn Brush or collected by a physician using liquid-based cytology (LBC). We found no significant differences in the α-diversity estimates between the SCS and LBC samples. Similarly, when analysing ß-diversity, we observed a close grouping of paired samples, indicating that both collection methods detected the same microbiome features. The identification of genera and Lactobacillus species in each sample allowed for their classification into community state types (CSTs). Notably, paired samples had the same CST, while HPV-positive and -negative samples belonged to distinct CSTs. As previously described in other studies, HPV-positive samples exhibited heightened bacterial diversity, reduced Lactobacillus abundance, and an increase in genera like Sneathia or Dialister. Altogether, this study showed comparable results between the SCS and LBC samples, underscoring the potential of self-sampling for analysing the microbiome composition in cervicovaginal samples initially collected for HPV testing in the context of cervical cancer screening.

Assessing Diagnosis of Candida Infections: A Study on Species Prevalence and Antifungal Resistance in Northern Morocco.

The incidence of Candida infections has increased in the last decade, posing a serious threat to public health. Appropriately facing this challenge requires precise epidemiological data on species and antimicrobial resistance incidence, but many countries lack appropriate surveillance programs. This study aims to bridge this gap for Morocco by identifying and phenotyping a year-long collection of clinical isolates (n = 93) from four clinics in Tetouan. We compared the current standard in species identification with molecular methods and assessed susceptibility to fluconazole and anidulafungin. Our results identified limitations in currently used diagnostics approaches, and revealed that C. albicans ranks as the most prevalent species with 60 strains (64.52%), followed by C. glabrata with 14 (15.05%), C. parapsilosis with 6 (6.45%), and C. tropicalis with 4 (4.30%). In addition, we report the first identification of C. metapsilosis in Morocco. Susceptibility results for fluconazole revealed that some isolates were approaching MICs resistance breakpoints in C. albicans (2), and C. glabrata (1). Our study also identified anidulafungin resistant strains in C. albicans (1), C. tropicalis (1), and C. krusei (2), rendering the two strains from the latter species multidrug-resistant due to their innate resistance to fluconazole. These results raise concerns about species identification and antifungal resistance in Morocco and highlight the urgent need for more accurate methods and preventive strategies to combat fungal infections in the country.

Profiling Bacteria in the Lungs of Patients with Severe Influenza Versus COVID-19 with or without Aspergillosis.

RATIONALE: The influence of the lung bacterial microbiome, including potential pathogens, in patients with influenza- or COVID-19-associated pulmonary aspergillosis (IAPA or CAPA) is yet to be explored. OBJECTIVES: To explore the composition of the lung bacterial microbiome and its association with viral and fungal infection, immunity and outcome in severe influenza versus COVID-19 with or without aspergillosis. METHODS: We performed a retrospective study in mechanically ventilated influenza and COVID-19 patients with or without invasive aspergillosis in whom bronchoalveolar lavage (BAL) for bacterial culture (with or without PCR) was obtained within two weeks after ICU admission. Additionally, 16S rRNA gene sequencing data and viral and bacterial load of BAL samples from a subset of these patients, and of patients requiring non-invasive ventilation, were analyzed. We integrated 16S rRNA gene sequencing data with existing immune parameter datasets. MEASUREMENTS AND MAIN RESULTS: Potential bacterial pathogens were detected in 20% (28/142) of influenza and 37% (104/281) of COVID-19 patients, while aspergillosis was detected in 38% (54/142) of influenza and 31% (86/281) of COVID-19 patients. A significant association between bacterial pathogens in BAL and 90-day mortality was found only in influenza patients, particularly IAPA patients. COVID-19 but not influenza patients showed increased pro-inflammatory pulmonary cytokine responses to bacterial pathogens. CONCLUSIONS: Aspergillosis is more frequently detected in lungs of severe influenza patients than bacterial pathogens. Detection of bacterial pathogens associates with worse outcome in influenza patients, particularly in those with IAPA, but not in COVID-19 patients. The immunological dynamics of tripartite viral-fungal-bacterial interactions deserve further investigation. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Hybrids unleashed: exploring the emergence and genomic insights of pathogenic yeast hybrids.

Hybridisation is the crossing of two divergent lineages that give rise to offspring carrying an admixture of both parental genomes. Genome sequencing has revealed that this process is common in the Saccharomycotina, where a growing number of hybrid strains or species, including many pathogenic ones, have been recently described. Hybrids can display unique traits that may drive adaptation to new niches, and some pathogenic hybrids have been shown to have higher prevalence over their parents in human and environmental niches, suggesting a higher fitness and potential to colonise humans. Here, we discuss how hybridisation and its genomic and phenotypic outcomes can shape the evolution of fungal species and may play a role in the emergence of new pathogens.

An inconspicuous identification: Isolation and identification of a novel Pichia species presenting as fungemia following cardiac surgery.

Fungemia is common in critically ill patient populations, and is associated with a high rate of mortality, especially when caused by nonalbicans Candida species. Herein, we describe a fatal case of fungemia following cardiothoracic surgery in which the organism, initially identified as Candida inconspicua, represents a novel species: Pichia alaskaensis.

On the impact of incomplete taxon sampling on the relative timing of gene transfer events.

A recent study questioned the use of branch length methods to assess the relative timing of horizontal gene transfers because of the effects of so-called "ghost" lineages. This Formal Comment discusses key considerations regarding the potential effect of missing lineages when assessing relative timing of evolutionary events.

Probing RNA structural landscapes across Candida yeast genomes.

Understanding the intricate roles of RNA molecules in virulence and host-pathogen interactions can provide valuable insights into combatting infections and improving human health. Although much progress has been achieved in understanding transcriptional regulation during host-pathogen interactions in diverse species, more is needed to know about the structure of pathogen RNAs. This is particularly true for fungal pathogens, including pathogenic yeasts of the Candida genus, which are the leading cause of hospital-acquired fungal infections. Our work addresses the gap between RNA structure and their biology by employing genome-wide structure probing to comprehensively explore the structural landscape of mRNAs and long non-coding RNAs (lncRNAs) in the four major Candida pathogens. Specifically focusing on mRNA, we observe a robust correlation between sequence conservation and structural characteristics in orthologous transcripts, significantly when sequence identity exceeds 50%, highlighting structural feature conservation among closely related species. We investigate the impact of single nucleotide polymorphisms (SNPs) on mRNA secondary structure. SNPs within 5' untranslated regions (UTRs) tend to occur in less structured positions, suggesting structural constraints influencing transcript regulation. Furthermore, we compare the structural properties of coding regions and UTRs, noting that coding regions are generally more structured than UTRs, consistent with similar trends in other species. Additionally, we provide the first experimental characterization of lncRNA structures in Candida species. Most lncRNAs form independent subdomains, similar to human lncRNAs. Notably, we identify hairpin-like structures in lncRNAs, a feature known to be functionally significant. Comparing hairpin prevalence between lncRNAs and protein-coding genes, we find enrichment in lncRNAs across Candida species, humans, and Arabidopsis thaliana, suggesting a conserved role for these structures. In summary, our study offers valuable insights into the interplay between RNA sequence, structure, and function in Candida pathogens, with implications for gene expression regulation and potential therapeutic strategies against Candida infections.

Positive selection in gamete interaction proteins in Carnivora.

The absence of robust interspecific isolation barriers among pantherines, including the iconic South American jaguar (Panthera onca), led us to study molecular evolution of typically rapidly evolving reproductive proteins within this subfamily and related groups. In this study, we delved into the evolutionary forces acting on the zona pellucida (ZP) gamete interaction protein family and the sperm-oocyte fusion protein pair IZUMO1-JUNO across the Carnivora order, distinguishing between Caniformia and Feliformia suborders and anticipating few significant diversifying changes in the Pantherinae subfamily. A chromosome-resolved jaguar genome assembly facilitated coding sequences, enabling the reconstruction of protein evolutionary histories. Examining sequence variability across more than 30 Carnivora species revealed that Feliformia exhibited significantly lower diversity compared to its sister taxa, Caniformia. Molecular evolution analyses of ZP2 and ZP3, subunits directly involved in sperm-recognition, unveiled diversifying positive selection in Feliformia, Caniformia and Pantherinae, although no significant changes were linked to sperm binding. Structural cross-linking ZP subunits, ZP4 and ZP1 exhibited lower levels or complete absence of positive selection. Notably, the fusion protein IZUMO1 displayed prominent positive selection signatures and sites in basal lineages of both Caniformia and Feliformia, extending along the Caniformia subtree but absent in Pantherinae. Conversely, JUNO did not exhibit any positive selection signatures across tested lineages and clades. Eight Caniformia-specific positive selected sites in IZUMO1 were detected within two JUNO-interaction clusters. Our findings provide for the first time insights into the evolutionary trajectories of ZP proteins and the IZUMO1-JUNO gamete interaction pair within the Carnivora order.

Genomics of the expanding pine pathogen Lecanosticta acicola reveals patterns of ongoing genetic admixture.

Lecanosticta acicola is the causal agent for brown spot needle blight that affects pine trees across the northern hemisphere. Based on marker genes and microsatellite data, two distinct lineages have been identified that were introduced into Europe on two separate occasions. Despite their overall distinct geographic distribution, they have been found to coexist in regions of northern Spain and France. Here, we present the first genome-wide study of Lecanosticta acicola, including assembly of the reference genome and a population genomics analysis of 70 natural isolates from northern Spain. We show that most of the isolates belong to the southern lineage but show signs of introgression with northern lineage isolates, indicating mating between the two lineages. We also identify phenotypic differences between the two lineages based on the activity profiles of 20 enzymes, with introgressed strains being more phenotypically similar to members of the southern lineage. In conclusion, we show undergoing genetic admixture between the two main lineages of L. acicola in a region of recent expansion. IMPORTANCE: Lecanosticta acicola is a fungal pathogen causing severe defoliation, growth reduction, and even death in more than 70 conifer species. Despite the increasing incidence of this species, little is known about its population dynamics. Two divergent lineages have been described that have now been found together in regions of France and Spain, but it is unknown how these mixed populations evolve. Here we present the first reference genome for this important plant pathogenic fungi and use it to study the population genomics of 70 isolates from an affected forest in the north of Spain. We find signs of introgression between the two main lineages, indicating that active mating is occurring in this region which could propitiate the appearance of novel traits in this species. We also study the phenotypic differences across this population based on enzymatic activities on 20 compounds.

Recent gene selection and drug resistance underscore clinical adaptation across Candida species.

Understanding how microbial pathogens adapt to treatments, humans and clinical environments is key to infer mechanisms of virulence, transmission and drug resistance. This may help improve therapies and diagnostics for infections with a poor prognosis, such as those caused by fungal pathogens, including Candida. Here we analysed genomic variants across approximately 2,000 isolates from six Candida species (C. glabrata, C. auris, C. albicans, C. tropicalis, C. parapsilosis and C. orthopsilosis) and identified genes under recent selection, suggesting a highly complex clinical adaptation. These involve species-specific and convergently affected adaptive mechanisms, such as adhesion. Using convergence-based genome-wide association studies we identified known drivers of drug resistance alongside potentially novel players. Finally, our analyses reveal an important role of structural variants and suggest an unexpected involvement of (para)sexual recombination in the spread of resistance. Our results provide insights on how opportunistic pathogens adapt to human-related environments and unearth candidate genes that deserve future attention.

Performance of a Shotgun Prediction Model for Colorectal Cancer When Using 16S rRNA Sequencing Data.

Colorectal cancer (CRC), the third most common cancer globally, has shown links to disturbed gut microbiota. While significant efforts have been made to establish a microbial signature indicative of CRC using shotgun metagenomic sequencing, the challenge lies in validating this signature with 16S ribosomal RNA (16S) gene sequencing. The primary obstacle is reconciling the differing outputs of these two methodologies, which often lead to divergent statistical models and conclusions. In this study, we introduce an algorithm designed to bridge this gap by mapping shotgun-derived taxa to their 16S counterparts. This mapping enables us to assess the predictive performance of a shotgun-based microbiome signature using 16S data. Our results demonstrate a reduction in performance when applying the 16S-mapped taxa in the shotgun prediction model, though it retains statistical significance. This suggests that while an exact match between shotgun and 16S data may not yet be feasible, our approach provides a viable method for comparative analysis and validation in the context of CRC-associated microbiome research.

JLOH: Inferring loss of heterozygosity blocks from sequencing data.

Heterozygosity is a genetic condition in which two or more alleles are found at a genomic locus. Individuals that are the offspring of genetically divergent yet still interfertile parents (e.g. hybrids) are highly heterozygous. One of the most studied aspects in the genomes of these individuals is the loss of their original heterozygosity (LOH) when multi-allelic sites lose one of their two alleles by converting it to the other, or by remaining hemizygous at that site. The region undergoing LOH may involve a single nucleotide polymorphism (SNP) or a longer stretch of DNA. LOH is deeply interconnected with adaptation but the in silico techniques to infer evolutionary relevant LOH blocks are hardly standardised, and a general tool to infer and analyse them across genomic contexts and species is missing. Here, we present JLOH, a computational toolkit for the inference and exploration of LOH blocks in genomes with at least 1% heterozygosity. JLOH only requires commonly available genomic sequencing data as input. Starting from mapped reads, called variants and a reference genome sequence, JLOH infers candidate LOH blocks based on SNP density (SNPs/kbp) and read coverage per position. Considering that most organisms that undergo extensive LOH are hybrids, JLOH has been designed to capture any subgenomic LOH pattern, assigning each LOH block to its subgenome of origin.

Origin of fungal hybrids with pathogenic potential from warm seawater environments.

Hybridisation is a common event in yeasts often leading to genomic variability and adaptation. The yeast Candida orthopsilosis is a human-associated opportunistic pathogen belonging to the Candida parapsilosis species complex. Most C. orthopsilosis clinical isolates are hybrids resulting from at least four independent crosses between two parental lineages, of which only one has been identified. The rare presence or total absence of parentals amongst clinical isolates is hypothesised to be a consequence of a reduced pathogenicity with respect to their hybrids. Here, we sequence and analyse the genomes of environmental C. orthopsilosis strains isolated from warm marine ecosystems. We find that a majority of environmental isolates are hybrids, phylogenetically closely related to hybrid clinical isolates. Furthermore, we identify the missing parental lineage, thus providing a more complete overview of the genomic evolution of this species. Additionally, we discover phenotypic differences between the two parental lineages, as well as between parents and hybrids, under conditions relevant for pathogenesis. Our results suggest a marine origin of C. orthopsilosis hybrids, with intrinsic pathogenic potential, and pave the way to identify pre-existing environmental adaptations that rendered hybrids more prone than parental lineages to colonise and infect the mammalian host.