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.

Differential Gene Expression of Malaria Parasite in Response to Red Blood Cell-Specific Glycolytic Intermediate 2,3-Diphosphoglycerate (2,3-DPG).

Innovative strategies to control malaria are urgently needed. Exploring the interplay between Plasmodium sp. parasites and host red blood cells (RBCs) offers opportunities for novel antimalarial interventions. Pyruvate kinase deficiency (PKD), characterized by heightened 2,3-diphosphoglycerate (2,3-DPG) concentration, has been associated with protection against malaria. Elevated levels of 2,3-DPG, a specific mammalian metabolite, may hinder glycolysis, prompting us to hypothesize its potential contribution to PKD-mediated protection. We investigated the impact of the extracellular supplementation of 2,3-DPG on the Plasmodium falciparum intraerythrocytic developmental cycle in vitro. The results showed an inhibition of parasite growth, resulting from significantly fewer progeny from 2,3-DPG-treated parasites. We analyzed differential gene expression and the transcriptomic profile of P. falciparum trophozoites, from in vitro cultures subjected or not subjected to the action of 2,3-DPG, using Nanopore Sequencing Technology. The presence of 2,3-DPG in the culture medium was associated with the significant differential expression of 71 genes, mostly associated with the GO terms nucleic acid binding, transcription or monoatomic anion channel. Further, several genes related to cell cycle control were downregulated in treated parasites. These findings suggest that the presence of this RBC-specific glycolytic metabolite impacts the expression of genes transcribed during the parasite trophozoite stage and the number of merozoites released from individual schizonts, which supports the potential role of 2,3-DPG in the mechanism of protection against malaria by PKD.

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.

Viral genetic clustering and transmission dynamics of the 2022 mpox outbreak in Portugal.

Pathogen genome sequencing during epidemics enhances our ability to identify and understand suspected clusters and investigate their relationships. Here, we combine genomic and epidemiological data of the 2022 mpox outbreak to better understand early viral spread, diversification and transmission dynamics. By sequencing 52% of the confirmed cases in Portugal, we identified the mpox virus sublineages with the highest impact on case numbers and fitted them into a global context, finding evidence that several international sublineages probably emerged or spread early in Portugal. We estimated a 62% infection reporting rate and that 1.3% of the population of men who have sex with men in Portugal were infected. We infer the critical role played by sexual networks and superspreader gatherings, such as sauna attendance, in the dissemination of mpox virus. Overall, our findings highlight genomic epidemiology as a tool for the real-time monitoring and control of mpox epidemics, and can guide future vaccine policy in a highly susceptible population.

Candida auris in Intensive Care Setting: The First Case Reported in Portugal.

Candida auris is an opportunistic human pathogen that has rapidly spread to multiple countries and continents and has been associated with a high number of nosocomial outbreaks. Herein, we report the first case of C. auris in Portugal, which was associated with a patient transferred from Angola to an ICU in Portugal for liver transplantation after a SARS-CoV-2 infection. C. auris was isolated during the course of bronchoalveolar lavage, and it was subjected to antifungal susceptibility testing and whole-genome sequence analysis. This isolate presents low susceptibility to azoles and belongs to the genetic clade III with a phylogenetic placement close to African isolates. Although clade III has already been reported in Europe, taking into account the patient's clinical history, we cannot discard the possibility that the patient's colonization/infection occurred in Angola, prior to admission in the Portuguese hospital. Considering that C. auris is a fungal pathogen referenced by WHO as a critical priority, this case reinforces the need for continuous surveillance in a hospital setting.

ReporTree: a surveillance-oriented tool to strengthen the linkage between pathogen genetic clusters and epidemiological data.

BACKGROUND: Genomics-informed pathogen surveillance strengthens public health decision-making, playing an important role in infectious diseases' prevention and control. A pivotal outcome of genomics surveillance is the identification of pathogen genetic clusters and their characterization in terms of geotemporal spread or linkage to clinical and demographic data. This task often consists of the visual exploration of (large) phylogenetic trees and associated metadata, being time-consuming and difficult to reproduce. RESULTS: We developed ReporTree, a flexible bioinformatics pipeline that allows diving into the complexity of pathogen diversity to rapidly identify genetic clusters at any (or all) distance threshold(s) or cluster stability regions and to generate surveillance-oriented reports based on the available metadata, such as timespan, geography, or vaccination/clinical status. ReporTree is able to maintain cluster nomenclature in subsequent analyses and to generate a nomenclature code combining cluster information at different hierarchical levels, thus facilitating the active surveillance of clusters of interest. By handling several input formats and clustering methods, ReporTree is applicable to multiple pathogens, constituting a flexible resource that can be smoothly deployed in routine surveillance bioinformatics workflows with negligible computational and time costs. This is demonstrated through a comprehensive benchmarking of (i) the cg/wgMLST workflow with large datasets of four foodborne bacterial pathogens and (ii) the alignment-based SNP workflow with a large dataset of Mycobacterium tuberculosis. To further validate this tool, we reproduced a previous large-scale study on Neisseria gonorrhoeae, demonstrating how ReporTree is able to rapidly identify the main species genogroups and characterize them with key surveillance metadata, such as antibiotic resistance data. By providing examples for SARS-CoV-2 and the foodborne bacterial pathogen Listeria monocytogenes, we show how this tool is currently a useful asset in genomics-informed routine surveillance and outbreak detection of a wide variety of species. CONCLUSIONS: In summary, ReporTree is a pan-pathogen tool for automated and reproducible identification and characterization of genetic clusters that contributes to a sustainable and efficient public health genomics-informed pathogen surveillance. ReporTree is implemented in python 3.8 and is freely available at https://github.com/insapathogenomics/ReporTree .

Evolution of loss of heterozygosity patterns in hybrid genomes of Candida yeast pathogens.

BACKGROUND: Hybrids are chimeric organisms with highly plastic heterozygous genomes that may confer unique traits enabling the adaptation to new environments. However, most evolutionary theory frameworks predict that the high levels of genetic heterozygosity present in hybrids from divergent parents are likely to result in numerous deleterious epistatic interactions. Under this scenario, selection is expected to favor recombination events resulting in loss of heterozygosity (LOH) affecting genes involved in such negative interactions. Nevertheless, it is so far unknown whether this phenomenon actually drives genomic evolution in natural populations of hybrids. To determine the balance between selection and drift in the evolution of LOH patterns in natural yeast hybrids, we analyzed the genomic sequences from fifty-five hybrid strains of the pathogenic yeasts Candida orthopsilosis and Candida metapsilosis, which derived from at least six distinct natural hybridization events. RESULTS: We found that, although LOH patterns in independent hybrid clades share some level of convergence that would not be expected from random occurrence, there is an apparent lack of strong functional selection. Moreover, while mitosis is associated with a limited number of inter-homeologous chromosome recombinations in these genomes, induced DNA breaks seem to increase the LOH rate. We also found that LOH does not accumulate linearly with time in these hybrids. Furthermore, some C. orthopsilosis hybrids present LOH patterns compatible with footprints of meiotic recombination. These meiotic-like patterns are at odds with a lack of evidence of sexual recombination and with our inability to experimentally induce sporulation in these hybrids. CONCLUSIONS: Our results suggest that genetic drift is the prevailing force shaping LOH patterns in these hybrid genomes. Moreover, the observed LOH patterns suggest that these are likely not the result of continuous accumulation of sporadic events-as expected by mitotic repair of rare chromosomal breaks-but rather of acute episodes involving many LOH events in a short period of time.

Pathogenic Escherichia coli, Salmonella spp. and Campylobacter spp. in Two Natural Conservation Centers of Wildlife in Portugal: Genotypic and Phenotypic Characterization.

Human-wildlife coexistence may increase the potential risk of direct transmission of emergent or re-emergent zoonotic pathogens to humans. Intending to assess the occurrence of three important foodborne pathogens in wild animals of two wildlife conservation centers in Portugal, we investigated 132 fecal samples for the presence of Escherichia coli (Shiga toxin-producing E. coli (STEC) and non-STEC), Salmonella spp. and Campylobacter spp. A genotypic search for genes having virulence and antimicrobial resistance (AMR) was performed by means of PCR and Whole-Genome Sequencing (WGS) and phenotypic (serotyping and AMR profiles) characterization. Overall, 62 samples tested positive for at least one of these species: 27.3% for STEC, 11.4% for non-STEC, 3.0% for Salmonella spp. and 6.8% for Campylobacter spp. AMR was detected in four E. coli isolates and the only Campylobacter coli isolated in this study. WGS analysis revealed that 57.7% (30/52) of pathogenic E. coli integrated genetic clusters of highly closely related isolates (often involving different animal species), supporting the circulation and transmission of different pathogenic E. coli strains in the studied areas. These results support the idea that the health of humans, animals and ecosystems are interconnected, reinforcing the importance of a One Health approach to better monitor and control public health threats.

Karyon: a computational framework for the diagnosis of hybrids, aneuploids, and other nonstandard architectures in genome assemblies.

BACKGROUND: Recent technological developments have made genome sequencing and assembly highly accessible and widely used. However, the presence in sequenced organisms of certain genomic features such as high heterozygosity, polyploidy, aneuploidy, heterokaryosis, or extreme compositional biases can challenge current standard assembly procedures and result in highly fragmented assemblies. Hence, we hypothesized that genome databases must contain a nonnegligible fraction of low-quality assemblies that result from such type of intrinsic genomic factors. FINDINGS: Here we present Karyon, a Python-based toolkit that uses raw sequencing data and de novo genome assembly to assess several parameters and generate informative plots to assist in the identification of nonchanonical genomic traits. Karyon includes automated de novo genome assembly and variant calling pipelines. We tested Karyon by diagnosing 35 highly fragmented publicly available assemblies from 19 different Mucorales (Fungi) species. CONCLUSIONS: Our results show that 10 (28.57%) of the assemblies presented signs of unusual genomic configurations, suggesting that these are common, at least for some lineages within the Fungi.

Phylogenomic characterization and signs of microevolution in the 2022 multi-country outbreak of monkeypox virus.

The largest monkeypox virus (MPXV) outbreak described so far in non-endemic countries was identified in May 2022 (refs. 1-6). In this study, shotgun metagenomics allowed the rapid reconstruction and phylogenomic characterization of the first MPXV outbreak genome sequences, showing that this MPXV belongs to clade 3 and that the outbreak most likely has a single origin. Although 2022 MPXV (lineage B.1) clustered with 2018-2019 cases linked to an endemic country, it segregates in a divergent phylogenetic branch, likely reflecting continuous accelerated evolution. An in-depth mutational analysis suggests the action of host APOBEC3 in viral evolution as well as signs of potential MPXV human adaptation in ongoing microevolution. Our findings also indicate that genome sequencing may provide resolution to track the spread and transmission of this presumably slow-evolving double-stranded DNA virus.

Genome analysis of five recently described species of the CUG-Ser clade uncovers Candida theae as a new hybrid lineage with pathogenic potential in the Candida parapsilosis species complex.

Candida parapsilosis species complex comprises three important pathogenic species: Candida parapsilosis sensu stricto, Candida orthopsilosis and Candida metapsilosis. The majority of C. orthopsilosis and all C. metapsilosis isolates sequenced thus far are hybrids, and most of the parental lineages remain unidentified. This led to the hypothesis that hybrids with pathogenic potential were formed by the hybridization of non-pathogenic lineages that thrive in the environment. In a search for the missing hybrid parentals, and aiming to get a better understanding of the evolution of the species complex, we sequenced, assembled and analysed the genome of five close relatives isolated from the environment: Candida jiufengensis, Candida pseudojiufengensis, Candida oxycetoniae, Candida margitis and Candida theae. We found that the linear conformation of mitochondrial genomes in Candida species emerged multiple times independently. Furthermore, our analyses discarded the possible involvement of these species in the mentioned hybridizations, but identified C. theae as an additional hybrid in the species complex. Importantly, C. theae was recently associated with a case of infection, and we also uncovered the hybrid nature of this clinical isolate. Altogether, our results reinforce the hypothesis that hybridization is widespread among Candida species, and potentially contributes to the emergence of lineages with opportunistic pathogenic behaviour.

Multiple Hybridization Events Punctuate the Evolutionary Trajectory of Malassezia furfur.

Malassezia species are important fungal skin commensals and are part of the normal microbiota of humans and other animals. However, under certain circumstances these fungi can also display a pathogenic behavior. For example, Malassezia furfur is a common commensal of human skin and yet is often responsible for skin disorders but also systemic infections. Comparative genomics analysis of M. furfur revealed that some isolates have a hybrid origin, similar to several other recently described hybrid fungal pathogens. Because hybrid species exhibit genomic plasticity that can impact phenotypes, we sought to elucidate the genomic evolution and phenotypic characteristics of M. furfur hybrids in comparison to their parental lineages. To this end, we performed a comparative genomics analysis between hybrid strains and their presumptive parental lineages and assessed phenotypic characteristics. Our results provide evidence that at least two distinct hybridization events occurred between the same parental lineages and that the parental strains may have originally been hybrids themselves. Analysis of the mating-type locus reveals that M. furfur has a pseudobipolar mating system and provides evidence that after sexual liaisons of mating compatible cells, hybridization involved cell-cell fusion leading to a diploid/aneuploid state. This study provides new insights into the evolutionary trajectory of M. furfur and contributes with valuable genomic resources for future pathogenicity studies. IMPORTANCEMalassezia furfur is a common commensal member of human/animal microbiota that is also associated with several pathogenic states. Recent studies report involvement of Malassezia species in Crohn's disease, a type of inflammatory bowel disease, pancreatic cancer progression, and exacerbation of cystic fibrosis. A recent genomics analysis of M. furfur revealed the existence of hybrid isolates and identified their putative parental lineages. In this study, we explored the genomic and phenotypic features of these hybrids in comparison to their putative parental lineages. Our results revealed the existence of a pseudobipolar mating system in this species and showed evidence for the occurrence of multiple hybridization events in the evolutionary trajectory of M. furfur. These findings significantly advance our understanding of the evolution of this commensal microbe and are relevant for future studies exploring the role of hybridization in the adaptation to new niches or environments, including the emergence of pathogenicity.

Genome analysis of Candida subhashii reveals its hybrid nature and dual mitochondrial genome conformations.

Candida subhashii belongs to the CUG-Ser clade, a group of phylogenetically closely related yeast species that includes some human opportunistic pathogens, such as Candida albicans. Despite being present in the environment, C. subhashii was initially described as the causative agent of a case of peritonitis. Considering the relevance of whole-genome sequencing and analysis for our understanding of genome evolution and pathogenicity, we sequenced, assembled and annotated the genome of C. subhashii type strain. Our results show that C. subhashii presents a highly heterozygous genome and other signatures that point to a hybrid ancestry. The presence of functional pathways for assimilation of hydroxyaromatic compounds goes in line with the affiliation of this yeast with soil microbial communities involved in lignin decomposition. Furthermore, we observed that different clones of this strain may present circular or linear mitochondrial DNA. Re-sequencing and comparison of strains with differential mitochondrial genome topology revealed five candidate genes potentially associated with this conformational change: MSK1, SSZ1, ALG5, MRPL9 and OYE32.

Extreme diversification driven by parallel events of massive loss of heterozygosity in the hybrid lineage of Candida albicans.

Candida albicans is the most commonly reported species causing candidiasis. The taxonomic classification of C. albicans and related lineages is controversial, with Candida africana (syn. C. albicans var. africana) and Candida stellatoidea (syn. C. albicans var. stellatoidea) being considered different species or C. albicans varieties depending on the authors. Moreover, recent genomic analyses have suggested a shared hybrid origin of C. albicans and C. africana, but the potential parental lineages remain unidentified. Although the genomes of C. albicans and C. africana have been extensively studied, the genome of C. stellatoidea has not been sequenced so far. In order to get a better understanding of the evolution of the C. albicans clade, and to assess whether C. stellatoidea could represent one of the unknown C. albicans parental lineages, we sequenced C. stellatoidea type strain (CBS 1905). This genome was compared to that of C. albicans and of the closely related lineage C. africana. Our results show that, similarly to C. africana, C. stellatoidea descends from the same hybrid ancestor as other C. albicans strains and that it has undergone a parallel massive loss of heterozygosity.

Factors enforcing the species boundary between the human pathogens Cryptococcus neoformans and Cryptococcus deneoformans.

Hybridization has resulted in the origin and variation in extant species, and hybrids continue to arise despite pre- and post-zygotic barriers that limit their formation and evolutionary success. One important system that maintains species boundaries in prokaryotes and eukaryotes is the mismatch repair pathway, which blocks recombination between divergent DNA sequences. Previous studies illuminated the role of the mismatch repair component Msh2 in blocking genetic recombination between divergent DNA during meiosis. Loss of Msh2 results in increased interspecific genetic recombination in bacterial and yeast models, and increased viability of progeny derived from yeast hybrid crosses. Hybrid isolates of two pathogenic fungal Cryptococcus species, Cryptococcus neoformans and Cryptococcus deneoformans, are isolated regularly from both clinical and environmental sources. In the present study, we sought to determine if loss of Msh2 would relax the species boundary between C. neoformans and C. deneoformans. We found that crosses between these two species in which both parents lack Msh2 produced hybrid progeny with increased viability and high levels of aneuploidy. Whole-genome sequencing revealed few instances of recombination among hybrid progeny and did not identify increased levels of recombination in progeny derived from parents lacking Msh2. Several hybrid progeny produced structures associated with sexual reproduction when incubated alone on nutrient-rich medium in light, a novel phenotype in Cryptococcus. These findings represent a unique, unexpected case where rendering the mismatch repair system defective did not result in increased meiotic recombination across a species boundary. This suggests that alternative pathways or other mismatch repair components limit meiotic recombination between homeologous DNA and enforce species boundaries in the basidiomycete Cryptococcus species.

Differential Expression of Fungal Genes Determines the Lifestyle of Plectosphaerella Strains During Arabidopsis thaliana Colonization.

The fungal genus Plectosphaerella comprises species and strains with different lifestyles on plants, such as P. cucumerina, which has served as model for the characterization of Arabidopsis thaliana basal and nonhost resistance to necrotrophic fungi. We have sequenced, annotated, and compared the genomes and transcriptomes of three Plectosphaerella strains with different lifestyles on A. thaliana, namely, PcBMM, a natural pathogen of wild-type plants (Col-0), Pc2127, a nonpathogenic strain on Col-0 but pathogenic on the immunocompromised cyp79B2 cyp79B3 mutant, and P0831, which was isolated from a natural population of A. thaliana and is shown here to be nonpathogenic and to grow epiphytically on Col-0 and cyp79B2 cyp79B3 plants. The genomes of these Plectosphaerella strains are very similar and do not differ in the number of genes with pathogenesis-related functions, with the exception of secreted carbohydrate-active enzymes (CAZymes), which are up to five times more abundant in the pathogenic strain PcBMM. Analysis of the fungal transcriptomes in inoculated Col-0 and cyp79B2 cyp79B3 plants at initial colonization stages confirm the key role of secreted CAZymes in the necrotrophic interaction, since PcBMM expresses more genes encoding secreted CAZymes than Pc2127 and P0831. We also show that P0831 epiphytic growth on A. thaliana involves the transcription of specific repertoires of fungal genes, which might be necessary for epiphytic growth adaptation. Overall, these results suggest that in-planta expression of specific sets of fungal genes at early stages of colonization determine the diverse lifestyles and pathogenicity of Plectosphaerella strains.

Genomic evidence for a hybrid origin of the yeast opportunistic pathogen Candida albicans.

BACKGROUND: Opportunistic yeast pathogens of the genus Candida are an important medical problem. Candida albicans, the most prevalent Candida species, is a natural commensal of humans that can adopt a pathogenic behavior. This species is highly heterozygous and cannot undergo meiosis, adopting instead a parasexual cycle that increases genetic variability and potentially leads to advantages under stress conditions. However, the origin of C. albicans heterozygosity is unknown, and we hypothesize that it could result from ancestral hybridization. We tested this idea by analyzing available genomes of C. albicans isolates and comparing them to those of hybrid and non-hybrid strains of other Candida species. RESULTS: Our results show compelling evidence that C. albicans is an evolved hybrid. The genomic patterns observed in C. albicans are similar to those of other hybrids such as Candida orthopsilosis MCO456 and Candida inconspicua, suggesting that it also descends from a hybrid of two divergent lineages. Our analysis indicates that most of the divergence between haplotypes in C. albicans heterozygous blocks was already present in a putative heterozygous ancestor, with an estimated 2.8% divergence between homeologous chromosomes. The levels and patterns of ancestral heterozygosity found cannot be fully explained under the paradigm of vertical evolution and are not consistent with continuous gene flux arising from lineage-specific events of admixture. CONCLUSIONS: Although the inferred level of sequence divergence between the putative parental lineages (2.8%) is not clearly beyond current species boundaries in Saccharomycotina, we show here that all analyzed C. albicans strains derive from a single hybrid ancestor and diverged by extensive loss of heterozygosity. This finding has important implications for our understanding of C. albicans evolution, including the loss of the sexual cycle, the origin of the association with humans, and the evolution of virulence traits.

HaploTypo: a variant-calling pipeline for phased genomes.

SUMMARY: An increasing number of phased (i.e. with resolved haplotypes) reference genomes are available. However, the most genetic variant calling tools do not explicitly account for haplotype structure. Here, we present HaploTypo, a pipeline tailored to resolve haplotypes in genetic variation analyses. HaploTypo infers the haplotype correspondence for each heterozygous variant called on a phased reference genome. AVAILABILITY AND IMPLEMENTATION: HaploTypo is implemented in Python 2.7 and Python 3.5, and is freely available at https://github.com/gabaldonlab/haplotypo, and as a Docker image. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Genome Assemblies of Two Rare Opportunistic Yeast Pathogens: Diutina rugosa (syn. Candida rugosa) and Trichomonascus ciferrii (syn. Candida ciferrii).

Infections caused by opportunistic yeast pathogens have increased over the last years. These infections can be originated by a large number of diverse yeast species of varying incidence, and with distinct clinically relevant phenotypic traits, such as different susceptibility profiles to antifungal drugs, which challenge diagnosis and treatment. Diutina rugosa (syn. Candida rugosa) and Trichomonascus ciferrii (syn. Candida ciferrii) are two opportunistic rare yeast pathogens, which low incidence (< 1%) limits available clinical experience. Furthermore, these yeasts have elevated Minimum Inhibitory Concentration (MIC) levels to at least one class of antifungal agents. This makes it more difficult to manage their infections, and thus they are associated with high rates of mortality and clinical failure. With the aim of improving our knowledge on these opportunistic pathogens, we assembled and annotated their genomes. A phylogenomics approach revealed that genes specifically duplicated in each of the two species are often involved in transmembrane transport activities. These genomes and the reconstructed complete catalog of gene phylogenies and homology relationships constitute useful resources for future studies on these pathogens.