Phylogenomic and genomic analysis reveals unique and shared genetic signatures of Mycobacterium kansasii complex species.

Species belonging to the Mycobacterium kansasii complex (MKC) are frequently isolated from humans and the environment and can cause serious diseases. The most common MKC infections are caused by the species M. kansasii (sensu stricto), leading to tuberculosis-like disease. However, a broad spectrum of virulence, antimicrobial resistance and pathogenicity of these non-tuberculous mycobacteria (NTM) are observed across the MKC. Many genomic aspects of the MKC that relate to these broad phenotypes are not well elucidated. Here, we performed genomic analyses from a collection of 665 MKC strains, isolated from environmental, animal and human sources. We inferred the MKC pangenome, mobilome, resistome, virulome and defence systems and show that the MKC species harbours unique and shared genomic signatures. High frequency of presence of prophages and different types of defence systems were observed. We found that the M. kansasii species splits into four lineages, of which three are lowly represented and mainly in Brazil, while one lineage is dominant and globally spread. Moreover, we show that four sub-lineages of this most distributed M. kansasii lineage emerged during the twentieth century. Further analysis of the M. kansasii genomes revealed almost 300 regions of difference contributing to genomic diversity, as well as fixed mutations that may explain the M. kansasii's increased virulence and drug resistance.

Comparative Genomics of the First Resistant Candida auris Strain Isolated in Mexico: Phylogenomic and Pan-Genomic Analysis and Mutations Associated with Antifungal Resistance.

Candida auris is an emerging multidrug-resistant and opportunistic pathogenic yeast. Whole-genome sequencing analysis has defined five major clades, each from a distinct geographic region. The current study aimed to examine the genome of the C. auris 20-1498 strain, which is the first isolate of this fungus identified in Mexico. Based on whole-genome sequencing, the draft genome was found to contain 70 contigs. It had a total genome size of 12.86 Mbp, an N50 value of 1.6 Mbp, and an average guanine-cytosine (GC) content of 45.5%. Genome annotation revealed a total of 5432 genes encoding 5515 proteins. According to the genomic analysis, the C. auris 20-1498 strain belongs to clade IV (containing strains endemic to South America). Of the two genes (ERG11 and FKS1) associated with drug resistance in C. auris, a mutation was detected in K143R, a gene located in a mutation hotspot of ERG11 (lanosterol 14-α-demethylase), an antifungal drug target. The focus on whole-genome sequencing and the identification of mutations linked to the drug resistance of fungi could lead to the discovery of new therapeutic targets and new antifungal compounds.

Comparative genomics reveals high genetic similarity among strains of Salmonella enterica serovar Infantis isolated from multiple sources in Brazil.

Background: Salmonella enterica serovar Infantis (Salmonella Infantis) is a zoonotic, ubiquitous and foodborne pathogen of worldwide distribution. Despite Brazil's relevance as a major meat exporter, few studies were conducted to characterize strains of this serovar by genomic analyses in this country. Therefore, this study aimed to assess the diversity of 80 Salmonella Infantis strains isolated from veterinary, food and human sources in Brazil between 2013 and 2018 by comparative genomic analyses. Additional genomes of non-Brazilian countries (n = 18) were included for comparison purposes in some analyses. Methods: Analyses of whole-genome multi-locus sequence typing (wgMLST), using PGAdb-builder, and of fragmented genomes, using Gegenees, were conducted to compare the 80 Brazilian strains to the 18 non-Brazilian genomes. Pangenome analyses and calculations were performed for all Salmonella Infantis genomes analyzed. The presence of prophages was determined using PHASTER for the 80 Brazilian strains. The genome plasticity using BLAST Ring Image Generator (BRIG) and gene synteny using Mauve were evaluated for 20 selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Unique orthologous protein clusters were searched in ten selected Salmonella Infantis genomes from Brazil and ten from non-Brazilian countries. Results: wgMLST and Gegenees showed a high genomic similarity among some Brazilian Salmonella Infantis genomes, and also the correlation of some clusters with non-Brazilian genomes. Gegenees also showed an overall similarity >91% among all Salmonella Infantis genomes. Pangenome calculations revealed an open pangenome for all Salmonella Infantis subsets analyzed and a high gene content in the core genomes. Fifteen types of prophages were detected among 97.5% of the Brazilian strains. BRIG and Mauve demonstrated a high structural similarity among the Brazilian and non-Brazilian isolates. Unique orthologous protein clusters related to biological processes, molecular functions, and cellular components were detected among Brazilian and non-Brazilian genomes. Conclusion: The results presented using different genomic approaches emphasized the significant genomic similarity among Brazilian Salmonella Infantis genomes analyzed, suggesting wide distribution of closely related genotypes among diverse sources in Brazil. The data generated contributed to novel information regarding the genomic diversity of Brazilian and non-Brazilian Salmonella Infantis in comparison. The different genetically related subtypes of Salmonella Infantis from Brazil can either occur exclusively within the country, or also in other countries, suggesting that some exportation of the Brazilian genotypes may have already occurred.

Step-by-Step Bacterial Genome Comparison.

Thanks to advancements in genome sequencing and bioinformatics, thousands of bacterial genome sequences are available in public databases. This presents an opportunity to study bacterial diversity in unprecedented detail. This chapter describes a complete bioinformatics workflow for comparative genomics of bacterial genomes, including genome annotation, pangenome reconstruction and visualization, phylogenetic analysis, and identification of sequences of interest such as antimicrobial-resistance genes, virulence factors, and phage sequences. The workflow uses state-of-the-art, open-source tools. The workflow is presented by means of a comparative analysis of Salmonella enterica serovar Typhimurium genomes. The workflow is based on Linux commands and scripts, and result visualization relies on the R environment. The chapter provides a step-by-step protocol that researchers with basic expertise in bioinformatics can easily follow to conduct investigations on their own genome datasets.

Genomic-wide analysis of Salmonella enterica strains isolated from peanuts in Brazil.

Peanut-based products have been associated with Salmonella foodborne outbreaks and/or recalls worldwide. The ability of Salmonella to persist for a long time in a low moisture environment can contribute to this kind of contamination. The objective of this study was to analyse the genome of five S. enterica enterica strains isolated from the peanut supply chain in Brazil, as well as to identify genetic determinants for survival under desiccation and validate these findings by phenotypic test of desiccation stress. The strains were in silico serotyped using the platform SeqSero2 as Miami (M2851), Javiana (M2973), Oranienburg (M2976), Muenster (M624), and Glostrup/Chomedey (M7864); with phylogenomic analysis support. Based on Multilocus Sequence Typing (MLST) the strains were assigned to STs 140, 1674, 321, 174, and 2519. In addition, eight pathogenicity islands were found in all the genomes using the SPIFinder 2.0 (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, SPI-13, SPI-14). The absence of a SPI-4 may indicate a loss of this island in the surveyed genomes. For the pangenomic analysis, 49 S. enterica genomes were input into the Roary pipeline. The majority of the stress related genes were considered as soft-core genes and were located on the chromosome. A desiccation stress phenotypic test was performed in trypticase soy broth (TSB) with four different water activity (aw) values. M2976 and M7864, both isolated from the peanut samples with the lowest aw, showed the highest OD570nm in TSB aw 0.964 and were statistically different (p < 0.05) from the strain isolated from the peanut sample with the highest aw (0.997). In conclusion, genome analyses have revealed signatures of desiccation adaptation in Salmonella strains, but phenotypic analyses suggested the environment influences the adaptive ability of Salmonella to overcome desiccation stress.

Analysis of twelve genomes of the bacterium Kerstersia gyiorum from brown-throated sloths (Bradypus variegatus), the first from a non-human host.

Kerstersia gyiorum is a Gram-negative bacterium found in various animals, including humans, where it has been associated with various infections. Knowledge of the basic biology of K. gyiorum is essential to understand the evolutionary strategies of niche adaptation and how this organism contributes to infectious diseases; however, genomic data about K. gyiorum is very limited, especially from non-human hosts. In this work, we sequenced 12 K. gyiorum genomes isolated from healthy free-living brown-throated sloths (Bradypus variegatus) in the Parque Estadual das Fontes do Ipiranga (São Paulo, Brazil), and compared them with genomes from isolates of human origin, in order to gain insights into genomic diversity, phylogeny, and host specialization of this species. Phylogenetic analysis revealed that these K. gyiorum strains are structured according to host. Despite the fact that sloth isolates were sampled from a single geographic location, the intra-sloth K. gyiorum diversity was divided into three clusters, with differences of more than 1,000 single nucleotide polymorphisms between them, suggesting the circulation of various K. gyiorum lineages in sloths. Genes involved in mobilome and defense mechanisms against mobile genetic elements were the main source of gene content variation between isolates from different hosts. Sloth-specific K. gyiorum genome features include an IncN2 plasmid, a phage sequence, and a CRISPR-Cas system. The broad diversity of defense elements in K. gyiorum (14 systems) may prevent further mobile element flow and explain the low amount of mobile genetic elements in K. gyiorum genomes. Gene content variation may be important for the adaptation of K. gyiorum to different host niches. This study furthers our understanding of diversity, host adaptation, and evolution of K. gyiorum, by presenting and analyzing the first genomes of non-human isolates.

A pan-genomic assessment: Delving into the genome of the marine epiphyte Bacillus altitudinis strain 19_A and other very close Bacillus strains from multiple environments.

Marine macroalgae are the habitat of epiphytic bacteria and provide several conditions for a beneficial biological interaction to thrive. Although Bacillus is one of the most abundant epiphytic genera, genomic information on marine macroalgae-associated Bacillus species remains scarce. In this study, we further investigated our previously published genome of the epiphytic strain Bacillus altitudinis 19_A to find features that could be translated to potential metabolites produced by this microorganism, as well as genes that play a role in its interaction with its macroalgal host. To achieve this goal, we performed a pan-genome analysis of Bacillus sp. and a codon bias assessment, including the genome of the strain Bacillus altitudinis 19_A and 29 complete genome sequences of closely related Bacillus strains isolated from soil, marine environments, plants, extreme environments, air, and food. This genomic analysis revealed that Bacillus altitudinis 19_A possessed unique genes encoding proteins involved in horizontal gene transfer, DNA repair, transcriptional regulation, and bacteriocin biosynthesis. In this comparative analysis, codon bias was not associated with the habitat of the strains studied. Some accessory genes were identified in the Bacillus altitudinis 19_A genome that could be related to its epiphytic lifestyle, as well as gene clusters for the biosynthesis of a sporulation-killing factor and a bacteriocin, showing their potential as a source of antimicrobial peptides. Our results provide a comprehensive view of the Bacillus altitudinis 19_A genome to understand its adaptation to the marine environment and its potential as a producer of bioactive compounds.

Genome-based approach to evaluate the metabolic potentials and exopolysaccharides production of Bacillus paralicheniformis CamBx3 isolated from a Chilean hot spring.

In the present study, a thermophilic strain designated CamBx3 was isolated from the Campanario hot spring, Chile. Based on 16S rRNA gene sequence, phylogenomic, and average nucleotide identity analysis the strain CamBx3 was identified as Bacillus paralicheniformis. Genome analysis of B. paralicheniformis CamBx3 revealed the presence of genes related to heat tolerance, exopolysaccharides (EPS), dissimilatory nitrate reduction, and assimilatory sulfate reduction. The pangenome analysis of strain CamBx3 with eight Bacillus spp. resulted in 26,562 gene clusters, 7,002 shell genes, and 19,484 cloud genes. The EPS produced by B. paralicheniformis CamBx3 was extracted, partially purified, and evaluated for its functional activities. B. paralicheniformis CamBx3 EPS with concentration 5 mg mL-1 showed an optimum 92 mM ferrous equivalent FRAP activity, while the same concentration showed a maximum 91% of Fe2+ chelating activity. B. paralicheniformis CamBx3 EPS (0.2 mg mL-1) demonstrated ß-glucosidase inhibition. The EPS formed a viscoelastic gel at 45°C with a maximum instantaneous viscosity of 315 Pa.s at acidic pH 5. The present study suggests that B. paralicheniformis CamBx3 could be a valuable resource for biopolymers and bioactive molecules for industrial applications.

Acidithiobacillia class members originating at sites within the Pacific Ring of Fire and other tectonically active locations and description of the novel genus 'Igneacidithiobacillus'.

Recent studies have expanded the genomic contours of the Acidithiobacillia, highlighting important lacunae in our comprehension of the phylogenetic space occupied by certain lineages of the class. One such lineage is 'Igneacidithiobacillus', a novel genus-level taxon, represented by 'Igneacidithiobacillus copahuensis' VAN18-1T as its type species, along with two other uncultivated metagenome-assembled genomes (MAGs) originating from geothermally active sites across the Pacific Ring of Fire. In this study, we investigate the genetic and genomic diversity, and the distribution patterns of several uncharacterized Acidithiobacillia class strains and sequence clones, which are ascribed to the same 16S rRNA gene sequence clade. By digging deeper into this data and contributing to novel MAGs emerging from environmental studies in tectonically active locations, the description of this novel genus has been consolidated. Using state-of-the-art genomic taxonomy methods, we added to already recognized taxa, an additional four novel Candidate (Ca.) species, including 'Ca. Igneacidithiobacillus chanchocoensis' (mCHCt20-1TS), 'Igneacidithiobacillus siniensis' (S30A2T), 'Ca. Igneacidithiobacillus taupoensis' (TVZ-G3 TS), and 'Ca. Igneacidithiobacillus waiarikiensis' (TVZ-G4 TS). Analysis of published data on the isolation, enrichment, cultivation, and preliminary microbiological characterization of several of these unassigned or misassigned strains, along with the type species of the genus, plus the recoverable environmental data from metagenomic studies, allowed us to identify habitat preferences of these taxa. Commonalities and lineage-specific adaptations of the seven species of the genus were derived from pangenome analysis and comparative genomic metabolic reconstruction. The findings emerging from this study lay the groundwork for further research on the ecology, evolution, and biotechnological potential of the novel genus 'Igneacidithiobacillus'.

Comparative genomic analysis of Pseudomonas aeruginosa strains susceptible and resistant to carbapenems and aztreonam isolated from patients with healthcare-associated infections in a Mexican hospital.

Pseudomonas aeruginosa (PA) is an important opportunistic pathogen that causes different infections on immunocompromised patients. Within PA accessory genome, differences in virulence, antibiotic resistance and biofilm formation have been described between strains, leading to the emergence of multidrug-resistant strains. The genome sequences of 17 strains isolated from patients with healthcare-associated infections in a Mexican hospital were genomically and phylogenetically analyzed and antibiotic resistance genes, virulence genes, and biofilm formation genes were detected. Fifteen of the 17 strains were resistant to at least two of the carbapenems meropenem, imipenem, and the monobactam aztreonam. The antibiotic resistance (mexA, mexB, and oprM) and the biofilm formation (pslA and pslD) genes were detected in all strains. Differences were found between strains in accessory genome size. The strains had different sequence types, and seven strains had sequence types associated with global high risk epidemic PA clones. All strains were represented in two groups among PA global strains. In the 17 strains, horizontally acquired resistance genes to aminoglycosides and beta-lactams were found, mainly, and between 230 and 240 genes that encode virulence factors. The strains under study were variable in terms of their accessory genome, antibiotic resistance, and virulence genes. With these characteristics, we provide information about the genomic diversity of clinically relevant PA strains.

Diversification of gene content in the Mycobacterium tuberculosis complex is determined by phylogenetic and ecological signatures.

We analyzed the pan-genome and gene content modulation of the most diverse genome data set of the Mycobacterium tuberculosis complex (MTBC) gathered to date. The closed pan-genome of the MTBC was characterized by reduced accessory and strain-specific genomes, compatible with its clonal nature. However, significantly fewer gene families were shared between MTBC genomes as their phylogenetic distance increased. This effect was only observed in inter-species comparisons, not within-species, which suggests that species-specific ecological characteristics are associated with changes in gene content. Gene loss, resulting from genomic deletions and pseudogenization, was found to drive the variation in gene content. This gene erosion differed among MTBC species and lineages, even within M. tuberculosis, where L2 showed more gene loss than L4. We also show that phylogenetic proximity is not always a good proxy for gene content relatedness in the MTBC, as the gene repertoire of Mycobacterium africanum L6 deviated from its expected phylogenetic niche conservatism. Gene disruptions of virulence factors, represented by pseudogene annotations, are mostly not conserved, being poor predictors of MTBC ecotypes. Each MTBC ecotype carries its own accessory genome, likely influenced by distinct selective pressures such as host and geography. It is important to investigate how gene loss confer new adaptive traits to MTBC strains; the detected heterogeneous gene loss poses a significant challenge in elucidating genetic factors responsible for the diverse phenotypes observed in the MTBC. By detailing specific gene losses, our study serves as a resource for researchers studying the MTBC phenotypes and their immune evasion strategies.IMPORTANCEIn this study, we analyzed the gene content of different ecotypes of the Mycobacterium tuberculosis complex (MTBC), the pathogens of tuberculosis. We found that changes in their gene content are associated with their ecological features, such as host preference. Gene loss was identified as the primary driver of these changes, which can vary even among different strains of the same ecotype. Our study also revealed that the gene content relatedness of these bacteria does not always mirror their evolutionary relationships. In addition, some genes of virulence can be variably lost among strains of the same MTBC ecotype, likely helping them to evade the immune system. Overall, our study highlights the importance of understanding how gene loss can lead to new adaptations in these bacteria and how different selective pressures may influence their genetic makeup.

RdJ detection tests to identify a unique MRSA clone of ST105-SCCmecII lineage and its variants disseminated in the metropolitan region of Rio de Janeiro.

Hospital bloodstream infection (BSI) caused by methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of morbidity and mortality and is frequently related to invasive procedures and medically complex patients. An important feature of MRSA is the clonal structure of its population. Specific MRSA clones may differ in their pathogenic, epidemiological, and antimicrobial resistance profiles. Whole-genome sequencing is currently the most robust and discriminatory technique for tracking hypervirulent/well-adapted MRSA clones. However, it remains an expensive and time-consuming technique that requires specialized personnel. In this work, we describe a pangenome protocol, based on binary matrix (1,0) of open reading frames (ORFs), that can be used to quickly find diagnostic, apomorphic sequence mutations that can serve as biomarkers. We use this technique to create a diagnostic screen for MRSA isolates circulating in the Rio de Janeiro metropolitan area, the RdJ clone, which is prevalent in BSI. The method described here has 100% specificity and sensitivity, eliminating the need to use genomic sequencing for clonal identification. The protocol used is relatively simple and all the steps, formulas and commands used are described in this work, such that this strategy can also be used to identify other MRSA clones and even clones from other bacterial species.

In silico prospection of Lactobacillus acidophilus strains with potential probiotic activity.

Lactic acid bacteria (LAB) are fermentative microorganisms and perform different roles in biotechnological processes, mainly in the food and pharmaceutical industries. Among the LAB, Lactobacillus acidophilus is a species that deserves to be highlighted for being used both in prophylaxis and in the treatment of pathologies. Most of the metabolites produced by this species are linked to the inhibition of pathogens. In this study, we utilized a pangenomic and metabolic annotation analysis using Roary and BlastKOALA, ML-based probiotic activity prediction with iProbiotic and whole-genome similarity using ANI to identify strains of L. acidophilus with potential probiotic activity. According to the results in BlastKOALA and iProbiotics, L. acidophilus NCTC 13721 had the greatest potential among the 64 strains tested, both in terms of its ability to be a Lactobacillus spp. probiotic, when in the amount of genes involved in the metabolism of organic acids and quorum sensing. In addition, DSM 20079 proved to be promising for prospecting new probiotic Lactobacillus from BlastKOALA analyses, as they presented similar results in the number of genes involved in the production of lactic acid, acetic acid, hydrogen peroxide, except for quorum sensing where the NCTC 13721 strain had 14 more genes. L. acidophilus NCTC 13721 and L. acidophilus La-5 strains showed greater ability to be Lactobacillus spp. probiotic capacity, showing 84.8% and 51.9% capacity in the iProbiotics tool, respectively. When analyzed in ANI, none of the evaluated strains showed genomic similarity with NCTC 13721. In contrast, the DSM 20079 strain showed genomic similarity with all evaluated strains except NCTC 13721. Furthermore, eight strains with characteristics with approximately 100% genomic similarity to La-5 were listed: S20_1, LA-5, FSI4, APC2845, LA-G80-111, DS1_1A, LA1, and BCRC 14065. Therefore, according to the findings in iProbiotics and BlastKoala, among the 64 strains evaluated, NCTC 13721 is the most promising strain to be used for future in vitro studies.

Insights into early evolutionary adaptations of the Akkermansia genus to the vertebrate gut.

Akkermansia, a relevant mucin degrader from the vertebrate gut microbiota, is a member of the deeply branched Verrucomicrobiota, as well as the only known member of this phylum to be described as inhabitants of the gut. Only a few Akkermansia species have been officially described so far, although there is genomic evidence addressing the existence of more species-level variants for this genus. This niche specialization makes Akkermansia an interesting model for studying the evolution of microorganisms to their adaptation to the gastrointestinal tract environment, including which kind of functions were gained when the Akkermansia genus originated or how the evolutionary pressure functions over those genes. In order to gain more insight into Akkermansia adaptations to the gastrointestinal tract niche, we performed a phylogenomic analysis of 367 high-quality Akkermansia isolates and metagenome-assembled genomes, in addition to other members of Verrucomicrobiota. This work was focused on three aspects: the definition of Akkermansia genomic species clusters and the calculation and functional characterization of the pangenome for the most represented species; the evolutionary relationship between Akkermansia and their closest relatives from Verrucomicrobiota, defining the gene families which were gained or lost during the emergence of the last Akkermansia common ancestor (LAkkCA) and; the evaluation of the evolutionary pressure metrics for each relevant gene family of main Akkermansia species. This analysis found 25 Akkermansia genomic species clusters distributed in two main clades, divergent from their non-Akkermansia relatives. Pangenome analyses suggest that Akkermansia species have open pangenomes, and the gene gain/loss model indicates that genes associated with mucin degradation (both glycoside hydrolases and peptidases), (micro)aerobic metabolism, surface interaction, and adhesion were part of LAkkCA. Specifically, mucin degradation is a very ancestral innovation involved in the origin of Akkermansia. Horizontal gene transfer detection suggests that Akkermansia could receive genes mostly from unknown sources or from other Gram-negative gut bacteria. Evolutionary metrics suggest that Akkemansia species evolved differently, and even some conserved genes suffered different evolutionary pressures among clades. These results suggest a complex evolutionary landscape of the genus and indicate that mucin degradation could be an essential feature in Akkermansia evolution as a symbiotic species.

Non-Structural Proteins (Nsp): A Marker for Detection of Human Coronavirus Families.

SARS-CoV-2 was the cause of the global pandemic that caused a total of 14.9 million deaths during the years 2020 and 2021, according to the WHO. The virus presents a mutation rate between 10-5 and 10-3 substitutions per nucleotide site per cell infection (s/n/c). Due to this, studies aimed at knowing the evolution of this virus could help us to foresee (through the future development of new detection strategies and vaccines that prevent the infection of this virus in human hosts) that a pandemic caused by this virus will be generated again. In this research, we performed a functional annotation and identification of changes in Nsp (non-structural proteins) domains in the coronavirus genome. The comparison of the 13 selected coronavirus pangenomes demonstrated a total of 69 protein families and 57 functions associated with the structural domain's differentials between genomes. A marked evolutionary conservation of non-structural proteins was observed. This allowed us to identify and classify highly pathogenic human coronaviruses into alpha, beta, gamma, and delta groups. The designed Nsp cluster provides insight into the trajectory of SARS-CoV-2, demonstrating that it continues to evolve rapidly. An evolutionary marker allows us to discriminate between phylogenetically divergent groups, viral genotypes, and variants between the alpha and betacoronavirus genera. These types of evolutionary studies provide a window of opportunity to use these Nsp as targets of viral therapies.

Emergence of Raoultella ornithinolytica in human infections from different hospitals in Ecuador with OXA-48-producing resistance.

Purpose: The purpose of this study was to highlight the clinical and molecular features of 13 Raoultella ornithinolytica strains isolated from clinical environments in Ecuador, and to perform comparative genomics with previously published genomes of Raoultella spp. As Raoultella is primarily found in environmental, clinical settings, we focused our work on identifying mechanisms of resistance that can provide this bacterium an advantage to establish and persist in hospital environments. Methods: We analyzed 13 strains of Raoultella ornithinolytica isolated from patients with healthcare associated infections (HAI) in three hospitals in Quito and one in Santo Domingo de Los Tsáchilas, Ecuador, between November 2017 and April 2018. These isolates were subjected to phenotypic antimicrobial susceptibility testing, end-point polymerase chain reaction (PCR) to detect the presence of carbapenemases and whole-genome sequencing. Results: Polymerase chain reaction revealed that seven isolates were positive isolates for blaOXA-48 and one for blaKPC-2 gene. Of the seven strains that presented the blaOXA-48 gene, six harbored it on an IncFII plasmid, one was inserted into the bacterial chromosome. The blaKPC gene was detected in an IncM2/IncR plasmid. From the bioinformatics analysis, nine genomes had the gene blaOXA-48, originating from Ecuador. Moreover, all R. ornithinolytica strains contained the ORN-1 gene, which confers resistance for ß-lactams, such as penicillins and cephalosporins. Comparative genome analysis of the strains showed that the pangenome of R. ornithinolytica is considered an open pangenome, with 27.77% of core genes, which could be explained by the fact that the antibiotic resistance genes in the ancestral reconstruction are relatively new, suggesting that this genome is constantly incorporating new genes. Conclusion: These results reveal the genome plasticity of R. ornithinolytica, particularly in acquiring antibiotic-resistance genes. The genomic surveillance and infectious control of these uncommon species are important since they may contribute to the burden of antimicrobial resistance and human health.

An Efficient Triplex TaqMan Quantitative PCR to Detect a Blackleg-Causing Lineage of Pectobacterium brasiliense in Potato Based on a Pangenome Analysis.

P. brasiliense is an important bacterial pathogen causing blackleg (BL) in potatoes. Nevertheless, P. brasiliense is often detected in seed lots that do not develop any of the typical blackleg symptoms in the potato crop when planted. Field bioassays identified that P. brasiliense strains can be categorized into two distinct classes, some able to cause blackleg symptoms and some unable to do it. A comparative pangenomic approach was performed on 116 P. brasiliense strains, of which 15 were characterized as BL-causing strains and 25 as non-causative. In a genetically homogeneous clade comprising all BL-causing P. brasiliense strains, two genes only present in the BL-causing strains were identified, one encoding a predicted lysozyme inhibitor Lprl (LZI) and one encoding a putative Toll/interleukin-1 receptor (TIR) domain-containing protein. TaqMan assays for the specific detection of BL-causing P. brasiliense were developed and integrated with the previously developed generic P. brasiliense assay into a triplex TaqMan assay. This simultaneous detection makes the scoring more efficient as only a single tube is needed, and it is more robust as BL-causing strains of P. brasiliense should be positive for all three assays. Individual P. brasiliense strains were found to be either positive for all three assays or only for the P. brasiliense assay. In potato samples, the mixed presence of BL-causing and not BL-causing P. brasiliense strains was observed as shown by the difference in Ct value of the TaqMan assays. However, upon extension of the number of strains, it became clear that in recent years additional BL-causing lineages of P. brasiliense were detected for which additional assays must be developed.

Analysis of the Genomic Features and Evolutionary History of Pithovirus-Like Isolates Reveals Two Major Divergent Groups of Viruses.

New representatives of the phylum Nucleocytoviricota have been rapidly described in the last decade. Despite this, not all viruses of this phylum are allocated to recognized taxonomic families, as is the case for orpheovirus, pithovirus, and cedratvirus, which form the proposed family Pithoviridae. In this study, we performed comprehensive comparative genomic analyses of 8 pithovirus-like isolates, aiming to understand their common traits and evolutionary history. Structural and functional genome annotation was performed de novo for all the viruses, which served as a reference for pangenome construction. The synteny analysis showed substantial differences in genome organization between these viruses, with very few and short syntenic blocks shared between orpheovirus and its relatives. It was possible to observe an open pangenome with a significant increase in the slope when orpheovirus was added, alongside a decrease in the core genome. Network analysis placed orpheovirus as a distant and major hub with a large fraction of unique clusters of orthologs, indicating a distant relationship between this virus and its relatives, with only a few shared genes. Additionally, phylogenetic analyses of strict core genes shared with other viruses of the phylum reinforced the divergence of orpheovirus from pithoviruses and cedratviruses. Altogether, our results indicate that although pithovirus-like isolates share common features, this group of ovoid-shaped giant viruses presents substantial differences in gene contents, genomic architectures, and the phylogenetic history of several core genes. Our data indicate that orpheovirus is an evolutionarily divergent viral entity, suggesting its allocation to a different viral family, Orpheoviridae. IMPORTANCE Giant viruses that infect amoebae form a monophyletic group named the phylum Nucleocytoviricota. Despite being genomically and morphologically very diverse, the taxonomic categories of some clades that form this phylum are not yet well established. With advances in isolation techniques, the speed at which new giant viruses are described has increased, escalating the need to establish criteria to define the emerging viral taxa. In this work, we performed a comparative genomic analysis of representatives of the putative family Pithoviridae. Based on the dissimilarity of orpheovirus from the other viruses of this putative family, we propose that orpheovirus be considered a member of an independent family, Orpheoviridae, and suggest criteria to demarcate families consisting of ovoid-shaped giant viruses.

Pangenome Reconstruction of Mycobacterium tuberculosis as a Guide to Reveal Genomic Features Associated with Strain Clinical Phenotype.

Tuberculosis (TB) is one of the leading causes of human deaths worldwide caused by infectious diseases. TB infection by Mycobacterium tuberculosis can occur in the lungs, causing pulmonary tuberculosis (PTB), or in any other organ of the body, resulting in extrapulmonary tuberculosis (EPTB). There is no consensus on the genetic determinants of this pathogen that may contribute to EPTB. In this study, we constructed the M. tuberculosis pangenome and used it as a tool to seek genomic signatures associated with the clinical presentation of TB based on its accessory genome differences. The analysis carried out in the present study includes the raw reads of 490 M. tuberculosis genomes (PTB n = 245, EPTB n = 245) retrieved from public databases that were assembled, as well as ten genomes from Mexican strains (PTB n = 5, EPTB n = 5) that were sequenced and assembled. All genomes were annotated and then used to construct the pangenome with Roary and Panaroo. The pangenome obtained using Roary consisted of 2231 core genes and 3729 accessory genes. On the other hand, the pangenome resulting from Panaroo consisted of 2130 core genes and 5598 accessory genes. Associations between the distribution of accessory genes and the PTB/EPTB phenotypes were examined using the Scoary and Pyseer tools. Both tools found a significant association between the hspR, plcD, Rv2550c, pe_pgrs5, pe_pgrs25, and pe_pgrs57 genes and the PTB genotype. In contrast, the deletion of the aceA, esxR, plcA, and ppe50 genes was significantly associated with the EPTB phenotype. Rv1759c and Rv3740 were found to be associated with the PTB phenotype according to Scoary; however, these associations were not observed when using Pyseer. The robustness of the constructed pangenome and the gene-phenotype associations is supported by several factors, including the analysis of a large number of genomes, the inclusion of the same number of PTB/EPTB genomes, and the reproducibility of results thanks to the different bioinformatic tools used. Such characteristics surpass most of previous M. tuberculosis pangenomes. Thus, it can be inferred that the deletion of these genes can lead to changes in the processes involved in stress response and fatty acid metabolism, conferring phenotypic advantages associated with pulmonary or extrapulmonary presentation of TB. This study represents the first attempt to use the pangenome to seek gene-phenotype associations in M. tuberculosis.

Complete genome analysis of Tequatrovirus ufvareg1, a Tequatrovirus species inhibiting Escherichia coli O157:H7.

Introduction: Bacteriophages infecting human pathogens have been considered potential biocontrol agents, and studying their genetic content is essential to their safe use in the food industry. Tequatrovirus ufvareg1 is a bacteriophage named UFV-AREG1, isolated from cowshed wastewater and previously tested for its ability to inhibit Escherichia coli O157:H7. Methods: T. ufvareg1 was previously isolated using E. coli O157:H7 (ATCC 43895) as a bacterial host. The same strain was used for bacteriophage propagation and the one-step growth curve. The genome of the T. ufvareg1 was sequenced using 305 Illumina HiSeq, and the genome comparison was calculated by VIRIDIC and VIPTree. Results: Here, we characterize its genome and compare it to other Tequatrovirus. T. ufvareg1 virions have an icosahedral head (114 x 86 nm) and a contracted tail (117 x 23 nm), with a latent period of 25 min, and an average burst size was 18 phage particles per infected E. coli cell. The genome of the bacteriophage T. ufvareg1 contains 268 coding DNA sequences (CDS) and ten tRNA genes distributed in both negative and positive strains. T. ufvareg1 genome also contains 40 promoters on its regulatory regions and two rho-independent terminators. T. ufvareg1 shares an average intergenomic similarity (VIRIDC) of 88.77% and an average genomic similarity score (VipTree) of 88.91% with eight four reference genomes for Tequatrovirus available in the NCBI RefSeq database. The pan-genomic analysis confirmed the high conservation of Tequatrovirus genomes. Among all CDS annotated in the T. ufvareg1 genome, there are 123 core genes, 38 softcore genes, 94 shell genes, and 13 cloud genes. None of 268 CDS was classified as being exclusive of T. ufvareg1. Conclusion: The results in this paper, combined with other previously published findings, indicate that T. ufvareg1 bacteriophage is a potential candidate for food protection against E. coli O157:H7 in foods.