Campylobacter prophage diversity reveals pervasive recombination between prophages from different Campylobacter species.

IMPORTANCE: Prophages play an important role in shaping the genetic diversity and evolution of their hosts. Acquisition or loss of prophages can lead to genomic variations, including changes in the bacterial phenotype promoted by recombination events, genetic repertoire exchanges and dissemination of virulence factors, and antibiotic resistance. By studying prophages in Campylobacter species, scientists can gain insights into the evolutionary patterns, pathogenicity mechanisms, epidemiology, and population dynamics of these species. This has implications for public health, antibiotic resistance surveillance, and the development of targeted therapeutic approaches.

Genetic insights into the microevolutionary dynamics and early introductions of human monkeypox virus in Mexico.

The recent global outbreak of mpox, caused by monkeypox virus (MPV) emerged in Europe in 2022 and rapidly spread to over 40 countries. The Americas are currently facing the highest impact, reporting over 50,000 cases by early 2023. In this study, we analyzed 880 MPV isolates worldwide to gain insights into the evolutionary patterns and initial introduction events of the virus in Mexico. We found that MPV entered Mexico on multiple occasions, from the United Kingdom, Portugal, and Canada, and subsequently spread locally in different regions of Mexico. Additionally, we show that MPV has an open pangenome, highlighting the role of gene turnover in shaping its genomic diversity, rather than single-nucleotide polymorphisms (SNPs), which do not contribute significantly to genome diversity. Although the genome contains multiple SNPs in coding regions, these remain under purifying selection, suggesting their evolutionary conservation. One notable exception is amino acid position 63 of the protein encoded by the Cop-A4L gene, which is intricately related to viral maturity, which we found to be under strong positive selection. Ancestral state reconstruction indicated that the ancestral state at position 63 corresponds to the amino acid valine, which is present only in isolates of clade I. However, the isolates from the current outbreak contained threonine at position 63. Our findings contribute new information about the evolution of monkeypox virus.

Genome Evolution and Early Introductions of the SARS-CoV-2 Omicron Variant in Mexico.

A new variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), named Omicron (Pango lineage designation B.1.1.529), was first reported to the World Health Organization by South African health authorities on 24 November 2021. The Omicron variant possesses numerous mutations associated with increased transmissibility and immune escape properties. In November 2021, Mexican authorities reported Omicron's presence in the country. In this study, we infer the first introductory events of Omicron and the impact that human mobility has had on the spread of the virus. We also evaluated the adaptive evolutionary processes in Mexican SARS-CoV-2 genomes during the first month of the circulation of Omicron. We inferred 160 introduction events of Omicron in Mexico since its first detection in South Africa; subsequently, after the first introductions there was an evident increase in the prevalence of SARS-CoV-2 during January. This higher prevalence of the novel variant resulted in a peak of reported cases; on average 6 weeks after, a higher mobility trend was reported. During the peak of cases in the country from January to February 2022, the Omicron BA.1.1 sub-lineage dominated, followed by the BA.1 and BA.15 sub-lineages. Additionally, we identified the presence of diversifying natural selection in the genomes of Omicron and found six non-synonymous mutations in the receptor binding domain of the spike protein, all of them related to evasion of the immune response. In contrast, the other proteins in the genome are highly conserved; however, we identified homoplasic mutations in non-structural proteins, indicating a parallel evolution.

Mining of Thousands of Prokaryotic Genomes Reveals High Abundance of Prophages with a Strictly Narrow Host Range.

Phages and prophages are one of the principal modulators of microbial populations. However, much of their diversity is still poorly understood. Here, we extracted 33,624 prophages from 13,713 complete prokaryotic genomes to explore the prophage diversity and their relationships with their host. Our results reveal that prophages were present in 75% of the genomes studied. In addition, Enterobacterales were significantly enriched in prophages. We also found that pathogens are a significant reservoir of prophages. Finally, we determined that the prophage relatedness and the range of genomic hosts were delimited by the evolutionary relationships of their hosts. On a broader level, we got insights into the prophage population, identified in thousands of publicly available prokaryotic genomes, by comparing the prophage distribution and relatedness between them and their hosts. IMPORTANCE Phages and prophages play an essential role in controlling their host populations either by modulating the host abundance or providing them with genes that benefit the host. The constant growth in next-generation sequencing technology has caused the development of powerful computational tools to identify phages and prophages with high precision. Making it possible to explore the prophage populations integrated into host genomes on a large scale. However, it is still a new and under-explored area, and efforts are still required to identify prophage populations to understand their dynamics with their hosts.

Computational Tools for the Analysis of Uncultivated Phage Genomes.

Over a century of bacteriophage research has uncovered a plethora of fundamental aspects of their biology, ecology, and evolution. Furthermore, the introduction of community-level studies through metagenomics has revealed unprecedented insights on the impact that phages have on a range of ecological and physiological processes. It was not until the introduction of viral metagenomics that we began to grasp the astonishing breadth of genetic diversity encompassed by phage genomes. Novel phage genomes have been reported from a diverse range of biomes at an increasing rate, which has prompted the development of computational tools that support the multilevel characterization of these novel phages based solely on their genome sequences. The impact of these technologies has been so large that, together with MAGs (Metagenomic Assembled Genomes), we now have UViGs (Uncultivated Viral Genomes), which are now officially recognized by the International Committee for the Taxonomy of Viruses (ICTV), and new taxonomic groups can now be created based exclusively on genomic sequence information. Even though the available tools have immensely contributed to our knowledge of phage diversity and ecology, the ongoing surge in software programs makes it challenging to keep up with them and the purpose each one is designed for. Therefore, in this review, we describe a comprehensive set of currently available computational tools designed for the characterization of phage genome sequences, focusing on five specific analyses: (i) assembly and identification of phage and prophage sequences, (ii) phage genome annotation, (iii) phage taxonomic classification, (iv) phage-host interaction analysis, and (v) phage microdiversity.

Clinical and pathological characteristics associated with the presence of the IS6110 Mycobacterim tuberculosis transposon in neoplastic cells from non-small cell lung cancer patients.

Lung cancer (LC) and pulmonary tuberculosis (TB) are the deadliest neoplastic and bacterial infectious diseases worldwide, respectively. Clinicians and pathologists have long discussed the co-existence of LC and TB, and several epidemiologic studies have presented evidence indicating that TB could be associated with the development of LC, particularly adenocarcinoma. Nonetheless, this data remains controversial, and the mechanism which could underlie the association remains largely unexplored. Some bioinformatic studies have shown that human cancer biopsies have a very high frequency of bacterial DNA integration; since Mycobacterium Tuberculosis (MTb) is an intracellular pathogen, it could play an active role in the cellular transformation. Our group performed an exploratory study in a cohort of 88 LC patients treated at the Instituto Nacional de Cancelorogía (INCan) of Mexico City to evaluate the presence of MTb DNA in LC tissue specimens. For the first time, our results show the presence of the MTb IS6110 transposon in 40.9% (n = 36/88) of patients with lung adenocarcinomas. Additionally, through in-situ PCR we identified the presence of IS6110 in the nuclei of tumor cells. Furthermore, shotgun sequencing from two samples identified traces of MTb genomes present in tumor tissue, suggesting that similar Mtb strains could be infecting both patients.

Targeted RNA-Seq Reveals the M. tuberculosis Transcriptome from an In Vivo Infection Model.

The study of host-pathogen interactions using in vivo models with intracellular pathogens like Mycobacterium tuberculosis (Mtb) entails technical limitations, such as: (i) Selecting an efficient differential lysis system to enrich the pathogen cells; (ii) obtaining sufficient high-quality RNA; and (iii) achieving an efficient rRNA depletion. Thus, some authors had used flow cytometers to separate infected cells or significantly increase the sequencing depth of host-pathogen RNA libraries to observe the pathogens' gene expression. However, these options carry additional expenses in specialized equipment typically not available for all laboratories. Here, we propose an experimental protocol involving differential cell lysis and a probe-based ribosomal depletion to determine the gene expression of Mtb and its host during in vivo infection. This method increased the number of observed pathogen-expressed genes from 13 using the traditional RNA-seq approach to 702. After eliminating rRNA reads, we observed that 61.59% of Mtb sequences represented 702 genes, while 38.41% represented intergenic regions. Some of the most expressed genes codified for IS1081 (Rv2512c) transposase and eight PE-PGRS members, such as PGRS49 and PGRS50. As expected, a critical percent of the expressed genes codified for secreted proteins essential for infection, such as PE68, lppN, and LpqH. Moreover, three Mtb ncRNAs were highly expressed (small RNA MTS2823, transfer-messenger RNA RF00023, and ribozyme RF00010). Many of the host-expressed genes were related to the inflammation process and the expression of surfactant proteins such as the Sftpa and Sftpc, known to bind Mtb to alveolar macrophages and mi638, a microRNA with no previous associations with pulmonary diseases. The main objective of this study is to present the method, and a general catalog of the Mtb expressed genes at one point of the in vivo infection. We believe our method represents a different approach to the existing ones to study host-pathogen interactions in tuberculosis and other similar intracellular infections, without the necessity of specialized equipment.

Gut dsDNA virome shows diversity and richness alterations associated with childhood obesity and metabolic syndrome.

Changes in the human gut microbiome are associated with obesity and metabolic syndrome, but the role of the gut virome in both diseases remains largely unknown. We characterized the gut dsDNA virome of 28 school-aged children with healthy normal-weight (NW, n = 10), obesity (O, n = 10), and obesity with metabolic syndrome (OMS, n = 8), using metagenomic sequencing of virus-like particles (VLPs) from fecal samples. The virome classification confirmed the bacteriophages' dominance, mainly composed of Caudovirales. Notably, phage richness and diversity of individuals with O and OMS tended to increase, while the VLP abundance remained the same among all groups. Of the 4,611 phage contigs composing the phageome, 48 contigs were highly prevalent in ≥80% of individuals, suggesting high inter-individual phage diversity. The abundance of several contigs correlated with gut bacterial taxa; and with anthropometric and biochemical parameters altered in O and OMS. To our knowledge, this gut phageome represents one of the largest datasets and suggests disease-specific phage alterations.

Reassessing the evolution of the 1-deoxy-D-xylulose 5-phosphate synthase family suggests a possible novel function for the DXS class 3 proteins.

The methylerythritol 4-phosphate (MEP) pathway is of paramount importance for generating plastidial isoprenoids. The first enzyme of the MEP pathway, 1-deoxy-D-xylulose-5-phosphate synthase (DXS), catalyzes a flux-controlling step. In plants the DXS gene family is composed of three distinct classes with non-redundant functions. Although the DXS1 and DXS2 subfamilies have been well characterized, the DXS3 subfamily has been considerably understudied. Here, we carried out in silico and functional analyses to better understand the DXS3 class. Our phylogenetic analysis showed high variation in copy number among the different DXS classes, with the apparent absence of DXS1 class in some species. We found that DXS3 subfamily emerged later than DXS1 and DXS2 and it is under less intense purifying selection. Furthermore, in the DXS3 subfamily critical amino acids positions in the thiamine pyrophosphate binding pocket are not conserved. We demonstrated that the DXS3 proteins from Arabidopsis, Maize, and Rice lack functional DXS activity. Moreover, the Arabidopsis DXS3 protein displayed distinctive sub-organellar chloroplast localization not observed in any DXS1 or DXS2 proteins. Co-expression analysis of the DXS3 from Arabidopsis showed that, unlike DXS1 and DXS2 proteins, it co-expresses with genes related to post-embryonic development and reproduction and not with primary metabolism and isoprenoid synthesis.

A novel vieuvirus from multidrug-resistant Acinetobacter baumannii.

Bacteriophages are considered the most abundant biological entities on earth, and they are able to modulate the populations of their bacterial hosts. Although the potential of bacteriophages has been accepted as an alternative strategy to combat multidrug-resistant pathogenic bacteria, there still exists a considerable knowledge gap regarding their genetic diversity, which hinders their use as antimicrobial agents. In this study, we undertook a genomic and phylogenetic characterization of the phage Ab11510-phi, which was isolated from a multidrug-resistant Acinetobacter baumannii strain (Ab11510). We found that Ab11510-phi has a narrow host range and belongs to a small group of transposable phages of the genus Vieuvirus that have only been reported to infect Acinetobacter bacteria. Finally, we showed that Ab11510-phi (as well as other vieuvirus phages) has a high level of mosaicism. On a broader level, we demonstrate that comparative genomics and phylogenetic analysis are necessary tools for the proper characterization of phage diversity.

Evidence for the Effect of Vaccination on Host-Pathogen Interactions in a Murine Model of Pulmonary Tuberculosis by Mycobacterium tuberculosis.

The global control of Tuberculosis remains elusive, and Bacillus Calmette-Guérin (BCG) -the most widely used vaccine in history-has proven insufficient for reversing this epidemic. Several authors have suggested that the mass presence of vaccinated hosts might have affected the Mycobacterium tuberculosis (MTB) population structure, and this could in turn be reflected in a prevalence of strains with higher ability to circumvent BCG-induced immunity, such as the recent Beijing genotype. The effect of vaccination on vaccine-escape variants has been well-documented in several bacterial pathogens; however the effect of the interaction between MTB strains and vaccinated hosts has never been previously described. In this study we show for the first time the interaction between MTB Beijing-genotype strains and BCG-vaccinated hosts. Using a well-controlled murine model of progressive pulmonary tuberculosis, we vaccinated BALB/c mice with two different sub-strains of BCG (BCG-Phipps and BCG-Vietnam). Following vaccination, the mice were infected with either one of three selected MTB strains. Strains were selected based on lineage, and included two Beijing-family clinical isolates (strains 46 and 48) and a well-characterized laboratory strain (H37Rv). Two months after infection, mice were euthanized and the bacteria extracted from their lungs. We characterized the genomic composite of the bacteria before and after exposure to vaccinated hosts, and also characterized the local response to the bacteria by sequencing the lung transcriptome in animals during the infection. Results from this study show that the interaction within the lungs of the vaccinated hosts results in the selection of higher-virulence bacteria, specifically for the Beijing genotype strains 46 and 48. After exposure to the BCG-induced immune response, strains 46 and 48 acquire genomic mutations associated with several virulence factors. As a result, the bacteria collected from these vaccinated hosts have an increased ability for immune evasion, as shown in both the host transcriptome and the histopathology studies, and replicates far more efficiently compared to bacteria collected from unvaccinated hosts or to the original-stock strain. Further research is warranted to ascertain the pathways associated with the genomic alterations. However, our results highlight novel host-pathogen interactions induced by exposure of MTB to BCG vaccinated hosts.

Mucosal Microbiome Profiles Polygenic Irritable Bowel Syndrome in Mestizo Individuals.

Irritable bowel syndrome (IBS) is the most frequent functional gastrointestinal disorder, worldwide, with a high prevalence among Mestizo Latin Americans. Because several inflammatory disorders appear to affect this population, a further understanding of host genomic background variants, in conjunction with colonic mucosa dysbiosis, is necessary to determine IBS physiopathology and the effects of environmental pressures. Using a simple polygenic model, host single nucleotide polymorphisms (SNPs) and the taxonomic compositions of microbiota were compared between IBS patients and healthy subjects. As proof of concept, five IBS-Rome III patients and five healthy controls (HCs) were systematically studied. The human and bacterial intestinal metagenome of each subject was taxonomically annotated and screened for previously annotated IBS, ulcerative colitis, and Crohn's disease-associated SNPs or taxon abundance. Dietary data and fecal markers were collected and associated with the intestinal microbiome. However, more than 1,000 variants were found, and at least 76 SNPs differentiated IBS patients from HCs, as did associations with 4 phyla and 10 bacterial genera. In this study, we found elements supporting a polygenic background, with frequent variants, among the Mestizo population, and the colonic mucosal enrichment of Bacteroides, Alteromonas, Neisseria, Streptococcus, and Microbacterium, may serve as a hallmark for IBS.

Phylogenomics Reveals Clear Cases of Misclassification and Genus-Wide Phylogenetic Markers for Acinetobacter.

The Gram-negative Acinetobacter genus has several species of clear medical relevance. Many fully sequenced genomes belonging to the genus have been published in recent years; however, there has not been a recent attempt to infer the evolutionary history of Acinetobacter with that vast amount of information. Here, through a phylogenomic approach, we established the most up-to-date view of the evolutionary relationships within this genus and highlighted several cases of poor classification, especially for the very closely related species within the Acinetobacter calcoaceticus-Acinetobacter baumannii complex (Acb complex). Furthermore, we determined appropriate phylogenetic markers for this genus and showed that concatenation of the top 13 gives a very decent reflection of the evolutionary relationships for the genus Acinetobacter. The intersection between our top markers and previously defined universal markers is very small. In general, our study shows that, although there seems to be hardly any universal markers, bespoke phylogenomic approaches can be used to infer the phylogeny of different bacterial genera. We expect that ad hoc phylogenomic approaches will be the standard in the years to come and will provide enough information to resolve intricate evolutionary relationships like those observed in the Acb complex.

Unexplored Genetic Diversity of Multidrug- and Extremely Drug-Resistant Acinetobacter baumannii Isolates from Tertiary Hospitals in Honduras.

Although Acinetobacter baumannii has become one of the most important nosocomial pathogens worldwide, very little is known about the genetic identity of isolates from less developed countries in Latin America. To alleviate this, we sequenced the genomes of 16 A. baumannii isolates from Honduras. Whole-genome sequencing was conducted on 16 isolates from five Honduran Hospitals. With the sequences of these Honduran isolates and other 42 publically available genomes, a maximum likelihood phylogeny was constructed to establish the relationship between the Honduran isolates and those belonging to the International Clones (ICs). In addition, sequence type (ST) assignation was conducted by the PubMLST, and antibiotic resistance genes were identified using ResFinder. The Honduran isolates are highly diverse and contain new allele combinations under the Bartual multilocus sequence typing scheme. The most common STs were STB447/STP10 and STB758/STP156. Furthermore, none of these isolates belongs to clonal complexes related to the ICs. Antibiotic susceptibility profiles of these isolates showed that they are multidrug resistant (MDR) or extensively drug resistant (XDR). In addition, the Honduran isolates had genes involved in resistance to seven antibiotic families. For instance, several blaOXA alleles were found, including blaOXA-23 and a gene encoding the metallo-beta-lactamase NDM-1. Notably, nine of the Honduran isolates have antibiotic resistance genes to three or more antibiotic families. In summary, in this study, we unveiled an untapped source of genetic diversity of MDR and XDR isolates; notably, these isolates did not belong to the well-known ICs.

Phylogenomics picks out the par excellence markers for species phylogeny in the genus Staphylococcus.

Although genome sequencing has become a very promising approach to conduct microbial taxonomy, few labs have the resources to afford this especially when dealing with data sets of hundreds to thousands of isolates. The goal of this study was to identify the most adequate loci for inferring the phylogeny of the species within the genus Staphylococcus; with the idea that those who cannot afford whole genome sequencing can use these loci to carry out species assignation confidently. We retrieved 177 orthologous groups (OGs) by using a genome-based phylogeny and an average nucleotide identity analysis. The top 26 OGs showed topologies similar to the species tree and the concatenation of them yielded a topology almost identical to that of the species tree. Furthermore, a phylogeny of just the top seven OGs could be used for species assignment. We sequenced four staphylococcus isolates to test the 26 OGs and found that these OGs were far superior to commonly used markers for this genus. On the whole, our procedure allowed identification of the most adequate markers for inferring the phylogeny within the genus Staphylococcus. We anticipate that this approach will be employed for the identification of the most suitable markers for other bacterial genera and can be very helpful to sort out poorly classified genera.

Functional and taxonomic classification of a greenhouse water drain metagenome.

Microbiome sequencing has become the standard procedure in the study of new ecological and human-constructed niches. To our knowledge, this is the first report of a metagenome from the water of a greenhouse drain. We found that the greenhouse is not a diverse niche, mainly dominated by Rhizobiales and Rodobacterales. The analysis of the functions encoded in the metagenome showed enrichment of characteristic features of soil and root-associated bacteria such as ABC-transporters and hydrolase enzymes. Additionally, we found antibiotic resistances genes principally for spectinomycin, tetracycline, and aminoglycosides. This study aimed to identify the bacteria and functional gene composition of a greenhouse water drain sample and also provide a genomic resource to search novel proteins from a previously unexplored niche. All the metagenome proteins and their annotations are available to the scientific community via http://microbiomics.ibt.unam.mx/tools/metagreenhouse/.

A novel thymidylate synthase from the Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) clade with altered nucleotide and folate binding sites.

Thymidylate synthase (TS, E.C. 2.1.1.45) is a crucial enzyme for de novo deoxythymidine monophosphate (dTMP) biosynthesis. The gene for this enzyme is thyA, which encodes the folate-dependent TS that converts deoxyuridine monophosphate group (dUMP) into (dTMP) using the cofactor 5,10-methylenetetrahydrofolate (mTHF) as a carbon donor. We identified the thyA gene in the genome of the Vibrio parahaemolyticus strain FIM-S1708+ that is innocuous to humans but pathogenic to crustaceans. Surprisingly, we found changes in the residues that bind the substrate dUMP and mTHF, previously postulated as invariant among all TSs known (Finer-Moore, Santi & Stroud, 2003). Interestingly, those amino acid changes were also found in a clade of microorganisms that contains Vibrionales, Alteromonadales, Aeromonadales, and Pasteurellales (VAAP) from the Gammaproteobacteria class. In this work, we studied the biochemical properties of recombinant TS from V. parahemolyticus FIM-S1708+ (VpTS) to address the natural changes in the TS amino acid sequence of the VAAP clade. Interestingly, the Km for dUMP was 27.3 ± 4.3 µM, about one-fold larger compared to other TSs. The Km for mTHF was 96.3 ± 18 µM, about three- to five-fold larger compared to other species, suggesting also loss of affinity. Thus, the catalytic efficiency was between one or two orders of magnitude smaller for both substrates. We used trimethoprim, a common antibiotic that targets both TS and DHFR for inhibition studies. The IC50 values obtained were high compared to other results in the literature. Nonetheless, this molecule could be a lead for the design antibiotics towards pathogens from the VAAP clade. Overall, the experimental results also suggest that in the VAAP clade the nucleotide salvage pathway is important and should be investigated, since the de novo dTMP synthesis appears to be compromised by a less efficient thymidylate synthase.

Evolution of a Sigma Factor: An All-In-One of Gene Duplication, Horizontal Gene Transfer, Purifying Selection, and Promoter Differentiation.

Sigma factors are an essential part of bacterial gene regulation and have been extensively studied as far as their molecular mechanisms and protein structure are concerned. However, their molecular evolution, especially for the alternative sigma factors, is poorly understood. Here, we analyze the evolutionary forces that have shaped the rpoH sigma factors within the alphaproteobacteria. We found that an ancient duplication gave rise to two major groups of rpoH sigma factors and that after this event horizontal gene transfer (HGT) occurred in rpoH 1 group. We also noted that purifying selection has differentially affected distinct parts of the gene; singularly, the gene segment that encodes the region 4.2, which interacts with the -35 motif of the RpoH-dependent genes, has been under relaxed purifying selection. Furthermore, these two major groups are clearly differentiated from one another regarding their promoter selectivity, as rpoH 1 is under the transcriptional control of σ(70) and σ(32), whereas rpoH 2 is under the transcriptional control of σ(24). Our results suggest a scenario in which HGT, gene loss, variable purifying selection and clear promoter specialization occurred after the ancestral duplication event. More generally, our study offers insights into the molecular evolution of alternative sigma factors and highlights the importance of analyzing not only the coding regions but also the promoter regions.

Complete Genome Sequence of Helicobacter pylori Strain 7C Isolated from a Mexican Patient with Chronic Gastritis.

Helicobacter pylori-induced gastritis is a risk factor for developing gastric pathologies. Here, we report the complete genome sequence of a multidrug-resistant H. pylori strain isolated from a chronic gastritis patient in Mexico City, Mexico. Nonvirulent VacA and cag-pathogenicity island (PAI) genotypes were found, but the presence of a potential mobilizable plasmid carrying an IS605 element is of outstanding interest.