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The review uses the Helicobacter pylori, the gastric bacterium that colonizes the human stomach, to address how to obtain information from bacterial genomes about prophage biology. In a time of continuous growing number of genomes available, this review provides tools to explore genomes for prophage presence, or other mobile genetic elements and virulence factors. The review starts by covering the genetic diversity of H. pylori and then moves to the biologic basis and the bioinformatics approaches used for studding the H. pylori phage biology from their genomes and how this is related with the bacterial population structure. Aspects concerning H. pylori prophage biology, evolution and phylogeography are discussed.

Helicobacter pylori lives in the human stomach and has a population structure resembling that of its host. However, H. pylori from Europe and the Middle East trace substantially more ancestry from modern African populations than the humans that carry them. Here, we use a collection of Afro-Eurasian H. pylori genomes to show that this African ancestry is due to at least three distinct admixture events. H. pylori from East Asia, which have undergone little admixture, have accumulated many more non-synonymous mutations than African strains. European and Middle Eastern bacteria have elevated African ancestry at the sites of these mutations, implying selection to remove them during admixture. Simulations show that population fitness can be restored after bottlenecks by migration and subsequent admixture of small numbers of bacteria from non-bottlenecked populations. We conclude that recent spread of African DNA has been driven by deleterious mutations accumulated during the original out-of-Africa bottleneck. Helicobacter pylori is a major human pathogen whose population structure is similar to that of its host. Here, the authors show that H. pylori has repeatedly spread out of Africa recently, replacing deleterious variants that accumulated during the original out of Africa migrations more than 50,000 years ago.

The Gram-negative bacterium Helicobacter pylori colonizes c.a. 50% of human stomachs worldwide and is the major risk factor for gastric adenocarcinoma. Its high genetic variability makes it difficult to identify biomarkers of early stages of infection that can reliably predict its outcome. Moreover, the increasing antibiotic resistance found in H. pylori defies therapy, constituting a major human health problem. Here, we review H. pylori virulence factors and genes involved in antibiotic resistance, as well as the technologies currently used for their detection. Furthermore, we show that next generation sequencing may lead to faster characterization of virulence factors and prediction of the antibiotic resistance profile, thus contributing to personalized treatment and management of H. pylori-associated infections. With this new approach, more and permanent data will be generated at a lower cost, opening the future to new applications for H. pylori biomarker identification and antibiotic resistance prediction.

Klebsiella pneumoniae is an increasing threat to public health and represents one of the most concerning pathogens involved in life-threatening infections. The resistant and virulence determinants are coded by mobile genetic elements which can easily spread between bacteria populations and co-evolve with its genomic host. In this study, we present the full genomic sequences, insertion sites and phylogenetic analysis of 150 prophages found in 40 K. pneumoniae clinical isolates obtained from an outbreak in a Portuguese hospital. All strains harbored at least one prophage and we identified 104 intact prophages (69.3%). The prophage size ranges from 29.7 to 50.6 kbp, coding between 32 and 78 putative genes. The prophage GC content is 51.2%, lower than the average GC content of 57.1% in K. pneumoniae. Complete prophages were classified into three families in the order Caudolovirales: Myoviridae (59.6%), Siphoviridae (38.5%) and Podoviridae (1.9%). In addition, an alignment and phylogenetic analysis revealed nine distinct clusters. Evidence of recombination was detected within the genome of some prophages but, in most cases, proteins involved in viral structure, transcription, replication and regulation (lysogenic/lysis) were maintained. These results support the knowledge that prophages are diverse and widely disseminated in K. pneumoniae genomes, contributing to the evolution of this species and conferring additional phenotypes. Moreover, we identified K. pneumoniae prophages in a set of endolysin genes, which were found to code for proteins with lysozyme activity, cleaving the β-1,4 linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in the peptidoglycan network and thus representing genes with the potential for lysin phage therapy.

Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium that presents resistance to several antibiotics, thus, representing a major threat to human and animal health. Phage-derived products, namely lysins, or peptidoglycan-hydrolyzing enzymes, can be an effective weapon against antibiotic-resistant bacteria. Whereas in Gram-positive bacteria, lysis from without is facilitated by the exposed peptidoglycan layer, this is not possible in the outer membrane-protected peptidoglycan of Gram-negative bacteria. Here, we suggest the encapsulation of lysins in liposomes as a delivery system against Gram-negative bacteria, using the model of P. aeruginosa. Bioinformatic analysis allowed for the identification of 38 distinct complete prophages within 66 P. aeruginosa genomes (16 of which newly sequenced) and led to the identification of 19 lysins of diverse sequence and function, 5 of which proceeded to wet lab analysis. The four purifiable lysins showed hydrolytic activity against Gram-positive bacterial lawns and, on zymogram assays, constituted of autoclaved P. aeruginosa cells. Additionally, lysins Pa7 and Pa119 combined with an outer membrane permeabilizer showed activity against P. aeruginosa cells. These two lysins were successfully encapsulated in DMPC:DOPE:CHEMS (molar ratio 4:4:2) liposomes with an average encapsulation efficiency of 33.33% and 32.30%, respectively. The application of the encapsulated lysins to the model P. aeruginosa led to a reduction in cell viability and resulted in cell lysis as observed in MTT cell viability assays and electron microscopy. In sum, we report here that prophages may be important sources of new enzybiotics, with prophage lysins showing high diversity and activity. In addition, these enzybiotics following their incorporation in liposomes were able to potentiate their antibacterial effect against the Gram-negative bacteria P. aeruginosa, used as the model.

Helicobacter pylori is a common component of the human stomach microbiota, possibly dating back to the speciation of Homo sapiens . A history of pathogen evolution in allopatry has led to the development of genetically distinct H. pylori subpopulations, associated with different human populations, and more recent admixture among H. pylori subpopulations can provide information about human migrations. However, little is known about the degree to which some H. pylori genes are conserved in the face of admixture, potentially indicating host adaptation, or how virulence genes spread among different populations. We analyzed H. pylori genomes from 14 countries in the Americas, strains from the Iberian Peninsula, and public genomes from Europe, Africa, and Asia, to investigate how admixture varies across different regions and gene families. Whole-genome analyses of 723 H. pylori strains from around the world showed evidence of frequent admixture in the American strains with a complex mosaic of contributions from H. pylori populations originating in the Americas as well as other continents. Despite the complex admixture, distinctive genomic fingerprints were identified for each region, revealing novel American H. pylori subpopulations. A pan-genome Fst analysis showed that variation in virulence genes had the strongest fixation in America, compared with non-American populations, and that much of the variation constituted non-synonymous substitutions in functional domains. Network analyses suggest that these virulence genes have followed unique evolutionary paths in the American populations, spreading into different genetic backgrounds, potentially contributing to the high risk of gastric cancer in the region.

Helicobacter pylori infection, a leading cause of gastric ulcers and gastric cancer, presents a major health challenge, exacerbated by rising antibiotic resistance. This study investigated the antibacterial potential of plant-derived compounds, isolated from different plant species, against H. pylori. Thus, a library of 153 natural compounds and derivatives, including monoterpene indole and bisindole alkaloids, obtained from the African medicinal plant Tabernaemontana elegans was screened in vitro for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against H. pylori. Active compounds (1–7) were tested for anti-biofilm activity and cytotoxicity on VERO cells to determine their half-maximal cytotoxic concentrations (CC50). Six monoterpene indole alkaloid azine derivatives (1–6) and vobasinyl-iboga type bisindole alkaloid (7) displayed antibacterial activity, with MICs between 10 and 20 µM. Compounds 2, 3, and 6 exhibited bactericidal activity, with MBCs of 20 µM. Notably, compounds 1 to 4 inhibited H. pylori biofilm formation at sub-inhibitory concentrations. Cytotoxicity assays revealed CC50 values above MICs, indicating a favorable safety profile for potential therapeutic use. This study highlights the potential of T. elegans monoterpene indole alkaloids as antibacterial agents and supports further exploration of plant-derived compounds as alternative treatments for H. pylori, offering a promising approach to address antibiotic resistance in gastrointestinal diseases.

The oral cavity may play a role as a reservoir and in the transmission and colonization of Helicobacter pylori. The route of transmission for H. pylori is not fully understood. The prevalence of this pathogen varies globally, affecting half of the world’s population, predominantly in developing countries. Here, we review the prevalence of H. pylori in the oral cavity, the characteristics that facilitate its colonization and dynamics in the oral microbiome, the heterogeneity and diversity of virulence of among strains, and noninvasive techniques for H. pylori detection in oral samples. The prevalence of H. pylori in the oral cavity varies greatly, being influenced by the characteristics of the population, regions where samples are collected in the oral cavity, and variations in detection methods. Although there is no direct association between the presence of H. pylori in oral samples and stomach infection, positive cases for gastric H. pylori frequently exhibit a higher prevalence of the bacterium in the oral cavity, suggesting that the stomach may not be the sole reservoir of H. pylori . In the oral cavity, H. pylori can cause microbiome imbalance and remodeling of the oral ecosystem. Detection of H. pylori in the oral cavity by a noninvasive method may provide a more accessible diagnostic tool as well as help prevent transmission and gastric re-colonization. Further research into this bacterium in the oral cavity will offer insights into the treatment of H. pylori infection, potentially developing new clinical approaches.

Background: Helicobacter pylori (H. pylori) is a Gram-negative bacterium capable of colonizing the human stomach, which can lead to various gastrointestinal conditions. Several invasive and non-invasive methods exist for diagnosing H. pylori; however, none can be considered the gold standard. This study aimed to evaluate the performance of three biopsy-based methods (rapid urease test - RUT, histopathology - HIST, and polymerase chain reaction - PCR) in diagnosing H. pylori, and to assess their combined effect in confirming the infection. Methodology: Eighty dyspeptic patients were recruited for this study, and gastric biopsies were collected from each of them using upper digestive endoscopy. H. pylori was diagnosed using three biopsy-based methods: RUT, HIST, and PCR. RUT was performed using the commercially available PYLO DRYTM Kit, HIST was conducted with Hematoxylin & Eosin and Giemsa staining, and PCR was performed by amplifying the 16S rRNA and 23S rRNA genes. The patient had to test positive in at least two combined diagnostic methods to be confirmed as a case. Results: The three biopsy-based methods (RUT, HIST, and PCR) showed positivity rates of 100% (80/80), 35% (28/80), and 65% (52/80), respectively. When all methods were combined to confirm H. pylori infection, 75% (60/80) of cases were confirmed, while the remaining 25% (20/80) were classified as undetermined, as they were positive only for RUT. Conclusions: Despite slight differences, RUT and PCR performed well in diagnosing H. pylori compared to HIST. However, when all three methods were combined, they improved the accuracy of H. pylori diagnosis and infection confirmation.