Antimicrobial activity of prophage endolysins against critical Enterobacteriaceae antibiotic-resistant bacteria.

Enterobacteriaceae species are part of the 2017 World Health Organization antibiotic-resistant priority pathogens list for development of novel medicines. Multidrug-resistant Klebsiella pneumoniae is an increasing threat to public health and has become a relevant human pathogen involved in life-threatening infections. Phage therapy involves the use of phages or their lytic endolysins as bioagents for the treatment of bacterial infectious diseases. Gram-negative bacteria have an outer membrane, making difficult the access of endolysins to the peptidoglycan. Here, three endolysins from prophages infecting three distinct Enterobacterales species, Kp2948-Lys from K. pneumoniae, Ps3418-Lys from Providencia stuartii, and Kaer26608-Lys from Klebsiella aerogenes, were purified and exhibited antibacterial activity against their specific bacterium species verified by zymogram assays. These three endolysins were successfully associated to liposomes composed of dimyristoyl phosphatidyl choline (DMPC), dioleoyl phosphatidyl ethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) at a molar ratio (4:4:2), with an encapsulation efficiency ranging from 24 to 27%. Endolysins encapsulated in liposomes resulted in higher antibacterial activity compared to the respective endolysin in the free form, suggesting that the liposome-mediated delivery system enhances fusion with outer membrane and delivery of endolysins to the target peptidoglycan. Obtained results suggest that Kp2948-Lys appears to be specific for K. pneumoniae, while Ps3418-Lys and Kaer26608-Lys appear to have a broader antibacterial spectrum. Endolysins incorporated in liposomes constitute a promising weapon, applicable in the several dimensions (human, animals and environment) of the One Health approach, against multidrug-resistant Enterobacteriaceae.

Molecular detection of Helicobacter pylori and its genotypic antimicrobial resistance patterns in dyspeptic Mozambican patients.

BACKGROUND: Helicobacter pylori strains show a high level of genotypic diversity and express several genes that contribute to their pathogenicity and resistance. In Mozambique, there is lack of information regarding its resistance pattern to antibiotics. In this study, we aimed to investigate the prevalence of H. pylori and its genotypic resistance to clarithromycin, metronidazole, and fluoroquinolones in Mozambican dyspeptic patients. Since appropriate eradication should be based on the local resistance rate, our data will guide clinicians in choosing the best drugs for the effective treatment of H. pylori-infected patients. METHODS: This is a cross-sectional descriptive study conducted between June 2017 and June 2020, in which 171 dyspeptic patients were recruited, and through upper gastrointestinal endoscopy, gastric biopsies were collected from those patients. Polymerase chain reaction was performed for the detection of H. pylori and its resistance mechanisms to clarithromycin (23S rRNA), metronidazole (rdxA), and fluoroquinolones (gyrA); mutations conferring resistance to these antibiotics were investigated by sequencing 23S rRNA, rdxA, and gyrA genes. RESULTS: Of the 171 samples tested, H. pylori was detected in 56.1% (96/171). The clarithromycin resistance rate was 10.4% (the responsible mutations were A2142G and A2143G), the metronidazole resistance rate was 55.2% (4 types of mutations responsible for metronidazole resistance were identified which include, D59N, R90K, H97T, and A118T. However, in many cases, they appeared in combination, with D59N + R90K + A118T being the most frequent combination), and the fluoroquinolones resistance rate was 20% (the responsible mutations were N87I and D91G). CONCLUSION: H. pylori infection remains common in dyspeptic Mozambican patients. High resistance to metronidazole and fluoroquinolones requires continuous monitoring of antibiotic resistance and adaptation of therapy to eradicate this infection.

Repeated out-of-Africa expansions of Helicobacter pylori driven by replacement of deleterious mutations.

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.

Liposomal Delivery of Newly Identified Prophage Lysins in a Pseudomonas aeruginosa Model.

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 DPPC: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.

Biomarker Characterization and Prediction of Virulence and Antibiotic Resistance from Helicobacter pylori Next Generation Sequencing Data.

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.

Cryptic Prophages Contribution for Campylobacter jejuni and Campylobacter coli Introgression.

Campylobacter coli and C. jejuni, the causing agents of campylobacteriosis, are described to be undergoing introgression events, i.e., the transference of genetic material between different species, with some isolates sharing almost a quarter of its genome. The participation of phages in introgression events and consequent impact on host ecology and evolution remain elusive. Three distinct prophages, named C. jejuni integrated elements 1, 2, and 4 (CJIE1, CJIE2, and CJIE4), are described in C. jejuni. Here, we identified two unreported prophages, Campylobacter coli integrated elements 1 and 2 (CCIE1 and CCIE2 prophages), which are C. coli homologues of CJIE1 and CJIE2, respectively. No induction was achieved for both prophages. Conversely, induction assays on CJIE1 and CJIE2 point towards the inducibility of these prophages. CCIE2-, CJIE1-, and CJIE4-like prophages were identified in a Campylobacter spp. population of 840 genomes, and phylogenetic analysis revealed clustering in three major groups: CJIE1-CCIE1, CJIE2-CCIE2, and CJIE4, clearly segregating prophages from C. jejuni and C. coli, but not from human- and nonhuman-derived isolates, corroborating the flowing between animals and humans in the agricultural context. Punctual bacteriophage host-jumps were observed in the context of C. jejuni and C. coli, and although random chance cannot be fully discarded, these observations seem to implicate prophages in evolutionary introgression events that are modulating the hybridization of C. jejuni and C. coli species.

Genomic Analysis of Prophages from Klebsiella pneumoniae Clinical Isolates.

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 ß-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.

Origin, phylogeny, variability and epitope conservation of SARS-CoV-2 worldwide.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses innumerous challenges, like understanding what triggered the emergence of this new human virus, how this RNA virus is evolving or how the variability of viral genome may impact the primary structure of proteins that are targets for vaccine. We analyzed 19471 SARS-CoV-2 genomes available at the GISAID database from all over the world and 3335 genomes of other Coronoviridae family members available at GenBank, collecting SARS-CoV-2 high-quality genomes and distinct Coronoviridae family genomes. Additionally, we analyzed 199,984 spike glycoprotein sequences. Here, we identify a SARS-CoV-2 emerging cluster containing 13 closely related genomes isolated from bat and pangolin that showed evidence of recombination, which may have contributed to the emergence of SARS-CoV-2. The analyzed SARS-CoV-2 genomes presented 9632 single nucleotide variants (SNVs) corresponding to a variant density of 0.3 over the genome, and a clear geographic distribution. SNVs are unevenly distributed throughout the genome and hotspots for mutations were found for the spike gene and ORF 1ab. We describe a set of predicted spike protein epitopes whose variability is negligible. Additionally, all predicted epitopes for the structural E, M and N proteins are highly conserved. The amino acid changes present in the spike glycoprotein of variables of concern (VOCs) comprise between 3.4% and 20.7% of the predicted epitopes of this protein. These results favors the continuous efficacy of the available vaccines targeting the spike protein, and other structural proteins. Multiple epitopes vaccines should sustain vaccine efficacy since at least some of the epitopes present in variability regions of VOCs are conserved and thus recognizable by antibodies.

Draft Genome Sequences of 29 Helicobacter pylori Strains Isolated from Colombia.

Here, we present the draft genome sequences of 29 Colombian Helicobacter pylori strains. These strains were isolated in Bogotá, Colombia, from patients diagnosed with chronic gastritis. The genomic characterization of these strains will provide more information on the genetic composition of H. pylori strains from Colombia.

Glutaredoxin: Discovery, redox defense and much more.

Glutaredoxin, Grx, is a small protein containing an active site cysteine pair and was discovered in 1976 by Arne Holmgren. The Grx system, comprised of Grx, glutathione, glutathione reductase, and NADPH, was first described as an electron donor for Ribonucleotide Reductase but, from the first discovery in E.coli, the Grx family has impressively grown, particularly in the last two decades. Several isoforms have been described in different organisms (from bacteria to humans) and with different functions. The unique characteristic of Grxs is their ability to catalyse glutathione-dependent redox regulation via glutathionylation, the conjugation of glutathione to a substrate, and its reverse reaction, deglutathionylation. Grxs have also recently been enrolled in iron sulphur cluster formation. These functions have been implied in various physiological and pathological conditions, from immune defense to neurodegeneration and cancer development thus making Grx a possible drug target. This review aims to give an overview on Grxs, starting by a phylogenetic analysis of vertebrate Grxs, followed by an analysis of the mechanisms of action, the specific characteristics of the different human isoforms and a discussion on aspects related to human physiology and diseases.

An American lineage of Helicobacter pylori prophages found in Colombia.

BACKGROUND: Helicobacter pylori is a human gastric carcinogen that is highly prevalent in Latin American. The prophages of H. pylori show a structured population and contribute to the diversity of this bacterium. However, H. pylori prophages present in American strains have not been described to date. In this study, we identified, characterized, and present the phylogenetic analysis of the prophages present in Colombian H. pylori strains. METHODS: To characterize Colombian H. pylori strains and their prophages, a Multilocus Sequences Typing (MLST) and a Prophage Sequences Typing (PST), using the integrase and holin genes, were performed. Furthermore, five Colombian H. pylori had their full genome sequenced, and six Colombian H.pylori retrieved from databases, allowing to determine the prophage complete genome and insertion site. RESULTS: The integrase gene frequency was 12.6% (27/213), while both integrase and holin genes were present in 4.2% (9/213) of the samples analyzed. The PST analysis showed that Colombian prophages belong to different populations, including hpSWEurope, hpNEurope, hpAfrica1, and a new population, named hpColombia. The MLST analysis classified most of the Colombia strains in the hpEurope population. CONCLUSIONS: The new H. pylori prophage population revealed that Colombian prophages follow a unique evolutionary trajectory, contributing to bacterial diversity. The global H. pylori prophage phylogeny highlighted five phylogenetic groups, one more than previously reported. After the arrival of Europeans, the Colombian H. pylori bacteria and their prophages formed an independent evolutionary line to adapt to the new environment and new human hosts.

A 500-year tale of co-evolution, adaptation, and virulence: Helicobacter pylori in the Americas.

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.

Bacteriophages of Helicobacter pylori.

The bacterium Helicobacter pylori colonize the stomach in approximately half of the world's population. Infection with this bacterium is associated with gastritis, peptic ulcer, adenocarcinoma, and gastric mucosa-associated lymphoid tissue lymphoma. Besides being a pathogen with worldwide prevalence, H. pylori show increasingly high antibiotic resistance rates, making the development of new therapeutic strategies against this bacterium challenging. Furthermore, H. pylori is a genetically diverse bacterium, which may be influenced by the presence of mobile genomic elements, including prophages. In this review, we analyze these issues and summarize various reports and findings related to phages and H. pylori, discussing the relationship between the presence of these elements and the genomic diversity, virulence, and fitness of this bacterium. We also analyze the state of the knowledge on the potential utility of bacteriophages as a therapeutic strategy for H. pylori.

Trends in Helicobacter pylori resistance to clarithromycin: from phenotypic to genomic approaches.

For a long time Helicobacter pylori infections have been treated using the macrolide antibiotic, clarithromycin. Clarithromycin resistance is increasing worldwide and is the most common cause of H. pylori treatment failure. Here we review the mechanisms of antibiotic resistance to clarithromycin, detailing the individual and combinations of point mutations found in the 23S rRNA gene associated with resistance. Additionally, we consider the methods used to detect clarithromycin resistance, emphasizing the use of high-throughput next-generation sequencing methods, which were applied to 17 newly sequenced pairs of H. pylori strains isolated from the antrum and corpus of a recent colonized paediatric population. This set of isolates was composed of six pairs of resistant strains whose phenotype was associated with two point mutations found in the 23S rRNA gene: A2142C and A2143G. Other point mutations were found simultaneously in the same gene, but, according to our results, it is unlikely that they contribute to resistance. Further, among susceptible isolates, genomic variations compatible with mutations previously associated with clarithromycin resistance were detected. Exposure to clarithromycin may select low-frequency variants, resulting in a progressive increase in the resistance rate due to selection pressure.

Rapid Characterization of Virulence Determinants in Helicobacter pylori Isolated from Non-Atrophic Gastritis Patients by Next-Generation Sequencing.

Helicobacter pylori is a major human pathogen that causes a wide range of gastrointestinal pathology. Progression of H. pylori induced gastritis to more severe disease has been found to highly correlate with the array of virulence factors expressed by the pathogen. The objective of this study was twofold: first, to characterize the genetic diversity of H. pylori strains isolated from 41 non-atrophic gastritis patients in Switzerland, an issue that has not been investigated to date. And second, to assess the prevalence and sequence variation of H. pylori virulence factors (cagA, vacA, iceA and dupA) and genes encoding outer membrane proteins (OMPs; babA, babB, sabA, sabB, hopZ, hopQ and oipA) by whole genome sequencing (WGS) using an Illumina MiSeq platform. WGS identified high genetic diversity in the analyzed H. pylori strains. Most H. pylori isolates were assigned to hpEurope (95.0%, 39/41), and the remaining ones (5.0%, 2/41) to hpEastAsia, subpopulation hspEAsia. Analysis of virulence factors revealed that 43.9% of the strains were cagA-positive, and the vacA s1 allele was detected in 56.0% of the isolates. The presence of cagA was found to be significantly associated (P < 0.001) with the presence of vacA s1, babA2 and hopQ allele 1 as well as expression of oipA. Moreover, we found an association between the grade of gastritis and H. pylori abundance in the gastric mucosa, respectively and the presence of cagA, vacA s1 and hopQ allele 1. Among our 41 gastritis patients, we identified seven patients infected with H. pylori strains that carried a specific combination of virulence factors (i.e., cagA, vacA s1 allele and babA2 allele), recently implicated in the development of more severe gastrointestinal pathology, like peptic ulcer disease and even gastric cancer. To this end, WGS can be employed for rapid and detailed characterization of virulence determinants in H. pylori, providing valuable insights into the pathogenic capacity of the bacterium. This could ultimately lead to a higher level of personalized treatment and management of patients suffering from H. pylori associated infections.

Analysis of genetic recombination and the pan-genome of a highly recombinogenic bacteriophage species.

Bacteriophages are the most prevalent biological entities impacting on the ecosystem and are characterized by their extensive diversity. However, there are two aspects of phages that have remained largely unexplored: genetic flux by recombination between phage populations and characterization of specific phages in terms of the pan-genome. Here, we examined the recombination and pan-genome in Helicobacter pylori prophages at both the genome and gene level. In the genome-level analysis, we applied, for the first time, chromosome painting and fineSTRUCTURE algorithms to a phage species, and showed novel trends in inter-population genetic flux. Notably, hpEastAsia is a phage population that imported a higher proportion of DNA fragments from other phages, whereas the hpSWEurope phages showed weaker signatures of inter-population recombination, suggesting genetic isolation. The gene-level analysis showed that, after parameter tuning of the prokaryote pan-genome analysis program, H. pylori phages have a pan-genome consisting of 75 genes and a soft-core genome of 10 genes, which includes genes involved in the lytic and lysogenic life cycles. Quantitative analysis of recombination events of the soft-core genes showed no substantial variation in the intensity of recombination across the genes, but rather equally frequent recombination among housekeeping genes that were previously reported to be less prone to recombination. The signature of frequent recombination appears to reflect the host-phage evolutionary arms race, either by contributing to escape from bacterial immunity or by protecting the host by producing defective phages.

Relating Phage Genomes to Helicobacter pylori Population Structure: General Steps Using Whole-Genome Sequencing Data.

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.