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.

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.

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.

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.

Population genetic structure of Helicobacter pylori strains from Portuguese-speaking countries.

BACKGROUND: The human gastric colonizer Helicobacter pylori is useful to track human migrations given the agreement between the bacterium phylogeographic distribution and human migrations. As Portugal was an African and Brazilian colonizer for over 400 years, we hypothesized that Portuguese isolates were likely genetically closer with those from countries colonized by Portuguese in the past. We aimed to characterize the population structure of several Portuguese-speaking countries, including Portugal, Brazil, Angola, and Cape Verde. MATERIALS AND METHODS: We included strains isolated in Portugal from Portuguese and from former Portuguese colonies. These strains were typed by multilocus sequence typing (MLST) for seven housekeeping genes. We also retrieved from Multi Locus Sequence Typing Web site additional housekeeping gene sequences, namely from Angola and Brazil. RESULTS: We provided evidence that strains from Portuguese belong to hpEurope and that the introgression of hpEurope in non-European countries that speak Portuguese is low, except for Brazil and Cape Verde, where hpEurope accounted for one quarter and one half of the population, respectively. We found genetic similarity for all strains from Portuguese-speaking countries that belong to hpEurope population. Moreover, these strains showed a predominance of ancestral Europe 2 (AE2) over ancestral Europe 1 (AE1), followed by ancestral Africa 1. CONCLUSIONS: H. pylori is a useful marker even for relative recent human migration events and may become rapidly differentiated from founder populations. H. pylori from Portuguese-speaking countries assigned to hpEurope appears to be a hybrid population resulting from the admixture of AE1, AE2 and ancestral hpAfrica1.

Genomics of Helicobacter pylori.

As Helicobacter pylori infects half the world's population and displays an extensive intraspecies diversity, genomics is a powerful tool to understand evolution and disease, to identify factors that confer higher risk of severe sequelae, and to find new approaches for therapy both among bacterial and host targets. In line with these objectives, this review article summarizes the major findings in Helicobacter genomics in papers published between April 2016 and March 2017.

The expression of Helicobacter pylori tfs plasticity zone cluster is regulated by pH and adherence, and its composition is associated with differential gastric IL-8 secretion.

BACKGROUND: Helicobacter pylori virulence is associated with different clinical outcomes. The existence of an intact dupA gene from tfs4b cluster has been suggested as a predictor for duodenal ulcer development. However, the role of tfs plasticity zone clusters in the development of ulcers remains unclear. We studied several H. pylori strains to characterize the gene arrangement of tfs3 and tfs4 clusters and their impact in the inflammatory response by infected gastric cells. METHODS: The genome of 14 H. pylori strains isolated from Western patients, pediatric (n=10) and adult (n=4), was fully sequenced using the Illumina platform MiSeq, in addition to eight pediatric strains previously sequenced. These strains were used to infect human gastric cells, and the secreted interleukin-8 (IL-8) was quantified by ELISA. The expression of virB2, dupA, virB8, virB10, and virB6 was assessed by quantitative PCR in adherent and nonadherent fractions of H. pylori during in vitro co-infection, at different pH values. RESULTS: We have found that cagA-positive H. pylori strains harboring a complete tfs plasticity zone cluster significantly induce increased production of IL-8 from gastric cells. We have also found that the region spanning from virB2 to virB10 genes constitutes an operon, whose expression is increased in the adherent fraction of bacteria during infection, as well as in both adherent and nonadherent fractions at acidic conditions. CONCLUSIONS: A complete tfs plasticity zone cluster is a virulence factor that may be important for the colonization of H. pylori and to the development of severe outcomes of the infection with cagA-positive strains.

Prevalence, antibiotic resistance, and MLST typing of Helicobacter pylori in Algiers, Algeria.

BACKGROUND: Helicobacter pylori infection is common in Algeria, but there are few data on the characterization of isolated strains. The aim of this study was to update data on the prevalence of H. pylori in patients submitted to endoscopy, antibiotic resistance, and phylogeography of H. pylori strains isolated in Algiers. MATERIALS AND METHODS: This is a prospective study carried out between November 2015 and August 2016. The culture of H. pylori was performed on antral and fundic gastric biopsies of adult patients from 3 hospitals. A real-time PCR using the fluorescence resonance energy transfer (FRET) principle for the detection of H. pylori followed by a melting curve analysis for the detection of mutations associated with resistance to clarithromycin was applied. Differentiation between antral and fundic isolates of the same patient was also determined by RAPD, and an MLST typing was performed for characterization of the phylogeographic group of H. pylori. RESULTS: By real-time PCR, the prevalence of H. pylori infection among the 147 patients included was 57%. Culture was positive in only 29% of the cases. Twenty-seven percent of patients had received H. pylori eradication treatment. The primary and secondary resistance rates to clarithromycin were 23% and 36%, respectively, and to metronidazole, 45% and 71%, respectively. Only one isolate was resistant to levofloxacin, and no resistance to amoxicillin, tetracycline, and rifampicin was detected. A double population was present in 14 patients. The MLST analysis classified the 42 H. pylori strains from 38 patients in 2 haplotypes: hpEurope (33) and hpNEAfrica (9). CONCLUSION: The prevalence of H. pylori remains high in Algeria but appears to be decreasing in recent years. High resistance to clarithromycin requires increased monitoring of the evolution of antibiotic resistance and adaptation of eradication therapy.

Recent "omics" advances in Helicobacter pylori.

The development of high-throughput whole genome sequencing (WGS) technologies is changing the face of microbiology, facilitating the comparison of large numbers of genomes from different lineages of a same organism. Our aim was to review the main advances on Helicobacter pylori "omics" and to understand how this is improving our knowledge of the biology, diversity and pathogenesis of H. pylori. Since the first H. pylori isolate was sequenced in 1997, 510 genomes have been deposited in the NCBI archive, providing a basis for improved understanding of the epidemiology and evolution of this important pathogen. This review focuses on works published between April 2015 and March 2016. Helicobacter "omics" is already making an impact and is a growing research field. Ultimately these advances will be translated into a routine clinical laboratory setting in order to improve public health.

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.

Iridovirus-like viruses in erythrocytes of lacertids from Portugal.

Icosahedral nucleo-cytoplasmic large DNA viruses (NCLDV)-like viruses, which forminclusions in the erythrocyte cytoplasm of reptiles, were previously presented as candidates for a new genus of the Iridoviridae family. The present work describes the distribution of infected lizard hosts and ultrastructural characteristics of the viral inclusions of NCLDV-like viruses from Portugal and adjacent locations in Spain. Giemsa-stained blood smears of 235 Lacerta schreiberi from Portugal and Spain, 571 Lacerta monticola from the mountain Serra da Estrela (Portugal), 794 Podarcis hispanica from several localities in Portugal and Spain, and 25 Lacerta dugesii from Madeira Island, were studied. Infection in L. schreiberi was only found in mountain populations, up to 30% in Serra da Estrela and 9-11% elsewhere. It was absent in lizards from lowlands. Prevalence of infection among L. monticola in Serra da Estrela was 10%; infected lizards were found during March to July and October but not in August and September. Infection in P. hispanica was below 3.3%. Only one infected specimen of L. dugesii was identified by light microscopy. Ultrastructural examination of infected samples revealed that the inclusions are virus assembly sites of icosahedral cytoplasmic iridovirus-like virions. Virions from different host species have different ultrastructural features and probably represent different related viruses.

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.

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.

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.

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.