Helicobacter pylori prophages: screening, detection, induction and potential therapeutic use

Helicobacter pylori is a microaerophilic bacterium that chronically infects the human gastric mucosa. Infections caused by this pathogen are difficult to treat, mainly due to the increased resistance of this species to conventional antibiotics. Therefore, it is important to develop antibiotic alternative or complementary approaches to tackle H. pylori infections. Bacterio(phages) have proven to be efficient antibacterial agents, however it is very difficult to isolate strictly lytic phages infecting H. pylori. Nevertheless, this bacterial species presents prophages in their genomes and although strictly lytic phages have been consensually preferred for phage therapy purposes, temperate prophages holds a great but an exploited potential. In the present work, we developed a new PCR-based screening method to detect the presence of prophages genes in a set of H. pylori Portuguese clinical strains. The genomes of selected strains were then sequenced using a combined Illumina platform and MinION nanopore-based sequencing strategy. Prophages content was then analysed using the PHASTER tool. After sequencing analysis, UV light was used to induce phages, from which one was further characterized in terms of morphology, host range, stability on an in vitro gastric model, genome analysis and efficacy against a H. pylori culture. The complementarity between Illumina and Nanopore results, allowed us to identify a total of 10 intact, 7 questionable and 47 incomplete prophages on the 14 sequenced strains. One predicted intact prophage was induced successfully, and presents a genome length of 31 162 bp with 37.1 % G+C content. Interestingly, this new podovirus infects five H. pylori strains, and in the gastric in vitro model only a small loss of phage titer was observed in the gastric phase, suggesting that this phage could be adapted to the stomach environment. Farther, this phage demonstrated to be capable of maintaining the H. pylori population at low levels for up to 24 h post-infection with MOIs of 0.01, 0.1 and 1. Overall, a new PCR screening method was developed to detect prophages on H. pylori and positive correlations with sequencing results were observed. Moreover, this new isolated phage seems to have therapeutic potential to treat H. pylori gastric infections.This work was supported by the Portuguese Foundation for Science and Technology – Fundação para a Ciência e Tecnologia (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit, and Project PTDC/SAU-PUB/29182/2017 [POCI-01-0145-FEDER-029182]. Rute Ferreira is recipient of a FCT PhD grant with the reference SFRH/BD/146496/2019info:eu-repo/semantics/publishedVersio

Screening and in silico characterization of prophages in Helicobacter pylori genomes

Temperate bacterio(phages) play an important role on the evolution of pathogenic bacteria. Nevertheless, information on their role in Helicobacter pylori (an important gastric pathogen bacterium) is scarce. The present study developed a workflow for the identification of prophages in Portuguese H. pylori clinical strains, proposing the use of a new PCR-based screening method. The genome of strains with different PCR profiles were then sequenced. In the fourteen genomes analysed, nine intact prophages were identified by PHASTER. These prophages were annotated by analogy with other identified phages, where seven contained the integrase gene, corroborating the results obtained in the PCR screening, with only one exception. Still, in PCR screening, the holin gene was identified in 75 % of the strains containing intact phages, but BLASTp homologies only recognized this gene in one of the prophages. Fifty-six percent are podovirus, while in 44 % it was not possible to assign any family, according to the VirFam tool. Using the Resistance Gene Identifier of CARD it was identified the Acinetobacter mutant Lpx gene conferring resistance to colistin in two intact prophages. The BLASTp search identified a putative ABC binding cassette transporter in one of the intact prophages. On the bacterial genomes, 71 % have the CRISPR-Cas system classified as evidence level 1 by CRISPRCasFinder, which typically indicate potentially invalid CRISPR arrays. The use of an initial PCR screening method increased the identification of intact prophage-containing strains from 20 % to 57 %. Furthermore, the few virulence factors identified in prophages, and the possible inactivity of CRISPR-Cas in the bacterial genomes, allow the choice of strains for the isolation of phages for future studies. Overall, our results represent a significant contribution to the knowledge of prophages in H. pylori, and provide valuable insights into their potential use in phage therapy.info:eu-repo/semantics/publishedVersio

Comparison of bioinformatics tools to predict the presence of prophages in Helicobacter pylori genomes

Bacterio(phages) are specific viruses for bacteria, being their natural enemies. When the genome of a phage is integrated into the host bacterial genome, it is named prophage. These are a latent form of phages, in which the viral genes can increase the virulence and/or fitness characteristics of the host. This life cycle - lysogenic - does not cause the bacterial cell to rupture. Prophages have already been identified in most pathogenic bacteria, providing them better chances of survival. In the case of Helicobacter pylori, a human gastric pathogen that causes, among others, chronic gastritis, peptic ulcers, and adenocarcinoma, the presence of important prophage genes in their genomes has already been identified. In our work, a total of 109 complete genomes of human isolates of H. pylori and plasmids, deposited in NCBI archives, between November 5, 2015, and February 21, 2020, and 19 complete genomes of Portuguese clinical isolates, were screened, regarding the presence of prophages. For that, two of the most widely used web servers for identifying putative prophages in bacterial genomes were used: Phaster and Prophage Hunter. With the use of Phaster, 78 prophage sequences were identified, 6 of which were intact (7.7 %). Regarding Prophage Hunter, 199 prophages were identified, in a total of 17 active (8.5 %). The differences observed in the number of prophages identified by each tool is probably due to variances in the identification methods that each tool uses, as already reported. However, the intact sequences identified in Phaster were also predicted, in the same strains, in Prophage Hunter. These results suggest a high probability of these strains having inducible sequences of prophages in their genomes. The use of web servers for the rapid identification and annotation of prophage sequences in bacterial genomes and plasmids has been growing, helping to direct laboratory experiments more easily. In this work, we observed some differences in the results between the two tools used, concluding that new prophage prediction tools using Machine-Learning are required to predict more accurately this important viral sequences.info:eu-repo/semantics/publishedVersio

Clinical and molecular characterisation of metastatic papillary thyroid cancer according to radioiodine therapy outcomes

Funding Information: This study was funded by iNOVA4Health Research Unit (LISBOA-01-0145-FEDER-007344; UID/Multi/00462; UIDB/04462/2020), a program co-funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência e do Ensino Superior, Sociedade Portuguesa de Endocrinologia, Diabetes e Metabolismo (SPEDM), and Instituto Português de Oncologia de Lisboa Francisco Gentil (IPOLFG). J.S.-P. was supported by iNOVA4Health – UIDB/04462/2020. M.P. was granted by Liga Portuguesa Contra o Cancro, Núcleo Regional do Sul (LPCC-NRS). R.R. was granted with a PhD scholarship by iNOVA4Health Research Unit - UIDP/04462/2020; UI/BD/154256/2022. C.P. was granted with a PhD scholarship by FCT – 2020.07120.BD. Publisher Copyright: © 2023, The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.Purpose: Radioiodine (RAI) therapy remains the gold-standard approach for distant metastatic differentiated thyroid cancer (TC). The main objective of our work was to identify the clinical and molecular markers that may help to predict RAI avidity and RAI therapy response of metastatic lesions in a cohort of papillary thyroid cancer (PTC) patients. Methods: We performed a retrospective analysis of 122 PTC patients submitted to RAI therapy due to distant metastatic disease. We also analysed, through next-generation sequencing, a custom panel of 78 genes and rearrangements, in a smaller cohort of 31 metastatic PTC, with complete follow-up, available RAI therapy data, and existing tumour sample at our centre. Results: The most frequent outcome after RAI therapy was progression of disease in 59.0% of cases (n = 71), with median estimate progression-free survival of 30 months. RAI avidity was associated with PTC subtype, age and stimulated thyroglobulin at first RAI therapy for metastatic disease. The most frequently altered genes in the cohort of 31 PTC patients’ primary tumours were RAS isoforms (54.8%) and TERT promoter (TERTp) (51.6%). The presence of BRAF p.V600E or RET/PTC alterations was associated with lower avidity (p = 0.012). TERTp mutations were not associated with avidity (p = 1.000) but portended a tendency for a higher rate of progression (p = 0.063); similar results were obtained when RAS and TERTp mutations coexisted (p = 1.000 and p = 0.073, respectively). Conclusions: Early identification of molecular markers in primary tumours may help to predict RAI therapy avidity, the response of metastatic lesions and to select the patients that may benefit the most from other systemic therapies.publishersversionepub_ahead_of_prin

H. pylori phages: from genome release to hope for use as therapy

The increasing antibiotic-resistant Helicobacter pylori infections worldwide and the ineffectiveness of treatments led the World Health Organization to designate clarithromycin-resistant H. pylori as a high-priority bacterium for antibiotic research and development. (Bacterio)phages, viruses that infect bacteria, showing effectiveness in the treatment of pathogenic bacteria, could be a promising alternative strategy in the fight against H. pylori infections. Material and methods In this work, a collection of 74 Portuguese H. pylori-clinical strains was used to screen for the presence of phage genes, using a new PCR-based method. Selected strains were subsequently sequenced and prophage isolation was attempted using UV radiation. Three phages were isolated, one of which was further characterized genetically and biologically. Results PCR-based detection indicated the presence of target phage sequences in 14 strains, and the induction strategies resulted in the release of a new phage. It presents a genome length of 31,162 bp with a G+C content of 37.1 %. This podovirus showed capability to form phage plaques in five strains, was stable under an in vitro gastric digestion model, and was able to maintain a H. pylori population at low levels for up to 24h post-infection. Conclusion The new PCR screening method proved to be very effective in the selection of strains carrying prophages, resulting in the isolation of a new H. pylori phage. This phage presented very promising characteristics in terms of stability and efficacy, being therefore a small step towards the future use of phage therapy in the fight against H. pylori infections.info:eu-repo/semantics/publishedVersio

Sobrecarga no cuidar e suas repercussões nos cuidadores de pacientes em fim de vida: revisão sistemática da literatura

ResumoCuidar de um familiar com doença avançada e/ou em fim de vida pode representar uma grande sobrecarga emocional, física e financeira que afeta a qualidade de vida dos cuidadores. O presente estudo teve como objetivo realizar uma revisão sistemática da literatura sobre a sobrecarga no cuidar, os fatores relacionados e suas consequências nos cuidadores de pacientes com câncer avançado em fim de vida ou em cuidados paliativos. Foi realizada uma busca de artigos científicos publicados nas bases de dados EBSCO, Web of Knowledge e Bireme, desde os primeiros registros nas respectivas bases de dados sobre o tema até março de 2014. Dos 582 artigos encontrados, apenas 27 foram selecionados. A maioria dos artigos afirma que os cuidadores familiares estão sobrecarregados. Em alguns estudos, a sobrecarga no cuidar aparece associada a características do paciente e da sua doença; em outros, a um pior estado de saúde do cuidador, a uma maior sintomatologia psicopatológica (ansiedade, depressão, distress emocional) e também ao desenvolvimento de complicações no luto. Porém, a esperança, o apoio social, a capacidade do cuidador de atribuir um significado à experiência de cuidar e se sentir confortável com as tarefas de cuidar foram associados a menores níveis de sobrecarga