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OHEJP Project: FED-AMR (Final Report)

FED-AMR project aimed to understand the role of free extracellular DNA (exDNA) in dissemination of antimicrobial resistance (AMR) over ecosystem boundaries along the food/feed chain in one-year crop growing season. As transformation is an important driver for genetic plasticity of bacterial genes and genomes and natural transformation does not require physical contact between donor and recipient bacteria we hypothesized that this may facilitate antibiotic resistance genes (ARG) crossing ecosystem barriers and invading new habitats compared to HGT by conjugation. The WP1 aims were met and were mainly based on project coordination, including scientific and administrative management, organization of regular activities, facilitation of cooperation between the WPs and integration of their outputs and results. WP2 aimed to quantify and assess microbial and AMR diversity with next generation sequencing (metagenomics) in different compartments of food production chain, during one-year-crop growing season. In particular we was interested in studying the role of extracellular DNA in the dissemination of AMR genes. We had 476 samples from six European countries (Austria, Czech Republic, Estonia, Ireland, Portugal, United Kingdom), distributed in four EU regions from up to eleven compartments along the food/feed chain. Both extracellular free DNA (exDNA) and total DNA were subjected to 16S rDNA sequence-based microbial profiling to measure the bacterial diversity. The Phyla in exDNA are dominated by Proteobacteria and Firmicutes. The principal component analysis suggests a separation between three groups of compartments: 1) soil 2) crops, feeds and drinking water 3) farmers, pigs, wild animals, manure and wastewater, which may show e.g. a smaller shifting between the microorganisms found globally in the compartments of these three groups. No significant differences were detected between soil amended with manure and without it, which may be due to an history of exposure to manure in the preceding years. ExDNA and total DNA were also subjected to target enrichment (equivalent to gene capture) to characterize and quantify ARGs in the different compartments. The analysis started with samples obtained from the wild animal compartment where we detected ARG conferring resistance to about 30 antibiotic classes, where β-lactams, tetracyclines, aminoglycosides and fluoroquinolones were the most prevalent. In the same compartment, we identified e.g. 11 ARG-types conferring resistance to fluoroquinolones. As for the competent bacteria the highest loads were observed in feed (exDNA), groundwater (total DNA) and field drainage (total DNA). We can conclude that bacteria known to be able to take up extracellular DNA are present in all environmental compartments and may serve as receptorsfor ARGs that are harboured on free exDNA. We also characterized genetically (WGS-based typing) and phenotypically (antimicrobial susceptibility testing) more than 500 cultivable bacteria. In all countries, isolates retrieved from wastewater, pig manure and pigs were those carrying more ARGs, including extended-spectrum beta-lactamases, but also ARGs associated to antimicrobials used usually in animal husbandry such as aminoglycosides, tetracyclines or sulfonamides. However, AMR data in our study (including those from metagenomics) are not thought to be a direct result of antimicrobial drug use, because 'globally' there is no direct linkage of AMR (associated with the antibiotics from the classes identified in WP4) across the various compartments and countries, therefore, there will be other risk factors (to be identified yet, if achievable). WP3 aimed at elucidating the role of Clostridioides difficile as a pathogen/ARGs transfer platform over ecosystems boundaries and the genetic overlap between human and non-human zoonotic reservoirs. At the farm level, dominant clones in environmental compartments associated with pig production were identified, suggesting a transmission chain between compartments involving these animals. The results contributed to unveil the role played by animal and environmental reservoirs within the C. difficile epidemiology. The studies assessing genetic overlap between human and non-human C. difficile lineages at different One Health settings support the zoonotic relevance of C. difficile and its presence in novel reservoirs. In WP4 we aimed at determining the selection pressures (antimicrobials, elements and herbicides) for antimicrobial resistance in environmental ecosystems (soil, water, faeces, manure, plants, feeds). We analysed samples collected by WP2. The highest concentrations of antimicrobials were detected in manure and faeces and are likely to select bacteria resistant to these compounds. The results of WP4 will be used for an impact evaluation of the analysed substances on the prevalence and quantities of ARGs encoded on extracellular DNA in exposed bacterial populations residing in the tested environmental compartments. In WP5 we successfully established and optimised (pH, temperature, flow rates and volumes) an in vitro model of the pig large intestine using pig faeces and conditions to represent the natural complex microbial community of the gut. We demonstrated sufficient growth and maintenance of an E. coli J53 strain to serve as a recipient of exDNA to study the impact of antibiotics and/or trace elements on the efficiency of natural transformation within the gut. Also, a soil microcosm setting was created using non-manured agricultural soil and Acinetobacter baylyi ADP1 as receptor of extracellular DNA. The results showed a repression of the mRNA levels of two competence genes (comA and dprA) during the first 6 hours after spiking if the microcosm was incubated at 35°C compared to 20°C. These results indicate that elevated environmental temperatures may diminish the ability of naturally competent soil bacteria to take up extracellular DNA and may reduce AMR spread by bacterial transformation. WP6 focused on generating a protocol for a systematic review on factors influencing the prevalence of antibiotic resistance in the environment and on formulating and validating a mathematical model for the dynamics of microbial communities. The protocol was registered in a public repository and accepted in Dec/2022 and published in Jan/2023 in an international journal (Environment International: https://doi.org/10.1016/j.envint.2022.107707). With respect to the task on modelling microbial communities, we have formulated a new model and validated it with in-silico data purposely developed. FED-AMR has a societal, policy and economic impact, as well as scientific impact through cross-sector communication of data contributing to the advancement of science. This project used the true concept of One Health, namely with an important environmental component (farmers, pigs, wild animals, manure, air of pig barns, feeds, crops, soil, water).

The changing epidemiology of Clostridioides diffcile reflects a well-established and intricate community transmission network. With rising numbers of reported community-acquired infections, recent studies tried to identify the role played by non-human reservoirs in the pathogen’s transmission chain. This study aimed at describing the C. dicile strains circulating in canine and feline populations, and to evaluate their genetic overlap with human strains to assess the possibility of interspecies transmission. &nbsp

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/publishedVersion

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/publishedVersion

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/146496/2019, info:eu-repo/semantics/publishedVersion

Helicobacter pylori, a Gram-negative microaerophilic bacterium in a spiral shape, is recognized as an etiological agent of active chronic gastritis and contributes to peptic ulceration1. Furthermore, some studies associate it to development of gastric carcinoma2. Currently, treatment of H. pylori is performed with conventional antibiotics that in addition to the several side effects, increase the appearance of antibiotic-resistant strains, which, consequently, increase their inefficiency3. Thus, novel strategies targeting H. pylori are a serious challenge. Bacterio(phages) have emerged in recent years as an effective alternative to fight bacterial infections4, but very little is known about H. pylori phages5. In this study, we described a novel H. pylori phage named HPy1R, induced by UV radiation from a Portuguese clinical strain, and characterized in terms of morphology, host range, stability, and efficacy against a H. pylori cultures. Moreover, the phage HPy1R was characterized in an in vitro gastric model with simulated salivary fluid and simulated gastric fluid6. Overall, the developed research allowed the isolation of a new H. pylori podovirus, that forms small uniform plaques (1mm diameter) in five of the 76 H. pylori strains tested. Additionally, in terms of genomic characterization, HPy1R has a genome length of 31 162 bp, and 37.1 % G+C content. HPy1R shares 80 % query coverage and 88.64 % identity with phage Helicobacter phage COL 23-PUJ. Moreover, using OrthoVenn2 software, we noticed that the COL 23-PUJ, Pt5771G, SwA626G and pHiHP33 phage genomes share between 66.67 % and 97.22 % orthologous proteins with HPyR1. Interestingly, HPy1R was stable from pH 3 to 11, and in in vitro gastric model small loss in the phage titer was observed in the gastric phase, suggesting that this phage could be adapted to the human stomach environment. Farther, this phage demonstrated to be capable of maintaining the H. pylori population at low levels for up to 24h post-infection with MOIs of 0.01, 0.1 and 1. In conclusion, in this work we present the first complete characterization of an H. pylori phage, which has features that envisage a great therapeutic potential against H. pylori gastric infections, if safety measures are guaranteed., info:eu-repo/semantics/publishedVersion

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/publishedVersion

This poster explores the diversity and dynamics of Clostridioides difficile in a farm environmentin Portugal. The recent increase in community acquired Clostridioides difficile infections discloses the shift in this bacterium epidemiology The commensal nature of this enteric pathogen reveals the importance of natural occurring animal and environmental reservoirs in the transmission cycle Our goal was to evaluate a possible transmission network based on a “one health” approach This is an outcome from the OHEJP FED-AMR project.

Funding This work was supported by the Familjen Erling-Perssons Stiftelse;Fundação para a Ciência e a Tecnologia [FCTPTDC/BIM-MEC/1051/2012];Swedish research council [521-2011-2370];Swedish research council [621-2014-4361]; Swedish cancer society;Åke Wibergs ;Ragnar Söderbergs stiftelse;Stiftelserna Wilhelm och Martina Lundgrens;Svenska Forskningsrådet Formas [221-2013-590];Svenska Forskningsrådet Formas [221-2011-1036]. Helicobacter pylori infection can result in non-ulcer dyspepsia (NUD), peptic ulcer disease (PUD), adenocarcinoma, and gastric lymphoma. H. pylori reside within the gastric mucus layer, mainly composed of mucins carrying an array of glycan structures that can serve as bacterial adhesion epitopes. The aim of the present study was to characterize the binding ability, adhesion modes, and growth of H. pylori strains from pediatric patients with NUD and PUD to gastric mucins. Our results showed an increased adhesion capacity of pediatric PUD H. pylori strains to human and rhesus monkey gastric mucins compared to the NUD strains both at neutral and acidic pH, regardless if the mucins were positive for Lewis b (Leb), Sialyl-Lewis x (SLex) or LacdiNAc. In addition to babA positive strains being more common among PUD associated strains, H. pylori babA positive strains bound more avidly to gastric mucins than NUD babA positive strains at acidic pH. Binding to Leb was higher among babA positive PUD H. pylori strains compared to NUD strains at neutral, but not acidic, pH. PUD derived babA-knockout mutants had attenuated binding to mucins and Leb at acidic and neutral pH, and to SLex and DNA at acidic pH. The results highlight the role of BabA-mediated adherence of pediatric ulcerogenic H. pylori strains, and points to a role for BabA in adhesion to charged structures at acidic pH, separate from its specific blood group binding activity. publishersversion published