Designing P. aeruginosa synthetic phages with reduced genomes

In the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48\% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB\_PaeP\\_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.Tis study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the project PTDC/SAU-PUB/29182/2017 (POCI-01-0145-FEDER-029182) and the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. DPP was supported by FCT through the grant SFRH/BPD/116187/2016. DPP also acknowledges the support from L’Oréal Portugal Medals of Honor for Women in Science 2019. Instituto for Bioengineering and Biosciences acknowledges the funding received from FCT (UID/BIO/04565/2020) and Programa Operacional Regional de Lisboa 2020 (Project N. 007317).info:eu-repo/semantics/publishedVersio

Antisense locked nucleic acid gapmers to control Candida albicans filamentation

Whereas locked nucleic acid (LNA) has been extensively used to control gene expression, it has never been exploited to control Candida virulence genes. Thus, the main goal of this work was to compare the efficacy of five different LNA-based antisense oligonucleotides (ASO) with respect to the ability to control EFG1 gene expression, to modulate filamentation and to reduce C. albicans virulence. In vitro, all LNA-ASOs were able to significantly reduce C. albicans filamentation and to control EFG1 gene expression. Using the in vivo Galleria mellonella model, important differences among the five LNA-ASOs were revealed in terms of C. albicans virulence reduction. The inclusion of PS-linkage and palmitoyl-2-amino-LNA chemical modification in these five LNA gapmers proved to be the most promising combination, increasing the survival of G. mellonella by 40%. Our work confirms that LNA-ASOs are useful tools for research and therapeutic development in the candidiasis field.This study was supported by the Portuguese Foundation forScience and Technology (FCT) under the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European RegionalDevelopment Fund under the scope of Norte2020-ProgramaOperacional Regional do Norte and Daniela Eira Araújo [SFRH/BD/121417/2016] PhD grant. The authors also acknowledge theproject funding by the“02/SAICT/2017–Projetos de Investiga-ção Científica e Desenvolvimento Tecnológico (IC&DT)–POCI-01-0145-FEDER-028893”. VILLUM Fonden is acknowledgedfor funding the Biomolecular Nano-scale Engineering Center(BioNEC), a Villum center of excellence, grant numberVKR18333. Funding received by iBB-Institute for Bioengineer-ing and Biosciences from FCT (UID/BIO/04565/2020) andPrograma Operacional Regional de Lisboa 2020 (Project No.007317) is also acknowledged. We acknowledge Dr. LucíliaGoreti Pinto, Life and Health Sciences Research Institute(ICVS), School of Medicine, University of Minho, forprocessing and sectioningG. mellonellatissue samples.The authors declare no conflict of interest.info:eu-repo/semantics/publishedVersio

Novel capsular depolymerases-based strategy to kill multidrug-resistant pathogenic bacteria

Multidrug resistant pathogens represent one of the greatest threats to human health of the new millennium. ESKAPE bacterial pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and other Enterobacteriaceae species) are the leading group among these socalled superbugs, which rapidly acquire resistances to several (and sometimes all) available antibiotics and cause a variety of nosocomial infections (e.g. bacteraemia and wound infections). Our research has been leading an innovative approach based on bacteriophage-derived enzymes (called capsular depolymerases) against A. baumannii (see video at ref 1). Previously, we found that some bacteriophages (i.e. viruses that specifically infect bacteria) acquired the ability to infect different Acinetobacter hosts through acquisition of different capsular depolymerases (2). These enzymes located at the bacteriophage tails bind and degrade specific bacterial capsules types (2). Recently, recombinantly expressed capsular depolymerases showed to be active in several environment conditions, non-nontoxic to mammalian cells and able to make A. baumannii fully susceptible to host complement effect, namely in i) Galleria mellonella caterpillar, ii) murine and iii) human serum models (3, 4). A single intraperitoneal injection of depolymerase protect 60% of mice from dead, with significant reduction of proinflammatory cytokine profile (4). We show that capsular depolymerases fit the new trend of antimicrobials needed, as they are highly specific, stable and refractory to resistance as they do not kill bacteria per se, instead they remove bacterial surface polysaccharides, diminishing bacterial virulence and exposing them to the host immune system. This innovative antimicrobial approach can be applied to other pathogenic bacteria.info:eu-repo/semantics/publishedVersio

Do Klebsiella pneumoniae environmental strains maintain clinically relevant genomic and phenotypic traits?

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Insights into the antimicrobial activities and metabolomes of Aquimarina (Flavobacteriaceae, Bacteroidetes) species from the rare marine biosphere

Two novel natural products, the polyketide cuniculene and the peptide antibiotic aquimarin, were recently discovered from the marine bacterial genus Aquimarina. However, the diversity of the secondary metabolite biosynthetic gene clusters (SM-BGCs) in Aquimarina genomes indicates a far greater biosynthetic potential. In this study, nine representative Aquimarina strains were tested for antimicrobial activity against diverse human-pathogenic and marine microorganisms and subjected to metabolomic and genomic profiling. We found an inhibitory activity of most Aquimarina strains against Candida glabrata and marine Vibrio and Alphaproteobacteria species. Aquimarina sp. Aq135 and Aquimarina muelleri crude extracts showed particularly promising antimicrobial activities, amongst others against methicillin-resistant Staphylococcus aureus. The metabolomic and functional genomic profiles of Aquimarina spp. followed similar patterns and were shaped by phylogeny. SM-BGC and metabolomics networks suggest the presence of novel polyketides and peptides, including cyclic depsipeptide-related compounds. Moreover, exploration of the ‘Sponge Microbiome Project’ dataset revealed that Aquimarina spp. possess low-abundance distributions worldwide across multiple marine biotopes. Our study emphasizes the relevance of this member of the microbial rare biosphere as a promising source of novel natural products. We predict that future metabologenomics studies of Aquimarina species will expand the spectrum of known secondary metabolites and bioactivities from marine ecosystems.info:eu-repo/semantics/publishedVersio

Erratum for Oliveira et al., "K2 Capsule Depolymerase Is Highly Stable, Is Refractory to Resistance, and Protects Larvae and Mice from Acinetobacter baumannii Sepsis"

Volume 85, no. 17, e00934-19, 2019, https://doi.org/10.1128/AEM.00934-19. Page 10, Acknowledgments, lines 4 and 5: POCI-01-0145-FEDER-016678 should read POCI-01-0145-FEDER-016643.info:eu-repo/semantics/publishedVersio

Controlling ETEC colonization on cultures of an intestinal pig cell line with a T4-like phage

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high producing costs. The rise of antibiotic selective pressure together with on-going limitation on their use demands news strategies to tackle this pathology. The pertinence of using phages to tackle this problematic is being explored, and in this work, the efficacy of a T4-like phage vB_EcoM_FJ1 (FJ1) in reducing the load of ETEC O9:H9 (Sta, F5/F41) was assessed. FJ1 has a 170,053 bp genome, and of the 270 coding sequences none corresponds to identified undesirable proteins, such as integrases or transposases. Envisaging the oral application to piglets, FJ1 was previously encapsulated on CaCO3/alginate. Assays were performed on 15-day cultures of the intestinal pig cell line IPEC-1 seeded in transwell inserts. Phage treatment occurred 2 hours after ETEC infection, when, in average, 5x105 CFU.cm-2 were adhered to cultured cells. Encapsulated phage provided reductions of, approximately, 2.3 Log CFU.cm-2 and 2.8 Log CFU.cm-2 on adhered bacteria, respectively 3 and 6 hours after administration. The repeated exposure of the host to FJ1 led to the emergence of phage-insensitive mutants, phenotype that brought fitness costs to the host strain: they were 70% more vulnerable to the pig complement system and less efficient in adhering to cultured cells (in about 90%). Overall, FJ1 is presented here as promising to fight against ETEC infections through oral administration to piglets.info:eu-repo/semantics/publishedVersio

Effect of phage vB_EcoM_FJ1 on the reduction of ETEC O9:H9 infection in a neonatal pig cell line

Enterotoxigenic Escherichia coli (ETEC) colonizes the intestine of young pigs causing severe diarrhoea and consequently bringing high production costs. The rise of antibiotic selective pressure together with ongoing limitations on their use, demands new strategies to tackle this pathology. The pertinence of using bacteriophages as an alternative is being explored, and in this work, the efficacy of phage vB\_EcoM\_FJ1 (FJ1) in reducing the load of ETEC EC43-Ph (serotype O9:H9 expressing the enterotoxin STa and two adhesins F5 and F41) was assessed. Foreseeing the oral application on piglets, FJ1 was encapsulated on calcium carbonate and alginate microparticles, thus preventing phage release under adverse conditions of the simulated gastric fluid (pH 3.0) and allowing phage availability in simulated intestinal fluid (pH 6.5). A single dose of encapsulated FJ1, provided to IPEC-1 cultured cells (from intestinal epithelium of piglets) previously infected by EC43, provided bacterial reductions of about 99.9\\% after 6 h. Although bacteriophage-insensitive mutants (BIMs) have emerged from treatment, the consequent fitness costs associated with this new phenotype were demonstrated, comparatively to the originating strain. The higher competence of the pig complement system to decrease BIMs' viability, the lower level of colonization of IPEC-1 cells observed with these mutants, and the increased survival rates and health index recorded in infected Galleria mellonella larvae supported this observation. Most of all, FJ1 established a proof-of-concept of the efficiency of phages to fight against ETEC in piglet intestinal cells.This study was mainly supported by the project Susphage, POCI-01–0247FEDER-033679, funded by FEDER through COMPETE 2020—Programa Operacional Competitividade e Internacionalização (POCI). The work was also supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic fund of UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01–0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte; by the project PTDC/CVT-CVT/29628/2017 [POCI-01–0145-FEDER-029628]; and by the project PhagoVet, H2020-EIC-FTI-2018–2020 funded by the European Union’s Horizon 2020 research and innovation programme under grant agreement No 820523. From Spain, this work was supported by the project PID2019-104439RB-C21/ AEI/10.13039/501100011033 from the Agencia Estatal de Investigación (AEI, Spain), co-funded by the European Regional Development Fund of the Euro‑ pean Union, a Way to Make Europe (ERDF). IG-M acknowledges the Xunta de Galicia for his post-doctoral grant ED481B-2021–006.info:eu-repo/semantics/publishedVersio

Marine sponge and octocoral-associated bacteria show versatile secondary metabolite biosynthesis potential and antimicrobial activities against human pathogens

Marine microbiomes are prolific sources of bioactive natural products of potential pharmaceutical value. This study inspected two culture collections comprising 919 host-associated marine bacteria belonging to 55 genera and several thus-far unclassified lineages to identify isolates with potentially rich secondary metabolism and antimicrobial activities. Seventy representative isolates had their genomes mined for secondary metabolite biosynthetic gene clusters (SM-BGCs) and were screened for antimicrobial activities against four pathogenic bacteria and five pathogenic Candida strains. In total, 466 SM-BGCs were identified, with antimicrobial peptide- and polyketide synthase-related SM-BGCs being frequently detected. Only 38 SM-BGCs had similarities greater than 70% to SM-BGCs encoding known compounds, highlighting the potential biosynthetic novelty encoded by these genomes. Cross-streak assays showed that 33 of the 70 genome-sequenced isolates were active against at least one Candida species, while 44 isolates showed activity against at least one bacterial pathogen. Taxon-specific differences in antimicrobial activity among isolates suggested distinct molecules involved in antagonism against bacterial versus Candida pathogens. The here reported culture collections and genome-sequenced isolates constitute a valuable resource of understudied marine bacteria displaying antimicrobial activities and potential for the biosynthesis of novel secondary metabolites, holding promise for a future sustainable production of marine drug leads.info:eu-repo/semantics/publishedVersio