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 β-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.F.F.V. is funded by Fundação para a Ciência e a Tecnologia (FCT) through an Assistant Researcher grant CEECIND/03023/2017, and a project grant (PTDC/BTM-SAL/28978/2017) that supported this work. The work is partially supported by National funds from FCT, projects UIDB/04138/2020 and UIDP/04138/2020.info:eu-repo/semantics/publishedVersio

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.F.F.V. was funded by Fundação para a Ciência e a Tecnologia (FCT) through a project grant (PTDC/BTM-SAL/28978/2017) that supported this work. The work is partially supported by National funds from FCT, projects UIDB/04138/2020, UIDP/04138/2020, UIDB/00211/2020 and UIDB/04046/2020 (DOI https://doi.org/10.54499/UIDB/0404 6/2020)info:eu-repo/semantics/publishedVersio

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.F.F.V. is funded by Fundação para a Ciência e a Tecnologia (FCT) through an Assistant Researcher grant CEECIND/03023/2017, and a project grant (PTDC/BTM-SAL/28978/2017) that supported this work. The work is partially supported by National funds from FCT, projects UIDB/04138/2020 and UIDP/04138/2020. Campylobacter strains were sequenced under the GenomePT project (POCI-01-0145-FEDER-022184), supported by COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Lisboa Portugal Regional Operational Programme (Lisboa2020), Algarve Portugal Regional Operational Programme (CRESC Algarve2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF), and by FCT. This work was also supported by Fundos FEDER through the Programa Operacional Factores de Competitividade—COMPETE and by Fundos Nacionais through the FCT within the scope of the project UID/BIM/00009/2019 (Centre for Toxicogenomics and Human Health-ToxOmics).info:eu-repo/semantics/publishedVersio

16S Genomics for Diagnosing Invasive Bacterial Infection

Invasive bacterial infections (IBI) are leading causes of global morbidity and mortality. Successful treatment of the patients depends on a rapid and accurate diagnosis. The current golden standard procedure for bacterial identification in clinical context requires bacterial culture and biochemical analysis—a slow, culture-dependent and possible subjective approach, presenting several limitations. Bacteria identification based on 16S rRNA gene represents a faster, culture-independent and objective alternative for diagnostic. Here, we give a general overview of 16S rRNA genomics as diagnosing tool for IBI, briefly presenting and discussing the most significant technical approaches for 16S rRNA-mediated diagnosis.info:eu-repo/semantics/submittedVersio

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.F.F.V. is funded by Fundação para a Ciência e a Tecnologia (FCT) through an Assistant Researcher grant CEECIND/03023/2017, and a project grant (project PTDC/BTM-TEC/3238/2020) that supported this work. JSV holds a research fellowship within the scope of project PTDC/BTM-TEC/3238/2020 (FCT). The work has been partially supported by National funds from FCT, projects UIDB/04138/2020 and UIDP/04138/2020.info:eu-repo/semantics/publishedVersio

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 DMPC: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.FFV is funded by Fundação para a Ciência e a Tecnologia (FCT) through an Assistant Researcher grant CEECIND/03023/2017, and a project grant (project PTDC/BTM-SAL/28978/2017) that supported this work. JSV holds a research fellowship within the scope of project PTDC/BTM-TEC/3238/2020 (FCT). The work has been partially supported by National funds from FCT, projects UIDB/04138/2020 and UIDP/04138/2020.info:eu-repo/semantics/publishedVersio