Browsing by Subject "bacteriophage"

Enterotoksigeeninen Escherichia coli (ETEC) -infektio on yksi merkittävimmistä kolibasilloosin aiheuttajista nuorilla eläimillä ja on siksi merkittävä syy E. colin aiheuttamaan ripuliin maailmanlaajuisesti. Välittömiä ratkaisuja tarvittaisiin tämän kriisin ratkaisemiseksi ja erityisesti faagiterapia olisi yksi mahdollinen vaihtoehto ETEC-infektioiden hoitoon. Jumbofageilla on hyvät ominaisuudet mahdolliseen terapiakäyttöön niiden laajan genomin ansiosta, mutta ne ovat vielä varsin huonosti tunnettuja. Tämän tutkielman tavoitteena oli karakterisoida äskettäin eristetty Escherichia coli faagi fPf-Eco01 keskittyen erityisesti faagiterapian kannalta keskeisiin ominaisuuksiin. Tavoitteena oli myös selvittää, soveltuuko faagi terapiakäyttöön. Faagin genomin koko oli 379 kb, mikä luokittelee sen jumbofageihin. fPf-Eco01 faagi on mahdollisesti Asteriusvirus-suvun edustaja kuuluen samalla Caudoviricetes-luokkaan. Genomisekvenssin analysoinnissa ei ilmennyt haitallisia geenejä, jotka voisivat koodata antibioottiresistenssiä tai toksiineja. Sekvenssin perusteella faagin elinkierto ei olisi lysogeeninen, mikä voisi estää faagin käytön faagiterapiassa. Läpäisyelektroni-mikroskopia paljasti, että faagi fPf-Eco01 on kooltaan jumbofagin kokoinen ja sillä on supistuva häntä sekä selkeä häntälevy ja häntäsäikeet. Faagi-partikkelin keskipituus on 227 nm. Faagilla on laaja isäntäkirjo, sillä se infektoi jopa 38 % testatuista kliinisistä E. coli -kannoista. Osa faagin isäntäkantana toimivista kannoista oli laajakirjoisia beetalaktamaasi (ESBL) kantoja, jotka olivat eristetty suomalaisilta sairaalapotilailta. Faagi ei kuitenkaan infektoinut muita ETEC-kantoja, kuin vain sen alkuperäisen eristysisännän. Faagi sieti matalia pH-olosuhteita ja selvisi infektiokykyisenä useissa säilytysliuoksissa, joita voidaan käyttää faagiterapiassa. Näin ollen faagi pysyisi mahdollisesti infektiokykyisenä, mikäli faagihoitoa annettaisiin suun kautta tai faageja annosteltaisiin suonensisäisen nesteen mukana. Faagi fPf-Eco01 vaikuttaa olevan hyvä kandidaatti faagiterapiakäyttöön. Faagin laajan isäntäkirjon vuoksi sitä voitaisiin mahdollisesti käyttää ihmisten E. coli -infektioiden hoidossa. Lisätutkimuksia tarvitaan, jotta voidaan selvittää faagin mahdollista käyttöä ETEC-infektioita vastaan.

Antibiotic-resistant bacteria are an increasing threat to global health, caused by the excessive use of antibiotics and the lack of new antimicrobial agents being introduced to the market. New approaches to prevent and cure bacterial infections are needed to halt the growing crisis. One of the most promising alternatives is phage therapy which utilizes bacteriophages to target and kill pathogens with specificity. Pseudomonas aeruginosa is a common opportunistic pathogen that is intrinsically resistant to antibiotics, making it one of the most heavily studied targets of phage therapy. In this study, I characterized four P. aeruginosa phages, fHo-Pae01, PA1P1, PA8P1 and PA11P1, and evaluate their potency in therapeutic applications. Bioinformatic analysis of the genomes revealed the phages to be genetically highly similar and belonging to the Pbunavirus genus of the Myoviridae family. No genes encoding harmful toxins, antibiotic-resistance, or lysogeny were predicted. On the other hand, many of the predicted genes had unknown functions. The host ranges of the phages were assessed using 47 clinical P. aeruginosa strains and predicted host receptor binding tail proteins were compared. Some correlation between the host ranges and mutations in the tail proteins were observed but this alone was not sufficient to explain the differences in the host ranges. The recently isolated vB_PaeM_fHoPae01 (fHo-Pae01) phage was further characterized by a one-step growth curve and imaged with a promising atomic force microscopy method that had not been used before in the Skurnik group. Though the imaging results failed to provide any further knowledge of the phage, the 70-minute-long latent period of infection could be determined from the growth curve. Anion- exchange chromatography was found inefficient in purifying the fHo-Pae01 phage, so alternative methods such as endotoxin columns should be used when purifying these phages for patient use. In conclusion, all four phages appeared to be safe for therapeutic use based on current knowledge, and PA1P1 and PA11P1 were the most promising candidates due to their broad host ranges.

The objective of this thesis was to isolate and characterize new bacteriophages (phages) against clinical Klebsiella pneumoniae strains for phage therapy. K. pneumoniae is causing an emerging threat to global health due to its broad antibiotic resistance profile and hypervirulent strains. New treatment options are urgently needed to defeat the crisis. Phage therapy could provide one option to treat multiresistant K. pneumoniae infections. In this thesis, five new phages were isolated and characterized from Finnish wastewater and Georgian river water against two clinical K. pneumoniae strains. The three phages from Georgian river water, fMtkKpn01, fMtkKpn03, and fMtkKpn04, resembled Drulisviruses based on phylogenetic analysis. The two phages from Finnish wastewater, fJoKpn03 and fJoKpn05 were phylogenetically distinct. fJoKpn03 couldn’t be classified. fJoKpn05 resembled Weberviruses. Based on sequence analysis, none of the phage genomes included any harmful genes that would prevent their use in phage therapy. All phages demonstrated a 6-hour total inhibition to host bacterial growth. Their host range was determined to be narrow, only infecting their respective host strains from the 80 bacterial strains tested. All the phages tolerated high pH well. fJoKpn03 was the only one tolerating very low pH. All phages showed a synergistic effect on the inhibition of bacterial growth when applied together with piperacillin. In conclusion, all five phages proved potential for phage therapy. They demonstrated inhibitory action against K. pneumoniae strains with capsule types against which there previously were no phages in our collection. Due to their narrow host range, they could be suited for personalized phage therapy or used in combination therapy with antibiotics to increase efficacy and duration of action. fJoKpn03 could provide an opportunity for oral administration due to its broad pH stability profile.

The increase of antibiotic resistance is one of the major healthcare threats globally. One potential way to battle against antibiotic resistant bacterial infections is to treat them with the natural opponents of bacteria, bacteriophages, known as phage therapy. The aim of this thesis was to identify new bacteriophages against clinically notable bacterial species such as Escherichia coli, Burkholderia cepacia, Enterococcus faecalis and Enterococcus faecium. Bacteriophages were screened from various origins such as hospital sewage samples, soil samples and manure samples, collected in between 2019 and 2022. The isolated bacteriophages were then initially characterized to evaluate their potential use in phage therapy. In this thesis, two phages (fHo-Eco16, fHo-Eco17) against clinical E. coli isolate and one phage (fHo-Efa06) against clinical E. faecalis isolate were found from the recently collected Finnish hospital sewage sample pool. Both E. coli phages were classified as Felixounaviruses belonging to family of Ounavirinae and class of Caudoviricetes. Enterococcus phage fHo-Efa06 was characterized as Saphexavirus belonging to class of Caudoviricetes. Preliminary genome annotation did not reveal any characteristics of lysogenic lifecycle, or antibiotic resistance or bacterial toxin genes, which would prevent the use of phages in phage therapy. Both E. coli phages (fHo-Eco16, fHo-Eco17) showed narrow host range infecting only the primary host bacterial isolate but none of 29 other tested clinical E. coli isolates. Phage fHo-Efa06 showed relatively broad host range properties infecting nine tested E. faecalis isolates out of 20 tested E. faecalis isolates but no infection capabilities against six tested clinical E. faecium isolates. In conclusion, freshly collected hospital sewage seemed to be optimal environment to find bacteriophages against clinical bacterial isolates. Furthermore, phages fHo-Eco16, fHo-Eco17 and fHo-Efa06 did not display any strictly unsuitable properties which could prevent their use in phage therapy. In turn, to obtain the definitive certainty on the usability of the phages in therapeutic use, in-depth host range screening together with detailed functional and structural annotation for the phage genomes of fHo-Efa06, fHo-Eco16 and fHo-Eco17 should be completed.

The objective of this thesis was to isolate and characterized phages from Beninese wastewater samples against clinical Acinetobacter baumannii strains for phage therapy use. A. baumannii is one of the most threatening nosocomial bacteria because most of the strains are resistant towards all commonly used antibiotics. One promising alternative treatment method could be phage therapy that utilizes lytic phages to dispose of specific bacteria. In this thesis, seven phages infecting clinical A. baumannii strains were isolated and two of them were characterized more in detail. Phages vB_AbaA_fBenAci001 (fBen-Aci001) and vB_Aba_fBenAci002 (fBen-Aci002) were members of the Friunavirus genus of the Autographiviridae family. In addition, they were the only phages characterised from their respective species to date. The genome analysis revealed 82.2% identity between the phages. No genes indicating lysogenic lifecycle, or genes encoding bacterial toxins or antibiotic resistance were identified from either of them. Phage fBen-Aci001 were infecting 4% and fBen-Aci002 were infecting 9% of tested 23 clinical A. baumannii isolates. Phylogenetic tree which was constructed based on whole genome sequences was compared to the trees that were made using tailspike proteins and capsid proteins. No correlation between genome-wide tree and trees built based on single genes were seen. In conclusion, the Beninese hospital wastewater appeared to be a good source for A. baumannii phages, as several phages were isolated and they were infecting clinical multidrug resistant strains isolated from Finnish patients. Phages fBen-Aci001 and fBen-Aci002 were concluded to be potential candidates to be used in the phage therapy though the narrow host range might negatively affect their usability.

Antibiotic-resistant bacteria present a severe threat to global health. The future treatment of common bacterial infections relies on the identification of novel antibiotics and targets in the present. One area of antimicrobial research is the study of bacteriophage (Petrovic Fabijan et al.) mechanisms and the identification of phage-derived antimicrobials. Sequenced phage genomes are largely (>70%) annotated as “hypothetical proteins of unknown function” (HPUFs) and investigation into HPUFs with a toxic effect on host bacteria (toxHPUFs) aims to reveal new antibacterial targets and antimicrobials. Next-generation sequencing and plating-based toxicity screening of Staphylococcus phage Stab21 HPUFs identified nine HPUFs that incurred toxicity to Escherichia coli. In this study, the tightly controlled tetracycline-inducible plasmid pRAB11N was used as a shuttle vector and verified the toxicity of five out of nine HPUFs to E. coli and revealed that no HPUFs caused toxicity to the Stab21 natural target and clinically relevant Staphylococcus aureus. These results suggest that screening for toxHPUFs should be carried out in closely related bacterial species or the phages’ natural host. The five toxHPUFs of E. coli were further characterised by protein function and structural predictions. Only one toxHPUF, g024, returned a reliable model with homology to Bacillus phage SPO1 homing endonuclease I-HmuI, yet the role of this DNase in bacterial host toxicity is still unknown. To determine the bacterial targets of the toxHPUFs, spontaneous toxin-insensitive mutants of the five toxHPUFs were investigated. For three toxHPUFs, the toxin insensitivity was ascribed to the elimination of the toxin-encoding gene. However, toxin-insensitive g172 and g187 sequences revealed mutations in the tetR gene of pRAB11N that led to the inability of tetracycline binding and thus no induction of gene expression and did not aid in identifying the bacterial targets of these toxHPUFs. This study highlights the experimental complexities of phage-derived antimicrobial research. It also maintains the value of this research strategy, with the verification of HPUFs with a toxic effect on E. coli and accompanied future studies of bacterial target determination having the potential to uncover novel antimicrobial mechanisms that can be exploited for therapeutic application.