Bakteriofagien eristäminen Beniniläisistä vesinäytteistä kliinisiä Acinetobacter baumannii kantoja vastaan

Tämän tutkielman tavoitteena oli eristää ja karakterisoida faageja Beniniläisitä jätevesinäytteistä kliinisiä Acinetobacter baumannii -kantoja vastaan käytettäväksi faagihoidoissa. A. baumannii on yksi vakavimmista sairaalabakteereista, koska suurin osa kannoista on resistenttejä kaikille yleisesti käytetyille antibiooteille. Yksi lupaavista vaihtoehtoisista hoitomenetelmistä on faagihoito, joka hyödyntää lyyttisiä faageja tiettyjen bakteerien hävittämiseksi. Tässä tutkielmassa eristettiin seitsemän faagia, jotka infektoivat kliinisiä A. baumannii -kantoja. Kaksi faageista karakterisoitiin tarkemmin. Faagit vB_AbaA_fBenAci001 (fBen-Aci001) ja vB_Aba_fBenAci002 (fBen-Aci002) kuuluivat Autographiviridae -heimoon ja Friunavirus-sukuun. Tähän mennessä tutkituista faageista ne olivat ainoat oman lajinsa edustajat. Genomianalyysin mukaan faagit ovat keskenään 82,2 % identtisiä. Faageista ei tunnistettu lysogeeniseen elinkiertoon viittaavia geenejä. Myöskään bakteeritoksiineja tai antibioottiresistenssiä koodaavia geenejä ei löydetty. Faagi fBen-Aci001 infektoi 4 % ja fBen-Aci002 infektoi 9 % testatuista 23 kliinisestä A. baumannii -kannasta. Koko genomin sekvenssin perusteella rakennettua fylogeneettistä puuta verrattiin puihin, jotka tehtiin käyttämällä häntäpiikkiproteiineja ja kapsidiproteiineja. Korrelaatiota koko genomin laajuisen puun ja yksittäisten geenien perusteella rakennettujen puiden välillä ei havaittu. Beniniläinen jätevesi vaikutti olevan hyvä lähde A. baumannii -faageille, koska siitä voitiin eristää useita faageja. Lisäksi eristetyt faagit infektoivat suomalaisista potilaista eristettyjä kliinisiä monilääkeresistenttejä kantoja. Faagit fBen-Aci001 ja fBen-Aci002 olivat lupaavia ehdokkaita faagihoidoissa käytettäväksi, mutta kapea isäntäkirjo voi vaikeuttaa niiden käyttöä.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

Isolation and Characterization of Klebsiella Phages for Phage Therapy

Introduction: Klebsiella is a clinically important pathogen causing a variety of antimicrobial resistant infections in both community and nosocomial settings, particularly pneumonia, urinary tract infection, and sepsis. Bacteriophage (phage) therapy is being considered a primary option for the treatment of drug-resistant infections of these types. Methods: We report the successful isolation and characterization of 30 novel, genetically diverse Klebsiella phages. Results: The isolated phages span six different phage families and nine genera, representing both lysogenic and lytic lifestyles. Individual Klebsiella phage isolates infected up to 11 of the 18 Klebsiella capsule types tested, and all 18 capsule-types were infected by at least one of the phages. Conclusions: Of the Klebsiella-infecting phages presented in this study, the lytic phages are most suitable for phage therapy, based on their broad host range, high virulence, short lysis period and given that they encode no known toxin or antimicrobial resistance genes. Phage isolates belonging to the Sugarlandvirus and Slopekvirus genera were deemed most suitable for phage therapy based on our characterization. Importantly, when applied alone, none of the characterized phages were able to suppress the growth of Klebsiella for more than 12 h, likely due to the inherent ease of Klebsiella to generate spontaneous phage-resistant mutants. This indicates that for successful phage therapy, a cocktail of multiple phages would be necessary to treat Klebsiella infections.Peer reviewe

Isolation and characterization of three novel Acinetobacter baumannii phages from Beninese hospital wastewater

Acinetobacter baumannii is an opportunistic pathogen that is mostly associated with hospital-acquired infections. The rapid emergence of multi- and pan-drug-resistant Acinetobacter strains poses an increasing challenge in hospitals. Phage therapy offers one treatment option for infections caused by A. baumannii. We isolated three phages from Beninese hospital wastewater – fBenAci001, fBenAci002, and fBenAci003 – that infected clinical A. baumannii strains from Finnish patients. Phylogenetic analysis showed that these phages resemble phages of the genus Friunavirus, family Autographiviridae. The isolated phages meet the requirements set for phages used for phage therapy. However, they were found to have a narrow host range, which may limit their therapeutic use.Peer reviewe