Bakteerien taudinaiheuttamismekanismeista. ja faagiterapiasta

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1 Bakteerien taudinaiheuttamismekanismeista ja faagiterapiasta Mikael Skurnik, FT Bakteriologian professori tai

2 Taustatietoja Tutkimustyö: halu ymmärtää miten bakteerit aiheuttavat taudin Tutkimuskohde: Yersinia-bakteerit, joiden aiheuttamat taudit vaihtelevat lievästä suolistoinfektiosta paiseruttoon Erityisesti kohteena Yersiniabakteerin pinnan virulenssitekijät YadA ja Ail (tarttumisproteiineja) ja LPS (lipopolysakkaridi, endotoksiini) Olen selvittänyt niiden toimintaa molekyylitasolla ja merkitystä bakteerin taudinaiheuttamiskyvylle Muuta: Tutkimuksestani on kuroutunut soveltavia hankkeita, joissa molekyylibiologiaa ja geeniteknologiaa on valjastettu mikrobidiagnostiikan käyttöön Bakteriofaagit ja faagiterapia

3 Miten bakteerit aiheuttavat tauteja? Yksinkertaista: Bakteerit kasvavat organismissa Tuhoavat kudoksia Aiheuttavat infektiotaudin

4 Mutta Kaikki bakteerit eivät aiheuta tauteja Vain pieni murto-osa tunnetuista bakteereista on tauteja aiheuttavia Useimmilla bakteereilla on ekologinen lokeronsa maaperässä tai vedessä Kaikki eläimet myös kantavat kilokaupalla hyödyllisiä bakteereita

5 Lisäksi Eri bakteerit aiheuttavat erilaisia tauteja Bakteerit ovat spesialisteja gonokokki sukupuolitauti meningokokki aivokalvon tulehdus, meningiitti pneumokokki keuhkokuume, pneumonia enterobakteerit suolistotulehdus Tartuntareitit vaihtelevat (limakalvo) kontakti aerosolit pilaantunut ruoka tai vesi hyönteisvektorit (rutto), borrelioosi, tularemia

6 Mikä tekee bakteerista pahan? Taudinaiheuttamiskyky, patogeenisyys riippuu taudinaiheuttamis- eli virulenssitekijöistä Virulenssitekijöiden avulla bakteerit selviävät hengissä ja lisääntyvät isäntäorganismissa Kullakin patogeenillä on oma spesifinen valikoima virulenssitekijöitä Bakteerit tarvitsevat eri tekijöitä infektioprosessin eri vaiheissa Virulenssitekijöiden kaksi pääluokkaa 1. Tekijät, jotka edistävät kolonisaatiota 2. Tekijät, jotka vahingoittavat isäntää

7 Eläinten puolustusmekanismit Evoluution aikana erilaisia puolustusmekanismeja on kehittynyt mikrobeja vastaan Iho ja limakalvot Mikrobisidisia aineita Kudoksissa ja kudosnesteissä Synnynnäinen immuniteetti Mikrobisidiset aineet Komplementtijärjestelmä seerumissa fagosyytit Viestintäjärjestelmä Bakteerien läsnäolo laukaisee kemiallisten välittäjäaineitten leviämisen ympäri elimistön veren ja kudosnesteitten kautta Houkuttelee vaikuttajasoluja infektiopaikalle Paikallinen tulehdusreaktio

8 Synnynnäinen immuniteetti Immuunivaste Epäspesifisiä antibakteerisia aineita ja soluja Tulehdus herättää spesifisen immuunivasteen Tehokas järjestelmä Valkoiset verisolut alkavat tuottaa spesifisiä vasta-aineita Soluvälitteinen immuniteetti Stimulaatio vie 5-7 vrk Siten vain synnynnäisellä vasteella on merkitystä infektion alkuvaiheessa

9 PATOGEENISYYS = Bakteerin kyky aiheuttaa tauti VIRULENSSI = Bakteerin taudinaiheuttamiskyvyn aste VIRULENSSITEKIJÄT = Rakenteita tai bakteerien osia, jotka ovat tarpeellisia patogeenisyydelle

10 Ruttobakteeri on hypervirulentti Tekijä LD 50 i.p. Fra Pur pyv Pst Pgm Hiiri Marsu Hiiri + Fe <10 <10 < < >10 7 > >10 7 >10 8 > ~10 5 ~ >10 7 >

11 Phage therapy

12 Phage therapy PubMed: 4411 hits 568 reviews Biotechnology Letters 29: ,

13 Phage therapy

14 Contents History of PT Bacteriophages Specific features of phage therapy (PT) Safety and control EVO-project

15 APU 33/2012 /Anuliina Savolainen

16 APU 33/2012 EVO

17 History Twort and d Herelle found bacterial viruses d Herelle started phage therapy (PT) France India (cholera, plague) Egypt Tbilisi Georgia, the Eliava Phage Institute late 1920 s Antibiotics PT stopped in West Politics, personal feuds, intrigues and conflicts Continued in Soviet Union (Georgia, Tblisi Eliava Phage Institute) Poland (Wroclaw)»

18 Bacteriophages Viruses that infect bacteria are called bacteriophages phages Phage variability: From a few to hundreds of genes (5 to 300 kb) Detection of phages as holes plaques - in a bacterial lawn

19 Bacteriophages Where. Bacterial viruses (bacteriophages) occupy all those habitats of world where bacteria thrive How many. For each bacterial cell there are ten bacteriophage particles, estimated in the world Archeophages. There are also viruses specific for archea Host range. Some phages are highly specific while others are extremely broad in their host range Classification. Bacteriophage taxonomy is based on their shape and size as well as on their nucleic acid Nucleic acid. Most known bacteriophages have dsdna, some have ssdna, dsrna or ssrna

20 Virus-like particles from geothermal environment of Kamchatka Bize et al, Res Microbiol. 159:358, 2008

21 Mycobacteriophages Hatfull et al, Res Microbiol. 159:332, 2008

22 Bacteriophage - lytic development cycle Similar to animal viruses 1. Phages need a host to replicate 2. Adsorption to phage-specific receptor 3. Phage injects its genes into a cell 4. Genes are expressed 5. Early genes expressed by host RNA polymerase production of phage RNA polymerase 6. Middle and late genes expressed by phage RNA polymerase phage-specific promoter 7. DNA-replication 8. Structural phage proteins 9. Assembly of virions - more viruses are made 10. Host cell lysis

23 Life cycle of phage T4 (adapted from Mosig and Eiserling, 2006) ~30 min

24 Why PT now? Sir Alexander Fleming prophesied >60 years ago: bacteria will develop resistance to antibiotics Antibiotic resistance multiresistant bacteria almost negligible discovery rate of novel antibiotics The exploration of alternative avenues PT is one possible rescue human, animal and plant infections preventive PT to eliminate pathogenic bacteria from food products or drinking water» Listeria, Vibrio cholerae etc

25 Specific features of PT The narrow specificity of bacteriophages Need for a prior diagnosis of the infecting bacterium not such a problem with antibiotics Phages are effective against multidrug-resistant pathogenic bacteria. Restricted host specificity phages generally will not affect beneficial bacteria Phage-associated side effects are uncommon Cost of developing a phage system is cheaper than that of developing a new antibiotic

26 Acceptable phages All new phages intended for PT have to be characterized in detail Structure genomic sequence Bioinformatic analyses including all relevant databases Proof that the phage(s) for a specified application are functional

27 Lysogenic vs lytic Many phages are temperate (= lysogenic) can integrate their genome into the host bacterial genomes may cause undesired phenotypical changes the prophage may carry genes encoding virulence factors Strictly lytic (i.e., virulent) phages lack the genetic factors required for integration always enter the lytic cycle kill and lyse the infected cells. Virulent phages preferred

28 Toxin and virulence factor genes in phage genomes Horizontal gene transfer Bacteriophages serve as driving force in bacterial evolution temperate phages carry genes beneficial to host bacterium for pathogens this means virulence factor, antibiotic resistance or toxin genes

29 For most toxins, the acquisition of the toxin gene is enough to turn a non-pathogenic strain into a pathogen Diphtheria toxin Botulinum toxin Cholera toxin Corynebacterium diphteriae -phage b Clostridium botulinum -phage c-st Vibrio cholerae -phage CTXf Shiga toxins Phospholipase A2 several phages of EHEC strains slaa gene on a phage genome of Streptococci

30 Which phage to use in PT? The target organism has to be known Conventional bacterial diagnostics provides the identity of the target organism for selection of appropriate phage Use of modern rapid diagnostics methods PCR Microarrays Sequencing Antigen detection

31 Selection of the therapeutic phage Effectiveness of a given phage Preferentially should be tested against the infecting bacterial strain Not always feasible for certain pathogens A comprehensive library of therapeutically approved phages kept in standardized phage banks should cover this need Custom-treatments are possible e.g. isolation of new phage for hard-to-treat bacterial infections

32 Phage resistance Loss of receptor = loss of surface structure attenuation (if virulence factor)? Phage cocktails

33 Pharmacokinetics and dynamics of PT Pharmacokinetic information Should be available for each phage to design the most effective ways to apply PT PT is challenging to current pharmacokinetic studies Phage behave as a self-amplifying drug which causes odd kinetic phenomena Modelling the phage in vivo The self-replicative nature of PT there is a reduced need for multiple doses

34 Phage safety Despite these risks phages can be considered safe >10 8 viable phage /g often present in fresh and processed meat and meat products We eat phages in our food in significant numbers Phages are normal commensals of humans and animals especially abundant in the gastrointestinal tract

35 Harald Brüssow In view of the growing antibiotic resistance crisis, a coordinated effort by the public health sector is needed to evaluate the potential of phage therapy as an adjunct to antibiotics

36 What is needed by FDA Food and Drug Administration (FDA): S. Stibitz -the regulatory standards requested by FDA for phage approval FDA: benefits Phages are highly specific They work where alternatives have failed Are self- dosing Can be highly purified Are generally non-toxic.

37 What is needed by FDA FDA: drawbacks -Phages are highly specific requires the identification of pathogen -Resistance might also develop against phages -Pharmacokinetics of phages might not be ideal -Immune response will limit the re-use of phages after i.v. use.

38 What is needed by FDA FDA: requirements -Only obligatory lytic, non-transducing phages are used -Do not avoid clearance by the body -Use preferentially virulence factors as receptors (such that resistant mutants will be less virulent) -Are derived from the natural environment FDA: product characterization -DNA sequence of each phage -Characterized seed lot system -Growth media not raising concerns about prion diseases -Toxicity testing in an animal system -Sterility and stability testing are also needed -Use of phage cocktails is fully accepted but each phage included must show activity and stability

39 What is needed by FDA FDA: requirements -Phages meeting FDA selection criteria are considered relatively low risk - High numbers of treated humans are not needed to demonstrate their safety -According to FDA registered controlled human clinical trials are the gold standard for the efficacy evaluation of phages FDA wants to develop pragmatic regulatory guidelines for phage use

40 Phage therapy initiative in Finland Aim: to set up phage therapy laboratory in Finland Funding: Modest 2-yr funding for MSc student -setting up phage collection against ESKAPEE Clinical ESKAPEE isolates (10 of each) from HUSLAB Clinical study of wound infections

41 Bacteriophage hunting in Finland Bacteriophages specific for: Enterococcus faecium- (Efm) Staphylococcus aureus- (Sau) Klebsiella pneumoniae-(kpn) Acinetobacter spp- (Aci) Pseudomonas aeruginosa- (Pae) Escherichia coli- (Eco) Enterobacter cloacae- (Ecl) Yersinia enterocolitica- (Yen3/8/9) fta-eco01 fta-eco02 fta-eco03 fta-eco04 fta-ecl01 fta-ecl03 fta-ecl06 fta-ecl07 fta-ecl08 fta-kpn01 fta-kpn02a fta-kpn03b ftu-eco01 ftu-ecl02 ftu-kpn01 ftu-kpn02 Tampere Helsinki fou-eco01 fou-ecl01 fou-ecl03 fou-kpn02 fou-kpn03 fku-eco01 fku-eco02 fku-ecl02 fku-kpn02a fku-kpn02b fku-kpn03a fku-kpn03b fku-kpn04 fhe-eco02 fhe-eco03 fhe-eco04 fhe-eco05 fhe-eco09 fhe-ecl05 fhe-ecl06 fhe-kpn01 fhe-kpn03a fhe-kpn03b fhe-yen3-01 fhe-yen8-01 fhe-yen9-01 fhe-yen9-02 fhe-yen9-03 fhe-yen9-04 fhe-yen9-05 fhe-yen9-06 fhe-yen9-07

42 Phage isolations from hospital sewages Hyvinkää hospital fhy-eco03 fhy-ecl01 fhy-ecl02 fhy-ecl04 fhy-aci01 Peijas hospital Porvoo hospital fpo-eco01 fpo-pae01 Lohja hospital flo-eco01 flo-ecl01, flo-pae01 flo-aci01 Midwife College hospital Jorvi hospital fjo-eco01 fjo-ecl01 Meilahti hospital fme-pae01 Surgery hospital Töölö hospital

43 Phage hunting summary Jätevesinäyte Rikastukset Näytteestä eristettyjen faagien lukumäärä Yhteenveto löydetyistä faageista Helsinki 3 18 Yen: 9, Eco: 5, Ecl: 2, Kpn: 2 Tampere 2 10 Eco: 3, Ecl: 4, Kpn: 3 Kuopio 2 6 Eco: 2, Ecl: 1, Kpn: 3 Turku 2 4 Eco: 1, Ecl: 1, Kpn: 2 Oulu 2 5 Eco: 1, Ecl: 2, Kpn: 2 Jyväskylä Ei vielä (1) Lahti Kesken (2) Rovaniemi 2 7 Kpn: 3, Pae: 3, Sau: 1 Kaikki kaupunkijätevedet 16 Yen: 9, Eco: 13, Ecl: 10, 51 Kpn: 15, Pae: 3, Sau: 1 Hyvinkään sairaala 2 5 Eco: 1, Ecl: 3, Aci: 1 Töölön sairaala Lohjan sairaala 2 4 Eco: 1, Ecl: 1, Pae: 1, Aci: 1 Jorvin sairaala, Espoo 2 2 Eco: 1, Ecl: 1 Peijaksen sairaala, Vantaa Meilahden tornisairaala 2 1 Pae: 1 Porvoon sairaala 2 2 Eco: 1, Pae: 1 Kätilöopiston sairaala Kesken (2) Kirurginen sairaala 2 3 Pae: 2, Sau: 1 Länsi-Uudenmaan Ei vielä (1) sairaala, Tammisaari Kaikki sairaalajätevedet Eco: 4, Ecl: 5, Pae: 5, Aci: 2, Sau: 1 Kaupungit + sairaalat 35 (osa pooleina) 68 (59 ESKAPEE) Yen: 9, Eco: 17, Ecl: 15, Kpn: 15, Pae: 8, Aci: 2, Sau: 2

44 Applications Tbilisi, Georgia Dermatology deep infections Furunculosis Abscesses Steel factory sanitary unit used phage to treat cuts, wounds and injuries 7-10 intradermal injections around the infected site. Phage in bandage. Spray Staphylococci Streptococci Clostridium perfringens 75% cure in 4-8 days more efficient in acute infections less scarring