adamianis paTogeni qoleras jgufis baqteriebis da maT ... · regionSi (Tbilisis SemogarenSi arsebuli tbebi, Savi zRva) Catarebulma kvlevebma aCvena saqarTvelos sarekreacio wyalsatevebSi

ivane javaxiSvilis saxelobis Tbilisis saxelmwifo universiteti

zust da sabunebismetyvelo mecnierebaTa fakulteti

mimarTuleba: imunologia, mikrobiologia

TinaTin elbaqiZe

adamianis paTogeni qoleras jgufis baqteriebis da maT mimarT

specifikuri baqteriofagebis gamoyofa, daxasiaTeba da

mravalferovneba

disertacia

biologiis doqtoris akademiuri xarisxis mosapoveblad

samecniero xelmZRvanelebi: marine TediaSvili, biologiis mecnierebaTa doqtori

nino gaCeCilaZe, biologiis mecnierebaTa doqtori

Tbilisi 2015

1

anotacia

Uukanasknel wlebSi klimatis cvlilebasTan dakavSirebiT did interess iwvevs wylis avtoqtonuri mikrofloris, gansakuTrebiT ki paTogenuri vibrionebis ekologiasa da biomravalferovnebasTan dakavSirebuli sakiTxebi. V. cholerae da misi monaTesave saxeoba V. mimicus-i zRvis, mtknar da mciremarilian wylian garemoSi gavrcelebuli baqteriebia. V. cholerae-s O1 da O139 serotipebs aqvT mkafiod gamoxatuli klinikuri da epidemiuri mniSvneloba, iwveven ra swrafad gavrcelebad, pandemiuri potencialis mqone, saSiS, nawlavur daavadebas-qoleras. saqarTvelos Tbili, subtropikuli klimati xelsayrel pirobas qmnis paTogenuri vibrionebis gamravlebisa da gavrcelebisaTvis. paTogenis drouli deteqcia garemoSi, Zalzed mniSvnelovania misi efeqturi zedamxedvelobisaTvis. qoleras jgufis vibrionebis mimarT specifikuri baqteriofagebis intensiuri daxsiaTeba saSualebas iZleva ukeT iyos Secnobili maTi populaciis struqturis cvlilebis da paTogenurobis meqanizmebis sakiTxebi.Qwylian garemoSi arsebuli baqteriuli virusebi zemoqmedeben wylismieri infeqciebis eTiologiur struqturaze. amdenad fagebis profili da raodenobrivi Semcveloba mniSvnelovan informacias iZleva paTogenebis Sesaxeb, am ukanasknelTa ararsebobis SemTxvevaSic ki. warmodgenili kvlevis ZiriTadi mizani iyo saqarTvelos sxvadasxva wylis rezervuarSi qoleras jgufis vibrionebis - V. cholerae-s da V. mimicus-is da maT mimarT aqtiuri baqteriofagebis gamovlena, gamoyofa da daxasiaTeba sxvadasxva mikrobiologiuri, molekuluri da imunologiuri meTodebis gamoyenebiT. Catarebuli kvlevisaTvis Seqmnili iyo Stamebis samuSao koleqcia, romelic moicavda V. cholerae-s 846 da V. mimicus-is 11 daxasiaTebul izolats, maT Soris V. cholerae-s epidemiuri serotipebis Stamebs. kvlevis procesSi gamoyofil iqna V. cholerae-s mimarT specifikuri 82 da V. mimicus-is mimarT specifikuri 7 pirveladi faguri izolati. maTi Semdgomi Seswavlis procesSi SerCeul iqna V. cholerae-s mimarT specifikuri 24 da V. mimicus-is mimarT specifikuri 7 baqteriofagi. moxda am baqteriofagebis detaluri daxasiaTeba da dajgufeba sxvadasxva biologiuri Tvisebebis (lizisuri speqtri, virionis morfologia, mgrZnobeloba fiziko-qimiuri faqtorebis mimarT) da genetikuri maxasiaTeblebis mixedviT. Ddadginda baqteriofagebis maRali mravalferovneba da maTi gamoyenebis potenciali patroni, paTogenuri baqteriebis gamovlenisa da baqteriuli Stamebis diferencirebis mizniT, aseve qoleris da msgavsi gastroenteritebis profilaqtikisa da Terapiis mizniT gamoyenebisaTvis.

sadisertacio naSromSi warmodgenili kvleva ganxorcielda giorgi eliavas sax.Bbaqteriofagiis, mikrobiologiis da virusologiis institutis mikrobuli ekoologiis laboratoriaSi, DTRA CBR GG-13 grantis farglebSi.

2

Iv. Javakhishvili Tbilisi State University Faculty of Exact and Natural Sciences

Department: Immunology, Microbiology

Tinatin Elbakidze

Isolation, Characterization and Diversity of Human Pathogenic Vibrios of V. cholerae Group and Their Corresponding Bacteriophages

This Dissertation is submitted in accordance with the requirements for the degree of Doctor of

Philosophy in Biology

Research Directors: Marina Tediashvili, Ph.D Nino Gachechiladze, Ph.D

Tbilisi 2015

Abstract

In recent years, there has been a renewed interest in understanding of the ecology and biodiversity of water autochthonous flora and human pathogenic vibrios, in particular -Vibrio cholerae and related species-V. mimicus-naturally occurring bacteria in marine, fresh-, and brackish water environments, causing various forms of gastroenterites. V. cholerae O1 and O139 is an etiological agent of the most serious diarrheal disease -cholera that has the potential to appear in explosive outbreak, epidemic or even pandemics. Warm and subtropical climate of Georgia may provide a favorable condition for reproduction and distribution of pathogenic vibrios. Timely detection of V. cholerae in environmental sources is of a great importance for efficient surveillance and contol of cholera. Intensive characterization of phages specific to V. cholerae and V. mimicus may provide a useful information on population structure of these clinically important bacteria and mechanisms of thier pathogenecity. Abundance and profile of V. cholerae-specific bacteriophages in water environment may have prognostic or retrospective indication for cholera outbreaks. The main goal of this study was to enumerate and characterize bacteriophages specific to V. cholerae and V. mimicus, naturally-occurring in Georgian water environment, by use of different microbiological, molecular and immunological methods. For the study purpose a working strain collection, consisting of 846 strains of V. cholerae, including strains belonging to O1 and O139 serotypes, and 11 strains of V. mimicus strains was created. Eighty two phages specific to V. cholerae and 7 phages specific to V. mimicus were collected and preliminarily characterized. Among them 24 phages specific to V. cholerae and 7 V. mimicus –specific phages were further characterized by basic biological features (lytic spectrum, virion morphology, life cycle phases, sensitivity to physical and chemical factors etc.) and genetic peculiarities. Obvious diversity of studied phages specific to cholera group vibrios was revealed, as well as their potential for as phage indicators, and means for subtyping of host bacterial population and the infection control.

The presented work was conducted in the laboratory of Microbial Ecology of George Eliava Institute of Bacteriophages, microbiology and Virology, Tbilisi, Georgia, supported by DTRA CBR grant GG-13

3

Sinaarsi Tavfurceli----------------------------------------------------------------------------------------------------------------1

anotacia----------------------------------------------------------------------------------------------------------------------2

Sinaarsi-----------------------------------------------------------------------------------------------------------------------4

Sesavali----------------------------------------------------------------------------------------------------------------------6

I. literaturuli mimoxilva----------------------------------------------------------------------------------9

1.1. qoleras jgufis vibrionebis zogadi daxasiaTeba ----------------------------------------9

1.1.1. gvaris Vibrio zogadi daxasiaTeba----------------------------------------------------------------------9 1.1.2. qoleras jgufis vibrionebis klasifikacia da tqsonomia---------------------------10

1.1.3.istoriuli cnobebi V. cholerae-ze----------------------------------------------------------------------11 1.1.4. V. cholerae-s serotipebi, paTogenoba da paTogenobis faqtorebi------------------12 1.1.5. V. cholerae ara- O1------------------------------------------------------------------------------------------------14 1.1.6. V. cholerae –gavrceleba da ekologia---------------------------------------------------------------14 1.1.7. daavadeba qolera da winaswarganwyoba-----------------------------------------------------------17

1.2. baqteriofagebi---------------------------------------------------------------------------------------------------18

1.2.1. baqteriofagebis aRmoCena --------------------------------------------------------------------------------18

1.2.2. baqteriofagebis klasifikacia -----------------------------------------------------------------------19

1.2.3. baqteriofagebis ZiriTadi tipebi--------------------------------------------------------------------22

1.2.4. baqteriofagis ganviTarebis ZiriTadi stadiebi--------------------------------------------23

1.2.4.1. baqteriofagis adsorbcia da dnm-is eJeqcia------------------------------------------------23

1.2.4.2. baqteriofagis ujredSida ganviTareba--------------------------------------------------------24

1.2.4.3. baqteriofagis moqmedebiT gamowveuli patronujredis lizisi---------------25

1.2.4.4 virulenturi baqteriofagebis sasicocxlo cikli ----------------------------------25

1.2.4.5. zomieri baqteriofagebis sacicoxlo cikli----------------------------------------------27

1.2.5. baqteriofagebis biologiuri Tvisebebi da garemo faqtorebis mimarT

rezistentoba----------------------------------------------------------------------------------------------------28

1.2.6. baqteriofagebis daxasiaTebisa da identifikaciis meTodebi----------------------28

1.2.7. Vibrio-s gvaris baqteriebis mimarT specifikuri baqteriofagebi---------------30

1.2.8. fagoTerapia da istoriuli konteqsti----------------------------------------------------------30

II.M masalebi da meTodebi---------------------------------------------------------------------------------------34

2.1. kvlevaSi gamoyenebuli masalebi --------------------------------------------------------------------34

2.2. kvlevaSi gamoyenebuli meTodebi---------------------------------------------------------------------38

2.2.1. Ria wyalsatevebidan aRebuli wylis sinjebis damuSaveba-------------------------38

2.2.2. Vibrio-s gvaris baqteriebis gamoyofa-------------------------------------------------------------38 2.2.3. baqteriuli Stamebis biologiuri Tvisebebis Seswavla------------------------------39

2.2.4. baqteriofagebis gamoyofa -----------------------------------------------------------------------------44

2.2.5. baqteriofagebis biologiuri Tvisebebis SeswavlaF------------------------------------44

2.2.6. baqteriofagebis gamravleba----------------------------------------------------------------------------46

2.2.7. fiziko-qimiuri faqtorebis zemoqmedeba baqteriofagebze -------------------------47

4

2.2.8. baqteriofagebis genetikuri Tvisebebis Seswavla---------------------------------------48

III kvlevis Sedegebi-------------------------------------------------------------------------------------------------49

3.1. qoleras jgufis vibrionebis koleqciis Seqmna----------------------------------------------49

3.2. qoleras jgufis vibrionebis izolatebis biologiuri Tvisebebis

Seswavla----------------------------------------------------------------------------------------------------------------52

3.3. V. cholerae-s toqsigenuri Stamebis pirdapiri gamovlena sxvadasxva

imunologiuri meTodiT---------------------------------------------------------------------------------------57

3.4. V. cholerae-s da V. mimicus-is mimarT specifikuri baqteriofagebis gamoyofa

da gavrceleba------------------------------------------------------------------------------------------------------66

3.5. V. cholerae-s da V. mimicus-is mimarT specifikuri baqteriofagebis

daxasiaTeba----------------------------------------------------------------------------------------------------------70

3.5.1. batqriofagebis morfologiuri parametrebi--------------------------------------------------70

3.5.2. fagebis lizisuri aqtivobis speqtri ------------------------------------------------------------73

3.5.3. patron-ujredTan urTierTqmedebis fazebi -------------------------------------------------76

3.5.4. baqteriofagebis serologiuri Tvisebebis Seswavla antifaguri Sratis

gamoyenebiT---------------------------------------------------------------------------------------------------------76

3.5.5. V. Cholerae O1–is mimarT specifikuri baqteriofagebis zegavlena Stamebis

mier qoleris toqsinis produqciaze--------------------------------------------------------------79 3.6. fiziko-qimiuri faqtorebis zegavlena baqteriofagebis

sicocxlisunarianobaze -----------------------------------------------------------------------------------80

3.7. baqteriofagebis genetikuri maxasiaTeblebi---------------------------------------------------90

IV Sedegebis ganxilva----------------------------------------------------------------------------------------------94

V daskvnebi--------------------------------------------------------------------------------------------------------------101

VI gamoyenebuli literatura---------------------------------------------------------------------------------102

5

Sesavali

sasmeli wylis da sarekreacio mizniT gamoyenebuli wyalsatevebis mikrobuli dabinZureba erT-erT mniSvnelovan problemas warmoadgens, rogorc ganviTarebadi, aseve ganviTarebuli qveynebisTvis da sazogadoebrivi jandacvis mTavar sakiTxad ganixileba. diareuli daavadebebis gamomwvevi sxvadasxva enteropaTogenebi avadobis da sikvdilianobis mniSvnelovan mizezad gvevlinebian, gansakuTrebiT, mcire asakis bavSvebSi. dabinZurebul wyalTan kontaqtis Sedegi SeiZleba iyos wylismieri infeqciebis gavrceleba. rekreaciuli mizniT gamoyenebuli wylebis mikrobuli flora, SesaZloa, moicavdes iseT mikrobul paTogenebs, rogoricaa: Salmonella spp., Shigella spp., Vibrio spp., Escherichia coli, Clostridium spp. da sxva, adamianis enterovirusebs da umartivesebs [82,85,188]. infeqciebis gamomwvevi avtoqtonuri organizmebis wyalSi gamravlebis erT-erTi ganmapirobebeli faqtoria garemos klimaturi pirobebi da aseve anTropogenuri gavleniT gamowveuli dabinZureba. Tbilisis SemogarenSi arsebul tbebSi da zRvis sanapiro zolSi regularuli monitoringis Sedegad dagrovili informacia endemur regionebSi gavrcelebuli enteropaTogenebis Semcvelobaze, maT epidemiologiur da klinikur mniSvnelobaze, gamoyenebuli antimikrobuli agentebis mimarT mgrZnobelobaze da sxv. saSualebas iZleva daigegmos prevenciuli zomebi wylismieri infeqciuri daavadebebis gavrcelebis Tavidan asacileblad. ukanasknel wlebSi klimatis cvlilebasTan dakavSirebiT gansakuTrebul interess iwvevs qoleras jgufis vibrionebis ekologiasa da biomravalferovnebasTan dakavSirebuli sakiTxebi. adamianis paTogeni arahalofiluri, qoleras jgufis vibrionebi V.cholerae da V. mimicus zRvis da mtknari wyalis bunebrivi binadaria, xSirad asocirebuli arian zRvis organizmebTan: TevzebTan, zooplanqtonTan, moluskebTan. V. cholerae O1-is serotipis “klasikuri” da “El Tor” biotipebi, aseve SemdgomSi aRmoCenili V. cholerae O139-is [154] serotipi mwvave infeqciuri daavadebis-qoleras eTiologiuri agentia. qolera iwvevs wvrili nawlavis dazianebas, diareas pirRebinebiT, dehidratacias, oligurias da kolafss. qolera vrceldeba dabinZurebuli wyliT, zRvis da sxva sakvebi produqtebiT. aSS-s CDC-is klasifikaciiT V. Cholerae gansakuTrebiT saSiSi paTogenebis me-2 kategorias ganekuTvneba radgan aqvs pandemiis ganviTarebis maRali potenciali. saxeoba- V. mimicus bioqimiuri parametrebiT axlos dgas V. cholerae-sTan, Tumca diareis SedarebiT ioli formis gamomwvevia. qolera seriozul problemas warmoadgens tropikuli klimatis, aseve cudi sayofacxovrebo pirobebisa da higienis mqone ganviTarebadi qveynebisTvis. ukanasknel wlebSi (2010-2014) V. cholerae-s epidemiuri serotipebis mier gamowveuli daavadebis yvelaze did afeTqebas adgili qonda haitize, kongos demokratiul respublikaSi, siera leoneSi, meqsikasa da samxreT sudanSi [45,189]. saqarTvelos Tbili, subtropikuli klimati xelsayrel pirobas qmnis vibironebis gamravlebisa da ganviTarebisTvis. dabali da saSualo marilianoba, aseve temperatura, romelic 18°C-s aRemateba, qmnis sasurveli

6

zrdis pirobebs Vibrio-s gvaris warmomadgenlebisTvis. adreul XX saukunis 70-ian wlebSi saqarTvelos teritoriaze adgili qonda qoleras 8 SemTxvevas, rac gamowveuli iyo “El Tor” biotipiT. daavadebis afeTqeba lokalizebuli iyo saTanado karantinuli zomebis miRebiT da risk jgufebis profilaqtikuri mkurnalobis gziT [39]. ukanasknel wlebSi samxreT kavkasiis regionSi (Tbilisis SemogarenSi arsebuli tbebi, Savi zRva) Catarebulma kvlevebma aCvena saqarTvelos sarekreacio wyalsatevebSi wylis xarisxis gauareseba. unda aRiniSnos, rom moqmed sasoflo-sameurneo arealSi arsebuli mciremariliani wylis rezervuaris-kumisis tbis dabinZurebis xarisxi Warbobda Tbilisis zRvis-mtkaniri wylis rezervuaris mikrobuli dabinZurebis xarisxs [68]. garemo-klimaturi pirobebi da maTi sezonuri cvlileba did zegavlenas axdens paTogenuri vibrionebis, aseve maTi

specifikuri baqteriofagebis gavrcelebaze. baqteriofagebi sicocxlis yvelaze gavrcelebuli formebia dedamiwaze. bunebriv pirobebSi baqteriofagebs did roli eniWebaT baqteriuli populaciis regulaciasa da genetikur mravalferovnebaSi, rac baqteriul Stamebs da saxeobebs Soris genetikuri gacvlis gziT xorcieldeba [91]. baqteriofagebi medicinasa da veterinariaSi ganixilebian anti-infeqciur agentebad, romelTac gamorCeuli antibaqteriuli moqmedeba axasiaTebT. antibiotikebis mimarT multirezistentuli baqteriuli Stamebis aRmocenebam Tanamedrove medicinaSi gazarda interesi fagebis mimarT. fagebs aqvT specifikuri moqmedeba, maTi samizne baqteriuli ujredia da ara adamianis an sxva eukariotuli ujredebi. es aris mniSvenlovani gansxvaveba faguri preparatisa antibiotikisgan, romelic moqmedebs, rogorc paTogenis, aseve normaluri mikrofloris winaaRmdeg, viTardeba mikrobuli disbalansi, rasac mivyavarT meoradi infeqciebis ganviTarebisken. baqteriofagebi gamoyeneboda baqteriuli infeqciebis prevenciisa da samkurnalod [17,148] antibiotikebis aRmoCenamdec. Terapiuli faguri preparatebi iwarmoeboda farmacevtuli industriis mier yofil sabWoTa kavSirSi (1923 wlidan) da aRmosavleT evropaSi (1952 wlidan). klinikurma fagoTerapiis cdebma aCvena fagebis profilaqtikuri da Terapiuli gamoyenebis dadebiTi mxare. adamianSi virulenturi fagebis gamoyenebis uaryofiTi maCveneblebi faqtobrivad ar iyo aRwerili [169]. miuxedavad fagebis Terapiuli mizniT gamoyenebis usafrTxoebisa, Cveulebriv maTi farTo moxmareba dasavleTis ganviTarebul qveynebSi ar xdeba da fagebis moqmedebis efeqturoba jer kidev sakamaTo sakiTxia. garda amisa erT-erT SezRudvas warmoadgens fagebis maRali specifikuroba samizne baqteriuli saxeobis mimarT. baqteriofagebis praqtikuli gamoyenebis areali gansxvavdeba, gamomdinare maTi lizisuri speqtridan. aRwerilia maTi indikatorebad da traserebad bunebrivi, Ria wyalsatevebis dabinZurebis xarisxis, wyarosa da gavrcelebis gzebis dadgenis mizniT gamoyeneba. V. cholerae O1-is mimarT specifikuri baqteriofagebi odiTganve interesis sferos warmoadgenda-infeqciebis sadiagnostikod. misi baqteriofagebis specifikuri nakrebebi dRemde gamoiyeneba V. cholerae O1-is serotipis “klasikuri” da “El Tor” biotipebis

7

gansasxvaveblad. qoleras epidemiis meTvalyureobisTvis fagotipireba farTod miRebuli meTodia [24]. saerTaSoriso fagotipirebis sqema, romelic mowodebulia basus da mukerjis [24] mier, moicavs 5 fags (I, II, III, IV da V). Tumca droTa ganmavlobaSi arsebuli sqema arasakmarisad specifikuri aRmoCnda da 1993 wels ”El Tor”-is biotipis StamebisTvis fagotipirebis axali sqema iyo mowodebuli [51]. daavadeba qolera misi aRmocenebis adgilidan sakmaod swrafad vrceldeba. epidemiologiuri TvalsazrisiT mniSvnelovania daavadebis gavrcelebis Tvalyuris devneba. Camdinare wylebSi V. cholerae O1-is Stamebisa da Sesabamisi fagebis deteqcia SesaZloa iyos specifikuri saSualeba qoleras afeTqebebis winaswar ganWvretisa manam, sanam, SemTxvevaTa mniSvnelovani raodenoba iCens Tavs an retrospeqtulad, daavadebis ukve Cavlili epizodis gamosavlenad. Cveni kvlevis ZiriTad mizans warmoadgenda saqarTvelos wylian garemoSi arahalofiluri (qoleras jgufis) vibrionebis mimarT specifikuri baqteriofagebis gamoyofa da daxasiaTeba maT mier gamowveuli infeqciuri daavadebebis riskis Sefasebisa da biomravalferovnebis dadgenis mizniT, aseve SemdgomSi samkurnalo-profilaqtikuri gamoyenebisaTvis. kvlevis ZiriTadi amocanebi iyo:

1. saqarTvelos wylian garemoSi gamoyofili (Savi zRva, lisis tba, kumisis tba, Tbilisis zRva) V. cholerae–s da V. mimicus–is daxasiaTebuli da identificirebuli baqteriuli Stamebis koleqciis Seqmna;

2. V. cholerae–s da V. mimicus–is mimarT specifikuri baqteriofagebis gamoyofa, gamravleba da klonireba;

3. baqteriofagebis morfologiuri da biologiuri Tvisebebis Seswavla;

4. baqteriofagebis stabilobis Seswavla sxvadasxva fiziko-qimiuri faqtorebis mimarT;

5. gamoyofili fagebis genetikuri daxasiaTeba da maTi mravalferovnebis Seswavla;

6. fagebis potencialis dadgena maT mier gamowveuli infeqciebis biokontrolis mizniT.

.

8

LI. literaturuli mimoxilva

1.1. qoleras jgufis vibrionebis zogadi daxasiaTeba

1.1.1. gvaris Vibrio zogadi daxasiaTeba

1964 wels mikrobiologTa gaerTianebis saerTaSoriso asociaciis mier Camoyalibda ojaxi saxelwodebiT Vibrionaceae [112] romelSic gaerTianebulia Semdegi gvaris warmomadgenelebi: Vibrio, Aeromonas, Plesiomonas, Photobacterium da Enhidrobacter. amgvari sistematika xelsayrelia Vibrionaceae-s ojaxis warmomadgenlebis (oqsidazadadebiTi, polaruli Soltis mqone, moZravi) diferenciaciisaTvis Enterobacteriacea-s ojaxis mikroorganizmTa warmomadgenlebisagan (oqsidazauaryofiTi, peritrixebi, naklebad moZravi da umoZrao). Gvari Vibrio –Vibrionaceae-s ojaxis warmomadgenelia. Ojaxi Vibrionaceae moicavs adamianisa da cxovelis oportunistul paTogenebs [63,177], aseve zRvis faunis, Tavisuflad mcxovreb qemoheterotrofebs da/an komensalebs [86,90]. Ojaxi Vibrionaceae-s warmomadgenlebi qmnian wyliani garemos predominantur heterotroful baqteriaTa jgufs [161]. vibrionebis roli wylian garemoSi moicavs biodegradacias, sakvebis regeneracias da biogeoqimiur cikls [58]. vibrionebi wylis mikrobiotas uxvi komponentebia [178]. Vibrio-s gvaris warmomadgenlebSi Sedis 6 saxeoba arahalofiluri baqteriebis, maT Soris aris adamianis paTogenebi V. cholerae da V. mimicus, aseve, 45 saxeoba halofiluri vibrionebis [28], romelTagan ramdenime adamianis paTogens warmoadgens (V. alginolyticus, V. cincinatensis, V. damsela. V. fluvialis, V. furnissii, V. parahaemolyticus, V. vulnificus, V. hollisae). vibrionebi gramuaryofiTi, moRunuli Cxirebia. maTi zoma meryeobs 1,4-3mkm sigrZeSi da 0,5-0,8mkm siganeSi. damaxasiaTebelia erTi an ramdenime polaruli Solti, romelic ujredis kedlis gare membranis gagrZelebas warmoadgens da uzrunvelyofs maT moZraobas. isini oqsidazadadebiTi (gaaCniaT indofenoloqsidazis gamoyofis unari) mikroorganizmebia (gamonakliss warmoadgens V. metschnikovii da V. gazogenes). vibrionebi fakultatur anaerobebs miekuTvnebian. maTi gamravlebisTvis xelsayreli temperatura 30 gradusia, Tumca umetesoba maTgani kargad mravldeba 15-dan 37 graduszec. am gvaris TiTqmis yvela warmomadgenlis cxovelmyofelobisaTvis saWiroa natriumis ionebi da wyalbad ionTa maRali koncentracia. kargad mravldebian pH 9 da wyalbad ionTa ufro maRali koncentraciis drosac. maTTvis damRupvelia mJava garemo.

9

1.1.2. qoleras jgufis vibrionebis klasifikacia da tqsonomia

cnobilia vibrionebis 70-ze meti saxeoba. isini pirobiTad or jgufad iyofa: arahalofilur da halofilur vibrionebad. arahalofilur vibrionebs miekuTvneba qoleras jgufis vibrioni- V.cholerae da V. mimicus. es ukanasknelni miekuTvnebian Proteobacteria-s tips, Gamma Proteobacteria-s klass, Vibrionales-is rigs, Vibrionaceae-s ojaxs da Vibrio-s gvars [73] (sur1). gvari Vibrio moicavs 45-mde saxeobas [28]. 12 saxeoba mudmivad gamoiyofa adamianis klinikuri sinjebidan, daavadebebi, romlebSic isini arian CarTulni moicavs: diareas, septicemias da WrilobiT infeqciebs [72,98,112]. berjis sistemuri baqteriologiis (2005) mixedviT gvari Vibrio-s 12 saxeobidan 11 adamianis aSkara paTogens warmoadgens iwvevs ra diareas da eqstraintestinalur infeqciebs. sami saxeoba: V. cholerae, V.parahaemolyticus da V.vulnificus-is mier adamianSi gamowveuli infeqciebis sixSire yvelaze didia. gvari Vibrio klinikuri daavadebis sami ZiriTadi sindromis gamomwvevia, esenia: gastroenteritebi, WrilobiTi infeqciebi da septicemia.

sur.1. V. cholera-s da V. mimicus-is taqsonomia

samefo• Bacteria

tipi• Proteobacteria

klasi• Gammaproteobacteria

rigi• Vibrionales

ojaxi• Vibrionaceae

gvari• Vibrio

saxeoba• V. cholerae• V. mimicus

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1.1.3. istoriuli cnobebi V. cholerae-ze

1854 wels italielma mecnierma filipo pacinim florenciaSi qoleris epidemiis mimdinareobisas aRmoaCina gvari Vibrio-s pirveli warmomadgeneli, qoleris daavadebis gamomwvevi V. cholerae. man mikroskopiT Seiswavla am daavadebiT daRupul adamianTa nawlavis lorwovanis mdgomareoba da yvela maTganSi V. cholerae aRmoaCina. mogvianebiT r.koxma [123] egvipteSi (1883w) da indoeTSi (1884w) qoleris me-5 afeTqebis mimdinareobisas, Jelatinian agarze gamoyo V. cholerae–s sufTa kultura. man da misma jgufma Seiswavles fataluri SemTxvevebis didi ricxvi da aRmoaCines, rom yvela daRupulis nawlavis qsovilebSi SeiniSneboda V. cholerae-s Cxiris arseboba. koxma da misma jgufa aramarto daadgina, rom daavadebis gamomwvevi swored es mikroorganizmi iyo, aramed isic, rom es daavadeba am mikroorganizmiT dabinZurebuli wylis meSveobiT vrceldeba. 1905 wels aCardma da bensaudma SeimuSaves qoleris SratiT aglutinaciis reaqciis meSveobiT, qoleris vibrionis identifikaciis meTodi, ramac migviyvana gvari Vibrio-s warmomadgenlebis or jgufad dayofamde: qoleris SratiT aglutinirebadi vibrionebi (qoleruli vibrionebi) da nag-vibrionebi. Tumca am ori jgufis warmomadgenlebis bioqimiuri Tvisebebis Seswavlam maTi diferenciaciis nacvlad isini metad daaaxlova erTmaneTs. f.gotSilixma 1906 wels eltoris sakarantino sadguridan kuW-nawlavis infeqciiT daRupul adamianTa nawlavebidan gamoyo vibrionis 38 Stami. 6 maTgani aglutinirdeboda qoleris SratiT. miuxedavad avtoris dasabuTebisa, rom es Stamebi kulturaluri da antigenuri TvisebebiT msgavsi iyo qoleris vibrionebis, epidemiologiuri TvalsazrisiT isini ar miakuTvnes qoleras, radgan ar dasturdeboda maTi xangrZlivi matarebloba. 1906 wels eltoris vibrionebTan dakavSirebul kvlevebSi mniSvnelovani wvlili Seitanes mecnierebma r.krausma da e.pribram, romlebmac Catarebuli cdebis safuZvelze eltoris vibrionebSi miuTiTes hemotoqsinisa da egzotoqsinis arseboba, rac im dros miRebuli kriteriumebiT araqoleruli vibrionebis maxasiaTeblad iTvleboda. amrigad zemoT aRwerili Stamebi miakuTvnes araqolerul vibrionebs da eltors paraqoleruli vibrionebi uwodes. aseTma mcdarma gadawyvetilebam tragikul SdemTxvevamde migviyvana. paraqolera eltoris pirveli msxvilmasStabiani afeTqeba 1938 wels sulavesis kunZulze dafiqsirda. daavadeba inficirebulTa 70%-Si letalurad dasrulda. V. cholerae- wylismieri daavadeba- qoleras eTiologiuri agentia, romlis ZiriTad gamomwvevad V. cholera O1 gvevlineba [60,143,146]. ukanaskneli aTwleulebis manZilze qoleras epidemia aRniSnulia samxreT amerikis afrikis da aziis 75 qveyanaSi. Yovelwliurad mTeli msoflios masStabiT xdeba sporaduli SemTxvevebis aRricxva. qoleras pirveli pandemia dafiqsirda 1817 wels. gasuli ori saukunis ganmavlobaSi 7 pandemiaa aRricxuli [118,152]. qoleras pandemiebi Cveulebriv saTaves indoeTis naxevarkunZulidan, gangis deltidan iRebda da Semdeg vrceldeboda sxva kontinentebze [75,159]. ukanaskneli (2010-2014), qoleras yvelaze mniSvnelovani afeTqeba moxda haitize, kongos demokratiul respublikaSi, siera leoneSi, meqsikasa da samxreT

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sudanSi [45,189]. qoleras me-5 (1881-1896) da me-6 (1899-1923) pandemiebis gamomwvevad miCneulia V. cholerae O1 “klasikuri” biotipi [118]. me-7 pandemia daiwyo 1961 wels da gamowveuli iyo sxva biotipis– “El Tor”-is mier. “El Tor”-is biotipi gamoyofili iyo piligrimebidan, “El Tor”-is sakarantino banakidan. sxva pandemiebisgan gansxvavebiT, romlebic saTaves indoeTis naxevarkunZulis gangis deltidan iRebdnen [48,131,152] me-7 pandemiam sulavesis kunZulze, indoneziaSi iCina Tavi [114], 1992 wlis bolos madrasSi (indoeTi) da SemdgomSi samxreT bangladeSSi mimdinare qoleris epidemiis Seswavlam

gamoavlina qoleris axali gamomwvevi- O139 “Bengal” serotipi [53,154]. epidemiuri Stamebis V. cholerae O1 da V. cholerae O139-s ZiriTadi damaxasiaTebeli Tviseba, maT mier qolera toqsinis (CT) producierebaa, rac diareis gamowvevis mTavari ganmsazRvreli faqtoria [118]. 1962 wels, roca V. cholera “El Tor”-is Stamma TiTqmis mTeli msoflio moicva da pandemiis saxe miiRo, msoflio jandacvis organizaciis generalurma ansambleam es daavadeba karantinulad gamoacxada da masze saerTaSoriso sanitaruli wesebi ganavrco.

1.1.4. V. cholerae-s serotipebi, paTogenoba da paTogenobis faqtorebi

rogorc qolerul ise araqolerul vibrionebs gaaCniaT saerTo tipospecifiuri, Termostabiluri O-antigeni da saxeobaspecifiuri H-antigeni. amis safuZvelze gvari VIbrio-s warmomadgenlebi dayves serojgufebad. romelTagan pirvelSi gaerTianda qoleris vibrioni da miiRo saxelwodeba V. cholera O1. dResdReisobiT 200-mde serojgufia cnobili. O1 serojgufs miekuTvnebian biotipebi “klasikuri” da “El-Tor”. G gvari VIbrio-s saxeobebis antigenur struqturas gansazRvravs baqteriuli antigenebis mTavari klasebi: somaturi (O), kafsularuli (K) da flagelaruli (F), romlebic asocirebulia baqteriuli lipopolisaqaridul eqstracelularul SresTan an kafsulasTan da flagelumTan. V. cholerae iyofa or serojgufad: V. cholerae O1 da V. cholerae ara-O1. es ukanaskneli morfologiurad da bioqimiurad msgavsia V. cholerae O1 –is, ar aglutinirdeba O1 jgufis specifikuri antiSratiT [56,71]. V. cholerae-s Soris 180 O antigenia cnobili, Tumca serojgufebi O1 da O139 moicaven yvela im Stams, romelTac gamoiwvies epidemiebi da pandemiebi [158,194]. V. cholerae O1-is serotipSi gaerTianebulia 3 serotipi: ogava, inaba da hikoSima, romelTa antigenuri frmulaa: AB, AC da ABC [87]. prearsebul epidemiur StamSi tipospecifiuri, Termostabiluri O-antigenis cvlilebam SesaZloa gamoiwvios axali paTogenis Camoyalibeba, romelsac epidemiuri Stamis mimarT imunur populaciaSi daavadebis gamowvevis unari eqneba [14,154]. amgvarad iCina Tavi V. cholerae O139-is (bengali) Stamma, V. cholerae O1-isepidemiuri Stamidan O antigenis biosinTezis regionebis genetikuri cvlilebis gziT [27,146,167]. V. cholerae O139 Stami iwvevs daavadebas iseT organizmSi, romelic imunuria O1 Stamis mimarT [29,145].

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V. cholerae toqsinis (CT) warmomqmneli baqteriuli mikroorganizmia. igi asinTezebs 2 tipis toqsins: endo da egzotoqsins (enterotoqsini). daavadebis ganviTarebaSi ZiriTadi roli enterotoqsins ekuTvnis. igi ori komponentisgan Sedgeba A (ctxA) da B (ctxB). B praqtikulad aratoqsiuria da monawileobs wvrili nawlavis epiTeluri ujredebis membranis specifikur receptorebze mimagrebaSi. A toqsikuria, igi aaqtiurebs specifikur fermentebs, rac iwvevs marilebis hipersekrecias da ujredebis gauwyloebas. genebi, romlebic

akodireben CTA da CTB sinTezs kodirebulia filamenturi fagiT CTXΦ. V. cholerae mwvave diareul daavadebas iwvevs da samxreT aziis, afrikisa da laTinuri amerikis endemur saxeobas warmoadgens. CDC–is klasifikaciiT V. cholerae gansakuTrebiT saSiS paTogenebis me-2 kategorias ganekuTvneba. qoleris daavadebis gamomwvevi wyaro am mikroorganizmiT dabinZurebuli wyali an sakvebia. es daavadeba ganviTarebuli qveynebisTvis aris damaxasiaTebeli. aseT qveynebSi qoleris afeTqebebi yovelwliurad 120000-ze meti adamianis sicocxles iwiravs. cnobilia qoleras didi afeTqebebi laTinur amerikaSi momxdari 1991 wels, indoeTSi 1992-1993 wlebSi, gomaSi, zairSi 1994 wels, romlis drosac 70000 daavadebis SemTxvevidan 12000 letalurad dasrulda. qoleras epidemiologiidan aRsaniSnavia is, rom am daavadebas V. cholerae-sTvis endemur adgilebSi sezonuri xasiaTi aqvs. inficirebulebis meti wili 1-idan 5 wlamde bavSvebze modis. wlidan wlamde icvleba am mikrobis antibiotikomgrZnobeloba da epidemiuri Stamebis klonebis nairsaxeoba. 1970 wels qoleras epidemias adgili hqonda yofili sabWoTa kavSiris teritoriazec (astraxani, odesa), romelmac garkveul wilad saqarTveloc moicva (aWara). saqarTvelos Tbili, subtropikuli klimati xelsayrelia vibrionebis gamravlebisa da gavrcelebisaTvis. 70-iani wlebis dasawyisSi saqarTvelos teritoriaze qoleris 8 SemTxveva iyo dafiqsirebuli, romelTagan erTi letalurad dasrulda, Tumca daavadebis afeTqeba lokalizebuli iyo saTanado karantinuli zomebis miRebiT da risk jgufebis profilaqtikuri mkurnalibs gziT [39]. yvela es SemTxveva O1 jgufis biotipiT-eltoriT iyo gamowveuli. 80-ian wlebSi sxvadasxva nag-vibrionebi iyo gamoyofili Woroxisa da maWaxelas estuariebidan. Tumca sabWoTa kavSiris daSlis Semdgom periodSi wylisa da kvebiTi infeqciebis intensiuri monitoringis Sesaxeb gamoqveynebuli cnobebi ar arsebobs. qoleras gacilebiT mniSvnelovan afeTqebas qonda adgili Savi zRvispireTis kidev erT qveyanaSi-TurqeTSi. saxeoba V. mimicus-i bioqimiuri parametrebiT axlos dgas V. cholerae-sTan. Tavdapirvelad aRwerili iyo, rogorc bioqimiurad atipiuri V. cholerae (ar axdens saqarozis fermentacias. TCBS–is seleqtiur niadagze mwvane koloniebis saxiT izrdeba). V. cholerae-sTan SedarebiT msubuqi diareis gamomwvevia, aseve iwvevs diareas an gastrointenstinaluri traqtis infeqciebs [154].

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1.1.5. V. cholerae ara-O1 V. cholerae-s zogierTi Stamebi ar aglutinirdebian O1 antiSratiT, Tumca iwveven diareul garTulebebs. V. cholera ara-O1–is mier gamowveuli infeqciis 130 SemTxveva iyo dafiqsirebuli floridaSi 1983-dan 1993 wlamde [63]. V. cholerae ara-O1 aRwerili iyo, rogorc qoleras msgavsi daavadebis mcire afeTqebebisa da sporaduli SemTxvevebis gamomwvevi agenti [29,122, 142,144]. V. cholerae ara-O1-is warmomadgenlebi ar Seicaven toqsinis genebs, farTod arian gavrcelebulni wylian garemoSi da warmoadgenen Tavisuflad mcxovreb organizmebs [32,190]. gansxvavebiT V. cholerae O1-sgan ar axasiaTebT epidemiuri an pandemuri potenciali [32], Tumca xSirad vlindebian qoleras msgavsi diareis sporaduli SemTxvevebis [100,139,165] da lokaluri afeTqebebis [16,64] mizezad, romelsac xSirad Tan sdevs temperaturis mateba da ganavalSi sisxlis da lorwos arseboba [64,100]. V. cholerae ara-O1-is warmomadgenlebi ZiriTadad CarTulni arian ariainvaziur daavadebebSi, rogoricaa septicaemia imunitet daqveiTebul organizmebSi [157,133]. V. cholerae ara-O1-is izolatebi SesaZloa gamoiyos diareis SemTxvevebis dros da aseve garemodan, sadac qoleras aqvs adgili. Stamebi xSir SemTxvevaSi hemolizuria. adamianis infeqcia, romelic V. cholerae ara-O1-iT aris gamowveuli zRvis produqtis miRebasTan, dabinZurebul garemosTan, mtknar, mciremarilian an zRvis wyalTan da aseve mogzaurobasTan [55,65,142] aris dakavSirebuli. italiaSi aRwerili iyo V. cholerae O158-is mier gamowveuli infeqcia [81]. italiaSi farTod gavrcelebuli serovarianti iyo aseve O40 (16%), romelsac mosdevda O6 (12%) da O2, O8, O41, aseve, O64da O107 (TiToeuli-6%), yvela izolati iyo CT genis mimarT negatiuri [81]. garemoSi V. cholerae ara-O1–is Stamebi ricxobrivad aWarbebs V. cholerae O1–is Stamebs da umravlesobas am Stamebisa ar aReniSnebaT iseTi virulenturi faqtorebis arseboba, rogoricaa qolera toqsini da koregulatori pili [25,119].

1.1.6. V. cholerae–gavrceleba da ekologia

V. cholerae warmoadgens, rogorc adamianis paTogens, aseve wylis garemos bunebriv binadars [54,61].kolvelma da Tanaavtorebma (1981) gamoTqves hipoTeza, rom V. cholerae warmoadgens mciremariliani wylis da estuariebis bunebrivad arsebul mikrobul floras. Singletonma da Tanaavtorebma (1982) daadastures V. cholerae-s, rogorc estuariebis mikrobuli Tanasazogadoebis wevris utyuaroba. V. cholerae-s avtoqtonuri arseboba qoleras endemuri regionebis wylian garemoSi dadginda sxvadasxva swavlebiT [58,102,83]. sawinaaRmdegod im mosazrebisa, rom V. cholerae warmoadgens mxolod klinikur baqterias, romelic mcire xniT cocxlobs mxolod wylian garmoSi V. cholerae amJamad cnobilia, rogorc mciremariliani wylebis binadari [57]. Kolvelma da Tnaavtorebma (1977) pirvelma gamoTqves hipoTeza, rom sanapiro wylebi V. cholerae-s mniSvnelovani rezervuaria. pirdapiri deteqciis meTodebiT Seswavlili iyo V.

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cholerae O1-is simbionturi asociacia planqtonTan [103]. V. cholerae nanaxi iyo, zRvis wyalSi da sxva garemoSi, rogorc qoleras endemur, aseve qoleras ararsebul regionebSi [55,105,113,119]. V. cholerae gamoyofili iyo mravali geografiuli arealis, rogorc kontinenturi, aseve zRvis wylidan da estuariebidan [23,54,55]. dReisobiT V. cholerae miCneulia avtoqtonuri baqteriebis [55,115,116,132], maTi gavrcelebis arealis wevrad da misi arseboba ar aris korelaciaSi koliformebTan da fekalur indikatorebTan [97,132]. dReisobiT V. cholerae sxvadasxva wyliani garemos, rogoricaa okeane, estuariebi, mdinareebi da tbebi bunebriv binadrad moisazreba [57,186]. wyali (da masTan asocirebuli mikroorganizmebi, cxovelebi da mcenareebi) bunebrivi garemoa arahalofiluri da halofiluri vibironebisTvis. garemo pirobebi, masSi arsebuli organuli da araorganuli qimikatebiT, pH-iT, temperaturiT, marilianobiT, Jangbadis simWidroviT da ultraiisferi sxivebis zemoqmedebiT aregulireben vibrionebis koncentracias da gavrcelebas, rogorc mtkanr, aseve zRvis wylis ekosistemaSi [47]. faqtorebi, romlebic gansazRvraven vibrionebis gavrcelebas moicaven adamianis, cxovelis an mcenaris maspinZel organizmebs, araorganul nivTierebebs da naxSirbadis wyaros, temperaturas, marilianobas, gaxsnil Jangbads da zedapiris qvemoT arsebul siRrmes saxeobebisTvis, romlebic arseboben okeaneebSi [162]. V. cholerae-s dadebiTi nimuSebi (wylis) korelaciaSia haeris temperaturasTan [135]. V. cholerae-s mikobuli ekologiis saukeTeso magaliTia mtknar da marilian wylebSi misi gadarCenis SesaZlebloba “Tavisuflad mcxovreb mdgomareobaSi”. gavrcelebis arealia: Camdinare da mciremariliani wylebi, estuariebi, sanapiro yureebi, dabinZurebuli dinebebi, mdinareebi da tbebi. V. cholerae aseve arsebobs epibiotur formaSi, mraval, mikroskopul cocxal arsebasTan [96]. mikroorganizmebi, romlebic asocirebulebi arian V. cholerae-sTan moicaven cianobaqteriebs, fitoplanqtons (diatomebi, mtknari wylis wyalmcenareebi) da zooplanqtons [47,104]. V. cholerae Ol da Ol39–is transmisias adamianebSi adgili aqvs ZiriTadad dabinZurebuli wylisa da sakvebis moxmarebis dros (gaurecxavi xili, bostneuli, umi zRvis produti). wyali warmoadgens mTavar rols qoleras gadacemaSi. sakvebismieri qoleras afeTqebebi ZiriTadad dakavSirebulia zRvis produqtTan. moluskebi, kibosnairni da Tevzebi dominireben, rogorc qoleras gamomwvevi wyaroebi [15,153]. toqsigenuri V. cholerae Ol aRmoCenili iyo sasmelad uvargis wylebSi satvirTo gemebidan [140]. wylis frinvelebi SesaZloa iyvnen veqtorebi V. cholerae-sTvis. V. cholerae “El Tor” biotipi koncentrirdeba wylis hiacintebis (Eichhornia crassipes) zedapirze, Sesabamisad Zlierdeba misi gadarCenis SesaZlebloba da wyliT gadacemis potenciali [166]. msgavsi kavSiri iyo aRwerili V. cholerae Ol39-is da Eichhornia crassipes SemTxvevaSi [30]. V. cholerae Ol-is specifikuri kavSiris SesaZlebloba cocxal Copepod-Tan dadasturebuli iyo eleqtronuli mikroskopiiT [101]. am organizms SeuZlia mtknar da zRvis wylebSi arseboba, rogorc cocxal da kultivirebad, aseve cocxal da arakultivirebad (VBNC) mdgomareobaSi [88]. VBNC aris mdgomareoba, roca metabolurad aqtiuri ujredebi ar kultivirdebian

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mikrobiologiur niadagze [59]. VBNC-is mdgomareobaSi ujredebi ganicdian mniSvnelovan Semcirebas zomaSi da morfologiur cvlilebas, gadadian ra kokis mdgomareobaSi. VBNC-is mdgomareobaSi gadasvla SesaZloa dakavSirebuli iyos sakvebi nivTierebebis simciresTan da fizikuri faqtorebiT gamowveul stresTan. mikrokosmis eqsperimentebma cxadyo, rom VBNC-is ujredebi inarCuneben garemoSi sicocxlisunarianobas wlebis ganmavlobaSi da aqvT daavadebis gamowvevis unari [59]. V. cholerae Ol-is unari kolonizirdes da daukavSirdes krevetebis da kobrCxalebis egzoskeletons, qmnis erTgvar potencilas wylis garemoSi gadarCenisTvis [43].Alami da Tanaavtorebi (2007) swavlobdnen cocxal da arakultivirebad V. cholerae Ol-is Stamebs wylis garemos biofilmebSi da maT rols qoleras gadacemaSi. am eqsperimentis Sedegad daginda, rom arsebuli Stamebi inarCunebdnen sicocxlisunarianobas 495 dRis ganmavlobaSi, matbarias (bangladeSi) wyalsatevSi. amrigad biofilmebi SesaZloa moqmedebdes, rogorc rezervuari V. cholerae Ol-is mier gamowveul epidemiaTa SualedebSi. V. cholerae- Tanaarsebobs kibosnairTa zooplanktonTan [101,94], aseve aRmoCenilia wyalmcenareebSic [70,108,109]. V. cholerae gamoyofilia mtknari da zRvis wylis makrofitebidan, aseve zRvis fskerze mcxovrebi organizmebidan, rogorebicaa: krevetebi, xamanwkebi [182], kiborCxalebi [31]. NnaCvenebia V. cholerae-s ujredSida gamravlebis unari Tavisuflad mcxovreb amebaSi [8]. V. cholerae cocxlobs wyalSi, rogorc Tavisuflad mcxovrebi organizmi planqtonis fazaSi [186,192]. Chironomus sp, diptera-s kvrcxis masebi navsayudelia V. cholerae-sTvis da moqmedebs, rogorc naxSirbadis erTaderTi wyaro, Sesabamisad qmnis bunebriv rezervuars qoleras vibrionebisTvis [189]. abdim da Tanaavtorebma (2007) aRweres V. cholerae O1-is Stamebi, rogorc fakultaturi ujredSida baqteriebi, romelTac aqvT unari gadarCnen da gamravldnen simbiozuri Tanacxovrebis Sedegad wylis amebas SigniT (Acanthamoeba castellanii). am urTierTkavSirma aCvena V. cholerae O1-is da “El Tor”-is Stamebis fakultaturi, Sidaujreduli qceva da A.castellanii-is SesaZlo roli, rogorc garemoSi arsebuli maspinZlis, V. cholerae-s StamebisTvis. amrigad, V. cholerae-s aqvs garemoSi, rogorc asocirebulad aseve Tavisuflad arsebobis unari. V. cholerae–s pandemuri buneba cxadyobs am organizmis adaptaciis da gadarCenis unars mravalferovan garemo pirobebSi [118]. qoleras epidemiologiis TvalsazrisiT didi mniSvneloba aqvs V. cholerae Ol-is gadarCenis unars wylian garemoSi, mcire sakvebi nivTierebebis pirobebSi. nanaxia qoleras Stamebis sicocxlisunarianoba Raribi sakvebi aris pirobebSi, inarCunebdnen ra cxovelmyofelobas 75 dRis ganmavlobaSi, kultivirebis maRali doziT dacemis gareSe [19].Ddadginda V. cholerae Ol-is bunebrivi arseboba epidemiebis ganviTarebebs Soris, gansakuTrebiT arakultivirebad formaSi da maTi arseboba agregatebis saxiT biofilmebSi [12]. adamianis nawlavisa da wyliani garmeos zedapirze qoleras vibrionebis adgezia mniSvnelovan rols asrulebs V. cholerae Ol-is, rogorc paTogenis aseve garemoSi arsebuli organizmis warmatebiT arsebobaSi [120].

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qoleras epidemiebi asocirebulia didi raodenobis naleqTan da zRvis zedapiris simaRlis zrdasTan [136,134]. orive dasaxelebuli mdgomareoba, mizezia estuariebSi organuli da araorganuli komponentebis cvlilebis, rac sawyisi zedapiria adamianTa da zRvis garemos Soris [91,150]. garemos da klinikuri monacemebis analizma cxadyo wylis temperaturis, siRrmis, uxvi naleqis, gamtarobis da copepod-ebis ricxvis korelacia qoleras toqsinis maproducirebel baqteriebis arsebobasTan [106]. distanciurad marTvadi satelitiT dadginda zRvis zedapiris temperaturisa da zRvis zedapiris simaRlis korelacia qoleras epidemiebTan [60,136]. ekologiurma kvlevebma gamoavlina wylis temperaturis, marilianobis, organuli nivTierebebis da sedimentebTan da organizmebTan asociaciis potenciuri tendenciis mniSvnelovani zegavlena garemoSi qoleras vibrionebis arsebobasa da ricxvze [185]. temperaturas da gaxsnili naxSirbadis xarisxs didi zegavlena aqvs V. cholerae–s vibrionebis zrdaze [121]. patelma da Tanaavtorebma (2004) Seiswavles V. cholerae Ol-is “klasikuri” da “El Tor” biotipis, aseve ara-Ol-is gadarCenis unari samrewvelo dabinZurebul wylebSi, rkinis ionebis koncentraciis xelisSemwyob damokidebulebaSi da gamoTqves varaudi, rom wylis qimiuri dabinZureba aris erTerTi yvelaze mniSvnelovani xelSemwyobi faqtori V. cholerae-s vibrionebis Semdgomi arsebobis, proliferaciisa da wylian garemoSi gadarCenis. 1.1.7. daavadeba qolera da winaswar ganwyoba V. cholerae Ol-iT gamowveul infeqcias Tan axlavs diarea, pirRebineba da krunCxvebi. daavadeba-qolera xasiaTdeba uxvi dehidrataciiT diareis fonze, rasac Tan sdevs siTxisa da eleqtrolitebis kargva. fekaliebis moculoba SesaZloa aRwevdes 1l/sT [95,151]. mwvave infeqcia asocirebulia kuWis dabal mJavianobasTan da O jgufis sisxlTan [33]. O jgufis sisxlis mqone adamianebi hospitalizaciis maRali riskis qveS imyofebian V. cholerae Ol-is “klasikuri” da “El Tor”, aseve V. cholerae Ol39-iT gamowveuli infeqciebis dros [22,74]. daavadeba-qolera xasiaTdeba "brinjis naxarSiseburi" sekrementebiT, sxeulis masis 10%-is (an meti) dakargviT, kanis normaluri turgoris dakargviT, Cavardnili TvalebiT, anuriiT, sustad gamoxatuli pulsiT, Secvlili cnobierebiT da kolafsiT. mwvave infeqcia SesaZloa damTavrdes letaluri SedegiT, dehidrataciis mkurnalobis, siTxeebisa da eleqtrolitebis Canacvlebis gareSe [63]. gamoiTqva hipoTeza, romlis mixedviTac V. cholerae–s Stamebis mier gamowveuli infeqcia aris evoluciuri Zala O jgufis sisxlis dabal gavrcelebasTan mimarTebaSi, gangis mdinaris deltaSi, romelic qoleras istoriul da globalur epicenters warmoadgens [118]. qolerisa da sxva vibrionebis mier gamowveuli mZime infeqciuri daavadebebis kontrolisa da prevenciis strategia damokidebulia am mikroorganizmebis bunebis, maTi paTogenobis arsis ufro Rrma da detalur Secnobaze da ra Tqma unda maTi saarsebo garemosa da gavrcelebis gzebis Seswavlaze.

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1.2. baqteriofagebi

1.2.1. baqteriofagebis aRmoCena

baqteriofagebis aRmoCena me-20 saukunis erT-erTi umniSvnelovanesi movlena iyo, romelic ori mecnieris, britaneli f.tuortis (1915 w.) da kanadeli mecnieris f.derelis (1917w.) saxelebTan aris dakavSirebuli. 1917 wels mikrobiologi f.dereli swavlobda dizinteriis eTiologiasa da paTogenzs, akvirdeboda avadmyofi adamianebis fekaluri masis, filtratis damatebiT gamowveul baqteriuli kulturis liziss. baqteriebis gamxsnel agents man baqteriofagi_ ”baqteiebis STanmTqmeli” (laT. Bacterium-baqteria, phagos-mSTanTqmeli) uwoda, xolo baqteriofagebis moqmedebas, romelic baqteriebis lizisiT mTavrdeba_baqteriofagiis fenomeni. baqteriofagiis fenomens akvirdebodnen sxva mkvlevrebic, magram am movlenis biologiuri arsi pirvelad swored f.derelma Seafasa. man gamoTqva mosazreba, rom mis mier aRmoCenili baqteriofagi baqteriis virusia, romelic baqteriuli ujredis SigniT mravldeba, ris gamoc es ukanaskneli iSleba da garemoSi gamodian axlad warmoqmnili virusuli STamomavlobis nawilakebi. aRsaniSnavia qarTveli mecnieris, giorgi eliavas damsaxureba, romelmac 1917 wels mtkvris wyalSi vibrionis aRmoCenasTan erTad aRwera baqteriofagiis fenomeni [40]. baqteriofagiis fenomenis Sesaxeb gamokvlevebs gansakuTrebuli adgili uWiravs mikrobiologiis istoriaSi. kultivirebis simartivem, generaciis mokle periodma, Taobebis didi raodenobiT miRebam, xeli Seuwyves rogorc virusebis nawilakebis struqturebis, aseve baqteriul ujredTan maTi urTierTqmedebis meqanizmebis warmatebiT Seswavlas. 50-60-ian wlebSi baqteriul virusebze Catarebulma gamokvlevebma SesaZlebeli gaxada mniSvnelovani Sedegebis miReba molekuluri biologiis umTavresi problemis gadasaWrelad. swored baqteriofagebi aRmoCndnen xelsayreli modelebi genebis ultrastruqturebis, mutagenezis molekuluri meqanizmebis, genetikuri kodis wakiTxvisa da organizmis memkvidreobiT struqturebze radiaciis gavlenis Sesaswavlad. f.dereli Tvlida, rom yvela baqteriofagi miekuTvneba erT saxeobas_Bacteriophagum intestinale, romelic iyofa mraval rasad, magram mogvianebiT daadgines, rom isini qmnian baqteriebis virusebis sxvadasxvagvar jgufebs, romlebic bunebaSi farTod arian gavrcelebulni. yvelgan, sadac mravldebian baqteriebi, aqtinomicetebi an mikoplazmebi SesaZlebelia fagebis aRmoCena. isini binadroben adamianis da cxovelis nawlavebSi, Camdinare wylebSi, niadagSi.

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1.2.2. baqteriofagebis klasifikacia

Baqteriofagebis pirveli klasifikacia [92] efuZneboda patroni baqteriuli ujredebis tipebs da maT mier gamowveuli daavadebebis simptomebs. 1962 wels lvofis, hornis da tunieris klasifikacias safuZvlad daedo virionis morfologia, kerZod, nukleinis mJavis tipi, kafsidis forma da kafsomerebis raodenoba [11,40,92]. es sqema mogvianebiT cnobili iyo LT-sistemis saxelwodebiT, romlis SemadgenlobaSic Sedioda ФX174 da ori kudiani fagi. mogvianebiT, praqtikulma aucileblobam ganapiroba mecnierebis miyvana im daskvnamde, rom baqteriofagebis klasifikacias safuZvlad unda daedos morfotipi da genetikuri Tvisebebi. morfotipebis mixedviT cnobilia bredlis da a.tixonenkos klasifikaciis sqemebi. 1967 wels bredlim miznad daisaxa baqteriuli virusebis sistemaSi moyvana, gaTvaliswinebuli iqna maTi morfologiuri niSnebi da nukleinis mJavis forma da amis mixedviT Camoyalibda fagebis eqvsi ZiriTadi jgufi: A. fagebi kumSvadi wanazardiT; B. mokle wanazardiani fagebi; C. kuburi formis fagebi; D. erTZafiani dnm-is Semcveli fagebi; E. rnm-is Semcveli fagebi; F. Zafiseburi (filamenturi) fagebi. baqteriofagebi, Semadgeneli nawilakebis formis mixedviT, arseboben sferosebrni, Cxirisebrni (Zafisebrni) da rTuli formis. ZiriTadad, Seswavlilia rTuli, spermatozoidis formis fagebi, romlebic Sedgebian Tavis da morCebisagan (wanazardebisagan). fagis Tavs aqvs swori an mogrZo forma, rac damokidebulia nukleinis mJavis mdebareobaze. Tavis saSualo zomaa: diametri 60-100nm. zogierT fags morCi Zalian mokle aqvs an saerTod ar gaaCniaT. mogrZo kudis zoma meryeobs 100-200nm-is farglebSi. kuds aqvs sxvadasxva sigrZe, sigane da agebuleba, rogorc wesi, spiraluri simetriis, swored amitom, Tu Tavis aRnagoba mraval fags erTnairi aqvs, maSin wanazardis struqturaSi aRiniSneba arsebiTi gansxvaveba. swored am gansxvavebis safuZvelze SeiZleba gaimijnos fagebis morfologiuri tipebi. gamokvleuli da klasificirebuli iyo daaxloebiT 2850 fagi eleqtronul-mikroskopulad [11]. am CamonaTvalSi ar arian gaerTianebuli defeqturi fagebi, baqteriocidulad msgavsi fagebi da aqamde arakultivirebuli fagebi. akermanma da aizensarkma mogvces saklasifikacio sqema. maT ganixiles aranakleb 1061 fagisa da gaafarToves bredlis originaluri klasifikacia axalaRmoCenili fagebiT. akermanma klasifikaciis dazustebisaTvis SemoiRo morfologiuri qvetipebi [10] sadac gaTvaliswinebulia fagis Tavisa da kudis SefardebiTi zomebi, Tavis forma da masze specifikuri warmonaqmnebis arseboba. 10 ojaxi dayofilia 4 morfologiur tipad: 1. kudiani fagebi 2. kuburi ukudo fagebi 3. Zafisebri fagebi 4. pleomorfuli ukafsido (lipidebiani) fagebi.

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eleqtronuli mikroskopiiT gamokvleul 5100 baqteriofags Soris 4950 (96%) wanazardiani (kudiani) fagia. kudiani (2 jaWviani dnm-iT) fagebis tipi Sedgeba 3 didi filogenetikuri ojaxisagan [11,37]: I mioviride- fagebi grZeli kumSvadi wanazardiT. am ojaxSi Sedis wanazardiani fagebis 25%. II sifoviride- fagebi grZeli, moqnili kudiT, romlis SaliTas ar gaaCnia SekumSvis unari.Aam ojaxs miekuTvneba kudiani fagebis 61% [11]. tipiuri warmomadgenlebia: T1, T5, L5, λ fagebi da sxv.Oojaxi moicavs 6 gvars. III podoviride- mokle wanazardiani fagebia. tipiuri warmomadgenlebi arian: P22, T7 fagebi da sxva. am ojaxSi gaerTianebulia 3 gvaris, kudiani fagebis 14%. kudiani fagebi daiyo maTi Tavebis sigrZis mixedviT. wanazardian fagebs gaaCniaT ikosaedris an oqtaedris formis Tavi. Figi Sedgeba Tavis, kudis da fakultaturi struqturebisgan, rogoricaa Tavis an kudis fibrilebi, sayelo, bazaluri firfita. kafsidSi moTavsebulia superspiralizebuli orZafiani dnm. wanazardiani fagebi SesaZloa iyos virulenturi an zomieri (50%-50%) [40]. Oojaxi Myoviridae warmodgenilia fagebis mravalferovani jgufiT, romlis ZiriTadi morfologiuri maxasiaTebelia kumSvadi wanazardi. aRniSnuli ojaxis warmomadgenlebs unari aqvT daainficiron, rogorc gram-dadebiTi, aseve gram-uaryofiTi baqteriebi da arqebaqteriebi. virusebis taqsonomiis saerTaSoriso komitetis klasifikaciis mixedviT, wanazardiani fagebis samive ojaxi aerTianebs 15 gvars, aqedan Myoviridae-s ojaxi dayofilia Semdeg gvarebad: T4-is msgavsi virusebi, P1-msgavsi virusebi, P2-msgavsi virusebi, Mu-msgavsi virusebi, SPO1-msgavsi virusebi, PBS1-msgavsi virusebi, H-msgavsi virusebi. aRniSnuli gvarebi mkveTrad gansxvavdebian erTmaneTisgan sasicocxlo ciklis da genomis struqturiT. mag.: P1-jgufi lizogenur fagebs aerTianebs, Mu-transpozonebs warmoadgens da ar eqvemdebareba P2-is msgavsad saitspecifikur rekombinaciebs, xolo T4 liTiuri baqteriuli virusia. maTi genomis zomebi varirebs 30000 nw-dan 170000 nw-mde. Myoviridae-s ojaxSi fagebi ZiriTadad gaerTianebulia morfologiuri niSnebis mixedviT. brusovis da Tanaavtorebis mier 1996 wels Catarebulma kvlevam cxadyo, rom fagis erTi morfotipis meored gardaqmna mutaciiT ar xdeba. erTi ojaxis fagebs Soris SemCneulia mxolod mcire struqturuli gansxvavebebi. mag.: maRali dnm-is homologiis mqone rZemJavuri streptokokuri fagebi gansxvavdebodnen mxolod sayelos morfologiuri SenebiT. zemoT CamoTvlili sami ojaxidan didi zomis kafsidiT da dnm-is maRali SemcvelobiT xasiaTdeba Myoviridae, xolo Podoviridae da Siphoviridae, am maxasiaTeblebiT ufro axlo msgavsebas avlenen [40]. morfologiuri kriteriumi aucilebelia, magram arasakmarisia, radgan arseboben morfologiurad identuri fagebi, romelTac maspinZlis gansxvavebuli speqtri aqvT da piriqiT. kudiani fagebis raodenoba aRwevs izolatebis 95%-s. kuburi da Zafisebri fagebi SedarebiT iSviaTobas warmoadgenen, amitom maTi dalageba ufro advilia, vidre zemoT aRniSnulis. genomSi G+C Semcveloba fagebSi da maT maspinZlebSi, wesisamebr, sakmaod msgavsia. igive SeiZleba iTqvas monaTesave fagebSi azotovani fuZeebis sixSireze. maRali taqsonomiuri Rirebulebis

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mqonea baqteriofagebis serologiuri Tvisebebi da neitralizaciis testebi rac saSualebas gvaZlevs, davajgufoT mWidrod monaTesave virusebi. axlad gamoyofili fagebis dasaxasiaTeblad aucilebelia maspinZlis arealis dadgena. Semdeg Sesamowmebelia morfologiuri da serologiuri Tvisebebi. wesisamebr, morfologiurad gansxvavebuli fagebi serologiuradac gansxvavdebian. sayuradReboa agreTve fiziko-qimiuri Tvisebebi, gansakuTrebiT, kuburi, Zafisebri da pleomorfuli fagebisTvis. amitom virusebis taqsonomiis saerTaSoriso komitetma zusti klasifikaciisaTvis daadgina, rom gamoyenebuli unda iqnes virionisa da misi nukleinis mJavis Tvisebebi: 1) virionis morfologia da zomebi, 2) nawilakis (molekuluri) masa, sedimentaciuri Tvisebebi, misi lRobis simkvrive, 3) molekuluri masa, nukleinis mJavis SefardebiTi wili da misi konformacia, 4) serologiuri Tvisebebi da nukleinis mJavis hibridizaciis monacemebi, 5) fiziologiuri testebi da lizisuri aqtivobis speqtri. gamoyofili naTesauri (msgavsi) fagebis saxelwodebis Sesaxeb erTnairi terminologia ar aris, zogi mecnieri saxeobas xmarobs, zogi ki ojaxs. baqteriofagebis klasifikaciis erT-erTi mniSvnelovani meTodia homologiuri doneebi da genomebSi maTi arseboba. erT-erTi meTodia agreTve restriqciuli profilebis Sedareba fagis dnm-Si. aranakleb mniSvnelovania fagis Semadgeneli struqturuli cilebis Seswavla. restriqciuli analizis meTodi SedarebiT iolia: restriqciuli endonukleazebis didi raodenoba iZleva fagebis diferencirebis saSualebas. Tu ori fagi restriqciuli analiziT msgavsia, es mowmobs maT naTesaobaze an msgavsebaze. fagebis msgavsebis dasamtkiceblad pirdapiri meTodic arsebobs. es aris maTi rekombinaciis da sicocxlisunariani hibridebis warmoqmnis aRmoCena, ase dadginda msgavseba P22 da S fags Soris [126]. baqteriofagebis SedarebiTi daxasiaTeba unda Catardes garkveuli sqemiT. kerZod, fagi jer unda miekuTvnos ama Tu im cnobil ojaxs da Semdeg unda moxdes misi Sedareba am ojaxis sxva fagebTan. aseTi Sedareba iZleva pasuxs, mocemuli fagi (Tu mutanti) ukve arsebuli fagis warmoebulia [36], Tu am ojaxis axali warmomadgeneli. brusovis da Tanaavtorebis mier 1996 wels Catarebulma kvlevam cxadyo, rom fagis erTi morfotipis meored gardaqmna mutaciiT ar xdeba. erTi ojaxis fagebs Soris SemCneulia mxolod mcire struqturuli gansxvavebebi. aucilebelia axlad gamoyofili fagis identifikacia da imis garkveva, romel ojaxs miekuTvneba igi, radgan erTi ojaxis fagebs gaaCniaT msgavsi genetikuri organiazacia, maTi genomebi SegviZlia warmovidginoT, rogorc genebis blokebis modulebis Semadgenloba. sayovelTaod cnobili faguri nomenklaturis ararsebobam ganapiroba is, rom axali fagis dasaxelebisaTvis 1983 wels akermanma SemogvTavaza wesebi: 1. uaryofa mxolod erTaderTi asosi an erTaderTi ricxvis, iseve aucilebelia, rogorc uaryofa Zalian xSirad gamoyenebuli asosi P da F. 2. maspinZlis da ricxvis, gvaris da saxeobis internacionaluri gamoyeneba. 3. ukve cnobili fagebis axali izolatebis aRniSvna gamoyofis adgilis inicialebis damatebiT.

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virusebis taqsonomiis saerTaSoriso komitetma (1992 ICTV), rogorc xerxemlianebis, uxerxemloebis da mcenaris virusebisTvis, aseve baqteriebis virusebisaTvis, gansazRvra virusebis ojaxebi. Tanamedrove klasifikaciis mixedviT baqteriofagebi gaerTianebulia Caudovirales klasis 13 ojaxSi da 31 gvarSi. klasifikacia damyarebulia Semdeg kriteriumebze: 1. virionis ultrastruqtura: binaluri simetriis (wanazardiani), kuburi,

helikaluri (spiraluri), filamenturian pleiomorfuli; 2. nukleinis mJavis tipi; 3. Semadgenloba da antigenuri Tvisebebi; 4. molekuluri masa; 5. patron-ujredebis wre.

1.2.3. baqteriofagebis ZiriTadi tipebi

baqteriofagebi, iseve rogorc virusebi, Sedgebian nukleinis mJavisa da cilisagan. maTi umetesoba Seicavs dnm-s, xolo zogierTi rnm-s. faguri dnm SesaZloa iyos erTZafiani an orZafiani; xazobrivi an Caketili, cikluri. nawili fagebis SemadgenlobaSi aRmoCenilia uCveulo azotovani fuZeebi, magaliTad: T2 fags aqvs 5-meTilcitozini, romelic citozins cvlis. B. subtilis-is fags Timinis Semcvleli 5-meTiluracili aqvs. fagis Tavis kafsidi da morCis SaliTa agebulia mowesrigebuli polipeptiduri suberTeulebisgan, romlebic TavSi ganlagebulni arian kuburi, xolo morCSi-spiraluri simetriiT. miuxedavad fagebis nairgvarobisa, maT bevri saerTo Tviseba aqvT. mag.: fagis ujredSi SeWris Semdeg ujredis metabolizmSi Careva da sxva. yvela baqteriofagis saerTo niSania ujredSida parazitizmi. fagisa da patron-ujredis urTierTqmedebis done sxvadasxva fagis SemTxvevaSi sxvadasxvaa. mag.: E.coli-s mimarT specifikur T-wyvil fagebs gaaCniaT genebi, romlebic funqciurad garkveuli baqteriuli genebis msgavsni arian. aseTi fagebi SedarebiT avtonomiurebi arian. baqteriofagis da ujredis urTierTobis mixedviT fagebi SeiZleba daiyos 3 tipad, kerZod: 1) WeSmaritad virulenturi fagebi. virulenturi fagiT ujredis inficirebis dros xdeba inficirebuli ujredis daSla da fagis STamomavlobis gamosvla (abortuli infeqciis garda). aseT fagebs uwodeben WeSmaritad virulentur fagebs, arsebobs zogierTi fagebis virulenturi mutantebic. 2) zomieri fagebi. zomieri fagiT ujredis inficireba SeiZleba ganviTarebis sxvadasxva gziT Camoyalibdes: 1) lizisuri, msgavsad virulenturi fagebis lizisuri ciklisa, 2) lizogenuri, roca zomieri fagis genomi gadadis gansakuTrebul – profagur mdgomareobaSi. lizogenur baqteriaSi garkveul pirobebSi SesaZloa moxdes fagis lizisuri gamravleba. 3) fagebi, romlebic ujredis inficirebis Semdeg ar iwveven mis daSlas. ujredi, romelic dasnebovnebulia aseTi fagiT, imyofeba ganuwyveteli (permanentuli) infeqciis mdgomareobaSi. fagis aseTi ganuwyveteli producirebis gamo ujredis gayofis siCqare ramdenadme Senelebulia. aseTi tipisaa Zafiseburi (filamenturi) fagebi.

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baqteriofagebi, romlebic iwveven produqciul infeqcias da fagis sicocxlisunarian STamomavlobas, iwodebian aradefeqtur fagebad. yvela aradefeqturi fagisaTvis cnobilia 2 an 3 mdgomareoba: 1) araujreduli (Tavisufali, momwifebuli fagis mdgomareoba); 2) vegetaciuri fagi; 3) zomieri fagisaTvis arsebobs profagis mdgomareoba.

1.2.4. baqteriofagis ganviTarebis ZiriTadi stadiebi

1.2.4.1 baqteriofagis adsorbcia da dnm-is eJeqcia

fagisa da baqteriis urTierTqmedeba iwyeba fagis mierTebiT (adsorbciiT) ujredis struqturebTan–receptorebTan. adsorbciis procesi (kinetika) sxvadasxva fagSi gansxvavebulad mimdinareobs. msgavsi fagis SemTxvevaSi receptoruli ubnebi SeiZleba gansxvavebuli iyos da piriqiT. aris fagebi, romlebic adsorbciisaTvis iyeneben baqteriis specifiur warmonaqmnebs e.w. pilebs. umetesoba fagebisaTvis adsorbciis procesi Seuqcevadia, romelic ujredSi fagis genomis eJeqciiT mTavrdeba. naCvenebia, rom SesaZlebelia zogierTi fagisaTvis eJeqciis procesis stimulireba. am dros mimdinareobs gravitaciuli urTierTqmedeba fagis kudis gamonazardsa da citoplazmur membranis cilovan komponentebs Soris, am saxis stimulacia xels uwyobs ujredSi fagis genomis droze adre SeWras. xSirad fagis adsorbciuli aparatis moqmedebiT xdeba ujredis membranuli SeRwevadobis potencialis cvlileba. am dros SeiZleba dairRves ujredis bioqimiuri aqtivoba. risi Tavidan asacilebladac fags gaaCnia genebi, romlebic axorcieleben inficirebuli ujredis membranis mTlianobis da daurRvevlobis kontrols. receptorebis qimiuri buneba erTi ojaxis fagebis SemTxvevaSic SeiZleba sxvadasxva iyos. receptori SeiZlebawarmoadgendes cilovan molekulas, polisaqarids, lipopolisaqaridebs, lipoproteinebs da sxva. adsorbciis procesis raodenobrivi daxasiaTebisaTvis iyeneben 2 ZiriTad meTods: a) narevis qloroformirebis meTodi; b) baqteriuli ujredebis daleqva centrifugebis gziT. am meTodebiT ganisazRvreba inficirebuli da arainficirebuli baqteriebis raodenoba, infeqciis mravlobiToba (faguri nawilakebis saSualo raodenoba 1 baqteriaze). rac Seexeba fagis genomis gamosvlas kafsididan da mis ujredSi SeRwevas, am process axorcielebs specialuri lizisuri fermentebis moqmedeba. maTi moqmedeba SeiZleba iseTi efeqturi iyos, rom infeqciis maRali mravlobiTobis dros ujredis garsi daiSalos da fagis STamomavloba ar iqnes miRebuli. am process uwodeben liziss, gamravlebis gareSe. fags eJeqciis Taviseburi meqanizmi aqvs. T4 fagis genomis Sesvla ujredSi kudis SaliTis SekumSviT xdeba. T5 fagis genomis Sesvla 2 stadiad mimdinareobs: jer dnm-is erTi nawili Sedis ujredSi, Semdeg meore. adsorbciuli receptorebis raodenoba yoveli fagis ojaxisaTvis gansazRvrulia, amitom fagis adsorbciuli Tvisebebis Seswavla ukve adre arsebuli fagogamZle

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baqteriul mutantebze SesaZleblobas iZleva, rom gadairCes gamZle, baqteriuli mutantebi. mecnierebis mier arsebobs mosazreba, rom koevolucia baqteria-fagi garkevulad SezRudulia. SezRudva ganpirobebulia fagis cilebis da baqteriis receptoruli aparatis TvisebebiT. zogierTma mutaciebma baqteriebSi SeiZleba gamoiwvion meqanikuri winaaRmdegoba adsorbciaSi. Tu magaliTad, receptoruli ubani baqteriuli kedlis zedapiris tipologiis SecvliT aRmoCndeba CaRrmavebuli, fagis adsorbciul aparats ar SeuZlia masTan pirdapiri Sexeba. Zalian Znelia gadairCes iseTi mutanturi fagebi, romelTac eqnebaT gafarToebuli moqmedebis speqtri im baqteriuli mutantebis mimarT, romlebsac receptoruli ubani saerTod dakarguli aqvT. (mag. “nonsence” _ mutaciis dros Sesabamis genSi).

1.2.4.2 baqteriofagis ujredSida ganviTareba

fagis genomis SeWris Semdeg iwyeba fagis vegetaturi ujredSida gamravleba. sxvadasxva fags ujredSida ganviTarebis etapi gansxvavebuli aqvs. fagebis ujredSida ganviTareba yvelaze kargad aris Seswavlili E.coli-s T-wyvili fagebis magaliTze. fagis dnm-is SeWris Semdeg ujredSi iwyeba misi transkrifcia (informaciuli rnm-is warmoqmna baqteriuli rnm-polimerazis saSualebiT). am dros mimdinareobs transkrifciis fazebis monacvleoba (adreulis wina, adreuli, gviani) da gansazRvruli cilebis Tanmimdevruli warmoqmna, romlebic, Tavis mxriv, xels uwyoben ganviTarebis etapebis swor msvlelobas. adreulis wina periodSi xdeba citoplazmuri membranis funqciebis gaZliereba. adreul periodSi xdeba genomis replikacia (mag. fagSi). replikaciis yoveli etapi da maTi sqema kontroldeba fagis genomis gansazRvruli produqtiT. replikaciis ganxorcielebisaTvis aucilebelia, rom fagis genomi iyos gansazRvrul formaSi, kerZod daculi iyos ujreduli nukleazebisagan da dnm-is molekulis boloebze ganxorcieldes genomis ubnebis replikacia. zogierT fags es forma uyalibdeba dnm-is ramdenime molekulis gaerTianebiT, romlebic pirveladi replikaciis dros warmoiqmneba. xdeba rekombinaciuli tipis - `Tavi da bolo~ formireba (mag. T tipis fagebSi). sxva fagebi, kerZod,İλ fagi warmoqmnian dnm-is rgolur formas molekulis boloebze arsebuli webovani daboloebebis saSualebiT (komplementaruli erTjaWviani nukleotidebis Tanmimdevroba). gansxvavebulia transpozonis dnm-is replikacia, rac xorcieldeba fagis genomis CajdomiT baqteriis genomSi (transpozicia).LLamis Sedegad fagis genomis daboloebebi mudmivad ukavSirdebian baqteriis dnm-Si nukleotidebis Tanmimdevrobas da daculni arian baqteriuli nukleazebis zemoqmedebisagan. dnm-is replikaciasTan erTad mimdinareobs fagis genomis rekombinacia, romelic SeiZleba aRmovaCinoT rekombinatebis arsebobiT, mag. Tu ujredi inficirdeba 2 genetikurad (markirebuliT) sxvadasxva fagiT. dnm-is sinTzis dawyebis Semdeg, rodesac warmoiqmneba fagis ramdenime genomi, iwyeba gviani genebis wakiTxva, anu gviani genebis pozitiuri regulacia. gviani genebi akontroleben sxvadasxva cilebs virusuli nawilakebis aucilebeli SenebisaTvis, rogoricaa: struqturuli cilebi, fermentebi: membranaza, endolizini da sxv..

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praqtikulad, TiTqmis yvela Seswavlili fagis genomSi mocemulia arsebiTi da araarsebiTi genebi. es ukanasknelni ar arian aucilebelni fagis ganviTarebis ciklis ganxorcielebisaTvis. Tumca zogjer, garkveul pirobebSi, am genebis arseboba aseve xdeba aucilebeli fagis srulyofilad ganviTarebisaTvis. fagis ujredSida ganviTarebis bolo etapze xdeba e.w. `lizisis drois~ CarTva. e.i. amoqmeddeba fagis funqcia daSalos ujredis garsi da gamoaTavisuflos momwifebuli faguri nawilakebi. sxvadasxva fagis SemTxvevaSi `lizisuri drois~ meqanizmi sxvadasxvanairad moqmedebs. nivTierebis (sekretis) gamoyofa uSualod fagis mier ar xdeba, aramed xdeba ori lizisuri fermentis warmoqmnis gakontroleba (T wyvil da λ fagebSi) erTi Slis ujredul membranas, meore ki – ujreduli garsis rigidul mukopolimerul Sres. λ da P22 fagebSi es genebi ganlagebulni arian erTmaneTis gverdiT genomSi da transkripciis erTi da igive erTeulSi Sedian. xolo T4 fagSi, lizisis makontrolirebeli fermentebis genebi regulirdeba cal-calke, magram SeTanxmebulad. lizisis aucilebeli pirobaa_ fosforilirebisa da membranis mTlianobis dacvis meqanizmebis Sewyveta.

1.2.4.3 baqteriofagis moqmedebiT gamowveuli patron ujredis lizisi

baqteriofagiT infeqcias moyveba fagiT gamowveuli patron ujredis lizisi. bevr baqteriofags amgvar procesSi sWirdeba ori, fagiT kodirebuli cilis moqmedeba. pirveli aris e.w. holini, patara transmembranuli proteini, romelic warmoSobs mcire zomis dazianebebs citoplazmur membranaSi. es dazianebebi funqcionireben, rogorc forebi da aZleven saSualebas ganTavisufldes meore cila_enzimi, romelsac aqvs peptidoglikanis-hidrolizis unari.

1.2.4.4 virulenturi baqteriofagebis sasicocxlo cikli

fagis baqteriul ujredze adsorbciiT iwyeba ganviTarebis lizisuri cikli [40,93]. baqteriofagis adsorbcia patron ujderze mkacrad specifikuria da zedapiruli struqturebis-receptorebis saSualebiT xorcieldeba. gram-uaryofiT baqteriaSi nebismieri cila, oligosaqaridi, lipopolisaqaridi SeiZleba warmoadgendes receptors fagisaTvis. morfologiurad msgavsi fagebis receptoruli ubnebi SeiZleba gansxvavebuli iyos da piriqiT. aris fagebi, romlebic adsorbciisaTvis iyeneben baqteriis specifikur warmonaqmnebs e.w. pilebs. adsorbciis procesi, misi kinetika, sxvadasxva fagis SemTxvevaSi gansxvavebulia. adsorbciis sixSire xSirad damokidebulia mraval garemo faqtorze da maspinZeli mikrobis fiziologiur mdgomareobaze.

magaliTad λ receptorebis eqspresia xdeba mxolod maltozis Saqris Tanaarsebobisas [129]. mravali fagisTvis sxvadasxva kofaqtorebis arsebobaa aucilebeli, magaliTad Ca2+, Mg2+ ionebis, an sxva martivi kaTionebis. mutaciebis dros, romelsac Tan axlavs baqteriuli receptorebis cvlileba,

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ujredi kargavs fagis adsorbciis unars da iZens rezistentobas mis mimarT. umetesoba fagebisaTvis adsorbciis procesi Seuqcevadia, romelic ujredSi fagis genomis eJeqciiT mTavrdeba. baqteriofagis genomi kudis gavliT Sedis ujedSi. es procesi ar ganixileba rogorc dnm-is martivi ineqcia, am procesSi CarTulia dnm-is transferis ramdenime meqanizmi [129]. fagis adsorbciis Semdeg, ujredis kedlis SemadgenlobaSi arsebuli TuTiis ionebi (Zn2+) iwvevs fagis kudis distaluri nawilis boWkoebis gaglejas. gamoTavisuflebuli bazaluri firfita, kudis distalur nawilSi moTavsebuli fermentis endolizinis (lizocimis) gamoyofiT axdens baqteriuli ujredis kedlis mimdebare fragmentis lizirebas. amavdroulad SaliTaSi gamoTavisuflebuli Ca2+ ionebi iwvevs atf-azas aqtivirebas, rac Tavis mxriv ganapirobebs SaliTis SekumSvas da ujredSi citoplazmaturi membranis gavliT kudis RerZis SeWras. amis Semdgom adgili aqvs virusuli dnm-is ujredis citoplazmaSi swraf gadasvlas [40,129]. gravitaciuli urTierTqmedebiT fagis kudis gamonazardsa da citoplazmur membranis cilovan komponentebs Soris, SesaZlebelia zogierTi fagisaTvis eJeqciis procesis stimulireba. msgavsi stimulacia xels uwyobs ujredSi fagis genomis droze adre SeWras [110,111]. xSirad fagis adsorbciuli aparatis moqmedebiT xdeba ujredis membranuli SeRwevadobis potencialis cvlileba. am dros SeiZleba dairRves ujredis bioqimiuri aqtivoba. ris Tavidan asacileblad fags gaaCnia genebi, romlebic axorcieleben inficirebuli ujredis membranis mTlianobis da daurRvevlobis kontrols [125]. ujredSi fagis dnm-is ineeqciis Semdeg sawyis safexurs warmoadgens maspinZlis rnm-polimerazas mier fagis promotoris Secnoba da adreuli genebis transkrifcia [125]. am genebis produqtebi icaven fagur dnm-s da axdenen maspinZeli ujredis restruqturizacias: inaqtivireben maspinZeli ujredis proteazebs, restriqciul enzimebs, Slian sxvadasxva cilebs. Sualeduri genebis transkrifcia mimarTulia fagis axali dnm-is sinTezisTvis. Sualedur genebs moyveba gviani genebi, romlebic ukve kodireben faguri nawilakebis asawyob struqturebs. axali fagebis Tavmoyras ujredSi ewodeba fagis momwifeba. fagis dnm-is ineeqciidan fagebis axali STamomavlobis gamoTavisuflebamde drois intervals ewodeba latenturi periodi [128]. latenturi periodi damokidebulia maspinZlis fiziologiaze da meryeobs 20 wT-dan 30-40 sT-mde. fagis sasicocxlo ciklis saboloo safexuri, romelic mohyveba baqteriofagiT infeqcias, aris fagis axali Taobis gamoTavisufleba da maspinZeli ujredis lizisi [129, 138]. kudian baqteriofagebs amgvar procesSi sWirdebaT fagiT kodirebuli ori cilis moqmedeba. pirveli aris e.w. holini, patara transmembranuli proteini, romelic warmoSobs mcire zomis dazianebebs citoplazmur membranaSi. es dazianebebi forebis funqcias asrulebs da aZleven saSualebas gaTavisufldes meore cila _ enzimi-lizini, romelsac aqvs peptidoglikanis hidrolizis unari. sxvadasxva fagis Svileul populaciebSi virionebis raodenoba met-naklebad mudmivia da iwodeba, rogorc fagis gamosavlianoba, anu fagis gamosavali.

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virulenturi fagi SesaZlebelia aRmoCndes e.w. fsevdolizogenur mdgomareobaSi, romelsac zogjer mtareblobas uwodeben [129]. cnobilia fsevdolizogeniis ramdenime meqanizmi. zogjer is viTardeba baqteriebis fagisagan droebiTi izolaciis Sedegad. es gamoyofa SesaZlebelia sufTa meqanikur xasiaTs atarebdes, magaliTad, romelime webovani substrati ujredebs gars ekvris da xels uSlis fagebiT maT swraf inficirebas. xSir SemTxvevaSi fsevdolizogenias gansazRvravs sakvebi nivTierebebis nakleboba. aseT dros ar aris sakmarisi energia imisTvis, rom fagma ganaxorcielos Tavisi sasicocxlo cikli da fagis genomi maspinZeli ujderis SigniT aris araaqtiur mdgomareobaSi.

1.2.4.5 zomieri baqteriofagebis sacicoxlo cikli zomieri fagebisTvis damaxasiaTebelia sasicocxlo ciklis 2 gza: lizisuri an zomieri. is, Tu romeli gziT warimarTeba sasicocxlo cikli, damokidebulia baqteriuli ujredis fiziologiuri mdgomareobis ganmsazRvrel mraval faqtorze [40,128]. lizisuri gzis SemTxvevaSi, lizogenuri fagebi gadian ganviTarebis igive etapebs, rasac litiuri fagebi. zomieri sasicocxlo ciklis SemTxvevaSi, faguri dnm integrirdeba baqteriul dnm-Si profagis saxiT da replicirdeba sinqronulad baqteriul genomTan erTad. swored am movlenas lizogenia ewodeba, xolo baqteriul kulturas lizogenuri. lizogenia sakmaod stabiluri Tvisebaa da mxolod garkveuli garemo faqtorebis gavleniT aRiniSneba fagis virulentobis ganmsazRvreli genebis stimulacia da fagis induqcia [40,128]. lizogeniis koncefcias gaaCnia cvalebadi istoria. fagis pirvel mkvlevrebs 1920-1930 wlebSi sjerodaT, rom fagi dakavSirebuli iyo baqteriasTan da Tavad baqterias hqonda fagebis spontanuri generaciis unari, romelic principSi am dros ufro fermentad miiCneoda da ara cocxal virusad [40,112]. SemdgomSi naxes, rom es ujredebi mravldebodnen fagis produqciis gareSe, Tumca garkveul SemTxvevaSi maT SeamCnies ujredis spontanuri lizisi da areSi Tavisufali fagi. lvofma fagis ujderSida mdgomareobas profagi uwoda da mogvianebiT aCvena, rom lizogenuri ujredis ultraiisferi dasxivebiT adgili hqonda profagis litiuri zrdis iniciacias da Sedegad fagis induqcias [128]. lizogeniis movlenas xSirad Tan axlavs e.w. lizogenuri konversia, romlis drosac aRiniSneba virusuli dnm-iT gamowveuli baqteriuli ujredis genetikuri Tvisebebis cvlileba [42,80]. lizogenur fagebs unari aqvT gadaitanon maspinZeli ujredis genebi erTi baqteriidan meoreSi (transduqcia) [41,128]. mraval lizogenur fags gaaCnia integrirebisTvis specifikuri saitebi. am SemTxvevaSi transduqcia moixsenieba, rogorc specializirebuli transduqcia. sxva fagebisTvis damxaxasiaTebeli ar aris integrirebisTvis specialuri ubnebis arseboba da isini integrirdebian baqteriuli dnm-is nebismier ubanSi, SemTxveviTad. aseTi saxis transduqcia ukve moiazreba, rogorc generalizirebuli transduqcia [128].

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fagebis roli bunebaSi, rogorc genebis horizontalurad gadamtanebisa, sakmaod didia, gansakuTrebiT baqteriebis evoluciis TvalsazrisiT [41]. mikrobTa virusebi miiCnevian baqteriebs Soris genebis, maT Soris antibiotikorezistentuli genebis horizontalur gadatanaSi monawile yvelaze metad gavrcelebul veqtorebad. Tanamedrove kvlevebi mowmobs, rom fagebi rezistentuli genebis rezervuars warmoadgenen da monawileoben maT gavrcelebaSi, filogenetikurad aramonaTesave mikrobebs Sorisac ki. fagebiT genebis horizontaluri gadatana sakmaod xSiri movlena unda iyos, rac axsnis antibiotikorezistentuli genebis aseT mravlobiTobas da gavrcelebis farTo areals. 1.2.5. baqteriofagebis biologiuri Tvisebebi da garemo faqtorebis mimarT

rezistentoba

baqteriofagebs axasiaTebT antigenuri Tvisebebi. organizmSi maTi Seyvanis Semdeg warmoiqmneba antisxeulebi, romlebic fagis aqtivobas aneitraleben da axasiaTebT maRali specifikuroba. fagebi Seicaven tipospecifikur da jgufospecifikur antigenebs. tipospecifikuri antigenebis mixedviT maT yofen serotipebad. fagebi fizikuri da qimiuri faqtorebisadmi ufro maRali gamZleobiT xasiaTdebian, vidre adamianis virusebi. maTi umravlesoba inaqtivirdeba aranakleb 65-70C-ze. kargad itanen gayinvas da Senaxvas dabal temperaturaze, aseve, gamoSrobas. sulemis 0,5%-iani, fenolis 1%-iani xsnarebi ver axdenen maTze SesamCnev gavlenas, xolo formalinis 1%-iani xsnari ramdenime wuTSi iwvevs maT inaqtivacias, maT aqvT maRali mgrZnobeloba mJavebisadmi. ultraiisferi sxivebi da maionizirebeli radiacia axdens fagebis inaqtivacias, xolo dabali dozebi iwveven maT mutacias.

1.2.6. baqteriofagebis daxasiaTebisa da identifikaciis meTodebi

baqteriofagebis kvlevaSi maTi daxasiaTebisaTvis gamoiyeneba Semdegi standartuli maxasiaTeblebi: fagis erTjeradi gamravlebis cikli (egc), negatiuri koloniebis daxasiaTeba, lizisuri aqtivobis speqtri, serologiuri maxasiaTeblebi, faguri nawilakebis fiziko-qimiuri Tvisebebi, fagis dnm-is tipi da restriqciuli profili [5,6]. fagis erTjeradi gamravlebis cikli. fagis erTjeradi gamravlebis dros unda iqnes uzrunvelyofili fagis ganviTarebis dawyeba erTdroulad inficirebuli baqteriebis umravlesobaze. amis miRweva SeiZleba sxvadasxva meTodiT. fagebi, romlebic kargad adsorbirdeba, Txevad areze adsorbcias ganicdian daaxloebiT 3-5- wT-is ganmavlobaSi. erTjeradi gamravlebis gza aCvenebs ganviTarebis TiToeuli periodis xangrZlivobas. fagis ujredSida gamravlebis dros yofen 3 periodad: eklifsuri (faruli), latenturi da lizisis dro. farul periodSi (dro infeqciis dawyebidan anu fagis genomis

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baqteriaSi Sesvlidan pirveli momwifebuli fagebis warmoqmnamde) xorcieldeba ujredSida ganviTarebis yvela procesi, romelic fagis momwifebisaTvis aris aucilebeli. faruli da latenturi periodebis xangrZlivoba Cveulebriv erTerTi SedarebiT damaxasiaTebeli Tvisebaa ama Tu im fagisaTvis. standartuli cdebis pirobebSi sakontrolo fagis Tanaobisas SeiZleba moxdes Sedareba kontrolTan. lizisis da latenturi periodis xangrZlivoba sxvadasxvaa. lizisis dro da infeqciis mravlobiToba ar iZleva fagis identifikaciis saSualebas. negatiuri kolonia (nk). es maxasiaTebeli axlad gamoyofili fagis pirveladi identifikaciis saSualebaa. fagis negatiuri kolonia myar an naxevradTxevad agarze warmoiqmneba fagis mier baqteriuli ujredis inficirebis Sedegad. fagis identifikaciisas Tundac naTesauri fagebis SemTxvevaSi negatiuri koloniis forma da zoma aris sakmaod specifikuri Tviseba. negatiuri koloniiT SeiZleba erTi fagis meorisagan gansxvaveba: centraluri lizisis zonis sididiT, simRvriviT, oreolis arsebobiT, misi formiT da zomiT, opalesenciis unariT. negatiuri koloniebis SedarebiT ar SeiZleba Sefasdes fagebis msgavseba, magram Tu ori fagis negatiuri kolonia erTmaneTisagan gansxvavdeba, es fagebi ar SeiZleba identurni iyvnen [3,4, 7, 180]. lizisuri aqtivobis speqtri, anu maspinZel baqteriaTa speqtri warmoadgens baqteriofagebis daxasiaTebisa da identifikaciis erT-erT saSualebas da mas didi saklasifikacio mniSvneloba aqvs. imisaTvis, rom lizisuri aqtivoba gamoyenebuli iqnes fagis saidentifikaciod aucilebelia: aRebul iqnes potenciuri maspinZlebis farTo speqtri da agreTve maTi

sxva naTesauri saxeobebis baqteriebi unda Semowmdes fagis aqtivoba sxvadasxva mutantebze_ Stamebze,

romlebic sxva fagebis mimarT rezistentulni arian; SesaZlebelia, dadgindes, Tu ratom ar aris fagi aqtiuri ama Tu im

Stamis mimarT; misi ganviTarebis romeli periodis daTrgunva xdeba maspinZel ujredSi (adsorbcia, ujredSida gamravleba) [7,11,49].

antifaguri Sratis gamoyeneba baqteriofagebis identifikaciisaTvis. specifikuri antifaguri Sratebi erTerTi umTavresi saSualebaa fagebis identifikaciisa da klasifikiciisaTvis. antifaguri Sratebis momzadebis meTodi martivia, xolo neitralizaciis jvaredini reaqcia_ sakmarisad zusti, romlis Sedegad SeiZleba dadgindes aris Tu ara ori fagi erTmaneTis msgavsi. Tu erTi da igive wyarodan mcire drois periodSi gamoyofili iyo 2 fagi, romelTac aqvT erTnairi mgrZnobeloba antifaguri Sratis mimarT, magram gansxvavdebian lizisuri aqtivobis speqtriT, miuTiTebs, rom gvian aRmoCenili fagi warmoadgens pirveli fagis mutants (fagis aqtivobis speqtris gazrdiT). msgavsi fagebi SeiZleba aRmoCndnen sxvadasxva serotipebSic. faguri nawilakebis fiziko-qimiuri Tvisebebi. fagis virionis morfologiuri Tvisebebi, romlebic Seiswavleba eleqtronuli mikroskopiis saSualebiT, aris

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fagis erTerTi mniSvnelovani damaxasiaTebeli Tavisebureba, magram saorientacio da ara zusti. baqteriofagebis daxasiaTebisaTvis metad mniSvnelovan maCveneblebs warmoadgens sxvadasxva fiziko-qimiuri faqtorebis moqmedeba, rogoricaa: qloroformis da sxva organuli gamxsnelebis moqmedeba, Termoinaqtivacia, UV gamosxiveba, aris sxvadasxva pH da mravali sxva.

1.2.7. Vibrio-s gvaris baqteriebis mimarT specifikuri baqteriofagebi

Vibrio-s gvaris mimarT specifikuri baqteriofagebi pirvelad identificirebul iqna derelis mier 1926 wels da 1950 wlisTvis aRweril iqna ramdenime, V. cholerae-s mimarT specifikuri baqteriofagi. adreuli Sromebi V. cholerae-s fagebis Sesaxeb Seexeboda ZiriTadad maT gamoyenebas mkurnalobis da profilaqtikis mizniT da naklebad ukavSirdeboda Stamebis diferenciacias. V. cholerae-s fagebis mimarT interesi ganaxlda VII pandemiis gavrcelebasTan da 1960-ian wlebSi “El Tor”-is biotipis aRmoCenasTan erTad. amJamad kvlevebs ukve ori mimarTuleba mieniWa, erTis mxriv fagotipirebis sqemis Seqmna litiuri fagebis da meores mxriv, klasifikacia- lizogenuri fagebis gamoyenebiT. aRmoCenilia VIbrio-s gvaris mimarT specifikuri 85 fagi, 84 maTgani miekuTvneba kudian fagebs, mxolod erTi aris filamenturi.Kkudiani fagebi mikuTvnebian 6 mTavar morfotips da Myoviridae-s, Siphoviridae-s, da Podoviridae-s ojaxebs. 63 fagi klasificirda 18 saxeobad. Ffilamenturi fagi aris Inovirus-is gvaris da Inoviridae-s ojaxidan. Vibrio-s fagebi Zalzed heterogenulia da moicavs zogierT morfologiurad saintereso virusebs. zogierTi fagi axlos dgas sxva gram (-) baqteriaTa fagebTan, SesaZloa es maT filogenezur naTesaobazec mianiSnebdes. V. cholerae–s filamenturi baqteriofagi- CTX iTvleba qolera-toqsinis genis klasteris (ctxAB) gadatanis meqanizmad.Mmocemuli fagi integrirdeba qromosomul dnm-Si attRS- mimagrebis saitis saSualebiT, xolo StamebSi, romelTac es saiti ar gaaCniaT, replicirebs plazmidis saxiT. vinaidan ctxAB CTX fagis struqturis nawilia, mas SeuZlia ctxAB-s horizontalurad gadatana toqsigenuridan aratoqsigenur Stamebze.

1.2.8. fagoTerapia da istoriuli konteqsti

f.derelis mier fagis aRmoCenidan maleve daiwyo misi gamoyeneba dizenteriis samkurnalod. swored es iyo fagis pirveladi Terapiuli gamoyeneba [50]. es kvleva mimdinareobda parizSi 1919 wels. Ppirveli eqsperimentebi sakmaod efeqturi aRmoCnda, Tumca warmatebuli Sedegebi maSinve ar gamoqveynebula. pirveli angariSi, sadac miTiTebulia fagoTerapiis warmatrebuli SemTxveva, gamoica 1921 wels, roca fagi gamoiyenes kanis stafilokokuri daavadebis samkurnalod [50]. maleve Catarda mravali msgavsi kvleva, romlis Sedegebic xazs usvamda fagebis did potencials sxvadasxva

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infeqciebis samkurnalod, ramac xeli Seuwyo ramdenime faguri preparatis warmoebas sxvadasxva kompaniebis mier. f.derelis laboratoria uSvebda 5 samkurnalo komerciul preparats sxvadasxva baqteriuli infeqciebis samkurnalod [9]. Terapiuli faguri preparatebi mniSvnelovani produqtebi iyo farmacevtul industriaSi, aRmosavleT evropasa (1952 wlidan) da yofil sabWoTa kavSirSi (1923 wlidan). am qveynebSi fagoTerapiuli kvlevebi da faguri Terapiis ganviTareba centrirebuli iyo poloneTis mecnierebaTa akademiis imunologiis da eqsperimentuli Treapiis hirscfildis institutSi (vroclavi, poloneTi), ufro farTod ki g.eliavas saxelobis baqteriofagiis, mikrobiologiis da virusologiis institutSi (Tbilisi, saqarTvelo). Terapiuli fagebi iwarmoeboda aseve aSS-Si. uiliam smitma da kolegebma TagvebSi ganxorcielebuli eqsperimentebis seriiT asaxes fagebis warmatebiT gamoyeneba E.coli-iT gamowveul infeqciebSi. arsebobs cnobebi, fagebis gamoyenebis eqsperimentul TagvebSi da zRvis goWebSi Pseudomonas aeruginosa da Acinetobacter-iT gamowveuli infeqciebis prevenciisa da mkurnalobis mizniT [21,34,164]. bogovazovam da mkvlevrebma [34] Seafases baqteriofagebis efeqturoba Klebsiella ozaenae, K.rhinoscleromatis da K.pneumoniae-iT gamowveuli infeqciebis mkurnalobaSi. faguri Terapia gmaoyenebuli iyo sxvadasxva infeqciis samkurnalod, rogoricaa: baqteriuli dizenteria [26,179], Wrilobebi [155], kuW-nawlavis traqtis infeqciebi [163], kanisa da nazaluri lorwovani garsis infeqciebi, salmonelozi, gamowveuli sxva eTiologiuri agentebiT, kerZod: Shigella, Salmonella, Proteus, Staphylococcus, Streptococcus. fagebi msgavsni arian antibiotikebis, imdenad, ramdenadac aqvT gamorCeuli antibaqteriuli moqmedeba. fagebs aqvT specifikuri moqmedeba, zemoqmedeben ra, mxolod samizne baqteriul ujredze da ara adamianze an sxva eukariotul ujredebze. V. cholerae- s mimarT specifikuri fagebi yovelTvis moqmedeben V. cholerae–s mimarT da ar axdenen sxva mag.: Shigella-s, Salmonella-s an E.coli-is baqteriis ujredis liziss. es aris mniSvenlovani gansxvaveba faguri preparatisa antibiotikisgan, romelic moqmedebs, rogorc paTogenis aseve normaluri mikrofloris winaaRmdeg. Sedegad pacientSi adgili aqvs mikrobul disbalanss, rasac mivyavarT meoradi infeqciebis ganviTarebisken. yofil sabWoTa kavSirSi da aRmosavleT evrobaSi Catarebulma klinikurma fagoTerapiis cdebma aCvena fagebis profilaqtikuri da Terapiuli gamoyenebis dadebiTi mxare. adamianSi liTiuri fagebis gamoyenebis uaryofiTi maCveneblebi jer ar aRwerila [169]. miuxedavad fagebis gamoyenebis usafrTxoebisa Terapiuli mizniT, Cveulebriv maTi farTo moxmareba ar xdeba da fagebis moqmedebis efeqturoba jer kidev kamaTis sakiTxia. erT-erTi SezRudva aris fagebis maRali specifikuroba samizne baqteriuli saxeobis mimarT. V. cholerae O1-is mimarT specifikuri baqteriofagebis Seswavla odiTganve interesis sferos warmoadgenda. fagebi gamoiyeneboda V. cholerae O1-is mier gamowveuli infeqciebis diagnozis dasamtkicebel saSualebad. baqteriofagebi dRemde gamoiyeneba V. cholerae O1-is serotipis “klasikuri” da “El Tor” biotipebis gansasxvaveblad. warsulSi E.coli-is mimarT specifikuri baqteriofagebi gamoiyeneboda wyalSi araenteropaTogenuri specifikurobis,

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fekaluri dabinZurebis deteqciisTvis [184]. qveynebSi, sadac qolera endemuria, V. cholerae O1-is mimarT specifikuri baqteriofagebi nanaxi iyo Camdinare wylebSi da SemdgomSi gamoyenebuli, rogorc Stamebis markerebi [17] da aseve V. cholerae O1-is da O139-is biotipirebisTvis [24,46,51,148]. qveynebSi, sadac qolera avlens sezonurobas, xasiaTdeba ra epizoduri gamovlinebebiT, garemos zedamxedvelobam SesaZloa mniSvnelovani roli iTamaSos qoleras kontrolSi. V. cholerae O1-is mier gamowveuli asimptomuri infeqciebi ufro xSiri xasiaTisaa aqtiur SemTxvevebTan SedarebiT [187], amgvarad Camdinare wylebSi V. cholerae O1-is Stamebisa da Sesabamisi fagebis deteqciis kontroli SesaZloa iyos misaRebi saSualeba qoleras afeTqebebis winaswar ganWvretisa manam, sanam SemTxvevaTa mniSvnelovani raodenoba iCens Tavs. araerTgzis iyo naTqvami, rom Camdinare wylebSi vibriofagebis arseboba dakavSirebulia qoleras SemTxvevebTan. antibiotikebis aRmoCena miCneulia medicinis sferoSi erTerT mniSvnelovan miRwevad. Tumca antibiotikebis mimarT multirezistentuli baqteriuli Stamebis dagrovebam Tanamedrove medicinaSi erT-erTi yvelaze kritikuli problema warmoSva. antibiotikebis aRmoCenamde da farTod gamoyenebamde SemoTavazebuli iyo baqteriofagebis gamoyeneba baqteriuli infeqciebis prevenciisa da samkurnalod. kanis stafilokokuri infeqciis warmatebuli mkurnaloba aRwerili iyo safrangeTSi [38]. britanelma baqteriologma ernest hankinma aRwera antibaqteriuli moqmedeba V. cholerae-s mimarT indoeTis mdinareebSi (gangi da iamuna) [92]. man ivarauda, rom ucxo agenti (romelic tardeboda Txel faifuris filtrSi da iyo Termolabiluri), iyo pasuxismgebeli am fenomenze da qoleras epidemiis gavrcelebis SezRudvaze [92]. hankinis kvlevebidan TiTqmis 20 wlis Semdeg ingliselma frederik tuortma gamoTqva hipoTeza, rom es agenti unda yofiliyo virusi [183]. me-20 saukunis ocian wlebSi dereli stumrobda indoeTs, kerZod hafkinis instituts, bombeiSi sadac muSaobda Savi Wiris sawinaaRmdego fagoTerapiaze, ramac xeli Seuwyo indoeTSi baqteriofagebis kvlevis dasabams, indoeTis kvlevis fondis asociaciis meSveobiT da aseve fagoTerapias, gansakuTrebiT qoleras epidemiebis dros religiuri festivalebis da piligrimebis arsebobisas. daavadeba qolera idealuri SemTxvevaa fagoTerapiisTvis. daavadebis transmisiis da epidemiologiis maxasiaTeblebi kargad aris cnobili, Tumca efeqturi vaqcina dRemde ar aris Seqmnili. daavadeba qolerasTvis arsebuli faguri Terapia ar aris mkafiod dadgenili pacientebis samkurnalod an prevenciisTvis, rogorc amas dereli imedovnebda [67]. gamokvleuli iyo, rom qoleras simptomebis simwvave da xangrZlivoba da am daavadebiT gamowveuli saerTo sikvdilianoba Semcirda mas Semdeg rac pacientebma oraluri gziT miiRes V. cholerae–s mimarT specifikuri fagebi [170]. 1960 wels msoflio jandacvis organizaciis (WHO) mier adgili qonda qoleras mkurnalobis saerTaSoriso mcdelobas dakaSi fagoTerapiis gziT. es mcdeloba Catarda farTod miRebuli saerTaSoriso standartebiT, jandacvis erovnuli institutebis (aSS) zedamxedvelobis qveS. WHO-s mier dafinansebul kvlevebSi 1970 wels, bangladeSSi fagoTerapia Sedarebuli iyo tetraciklinTan,

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rogorc Terapiul agentTan. naCvenebi iyo, rom didi doziT faguri Terapia msgavsi iyo tetraciklinis moqmedebasTan fekaliebidan vibrionebis gamoyofis Semcirebis TvalsazrisiT. infeqciur paTologiaSi baqteriofagis gamoyenebis rekomendaciebi dafuZnebulia im gamocdilebaze, rac dagrovda mravali wlis manZilze. dReisTvis baqteriofagebi warmatebiT gamoiyeneba Cirqovan-anTebiTi, infeqciebis, nawlavuri da Sard-sasqeso gzebis infeqciuri anTebiTi procesebis samkurnalod [99, 130, 141, 168, 169].

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II.Mmasalebi da meTodebi

2.1 kvlevaSi gamoyenebuli masalebi

wylis sinjebi baqteriuli Stamebisa da baqteriofagebis gamosayofad wylis sinjebs viRebdiT saqarTvelos sxvadasxva wyalsatevidan, kerZod: Tbilisis SemogarenSi arsebuli mtknari wyalsatevebidan (lisis da kumisis tbebi, Tbilisis zRva, md. mtkvari) da Savi zRvis sxvadasxva wertilidan (md. sufsis SesarTavi, md.Woroxis SesarTavi, mwvane koncxis sanapiro zoli da baTumis bulvari). baqteriuli Stamebi: Svidi standartuli Stami-mowodebuli saqarTvelos (Tbilisi) daavadebaTa kontrolis erovnuli centris (NCDC) koleqciidan. V. cholerae-s ori standartuli Stami: O1 “El Tor” (V.ch 890-M-878) da V. cholerae O1 “klasikuri” (V.ch 145-P-1) biotipebi. pasteris institutis (CIP) mier mowodebuli 4 standartuli Stami: V. cholerae O1 “klasikuri” (V.chCIP 55.91, V.chCIP 62.13) da V. cholerae O1 “El Tor” (V.ch CIP 106855) biotipebi da V. cholerae O139 (V.ch CIP 104151). aseve V. mimicus-is standartuli Stami (V. mimicus ATCC33653) mowodebuli iyo amerikis tipiur kulturaTa koleqciidan (ATCC, Manassas,VA USA). 2006-2009 wlebSi, monitoringis Sedegad saqarTvelos wyliani garemodan (lisis da kumisis tbebi, Tbilisis zRva, md. mtkvari da Savi zRvis wertilebi: md. sufsis SesarTavi, md.Woroxis SesarTavi, mwvane koncxis sanapiro zoli da baTumis bulvari) gamoyofili 846 V. cholerae-s da 11 V. mimicus-is izolati, romlebic SemdgomSi gamoyenebuli iyo specifikuri bateriofagebis gamosayofad da gasamravleblad. gamoyofili baqteriuli izolatebi inaxeba g.eliavas saxelobis baqteriofagiis, mikrobiologiisa da virusologiis institutSi. V. cholerae-s da V. mimicus-is mimarT specifikuri baqteriofagebi V. cholerae-s mimarT specifikuri standartuli baqteriofagebi: baqteriofagi 163 (I jgufis prototipi; ATCC 14100-B4) da baqteriofagi 138 (II jgufis prototipi;ATCC 14100-B1) mowodebuli iyo amerikis tipiur kulturaTa koleqciidan (ATCC, Manassas,VA USA). samuSaos Sesrulebis dros gamoyofili 82 baqteriofagis pirveladi izolati. V. cholerae-s mimarT specifikuri 24 izolati da V. mimicus-is mimarT specifikuri 7 izolati, Semdgomi eqsperimentuli kvlevisTvis.

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mikrobiologiuri areebi: Vibrio spp. mikroorganizmebis saidentifikacio: TCBS- Tiosulfat-citrat-naRvlis marilebis- saqarozas Semcveli myari

are. baqteriebisa da baqteriofagebis kultivirebisa da SenaxvisaTvis saWiro myari, Txieri da naxevradTxieri sakvebi areebi: LB (Luria-Bertani) luria-bertanis myari 2%-iani agari (Difco); LB (Luria-Bertani) luria-bertanis Txieri are (Difco); LB (Luria-Bertani) luria-bertanis naxevradTxieri 0.7%-iani are (Difco); soios agari da bulioni (“Difco”, “Biomereux”); peptoniani tute wyali; koncentrirebuli bulioni.

B baqteriebis fiziologiur-bioqimiuri Tvisebebis Sesaswavlad: T1N1 agari–tripton mariliani agari; T1N6 bulioni-tripton mariliani bulioni; T1N8 bulioni-tripton mariliani bulioni; GSA agari _3% natriumis qloridis Semcveli agari; GA agari_Jelatiniani, umarilo agari; hiu-leivsonis (HF) glukoziani are; aminomJavebi: arginini, lizini, orniTini; naxSirwylebi: saqaroza, manoza, arabinoza, laqtoza.

samuSao buferuli sistemebi da qimiuri reaqtivebi:

faguri buferi – 50mM Na2HPO4, 22mM KH2PO4, 85mM NaCl, 12mM MgSO4, 11mM CaCl2, pH 7,2;

PBS –dabuferebuli fiziologiuri xsnari, pH 7,4 ( Sigma) ; TA- tris acetaturi buferi pH 7,4( Sigma); 5M NaCl- xsnari; 10mM MgCl2- xsnari; 10mM Tris- 1mM EDTA buferuli xsnari pH 7,4; SM buferi (100mMNaCl, 8mM MgSO4•7H2O, 50mMTris-Cl (1 M, pH 7.5), 0.002% (w/v)

Jelatini); 1% SDS.

V. cholerae-s izolatebis serologiuri identifikaciisTvis: fluorescentulad moniSnuli antisxeulebi (New Horizon Diagnostics, MD)

garemoSi V. cholerae-s epidemiuri serotipebis gamosavlenad pirdapiri

fluorescentuli analizis (DFA -direct fluorescent antibody assay) meTodiT;

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qoleras poli-imunuri Srati (“Difco”) garemoSi V. cholerae-s epidemiuri serotipebis gamosavlenad;

Cholera O1 SMARTTM garemoSi V. cholerae-s epidemiuri serotipebis gamosavlenad;

baqteriofagebis serologiuri daxasiaTebisTvis: antifaguri (APS) Sratebi. miRebuli iyo bocvrebis imunizaciis Sedegad

V. cholerae-s mimarT specifikuri baqteriofagebis gamoyenebiT (sul 3 antifaguri Srati: APS 54, APS 10CH, APS 105);

adiuvanti (Freund’s adjuvant, Difco); laboratoriuli cxovelebi: SinSilas jiSis 20 bocveri. gamoyenebuli

iyo antifaguri Sratis misaRebad;

baqteriuli dnm-is gamosayofad da genetikuri identifikaciisTvis: specialuri komerciuli nakrebi (Bio-Rad Laboratories, Hercules, CA da Gen Elute,

Sigma ) baqteriuli dnm-is gamosayofad; polimerazuli jaWvuri reaqciis (ITS PCR) praimerebi V. cholerae-s da V.

mimicus-is izolatebis genetikuri identifikaciisTvis (cxr.1); V. cholerae-s epidemiuri serotipebis (V. cholerae O1 “klasikuri” da “El Tor“

biotipi, V. cholerae O139) da virulentobis faqtorebis Sesaswavlad (ctxA genebi) gamoyenebuli polimerazuli jaWvuri reaqciis (multipleqs-PCR) praimerebi (cxr.2).

cxr.1. polimerazuli jaWvuri reaqciis (ITS -PCR) praimerebi

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cxr.2. polimerazuli jaWvuri reaqciis (multipleqs-PCR) praimerebi

restriqciuli endonukleazebi: baqteriofagebis restriqciuli profilis Sesaswavlad (cxr.3):

cxr.3. restriqciuli endonukleazebi

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2.2. kvlevaSi gamoyenebuli meTodebi

2.2.1. Ria wyalsatevebidan aRebuli wylis sinjebis damuSaveba kvlevis miznisa da specifikis gaTvaliswinebiT sinjebis aRebis sixSire da damuSavebis procedura gansxvavdeboda. wylis sinjebis aReba warmoebda erTi ZiriTadi sqemis mixedviT. sinjebs viRebdiT dRis pirvel naxevarSi 11-15 saaTamde intervalSi. sinjebs viRebdiT wylis zedapiridan 15-30sm siRrmeze sterilur WurWelSi 1 litris moculobiT. paralelurad viniSnavdiT: aminds, temperaturas da sxva klimatur pirobebs. wylis nimuSebis analizs vanxorcielebdiT sinjis aRebidan 4 sT-is ganmavlobaSi. wylis fiziko-qimiuri parametrebis gansazRvra. wylis zogierT fiziko-qimiuri parametri, rogoricaa marilianoba, gamtaroba, wyalbad ionTa koncentracia, wyalSi gaxsnili Jangbadis raodenoba da myari nawilakebis raodenoba, isazRvreboda multiparametruli portatuli sazomi xelsawyos (YSI 556 MPS) saSualebiT. mikrobuli dabinZurebis xarisxis gansazRvra. sakvlev wyalSi saerTo mikrobuli ricxvis gansazRvrisaTvis viyenebdiT 1,8% -iani LB agaris finjnebs. sakvlevi wylis sawyisi da aTjeradi ganzavebebi 1 ml raodenobiT dagvqonda petris finjanze da vamatebdiT 15-20 ml gamlRval 45ºC-mde gagrilebul LB agars. vaxdendiT inkubacias 37oC-ze 24-48 saaTis ganmavlobaSi da viTvlidiT warmoqmnili baqteriuli koloniebis raodenobas. „Aerobic Count plates“ (3M, aSS) petrifilmebis meSveobiT vsazRvravdiT wylis nimuSSi aerobebis saerTo raodenobas. pertifilmze dagvqonda sakvlevi wylis sawyisi da aTjeradi ganzaveba 1 ml-is odenobiT. vaxdendiT inkubacias 37oC-ze 24 saaTis ganmavlobaSi. Sedegs viwerdiT Sesabamisi sqemis meSveobiT. 2.2.2. Vibrio-s gvaris baqteriebis gamoyofa 100 litri wyali tardeba 200mkm-ian da 64mkm-ian badeSi. 100ml gafiltruli wyali (PFW-plankton free water) ifiltreba 0,45mkm-ian filtrSi, Tavsdeba 100 ml peptonian wyalSi. 10-20 ml gafiltruli wyali ifiltreba 0,45mkm-ian filtrSi, Tavsdeba TCBS-is niadagze da orive varianti inaxeba TermostatSi 370C-ze 24 saaTis ganmavlobaSi. 9ml peptonian wyals emateba 1 ml planqtoni (64mkm da 200 mkm-ian badeSi gafiltruli) da inaxeba TermostatSi 370C-ze 24 saaTis ganmavlobaSi.

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2.2.3. baqteriuli Stamebis biologiuri Tvisebebis Seswavla sakvlevi Stamebis kulturalur-morfologiur Tvisebebs vswavlobdiT baqteriebis zrdis mixedviT myar, Txevad da naxevradTxevad areebze. bioqimiuri Tvisebebis Seswavla sakvlevi Stamebis bioqimiuri Tvisebebis Seswavlas vawarmoebdiT specializirebul sakveb areebze (sur.2), kerZod: proteolizur aqtivobas vswavlobdiT Jelatinis gajirjvebis mixedviT GSA agarze. hiu-leivsonis glukozian areSi mikroorganizmebis kultivirebisas glukozis moxmareba ganixileba ori meqanizmiT: glukozis fermentaciiT (zeTmosxmuli sinjara) an glukozis daJangviT (uzeTo sinjara). izolatebi mowmdeboda marilis moxmarebaze, naxSirwylebis moxmarebaze (saqaroza, laqtoza, manoza, arabinoza), oqsidazaze, lizin da ornitin dekarboqsilasa da arginin-dehidrolazaze. Bbioqimiur testebze dayrdnobiT identificirebuli V.cholerae-s da V. mimicus-is Stamebi Semdgom damatebiT mowmdeboda or saxeoba-specifiuri polimerazuli jaWvuri reaqciiT.

sakvlevi wylis nimuSebidan vibrionebis

gamoyofisa da identifikaciis sqema

sur.2. Vibrio-s gvaris baqteriebis identifikaciis sqema

wylis sinjebi

vibrionebis momravleba 1% -ian peptoniani wyalSi

kultivireba seleqtiur niadagze TCBS

oqsidaza testi

zrda 1% NaCl-isSemcvel Jelatinian agarze da 0%-10% NaCl-isSemcvel

Txevad niadagSi

hiu-leivsonis

glukoziani are

naxSirwylebis da aminomJavebis fermentacia

polimerazuli jaWvuri reaqcia (PCR)

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genetikuri identifikacia. Stamebis genetikuri daxasiaTeba xdeboda ITS-polimerazuli jaWvuri reaqciiT (ITS-PCR, Internal Transcribed Spacer PCR) specifikuri praimerebis gamoyenebiT. V. cholerae O1-is, V. cholerae O139-is da toqsogenuri genebis (ctxA, ctxB, omp) aRmosaCenad viyenebdiT multipleqsuri

polimerazuli jaWvuri reaqciis meTods (Multiplex-PCR). genomuri dnm-is gamoyofas vaxdendiT specialuri komerciuli nakrebis gamoyenebiT (Bio-Rad Laboratories, Hercules, CA). saxeoba-specifiuri praimerebis gamoyenebiT xdeboda bioqimiurad identificirebuli V. cholerae–s da V. mimicus-is izolatebis identifikacia polimerazuli jaWvuri reaqciis (pjr) saSualebiT. polimerazul jaWvur reaqciaSi gamoyenebuli iyo baqteriuli kulturebis dnm, Taq-dnm polimeraza, Tavisufali nukleotidebis narevi –deoqsinukleozid trifosfatebi (dATP, dCTP, dGTP. CITTP 10 mmoli TiToeuli koncentracia) da oligonukleotiduri praimeri (pirdapiri da uku-mimarTulebis). Ppjr-produqtebis analizs vaxdendiT 1-1,2% agarozas gelSi eleqtroforeziT.

Cholera O1 da Bengal DFA–pirdapiri fluirescentuli analizi. Cholera DFA-is testi moicavs V. cholerae O1–is gare membranaze arsebul O1 lipopolisaqaridis A antigenis mimarT da V. cholerae O139-is gare membranaze arsebul O139 lipopolisaqaridis zedapiruli antigenis mimarT specifikur moniklonur antisxeulebs, romlebic pirdapir arian dakavSirebulni fluorescentul izoTiocianitTan (FITC). meTodi gamoiyeneba V. cholerae O1 da V. cholerae O139-is swrafi, advili deteqciisa da aRricxvisTvis wylis, sakvebis da fekalur sinjebSi. masalas warmoadgenda wylis nimuSebi:koncentrirebuli, gamdidrebuli wyali, gamdidrebuli planqtoni (64mkm da 200mkm, 64mkm homogenizirebuli da 200mkm homogenizirebuli). vaxdendiT wylis nimuSis dafiqsirebas DFA-is preparatisTvis. gamdidrebuli wylis nimuSidan da gamdidrebuli planqtonis nimuSidan 1 ml gadagvaqonda ependorfSi. vatrialebdiT 5000g/wT 5wT-is ganmavlobaSi. supernatants vRvridiT da naleqs vxsnidiT 1ml fiziologiur xsnarSi. xelaxla vatrialebdiT 5000g/wT 5 wuTis ganmavlobaSi. supernatants kvlav vRvridiT, naleqs vxsnidiT 1ml fiziologiur xsnarSi da vafiqsirebdiT 10mkl 37%-iani formaldehidiT.

cdis dawyebamde yvela materiali Tavsdeboda oTaxis temperaturaze; sasagne minaze wveTdeba 5mkl sinjis nimuSi; calke sasagne minaze wveTdeboda V. cholerae O1 –is da Bengal-is pozitiuri

da negatiuri kontroli, 5mkl-is odenobiT; sasagne minebs vaSrobdiT 30 wuTis ganmavlobaSi; Semdeg etapze vamatebdiT 5mkl eTanols da kvlav vaSrobdiT; vamatebdiT 10mkl qolera DFA reagents (O1 da Bengal); sasagne minebs vaTavsebdiT nestian garemoSi da inkubacias vaxdendiT

370C temperaturaze; vrecxavdiT PBS-Si vaSrobdiT da vaTavsebdiT sibneleSi; vawveTebdiT TiTo wveT fluorescentul Mounting Medium-s;

40

sinjebs vnaxulobdiT fluorescentul mikroskopSi 100x gadidebaze imersiuli zeTiT.

aglutinaciis reaqcia B.D. DifcoTMV. cholerae-s antiSratiT. gamoiyeneboda qoleras poli-imunuri Srati (“B.D. DifcoTM”) garemoSi V. cholerae-s epidemiuri serotipebis gamosavlenad (cxr.4).

cxr.4. reagentebi

antiSrati O antisxeulebi

B.D. DifcoTM V. cholerae-s antiSrati Poly ABC

B.D. DifcoTMV. cholerae-s antiSrati Ogawa B

B.D. DifcoTMV. cholerae-s antiSrati Inaba C

B.D. DifcoTMV. cholerae-s antiSrati Poly gamoiyeneba V. cholerae-s izolatebis savaraudo dadgenisaTvis. Semdeg etapze aglutinaciis reaqcia tardeba B.D. DifcoTM V. cholerae-s antiSrati “ogava” da B.D. DifcoTM V. cholerae-s antiSrati “inaba”–s gamoyenebiT.

B.D. DifcoTM V. cholerae-s antiSrati 1 wveTis odenobiT daitaneba sasagne minaze;

antiSratze maryuJiT Tanabrad nawildeba myar areze gazrdili sakvlevi mikrobuli kultura;

aglutinaciis Sedegis wasakiTxad sasagne minas 1 wuTis ganmavlobaSi wriulad vamoZravebT.

Sedegebis wakiTxva:

4+ 100%-iani aglutinacia, ukana foni sufTaa,umniSvnelod bundovani; 3+ 75%-iani aglutinacia, ukana foni umniSvnelod bundovania; 2+ 50%-iani aglutinacia, ukana foni zomierad bundovania; 1+ 25%-iani aglutinacia, ukana foni bundovania; _ aglutinacia ar SeimCneva; pozitiuri kontroli aCvenebs 3+ an met aglutinaciis xarisxs; negatiuri kontrolis dros aglutinacia ar SeimCneva; sakvlevi izolatebis SemTxvevaSi 3+ an meti xarisxis aglutinacia 1

wT-is ganmavlobaSi, dadebiTi Sedegia.

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Cholera O1 SMARTTM_mgrZnobiare, membranuli antigenis swrafi testi. Cholera O1 SMARTTM moicavs monoklonur antisxeulebs, moTavsebuls specialuri formis horizontalur firfitebze. antisxeulebi specifiuria V. cholerae O1–is O1 lipopolisaqaridis A antigenis mimarT (cxr.5) anti- A antigen specifikuri antisxeuli dafaruli koloidaluri oqros nawilakebiT (wiTeli Seferilobis) moTavsebulia membranul zedapirze. nimuSi Tavsdeba specialur safiltracio fosoSi. rodesac sakvlevi sinji Seiwoveba (S) safiltracio nawilSi, oqros koniugati urTierTqmedebs nebismier anti –A antigenTan, romelic warmodgenilia membranis mTel sigrZeze, sadac aRiniSneba dakavSirebuli antisxeulis ori zona: T-testis da C-kontrolis zona. antisxeul-oqros koniugatebi, romlebic daukavSirdnen antigens mocemul nimuSSi, daikavSireba V. cholerae O1-is damWeri antisxeulebis zonaSi (T zona), rac gamoisaxeba vizualurad Seferili xiluli zolis saxiT da asaxavs pozitiur Sedegs (T). im SemTxvevaSi Tu nimuSSi V. cholerae O1 ar aris, warmodgenil T zonaSi ar warmoiqmneba Seferili zoli da sakvlevi sinji ganagrZobs migracias pozitiuri kontrolis zonamde (romelic ar aris specifikuri A antigenis mimarT), da daukavSirdeba nebismier arsebul antisxeul-oqro koniugats, Sedegad wiTeli zoli gamoisaxeba C-zonaSi. C zoli unda iyos xiluli, rac testis swori muSaobis maCvenebelia. testis Sesrulebis mTliani dro 20 wuTia.

cxr.5. Cholera O1 SMARTTM testi

dadebiTi Sedegi T da C zonaSi gamoisaxeba wiTeli zoli

uaryofiTi Sedegi Mmxolod C zonaSi gamoisaxeba wiTeli zoli

araswori Sedegi Mmxolod T zonaSi gamoisaxeba wiTeli zoli

araswori Sedegi feradi zoli arcerT zonaSi ar warmoiqmneba

GM1-ELISA-fermentdakavSirebuli imunosorbentuli analizi-qoleris toqsinis

deteqciisTvis [62,76]. eqsperimentisTvis viyenebdiT:

V. cholerae-s Stamebi (x103 ujr/ml kultura,kultivirebuli 16 sT 300C sanjRrevelaze, dacentrifugebuli da gafiltruli);

GM1 gangliozidi (2mg/ml samuSao konc. PBS-Si ganzavebuli)-moqmedebs, rogorc damakavSirebeli saiti qoleris toqsinisTvis (sekretirebuli

42

toqsini emagreba maspinZel organizmSi lorwovani garsis ujredebs GM1 -Tan dakavSirebis gziT);

BSA-Sratis cila (Bovine serum albumin, 1mg/ml samuSao konc. PBS Tween 20-Si ganzavebuli). axasiaTebs:stabiluroba, bioqimiur reaqciebSi efeqturobis

nakleboba. damakavSirebeli Tvisebebis gamo aqvs blokirebis unari,

amitom mniSvnelovnad amcirebs arasasurvel kavSirebs, rac

xelisSemSleli faqtors warmoadgens analizis Catarebis dros;

PBS –fosfaturi buferi; Tween 20- (polioqsieTileni) stabilurobis da aratoqsiurobis gamo

gamoiyeneba, rogorc detergenti. acilebs araspecifiurad dakavSirebul

proteinebs. blokavs proteinebis damakavSirebel ubnebs planSetis

fosoebSi (zavdeba TBS an PBS–Si); anti-CT (cholera toxin ) AB (1:2000 PBS Tween 20-Si); anti-Rabbit IMG Ab peroqsidaz koniugati; fosfat-citratuli buferi natriumis perboratiT (1 kafsula:100ml

distilati); OPD (o-phenylene diamine dihydrochloride) substrati (0.4mg/ml samuSao konc.).

mikrokosmebis eqsperimenti V.cholerae-s sicocxlisunarian, magram arakultivirebadi mdgomareobis (VBNC) ujredebze. kulturebs vzrdidiT dacerebul 1.8%-ian LB-agarze. 24 saaTiani inkubaciis Semdeg 370C-ze, nazrds vrecxavdiT 5 ml fiziologiuri xsnariT da Segvqonda sxvadasxva areebSi: LB-bulionSi, lisis tbis da zRvis wylebSi, T1N6-areSi. TiToeuls vaTavsebdiT macivris (40C) da oTaxis (250C) temperaturebze. mikrokosmebis TiToeuli sinjaridan drois garkveuli intervaliT vaxdendiT baqteriebis amoTesvas, rogorc LB, aseve TCBS-is niadagze.Uujredebis daTvla xdeboda arakultivirebad mdgomareobaSi, rodesac adgili hqonda 0.1 uj/ml-ze naklebi kultivirebadi ujredis gamovlenas.Gkultivirebadi ujredebis gamovlenis limiti dgindeboda mikrokosmis 10 ml-is gafiltvriT 0.22 mkm forovnebis filtrSi da Semdeg filtris moTavsebiT 1.8%-ian LB-agarze.

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2.2.4. baqteriofagebis gamoyofa

baqteriuli virusebis wylidan gamoyofas da kultivirebas vaxdendiT standartuli gamdidrebis meTodiT. TiToeuli saitidan 100 ml wyals vfiltravdiT 0.45 mkm-ian filtrSi. wylis sinjs vumatebdiT koncentrirebul bulions da baqteriuli kulturis narevs, TanafardobiT 10:1:1 da vaTavsebdiT TermostatSi mTeli RamiT 370C-ze. 5000 br/wT centrifugirebis Semdeg vamatebdiT qloroforms da vawveTebdiT Sesabamisi baqteriebis gazonebze. fagis lizisuri zonebis aRmosaCenad finjnebs vamowmebdiT 6-18-24 saaTSi. baqteriofagebis miReba Stamebidan xdeboda fiskis meTodiT [4], SerCeuli indikatori Stamebis meSveobiT. 24 sT-iani baqteriul kulturas vacentrifugebdiT 5,000 rpm-ze da vfiltravdiT 0.45 mkm-ian zomis filtrSi,Semdeg vawveTebdiT indikatori Stamis gazonze. 18 sT-iani inkubaciis Semdeg vnaxulobdiT lizisur zonebs, romelTac vWridiT gazonidan da vainkubirebdiT indikator StamTan erTad fagis Semdgomi gamravlebisa da gasufTavebisaTvis. 2.2.5 baqteriofagebis biologiuri Tvisebebis SeswavlaF fagebis sufTa xazebis misaRebad viyenebdiT orSriani agaris meTods. morfologiurad erTgvarovani koloniebidan 5-10 Tanmimdevruli pasaJis Sedegad. fagebis lizisuri speqtris Seswavlas vawarmoebdiT myar sakveb areze kreijis meTodis modifikaciis mixedviT. “spot testis” meSveobiT fagis suspenziis wveTebi dagvqonda logariTmul fazaSi arsebuli baqteriebis gazonebze [130]. sacdeli fagis samuSao titri Seadgenda 1X108 naw/ml. 370C t-ze 18-24 sT inkubaciis Semdeg aRvricxavdiT Sedegebs rasac Tan sdevda fagebis Semdgomi koncentrireba da gasufTaveba. fagebis lizisis stabilobis Seswavlas vaxdendiT Txier areSi “apelman”-is meTodis gamoyenebiT. 4,5ml LB bulionis Semcvel sinjarebSi Segvqonda 2X109

ujr/ml koncentraciis baqteriuli kulturis 10mkl da vumatebdiT 0,2mkm-ian membranul filtrSi gafiltrul 0,5ml sakvlev baqteriofags iseTi ganzavebebiT, rom faguri nawilakebis raodenoba pirveli sinjaridan bolo sinjarisken TanmimdevrobiT Semcirebuliyo 108naw/ml-101naw/ml. vaxdendiT inkubacias 370C-ze 4-6-24-48 saaTis ganmavlobaSi. sakvlevi fagis lizisis stabilobis maxasiaTeblad aRvricxavdiT inkubaciis dros da im bolo ganzavebas, romel sinjaraSic ar aRiniSneboda baqteriuli kulturis zrda. fagis zusti titris dadgenas mocemul Stamze vaxdendiT agaris orSriani fenebis meTodiT [4].

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fagis negatiuri koloniis morfologias vadgendiT standartuli Semadgenlobis niadagze (LB-niadagi) orSriani agaris meTodis saSualebiT. negatiuri koloniebis analizs vaxdendiT maTi formirebis dasrulebis Semdeg (18-24sT) [4]. fagis virionis morfologias vswavlobdiT eleqtronuli mikroskopis saSualebiT. Ppreparatebs vamzadebdiT negatiuri kontrastirebis meTodiT uranilacetatis 2%-iani wyliani xsnaris gamoyenebiT. Ffagis suspenzia an koncentrati dagvqonda sakontrasto nimuSebTan erTad kolodiumis apkebze-badeebze. standartul kvlevas vawarmoebdiT IEM-1200SX (Jeol, iaponia) da M10 (Opton-Zeiss, germania) eleqtronuli mikroskopiis saSualebiT. patron - ujredTan urTierTqmedebis fazebis Seswavla xdeboda TiToeuli parametris mixedviT, kerZod: a) adsorbciis siCqares da intensivobas baqteriul ujredebze vsazRvravdiT araadsorbcirebuli fagis raodenobis mixedviT, qloroformirebis meTodiT [86,90]. b) latenturi periodis xangrZlivobas da saSualo gamosavlis sidides vadgendiT elisis da delbrukis klasikuri meTodiT [4]. fagis efeqturi mravlobiTobis gansazRvrisaTvis vawarmoebdiT ganzavebebis or serias: 1) 4,5ml bulionian sinjarebSi Segvqonda myar areze gazrdili baqteriuli kulturis Camonarecxi ganzavebebis im angariSiT, rom mikrobTa ricxvi TanmimdevrobiT Semcirebuliyo 108ujr/ml-101ujr/ml. sinjarebs vamatebdiT 0,2 mkm forovnebis membranul filtrSi gafiltrul sakvlevi baqteriofagis 50mkl-s (108naw/ml). 2) igive moculobis bulionian sinjarebSi Segvqona 0,2mkm forovnebis membranul filtrSi gafiltruli sakvlevi baqteriofagi im angariSiT, rom faguri nawilakebis ricxvi TanmimdevrobiT Semcirebuliyo 108naw/ml-101naw/ml da TiToeul sinjaras vamatebdiT dacerebul agarze gazrdili baqteriuli kulturis Camonarecxis 50mkl-s. vaxdendiT inkubacias TermostatSi 18 saaTis ganmavlobaSi 370C-ze, ris Semdegac yvela sinjaraSi vamowmebdiT sakvlevi fagis titrs gracias meTodiT. efeqtur mravlobiTobad miviCnebdiT sakvlevi baqteriofagis da baqteriuli kulturis im Sefardebas, romelSic baqteriofagi maqsimalurad gamravlda. (mag. 1 faguri nawilaki : 100 baqteriuli ujredi). specifikuri imunuri SratebiT baqteriofagebis neitralizacia. specifikuri antifaguri Sratebi miRebuli iyo bocvrebis imunizaciis Sedegad V. cholerae-s mimarT specifikuri baqteriofagebiT: Vch105, Vch54, Vch10CH (sul 3 antifaguri Srati). specifikuri antifaguri Sratis misaRebad 2-3 kg wonis bocvrebis yuris venaSi Segvyavda sakvlevi fagis (1-5X1011 naw/ml) da froindis adiuvantis narevi (1:1, 3ml fagi: 3ml adiuvanti). es ganzaveba TiTo bocvers ukeTdeboda 1ml-is odenobiT. antisxeulebis titris dasadgenad, pirveli imunizaciidan yovel me-10, me-20 da 30-e dRes daigegma sisxlis aReba yuris venidan, risTvisac SeirCa V. cholerae-s mimarT aqtiuri 2 fagi: Vch54 da Vch10CH. me-40

45

dRes bocvrebs vuRebdiT sisxls gulidan, gamovyofdiT sisxlis Srats da vinaxavdiT Semdgomi kvlevebisTvis. neitralizaciisTvis fagi sWiroa 107 xarisxSi. unda damzaddes Sratis ganzavebebi: 1:50, 1:100, 1:500, 1:1000. neitralizaciamde fagi da Srati inaxeba wylian abazanaSi 280 C temperaturaze gansazRvruli droiT. saneitralizacio narevis misaRebad 0,1 ml fags emateba 0,9 ml Sesabamisi ganzavebis Srati da inaxeba TermostatSi 370C temperaturaze garkveuli xniT. neitralizaciis drois gavlis Semdeg 0,1 ml saneitralizacio narevi Segvaqvs 9,9ml fiz. xsnarSi da vtitravT gracias meTodiT. kontroli: 0,1ml fagi+0,9ml fiz.xsnari an LBbulioni gadagvaqvs finjnebze. fagis serologiur naTesaobas vswavlobdiT fagis neitralizaciis jvaredina reaqciis Catarebis gziT sxvadasxva specifiurobis antifaguri Sratis gamoyenebiT. fagi – antifaguri Sratis TiToeuli wyvilis SemTxvevaSi vsazRvravdiT neitralizaciis procents da konstantas [4]. neitralizaciis konstanta gamoiTvleba formuliT:

Kt=2,3xD/t x lgP0/Pt D-Sratis ganzaveba. t-neitralizaciis dro. P0-fagis raodenoba kontrolSi. Pt-fagis raodenoba Sesabamis droze.

2.2.6. baqteriofagebis gamravleba fagebis gamravleba myar sakveb areze da koncentratebis miReba xdeboda Semdegi meTodebis gamoyenebiT: myar areze koncentrats viRebdiT standartuli meTodiT, romelic gulisxmobs Txeli agaris fenaSi maRalkoncentrirebuli baqteriuli suspenziis fagolizisSi da minimaluri moculobis Txevad areSi fagis Semdgom eqstragirebas. faguri koncentratis momzadebisas 0,7%-ian LB-agarSi Segvqonda baqteriebi 1-5X108 da baqteriofagebi 1X105 naw/ml saboloo koncentraciiT. inkubacias vaxdendiT 370C-ze 18-24 sT, vxsnidiT agaris zeda fenas SpadeliT, vacentrifugirebdiT 5000 br/wT 30 wT da vamowmebdiT titrs gracias meTodiT. miRebuli fagolizatis titri iyo 1-5X1010-1011naw/ml. Txier areSi fagolizatis miRebisas kulturas vzrdidiT bulionian kolbaSi sanjRrevelaze 37C-ze logariTmul fazamde (1-5X108 uj/ml). Semdeg xdeboda fagis suspenziis damateba sxvadasxva mravlobiTobiT (1;10, 1:25, 1:50, 1:100) da kultivirebis gagrZeleba Semcirebuli aeraciiT. 280C -ze 1,5-2,0 sT-is Semdeg xdeboda baqteriebis sruli lizisi. qloroformiT damuSavebis Semdeg, lizats vtovebdiT 14-16 sT macivarSi sruli lizisisTvis. Llizats vacentrifugirebdiT 5000 br/wT 20-30 wT da vadgendiT titrs gracias meTodiT. miRebuli fagolizatis titri aRwevda 1-5X1010naw/ml koncentracias.

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2.2.7. fiziko-qimiuri faqtorebis zemoqmedeba baqteriofagebze fagebis stabiloba sxvadasxva areSi SeviswavleT arsebul sxvadasxva xsnarebSi 24 sT-is, 1-2 kviris, 1-3-6 Tvis ganmavlobaSi (LB _luria-bertanis bulioni, LB +MgSO4, LB+2% NaCl, PBS pH7.4, buferizebuli peptoniani wyali pH7, peptoniani wyali pH7.8, fiziologiuri xsnari, SM-buferi, lisis tbis wyali, Savi zRvis wyali, LB +Cacl2, tripton mariliani bulioni (T1N6, T1N8), LB+qloroformi). baqteriofagebi 1X107 naw/ml titrSi inaxeboda 40C t-ze sibneleSi da periodulad vadgendiT titrs agaris orSriani fenis meTodiT. fagebis Termoinaqtivacia Seiswavleboda fiziologiur xsnarSi. fagebs saboloo koncentraciiT 1X108 naw/ml vainkubirebdiT sasurvel temperaturaze (55, 60, 65, 700C) Termostatikulad kontrolirebad wylis abazanaSi sxvadasxva drois intervaliT (10,20,30 wT). drois gasvlis Semdeg xdeboda fagis myisieri ganzaveba da titris dadgena agaris orSriani fenis meTodiT. fagis aqtiuri nawilakebis procents vadarebdiT fagis kontrols sawyis droze. aseve ganisazRvra reaqciis siCqare. wyalbadionTa koncentraciis (pH) zegavlena ganisazRvra fagur buferSi SM (100 mMNaCl, 8 mM MgSO4•7H2O, 50 mMTris-Cl (1 M, pH 7.5), 0.002% (w/v) Jelatini) sxvadasxva pH-ze (pH=1,4,7,9,12) fagebi sawyisi koncentraaciiT 1X108naw/ml Segvqonda sacdel xsnarSi da 30-60 wT-is Semdeg xdeboda titris dadgena agaris orSriani fenis meTodiT. UV zegavlena. fagebi saboloo koncentraciiT 1X108naw/ml dagvqonda petris finjanze UV-is mokletalRovan gamosxivebaze (254nm), 30-35sm-is distanciiT, bnel pirobebSi. aqtiur fagur nawilakebs vsazRvravdiT drois gansazRvruli intervalebiT (30,60,90 wm). osmosuri Sokis zemoqmedeba SeviswavleT standartuli meTodiT [18]. fagi zavdeboda 3,5 M natriumis qloridis (NaCl) xsnarSi, inkubacias vaxdendiT 37°C t-ze 15 wT-is ganmavlobaSi, ris Semdegac 0.1 ml odenobis fagi zavdeboda 10 ml distilirebul wyalSi da fagis titris dadgena xdeboda agaris orSriani fenis meTodiT. natriumis citratis zemoqmedeba SeviswavleT natriumis citratis xsnarSi. fagebi zavdeboda natriumis citratis xsnarSi, romelic Tavdapirvelad ifiltreboda 0.22 mkm zomis filtrSi. sicocxlisunariani negatiuri koloniebis procentuli raodenoba dgindeboda agaris orSriani fenis meTodiT.

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2.2.8. baqteriofagebis genetikuri Tvisebebis Seswavla

faguri dnm-is gamoyofas vaxdendiT fenol/qloroformiT deproteinizaciis meTodiT. baqteriofagis gawmendil koncentrats (5x1011naw/ml) vumatebdiT sawyisi moculobis 1/10 lizisur bufers ( 1% SDS, 10mM EDTA), vayovnebdiT oTaxis temperaturaze 10 wuTis ganmavlobaSi. Semdeg vaxdendiT sametapian deproteinizacias fenol/qloroformiT. narevs vumatebdiT fenols (pH8.0) TanafardobiT 1:1, vanjRrevdiT da vacentrifugebdiT 15000br/wT 10wT. Semdgom etapze vumatebdiT fenol-qloroforms 1:1 SefardebiT, kvlav vanjRrevdiT da vacentrifugirebdiT 15000 br/wT 10wT. Bbolos ki- izoamil-qloroforms 1:24 TanafardobiT, vanjRrevdiT da vatrialebdiT igive reJimze. dnm-s gamoleqvas vaxdendiT 5M NaCl da izopropanoliT. narevs vacentrifugebdiT igive reJimze, supernatants vamatebdiT 70% eTanols (2:1). narevis centrifugirebis Semdeg daleqil dnm-s vaSrobdiT oTaxis temperaturaze, vukeTebdiT resuspendirebas TE-buferSi da vinaxavdiT macivarSi Semdgomi procedurebisaTvis [4]. faguri dnm-is restriqciuli analizis mizniT gamoyofil fagur dnm-s vamuSavebdiTY sxvadasxva restriqciuli enzimebiT (EcoRI, EcoRV, HindIII, BamH I, Pst I, Not I) mwarmoeblis mier mowodebul pirobebSi. Semdeg sinjebi Segvqonda 0,8%-ian agarozis gelSi da vawarmoebdiT eleqtroforezs 1,5-2 sT-is ganmavlobaSi. markerul preparatad viyenebdiT HindIII fermentiT daWril λ fagis dnm-s (Sigma). gels vRebavdiT eTidium bromidiT (0,5mkg/ml) sanjRrevelaze 45 wuTis ganmavlobaSi. Sedegs vnaxulobdiT UV-iluminatorze. miRebuli restriqiciuli fragmentebis molekulur masas vsazRvravdiT sakalibracio mrudis saSualebiT. monacemTa analizi. TiToeuli nimuSis SemTxvevaSi xdeboda sami sxvadasxva analizis Sedegad miRebuli monacemis damuSaveba, gasaSualoeba da SemdgomSi standartuli gadaxrebis daTvla. fagebs Soris naTesauri kavSiris dadgena da filogenezuri xeebis ageba xdeboda daSorebuli-matriqsuli meTodiT, kerZod ki UPGMA (Unweighted Pair Group Method with Arithmetic Mean)-dafuZnebuli analiziT (FreeTree) [110] da filogenezuri xeebis vizualizaciis programiT (TreeView) [173].statistikuri analizi Catarda Microsoft Excel 2010-is statistikur monacemTa damuSavebis meTodis gamoyenebiT, kerZod variaciuli (ANOVA) da regresiuli analizis meSveobiT. SedegebSi moyvanili korelaciebis statistikuri sarwmunoeba (Pearson’s r) aRwevda <0.05 mniSvnelobas.

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III kvlevis Sedegebi

3.1. qoleras jgufis vibrionebis koleqciis Seqmna

qolears jgufis vibrionebisa da Sesabamisi baqteriofagebis gamoyofa xorcieldeboda 2006-2008 wlebis ganmavlobaSi, 30 Tvis monitoringis Sedegad, Savi zRvis sanapiro zolidan (md.sufsis SesarTavi, md.Woroxis SesarTavi, mwvane koncxis sanapiro zoli da baTumis bulvari), aseve 2009 wels 3 Tvis monitoringis Sedegad, Tbilisis SemogarenSi arsebuli tbebidan (kumisis tba, lisis tba, Tbilisis zRva da md. mtkvari) aRebuli sinjebidan. 2006-2009 wlebSi monitoringis Sedegad wyliani garemodan sul 846 V. cholerae-s da 11 V. mimicus-is izolati gamoiyo, romlebic SemdgomSi gamoyenebuli iyo specifikuri baqteriofagebis gamosayofad da gasamravleblad. gamoyofas vawarmoebdiT, rogorc wylidan aseve fito- da zooplanqtonidan. Cvens mier Catarebulma monitoringma aCvena, rom qoleras jgufis vibrionebis gamoyofis sezonuri xasiaTi damokidebuli iyo temperaturaze, rogorc planqtonmocilebuli wylis, aseve fito- da zooplanqtonebis SemTxvevaSi. mocemuli Sedegi SeiZleba davukavSiroT imas, rom 2006-2009 ww Catarebuli monitoringis manZilze, wylis marilianoba, masSi gaxsnili Jangbadis raodenoba da mJavianoba, wylis ekosistemis, metnaklebad, ucvlel maxasiaTeblebs warmoadgenda, gansxvavebiT temperaturisagan, romelic sezonis mixedviT cvalebadobda. V. cholerae-s Stamebis intensiuri gamoyofa xdeboda zafxulis da adreuli Semodgomis TveebSi (ivlisi-seqtemberi). swored am periodSi gamoiyo V. cholerae-s izolatebis umravlesoba-475 izolati (56.1%). mniSvnelovnad mcirdeboda V. cholerae-s Stamebis gamoyofis sixSire zamTaris da adreuli gazafxulis periodSi (ianvari-marti). mxolod 21 izolati (2.5%) iyo gamoyofili aRniSnul TveebSi Catarebuli monitoringis Sedegad (sur.3). 2006-2007 wlebSi Catarebuli monitoringis Sedegad V. mimicus-is sul 11 izolati (7-PCR-iT identificirebuli) iyo gamoyofili. maT Soris 9 Stami gamoiyo oqtomberi-noembris TveSi aRebuli lisis tbis wylis sinjebidan, 1 Stami ivlisis TveSi aRebuli Tbilisis zRvis wylis sinjidan da 1 Stami aseve ivlisis TveSi aRebuli kus tbis wylis sinjidan (sur.4).

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sur.3. 2006-2009 wlebSi gamoyofili V. cholerae-s Stamebis gamoyofis

sixSire sezonis mixedviT

sur.4. 2006-2007 wlebSi gamoyofili V. mimicus-is Stamebis raodenobrivi

ganawileba sezonis mixedviT

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V. cholerae-s Stamebis gamoyofaze wylis temperaturis garda, aseve mniSvnelovan zegavlenas axdens wylis marilianoba. V. cholerae-s Stamebis umravlesoba-705 izolati (83.3%) gamoiyo Tbilisis SemogarenSi arsebuli mtknari wyalsatevebidan, sadac Sav zRvasTan SedarebiT marilianoba dabalia. Savi zRvis sxvadasxva saitidan V. cholerae-s Stamebis sul 141 izolati (16.7%) gamoiyo. gansxvavdeboda gamoyofis sixSire TiToeuli saitis SemTxvevaSi. yvelaze didi raodenobiT V. cholerae-s izolatebis gamoyofa dafiqsirda lisis tbidan-266 izolati (31.4%), xolo yvelaze mcire raodenoba- 20 izolati (2.4%) gamoiyo baTumis bulvaridan aRebuli wylis sinjebidan (sur.5).

sur.5. 2006-2009 wlebSi gamoyofili V. cholerae-s Stamebis sixSire gamoyofis

adgilis mixedviT rac Seexeba V. cholerae-s epidemiur serotipebs, 846 Stamidan V. cholerae O1-is serotipad multipleqs polimerazuli jaWvuri reaqciiT (PCR) identificirebuli iyo 46 baqteriuli izolati. umravlesoba V. cholerae O1-is izolatebisa-37 izolati (80.4%) gamoyofili iyo Savi zRvis wylis sinjebidan. V. cholerae O1-is serotipis mxolod 9 izolati (19.6%) gamoiyo Tbilisis SemogarenSi arsebuli tbebidan. baTumis bulvaridan gamoiyo V. cholerae O1-is serotipis yvelaze mcire-1 izolati (2.2%), xolo yvelaze didi raodenobiT V. cholerae O1-is serotipis izolatebis-19 Stami (41.3%) gamoyofili iyo mwvane koncxis sanapiro zolidan aRebuli wylis sinjebidan (sur.6) V. cholerae O1-is

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serotipis yvela izolati gamoiyo zafxulis da adreuli Semodgomis TveebSi (ivlisi-seqtemberi). SemdgomSi, bioqimiuri parametrebiT V. cholerae O1-is “klasikur” da “El Tor” biotips miekuTvna 29 baqteriuli izolati.

sur.6. 2007-2008 wlebSi gamoyofili V. cholerae O1-is Stamebis procentuli ganawileba

gamoyofis adgilis mixedviT

3.2. qoleras jgufis vibrionebis izolatebis biologiuri Tvisebebis Seswavla sakvlevi Stamebis kulturalur–morfologiur Tvisebebs vswavlobdiT baqteriebis zrdis xasiaTis mixedviT myar, Txevad da naxevradTxevad niadagze. Tavdapirvelad kultivirebas vaxdendiT sadiferenciacio-sadiagnostiko areebze 370C-ze. saqaroza-dadebiTi saxeobebi anu saxeobebi, romelTac aqvT saqarozas fermentaciis unari TCBS-is niadagze iZlevian yviTeli feris koloniebs, aseTia V. cholerae–s izolatebi, xolo saqaroza uaryofiTi saxeobebi, rogoricaa V. mimicus–is izolatebi TCBS-is niadagze iZlevian mwvane feris koloniebs (sur.7). myar LB-niadagze qoleras jgufis vibrionebi warmoqmnian 2-5 mm zomis, sferosebr koloniebs. Ppeptonian wyalSi izrdebian zedapirulad, apkis saxiT.

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sur.7. TCBS-is baqteriul areze kultivirebuli qoleras jgufis vibrionebi

V. cholerae-s zogierTi izolatis morfologia SeviswavleT M10 (Opton-Zeiss, germania) eleqtronuli mikroskopis saSualebiT (sur.8). V. cholerae-s vibrionebi moxrili Cxiris formisaa, zomiT, sigrZeSi daaxloebiT 1-1,5 mkm-ia, xolo siganeSi 0,4-0,8 mkm. Stamebis tinqtorialur Tvisebebs vswavlobdiT gramis wesiT SeRebvis mixedviT. Vibrio-s gvaris baqteriebi miekuTvnebian gram(_) baqteriebs.

sur.8. kumisis tbidan gamoyofili V. cholerae-s

izolatis (Km16) ujredis morfologia

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V. cholerae-s da V. mimicus–is Stamebis bioqimiuri Tvisebebis Seswavlas vawarmoebdiT specializirebul sakveb areebze. proteolizur aqtivobas vswavlobdiT am mikrobebis mier Jelatinis gajirjvebis unariT GSA da GA-s agarze. VIbrio-s gvaris baqteriebs aqvT rogorc glukozis fermentaciis, aseve misi daJangvis unari. triptoniani Txevadi are, natriumis qloridis sxvadasxva SemcvelobiT, gamoiyeneboda mikrobTa zrdis unaris dasadgenad sxvadasxva procentobis marilian areSi. V. cholerae-s da V. mimicus–is izolatebi VIbrio-s gvaris sxva saxeobebis msgavsad izrdebian 1%-ian marilianobis niadagSi. halofiluri vibrionebisgan gansxvavebiT izrdebian umarilo areSi. rogorc arahalofilur saxeobebs, ar axasiaTebT maRali procentobis marilis Semcvel areSi zrda (6-8%). SeviswavleT agreTve am baqteriebis mier amonomJavebis dekarboqsilirebis da naxSirwylebis fermentaciis unari sxvadasxva areebSi.

Stamebis genetikuri gamokvleva ganxorcielda polimerazuli jaWvuri reaqciis sxvadasxva saxeebis saSualebiTac (PCR): 1.polimerazuli jaWvuri reaqcia (ITS – PCR, Internal Transcribed Spacer PCR) specifikuri praimerebiT (cxr.1). 2.multipleqsuri polimerazuli jaWvuri reaqcia (Multiplex-PCR) (cxr.2) V. cholerae O1-is, V. cholerae O139-is, toqsigenuri genebis aRmosaCenad (ctxA).Ggamovlinda ctxA+ 8 Stami, maT Soris 6 Stami iyo V. cholerae ara-O1-is da 2 Stami- V. cholerae O1-is warmomadgeneli. kvlevebma aCvena bioqimiuri identifikaciis maRali sizuste. izolatebis 98.5%-Tvis bioqimiuri identifikacia aRmoCnda zusti (sur.9).

sur.9. polimerazuli jaWvuri reaqcia V. cholerae-s saxeobebis identifikaciisTvis

1. PCR-markeri; 2.V.ch 3; 3.V.ch 4.V.ch9;5.V.ch 10;6.V.ch 44; 7.V.ch 56; 8.V.ch 39; 9.V.ch46; 10. V.ch 48; 11.V.ch 69; 12.V.ch 70; 13 V.ch71; 14.V.ch73; 15.V.ch74; 16. V.ch75; 17.V.ch102; 18.V.ch103; 19.V.ch104; 20.Vch105; 21.V.ch 106; 22.V.ch 107; 23.V.ch108; 24.V.ch 109; 25.V.ch110; 26. PCR-markeri.

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qoleras jgufis izolatebis fagomgrZnobeloba. kvlevis ganmavlobaSi gamoyofili 135 V. cholerae-s da 10 V. mimicus-is baqteriuli Stami (g.eliavs bmv institutis koleqcia) Semowmda aseve, kvlevis farglebSi gamoyofili baqteriofagebis mimarT mgrZnobelobaze. 135 Stamidan (60 V. cholerae O1-is da 75 V. cholerae ara-O1-is izolati) 81 (60%) Stami mgrZnobiare iyo mocemuli fagebis mimarT. fagomgrZnobelobis profilis mixediT baqteriuli Stamebis Sedarebis da dajgufebis mizniT avageT filogenezuri xe (UPGMA analizi), rogorc V. cholerae ara-O1-is (sur.10), aseve V. cholerae O1-is Stamebis SemTxvevaSi(sur.11).

sur.10. UPGMA (Nei-Li daSorebuli analizi) filogenezuri xe V. cholerae ara-O1-is

Stamebis fagomgrZnobelobis mixedviT

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sur.11. UPGMA (Nei-Li daSorebuli analizi) filogenezuri xe V. cholerae O1-is Stamebis fagomgrZnobelobis mixedviT * 3 baqteriuli izolati miekuTvneba V. cholerae ara-O1-s da warmoadgens maspinZel Stams (Vch105, Vch89, Vch469).

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V. mimicus-is 10 Stami Semowmda fagomgrZnobelobaze, rogorc V. mimicus-is aseve V. cholerae O1-is da V. choleraeara-O1-is mimarT sepcifikur baqteriofagebze. 10-dan 3 Stami iyo rezistentuli yvela baqteriofagis mimarT, xolo 7 Stami metnaklebad fagomgrZnobiare aRmoCnda (cxr.6).

cxr.6.V. mimicus-is baqteriuli izolatebis fagomgrZnobeloba

*cl – sruli lizisi; ol- sruli lizisi, erTeuli baqteriuli koloniebiT; scl- arasruli

lizisi; tv- erTeuli negatiuri kolonia; ntv-mravali calkeuli negatiuri kolonia;

carieli ujrebi-uaryofiTi reaqcia/lizisi ar momxdara

3.3. V. cholerae-s toqsigenuri Stamebis pirdapiri gamovlena sxvadasxva imunologiuri meTodiT

V. cholerae–s epidemiuri serotipebis gamovlena pirdapiri fluerescentuli analiziT (DFA). pirdapiri kvleva fluorescentuli antisxeulebiT gamoiyenebida Savi zRvis sanapiro zolidan da mtknari tbebidan aRebuli sinjebis gamosakvlevad da maTSi V. cholerae O1-is da O139–is gamosavlenad, rogorc gamdidrebul da koncentrirebul wylis nimuSebSi, aseve gamdidrebul da koncentrirebul planqtonis nimuSebSi. DFA-iT damuSavda 267 sakvlevi nimuSi. wylis pirdapirma fluorescentulma analizma gamoavlina V. cholerae O1–is arseboba rogorc, zRvis, aseve tbebis wylis nimuSebSi (kumisisa da lisis tbebi, Tb.zRva). gamokvleuli sinjebidan 82-ma nimuSma (31%) dadebiTi Sedegi aCvena. dadebiTi nimuSebidan 74 (90%) nimuSi pozitiuri iyo V. cholerae O1-is SemTxvevaSi, 8(10%) nimuSi_ V. cholerae O139-is (savaraudod VBNC-

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sicocxlisunarian, magram arakultivirebad formaSi) SemTxvevaSi. wyalSi, V. cholerae-s aRniSnuli serojgufis baqteriebis arseboba umetesad, aRiniSneboda zafxulis periodSi, wylis gamdidrebul da planqtonis gamdidrebul nimuSebSi. dakoncentrirebul wyalSi V. cholerae O1 mcire raodenobiT iyo nanaxi (sur.12). tbebis SemTxvevaSi V. cholerae O1–is dadebiTi nimuSebis raodenoba Seadgenda 23-s (28%), saidanac V. cholerae O1 –is serotipisTvis dadebiTi Sedegi nanaxi iyo 22 nimuSisTvis, xolo erTi miekuTvneboda V. cholerae O139 –is serotips (sur.13). Savi zRvis wylis sinjebSi V. cholerae O1–is dadebiTi nimuSebis raodenoba aRwevda 59-s (72%), am SemTxvevaSic umetesi wili dadebiTi nimuSebis miekuTvneboda V. cholerae O1 –is serotips, kerZod 52 nimuSi. V. cholerae O139–is serotipisTvis ki dadebiTi Sedegi nanaxi iyo 7 gamokvleuli nimuSis SemTxvevaSi (sur.14). V. cholerae O1-is da O139-is serotipebis deteqcia xdeboda specifikuri fluorescentulad moniSnuli antisxeulebiT (New Horizon Diagnostics, MD) epifluorescentuli mikroskopiis meSveobiT (sur.15-17).

sur.12. DFA dadebiTi nimuSebis ganawileba nimuSis tipis mixedviT

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sur.13. V. cholerae-s epidemiuri serotipebis DFA dadebiTi nimuSebis

ganawileba tbebSi

sur.14. V. cholerae-s epidemiuri serotipebis DFA dadebiTi nimuSebis

ganawileba Sav zRvaSi

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sur.15. V. cholerae O1 serotipis dadebiTi DFA nimuSi. gamdidrebuli

wylis nimuSi Savi zRva, mwvane koncxi, ivlisi, 2008w.

sur. 16. V. cholerae O1 serotipis dadebiTi DFA nimuSi. Gamdidrebuli wylis nimuSi. kumisi, ivnisi 2008w.

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sur.17. V. cholerae O1 serotipis dadebiTi DFA nimuSi.64mkm

homogenizirebuli planqtonis nimuSi. Tbilisis zRva, ivlisi 2009w.

V. cholerae–s epidemiuri serotipebis gamovlena qoleras poli-imunuri Sratis (“B.D.DifcoTM”) gamoyenebiT. V. cholerae-s antiSrati “poli” warmoadgens serologiuri testis erT-erT magaliTs, romelic dafuZnebulia aglutinaciis reaqciaze da romlis meSveobiTac xdeba garemoSi V. cholerae-s toqsigenuri izolatebis pirdapiri detqcia. aglutinaciis reaqcia tardeba B.D. DifcoTM

V.cholerae-s antiSrati “ogava” da B.D. DifcoTM V.cholerae-s antiSrati “inabas” gamoyenebiT. Testma 73 Stamze aCvena dadebiTi Sedegi. Umetesoba V. cholera O1-is Stamebisa (43 Stami) gamoyofili iyo Savi zRvis sxvadasxva saitidan. aseve Savi zRvidan gamoyofilma 7-ma Stamma savaraudod mogvca dadebiTi Sedegi V. cholera O139-is serotipze. V. cholerae O1-is Stamebis ganawileba kumisisa da lisis tbis SemTxvevaSi metnaklebad msgavsi iyo da Seadgenda kumisis tbis sinjebSi 3 Stams, Tbilisis zRvis sinjebSi 7 Stams, xolo lisis tbis nimuSebSi 20 Stami iyo V. cholerae O1-is serotipis. slaid-aglutinaciis dros sakvlev nimuSebSi gamovlinda aseve inaba da ogavas serotipis izolatebi (sur.18). aRsaniSnavia, rom slaid-aglutinaciis Sedegebi dadebiT korelaciaSi modioda polimerazuli jaWvuri reaqciiT da pirdapiri fluorescentuli analiziT Catarebuli kvlevis Sedegad miRebul SedegebTan.

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sur.18. V. cholerae O1 serotipis ganawileba Sav zRvaSi da tbebSi slaid- aglutinaciis meTodis mixedviT

V. cholerae–s epidemiuri serotipebis gamovlena qolera smartis gamoyenebiT serologiuri testi-garemodan gamoyofili V. cholerae O1-is serotipisTvis tardeboda Cholera O1 SMARTTM _mgrZnobiare, membranuli antigenis swrafi testis gamoyenebiT. Cholera O1 SMARTTM -is specialuri formis horizontalur firfitebze datanili monoklonuri antisxeulebi specifikuria V. cholerae O1–is O1 lipopolisaqaridis A antigenis mimarT. aRniSnul testze xdeboda V. cholerae-s epidemiuri serotipebis gadamowmeba. aseve Semowmda da dadebiTi Sedegi aCvena V. cholerae O1–is standartuli Stamebis (NCDC, Tbilisi) Semowmebam (sur.19).

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sur. 19.qolera smartis testi V. cholerae O1-is serotipisTvis

V. cholerae–s Stamebis mier toqsinis gamoyofis Seswavla GM1 gangliozid damokidebuli enzimdakavSirebuli imunosorbentuli analiziT (GM1-ELIZA). qoleris toqsinis (CT) produqciis daTvla xdeboda standartul mrudTan Sedarebis mixedviT, romelic asaxavda V. cholerae O1–is standartuli Stamis (V.chCIP62.13, V. cholerae 4805-aziatikusi) filtratis sxvadasxva ganzavebebis SemTxvevaSi producirebul toqsinis raodenobas (OD-optikuri simkvrive). gamoyofili qolera toqsinis raodenoba, TiToeuli Stamis SemTxvevaSi iTvleboda sami sxvadasxva analizis Sedegad miRebuli pasuxis gasaSualoebiT. kvlevis Sedegad ganxorcielda 55 garemodan gamoyofili V. cholerae O1–is serotipis izolatis skriningi qolera toqsinis produqciaze maT Soris polimerazuli jaWvuri reaqciiT identificirebuli iyo V. cholerae O1–is serotipis 41 Stami. gamokvleuli Stamebidan 11 izolati ctx genis matarebeli iyo. kontrolis jgufi moicavda V. cholerae-s toqsigenuri Stamebis 5 standartul izolats kulturaTa sxvadasxva koleqciidan (NCDC-Tbilisi, CIP-pasteris institutis koleqcia) (sur.20). kvlevam aCvena, rom 6 garemos izolati dadebiTi iyo qolera toqsinis produqciaze AKI sakveb areSi kultivirebis SemTxvevaSi. aseve nanaxi iyo V. cholerae O1–is mimarT specifikuri baqteriofagebis garkveuli zegavlena Stamebis mier qolera toqsinis produqciaze (ix.Tavi 3.4.5) fagisa da Stamis sxvadasxva TanafardabiT urTierTqmedebisas drois sxvadasxva intervalis SemTxvevaSi.

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sur.20. V. cholerae O1-is serotipis standartuli Stamebis mier qolera

tqosinis gamoyofa.

baqteriebis sicocxlisunariani, magram arakultivirebadi mdgomareobis (VBNC) Seswavla mikrokosmebis cdebSi. V. cholerae–s baqteriebis gadasvlis unars VBNC-is mdgomareobaSi vswavlobdiT arasakmarisi sakvebis Semcvelobis da araxelsayreli temperaturuli reJimis SeqmniT sami Stamis magaliTze: V.ch O1 „klasikuri“ (V.ch 145-P-1, NCDC Tbilisi), V.ch 104 da V.ch123 (g.eliavas bmv-s koleqcia).viyenebdiT lisisa da Savi zRvis wylebs, aseve, T1N6–is Semcvel ares. Sesadareblad viyenebdiT mdidar sakveb ares LB-niadags da seleqtiur TCBS-is niadags. araxelsayreli temperaturis magaliTad SeirCa 40C-s, kontrolad ki optimaluri temperatura-250C.Uujredebi iTvleboda arakultivirebad mdgomareobaSi, rodesac xdeboda 0.1 uj/ml-ze naklebi kultivirebadi ujredis gamovlena, aseve VBNC-is mdgomareobaSi gadasul ujredebs vnaxulobdiT epifluorescentul mikroskopSi DFA-is meTodiT momzadebul masalebSi. kvlevam aCvena, rom 40C-ze lisis tbisa da zRvis wyalSi V. cholerae O1–is baqteriebis titris cvlileba metnaklebad msgavsi iyo. samive Stamis SemTxvevaSi arakultivirebad mdgomareobaSi gadasvla xdeboda 42 dRis Semdeg. unda aRiniSnos rom V.ch 104 daV.ch123 Stamebi Savi zRvis sinjebidan aris gamoyofili. V. cholerae O1–is baqteriebisTvis kritikuli aRmoCnda T1N6–is

64

Semcveli are, 7 dRis Semdeg maTi zrda sakveb niadagze aRar SeiniSneboda. mocemuli maCvenebeli dakavSirebulia V. cholerae O1–is, rogorc arahalofiluri saxeobis mier marilis nakleb moTxovnilebasTan. mdidar sakveb areSi, 250C temperaturaze, 7 kviriani periodis ganmavlobaSi kultivirebadi baqteriebis raodenoba mxolod 1 log-iT Semcirda (sur.21) Sesabamisad, ar moxda arakultivirebad mdgomareobaSi gadasvla. rac SesaZloa gamowveuli iyos areSi sakmarisi raodenobiT sakvebi nivTierebebis arsebobiT.

a)

b)

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g)

sur.21. V. cholerae O1 –is baqteriebis sicocxlisunarianobis Seswavla

a) V.ch O1 „klasikuri“; b) V.ch 104; g) V.ch 123

3.4. V. cholerae-s da V. mimicus-is mimarT specifikuri baqteriofagebis gamoyofa da gavrceleba

2006-2009 wlebis ganmavlobaSi, Savi zRvis sxvadasxva saitis (bulvaris sanapiro zoli, mwvane koncxis sanapiro zoli, md.Woroxis SesarTavi, md.sufsis SesarTavi) da Tbilisis SemogarenSi arsebuli tbebis (kumisis da lisis tbebi da Tbilisis zRva, aseve md.mtkvari) monitoringis Sedegad aRebuli wylis sinjebis 882 gamdidrebuli wylis nimuSidan V. cholerae-s mimarT specifikuri 71 da V. mimicus-is mimarT specifikuri 7 pirveladi izolati gamoiyo. gamoyofis

sixSire maRali iyo aprili-ivnisis periodSi (49.3%). oqtomberi–dekembris

periodSi fagebis yvelaze mcire (19.7%) raodenoba iyo gamoyofili (R2=0.9, p<0.04) (sur.22). daaxloebiTi sixSire V. cholerae-s mimarT specifikuri fagebis gamoyofisa Seadgenda 8%-dan 15%-s. fagebis gamoyofam cxadyo, rom maTi

umravlesoba (51.2%) gamoiyo Tbilisis SemogarenSi arsebuli mtknari wyalsatevebidan. fagebis gamoyofis siuxviT xasiaTdeboda kumisis tba, saidanac

yvelaze didi raodenobiT (24.4%) faguri izolatis gamoyofa moxda. fagebi naklebi siuxviT gamoirCeodnen Sav zRvaSi (22%). fagebis umciresi raodenoba gamoiyo kulturaTa supernatantebidan (13.4%) (sur.23). V. mimicus-is mimarT

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specifikuri 7 pirveladi izolatis gamoyofa moxda 2008-2009 wlebis ivlisi-agvistos TveebSi aRebuli wylis sinjebidan. 7 faguri izolatidan 3 fagis gamoyofa moxda kumisis tbidan aRebuli wylis sinjidan, 1-is lisis tbidan, 2-is md.mtkvridan da 1-is md.Woroxis SesarTavidan aRebuli wylis sinjidan. 3 faguri izolati gamoiyo ivlisis, xolo 4-agvistos TveSi Catarebuli monitoringis Sedegad aRebuli wylis nimuSebidan (cxr.7).

Kkvlevis ganmavlobaSi V.choelrae O1-is mimarT specifikuri 17 pirveladi izolati gamoiyo. gamoyofis sixSire V.choelrae O1-is mimarT specifikuri fagebis

SemTxvevSi maRali iyo (10 faguri izolati, 58.8%) ivlisi-seqtembris periodSi.

umetesoba fagebisa (13 faguri izolati, 76.5%) gamoiyo Tbilisis SemogarenSi arsebuli wyalsatevebidan aRebuli wylis sinjebidan (cxr.8). aRsaniSnavia, rom V. cholerae-s aqvs dabali faguri potenciali. mag.: 2009 wlis ivlisSi aRebuli sinjebis 24 gamididrebuli wylis nimuSidan mxolod 5 pirveladi faguri izolatis gamoyofa moxerxda. fagis gamoyofis sixSirem aCvena sezonuri gamoxatuleba. fagebis gamoyofa win uswrebda maspinZeli baqteriuli Stamebis gamoyofis piks (sur.24).

sur.22. V. cholerae-s mimarT specifikuri baqteriofagebis procentuli

ganawileba sezonis mixedviT

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sur. 23. V. cholerae-s mimarT specifikuri baqteriofagebis procentuli ganawileba saqarTvelos wylis aqvatoriaSi

cxr.7. V. mimicus-is mimarT specifikuri baqteriofagebis ganawileba

68

cxr.8. V. cholerae O1-is mimarT specifikuri baqteriofagebis ganawileba

69

sur.24. V. cholerae-s baqteriuli izolatebis da maT mimarT specifikuri baqteriofagebis gamoyofis procentuli ganawileba sezonis mixedviT

3.5. V. cholerae-s da V. mimicus-is mimarT specifikuri baqteriofagebis daxasiaTeba

3.5.1. batqriofagebis morfologiuri parametrebi baqteriofagebis negatiuri koloniis forma. baqteriuli izolatebis gazonebze maTi formirebiT vaxdendiT fagis negatiuri koloniis formis Semowmebas. sxvadasxva fags misTvis damaxasiaTebeli morfologiis negatiuri koloniebi axasiaTebda. negatiuri koloniebis zomebi varirebda 1-2.5 mm diametris farglebSi. sufTa faguri klonebis miReba xdeboda msgavsi klonebis 5-10 Tanmimdevruli klonirebis pasaJis Sedegad. zogierT SemTxvevaSi baqteriofagebi xasiaTdebian polimorfizmiT, rac negatiuri koloniebis araerTgvarovnebaSi vlindeba. faguri klonebis dazustebis mizniT sakvlevi baqteriofagebi mowmdeboda eleqtronuli mikroskopiT da agarozis gelSi eleqtroforeziT.

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baqteriofagebis virionis morfologia. baqteriofagis virionis morfologias vswavlobdiT transmisiuli eleqtronuli mikroskopiT M10 (Opton-Zeiss, germania) an Jeol SX100 (Jeol, iaponia). Seswavlam gamoavlina kudiani fagebis samive morfotipis arseboba V. cholerae-s mimarT specifikuri baqteriofagebis SemTxvevaSi, kerZod: mioviride (izometruli Tavi da grZeli kumSvadi kudi), sifoviride (arakumSvadi, grZeli kudi) da podoviride (mokle arakumSvadi kudi). V. mimicus-is mimarT specifikur baqteriofagebSi Warbobda sifovirides morfotipis fagebi. gansxvavdeboda aseve sakvlevi baqteriofagebis nukleokapsidisa da morCis zomebi (cxr.9), (sur.25-27).

cxrili 9. V. cholerae-s da V. mimicus-is mimarT specifikuri baqteriofagebis morfologia

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sur.25. V. cholerae ara-O1-is mimarT specifikuri baqteriofagebi:

1.Vch105; 2.Vch10CH; 3.VchBS3

sur.26. V. cholerae O1-ismimarT specifikuri baqteriofagebi:

1.Vch9K; 2.Vch16K-4805; 3.Vch20K-4805;

4.Vch25MT; 5.Vch54; 6.VchK1.

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sur.27. V. mimicus-is mimarT specifikuri baqteriofagebi: 1.Vmim9K; 2. Vmim29.08K; 3. Vmim L4.

3.5.2 fagebis lizisuri aqtivobis speqtri. standartuli procedurebidan gamomdinare moxda yvela sawyisi faguri izolatis Semdgomi momravleba, daklonva da Semowmeba antibaqteriul aqtivobaze. SerCeuli 32 baqteriofagis (V. cholerae O1-is mimarT specifikuri -21, V. cholerae ara-O1-is mimarT specifikuri -4 da V. mimicus-is mimarT specifikuri 7 baqteriofagi) klonebis daxasiaTeba moxda patron ujredis specifikurobis da lizisuri speqtris mixedviT. fagebis lizisuri speqtris saorientacio gansazRvra xdeboda myar sakveb areze [4]. sakvlevi fagis samuSao titri Seadgenda 107-108 naw/ml. skriningisTvis SeirCa 4 V. cholerae ara-O1-is da 21 V. cholerae O1-is mimarT specifikuri baqteriofagi. fagomgrZnobelobaze Sesamowmeblad SeirCa 135 V. cholerae–s Stami (60-V. cholerae O1 da75 -V. cholerae ara-O1) g.eliavas saxelobis bmv institutis koleqciidan, maT Soris 6 standartuli Stami sxvadasxva koleqciidan (NCDC, CIP). V. cholerae O1-is sacdeli baqteriuli izolatebis 85% (51, 60-dan) da V. cholerae ara-O1-is sacdeli baqteriuli izolatebis -40 % (30, 75-dan) aRmoCnda mgrZnobiare erTi da meti fagis mimarT. fagebis 36%–ma (9, 25-dan) aCvena viwro speqtri, aqtiurebi iyvnen mxolod maspinZeli Stamis mimarT. maT Soris V. cholerae ara-O1-is mimarT specifikuri 3 fagi (4-dan) da V. cholerae O1-is mimarT specifikuri 6 fagi (21-dan). V. cholerae O1-is mimarT specifikuri yvela sxva fagi (71%) gamoirCeoda lizisuri aqtivobis SedarebiT farTo speqtriT. fagebis garkveulma jgufebma gamoavlina msgavseba lizisuri speqtris mixedviT. V. mimicus-is mimarT specifikuri 7 baqteriofagis lizisuri speqtris Semowmeba moxda 10 baqteriul Stamze. maT Soris iyo V. mimicus-is 9, wylis nimuSidan gamoyofili izolati (g.eliavas bmv institutis koleqcia) da erTi standartuli Stami (ATTC-is koleqcia). fagebi aqtiurni iyvnen 10-dan 7 baqteriul Stamze. umetesobam gamoavlina farTo speqtri, aqtiuri iyvnen ara marto patron-ujredis, aramed sxva baqteriuli izolatebis mimarTac. erTi fagi (Vmim8.07K) iyo Stamspecifikuri, moqmedebda ra mxolod maspinZel ujredze. baqteriofagebis Sedarebis mizniT V. mimicus-is Stamebis imave

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nakrebze moxda V. cholerae O1-is da V. cholerae ara-O1-is mimarT specifikuri fagebis lizisuri speqtris Semowmebac. V. mimicus-is mimarT specifikuri fagebis lizisuri speqtri aseve Semowmda V. cholerae O1-is da V. cholerae ara-O1-is Stamebze. Sedegebma aCvena, rom V. cholerae-s mimarT specifikuri fagebi V. mimicus-is mimarT specifikuri fagebis msgavsad aseve aqtiurni iyvnen V. mimicus-is imave 7 Stamis mimarT. V. mimicus-is mimarT specifikuri 7 fagidan 5 iCenda aqtivobas V. cholerae-s izolatebis mimarT. fagebis moqmedebis areali met-naklebad gansxvavdeboda. Sedegebma gamoavlina V. cholerae O1-is da V. mimicus-is mimarT specifikuri fagebis farTo lizisuri speqtri, gansxvavebiT V. cholerae ara-O1-is mimarT specifikuri fagebisa (cxr.10), romlebic ZiriTadad Stam-specifikurobiT xasiaTdebodnen (Vch10CH, Vch105, Vch105S). lizisuri aqtivobis Sedegebidan gamomdinare fagebs Soris naTesaoba dadginda specialuri programis meSveobiT (UPGMA) (sur.28), sadac naCvenebia, rom V. cholerae ara-O1-is mimarT specifikuri baqteriofagebi: Vch105, Vch105S mcire, damoukidebel klasterSi erTiandebian, gansxvavebiT V. cholerae ara-O1-is mimarT specifikuri -VchBS3 fagisa, romelic V. mimicus-is mimarT specifikur -Vmim8.07K fagTan erTad qmnis klasters da momijnavea sxva, V. cholerae O1-is da V. mimicus-is mimarT specifikuri fagebis klasterTa jgufebis. aRsaniSnavia aseve V. cholerae ara-O1-is mimarT specifikuri fagi -Vch10CH, romelic mkveTrad aris gamijnuli yvela, sxva klasterisgan, iseve, rogorc V. cholerae O1-is mimarT sepcifikuri fagebi: VchK1, VchK3, Vch801, Vch802, Vch803, romlebic erT, damoukidebel klasterSi erTiandebian. unda aRiniSnos, rom sxvadasxva patron-ujredis mqone V. cholerae O1-is mimarT sepcifikuri fagebi axlomdgom, Tumca gansxvavebul klasterTa jgufebSi gaerTianda, gansxvavebiT erTi fagisa, kerZod V. cholerae O1-is mimarT sepcifikuri fagi-VchKm9-9004, romelic mkveTrad aris gamijnuli yvela, sxva klasterisgan. V. mimicus-is mimarT specifikuri fagebis klasterTa jgufebi V. cholerae O1-is mimarT sepcifikuri fagebis klasterebis momijnavea.

cxr.10 fagebis lizisuri aqtivobis speqtri

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sur.28.BUPGMA (Nei-Li daSorebuli analizi) filogenezuri xe baqteriofagebis lizisuri speqtris mixedviT

75

3.5.3. patron-ujredTan urTierTqmedebis fazebi patron-ujredTan urTierTqmedeba SeviswavleT V. cholerae-s da V. mimicus-is baqteriebis adsorbciisTvis optimalur 370C temperaturaze. Semdgom etapze SeviswavleT adsorbciis siCqare, latenturi periodi da saSualo gamosavli TiToeul ujredze. sakvlev baqteriofagebs Soris umniSvnelo gansxvaveba dafiqsirda maTi ujredSida gamravlebis ciklis maxasiaTeblebis mixedviT, rogoricaa: adsorbciis efeqturoba, latenturi periodis xangrZlivoba da fagur nawilakTa gamosavali. Catarebuli eqsperimentebis Sedegad dadginda, rom baqteriofagebis umetesoba xasiaTdeba Senelebuli, Tumca maRalprocentuli adsorbciis unariT, xangrZlivi latenturi periodiT da virusuli nawilakebis maRali gamosavaliT, gansakuTrebiT zogierTi fagis SemTxvevaSi (Vch105, Vch20K-4805, V.mim8.07K, VmimL4) (cxr.11).

cxr.11 baqteriofagebis ujredSida gamravlebis ciklis maxasia|Teblebi

3.5.4. baqteriofagebis serologiuri Tvisebebis Seswavla antifaguri Sratis gamoyenebiT baqteriofagebis serologiuri Tvisebebis Seswavlas vaxdendiT specifikuri antifaguri Sratebis gamoyenebiT da Semdgomi jvaredini neitralizaciis Sedegebis analiziT. cnobilia, rom baqteriofagebis daxasiaTebisa da diferencirebisTvis metad mniSvnelovan kriteriums warmoadgens maT Soris antagonisturi naTesaobis dadgena, rasac safuZvlad udevs jvaredini neitralizaciis reaqciebi.

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imunizaciisTvis SeirCa 10 fagi da 20 bocveri, maT Soris V. cholerae–s mimarT specifikuri 3 fagi: ori-V. cholerae ara-O1-is mimarT specifikuri, kerZod :Vch105 da Vch10CH da erTi-V. cholerae O1-is mimarT specifikuri, kerZod:Vch54. antisxeulebis titris dasadgenad, pirveli imunizaciidan yovel me-10, me-20 da 30-e dRes sakvlevi sisxlis aReba xdeboda yuris venidan, risTvisac SeirCa V. cholerae ara-O1-is da V. cholerae O1-is mimarT aqtiuri 2 fagi:Vch10CH da Vch54. Vch105-is antifaguri Sratis SemTxvevaSi mxolod me-40 dRes moxerxda sakvlevi sisxlis aReba. homologiur sistemaSi Catarebuli neitralizaciis Sedegebidan gamomdinare Vch54, Vch10CH –antifagur SratebSi yoveli 10 dRis intervaliT me-40 dRis CaTvliT SeiniSneboda antisxeulebis titris mateba, ris mixedviTac gamovTvaleT neitralizaciis siCqaris konstanta, rac asaxavda antisxeulebis titris zrdis dinamikas (sur.29). me-40 dRes bocvrebis gulidan aRebuli sisxlidan gamoyofil Srats viyenebdiT neitralizaciis reaqciebisTvis. fagis serologiur naTesaobas vswavlobdiT fagis neitralizaciis jvaredina reaqciis Catarebis gziT sxvadasxva specifikurobis antifaguri Sratis gamoyenebiT. fagi-antifaguri Sratis TiToeuli wyvilis SemTxvevaSi ganvsazRvreT neitralizaciis procenti da konstanta. fagebis mkvlevarTa mier aRiarebuli sqemidan gamomdinare, mxolod 70%-ze meti neitralizaciis SemTxvevaSi SeiZleboda gvevarauda fagebs Soris serologiur naTesaobaze. Cvens mier Catarebuli cdebis Sedegad, moxda 36 baqteriofagis gamokvleva. maT Soris 16 fagi iyo V. cholerae O1-is mimarT specifikuri (2 standartuli fagi), 13-V. cholerae ara-O1-is mimarT specifikuri, xolo 7-V. mimicus-is mimarT specifikuri. miRebulma Sedegebma saSualeba mogvca gvemsjela am fagebis serologiur naTesaobaze, kerZod, moxda baqteriofagebis dajgufeba 5 sxvadasxva jgufSi (cxr.12), rasac safuZvlad daedo jvaredini neitralizaciis Sedegad faguri nawilakebis infeqciurobis inhibiciis procentuli maCveneblebi. miRebuli Sedegebis analizma gamoavlina, rom antifaguri SratiT-Vch54-iT moxda V. cholerae O1-is mimarT specifikuri 11 fagis neitralizacia (70% da meti), ris mixedviTac isini gadanawildnen I jgufSi (jgufiI/Vch54). II jgufSi (jgufiII/Vch10CH) gaerTianebuli 9, V. cholerae ara-O1-is mimarT specifikuri fagis neitralizacia (90% da meti) moxda Vch10CH antifaguri SratiT. III jgufSi (jgufiIII/Vch105) ganawilebuli 5 fagidan 3 fagi iyo V. cholerae ara-O1-is xolo, 2-V. cholerae O1-is mimarT specifikuri da maTi neitralizacia (70% da meti) moxda Vch105 antifaguri SratiT. IV jgufis fagebi gansxvavdebodnen sxva fagebisgan. maTi neitralizaciis procentis maqsimumi aRwevda 36 %-s. V jgufSi ganawildnen V. mimicus-is mimarT specifikuri fagebi. maTi neitralizaciis procentis maqsimumi aRwevda 68%-s. Vch54-is antifaguri Sratis SemTxvevaSi 90% da meti neitralizaciis procentis arsebobisas moxda neitralizaciis konstantas daTvla (K ml/wT ) (sur.30). suraTze gavlebuli zRvari miuTiTebda neitralizaciis konstantas maqsimumis (homologiur sistemaSi) 70% da met maCvenebels. V. cholerae O1-is mimarT specifikuri fagebis erT jgufSi gaerTianeba miuTiTebs maT serologiur naTesaobaze, rac Seesabameba lizisuri speqtris monacemebsac.

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Vch105, Vch54, Vch10CH, iseve, rogorc lizisuri speqtris mixedviT, serologiuri analizidan gamomdinare erTmaneTisgan gansxvavebuli fagebi aRmoCnda. serologiuri TvalsazrisiT gansxvavebuli fagebi aRmoCnda aseve V. mimicus-is mimarT aqtiuri fagebi, iCenen, ra, serologiur naTesaobas erTmaneTTan, Tumca gansxvavdebian V. cholerae-s mimarT specifikuri fagebisagan. serologiurma analizma anaxa, rom V. cholerae O1-is mimarT specifikuri fagebi ar iCenen naTesaobas V. cholerae ara-O1-is mimarT specifikur fagebTan.

sur.29. antisxeulebis titris zrdis dinamika sami antifaguri Sratis homologiur sistemaSi

cxr.12. fagebis dajgufeba neitralizaciis maxasiaTeblis mixedviT

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sur.30. V. cholerae O1-is mimarT specifikuri fagebis neitralizaciis

maxasiaTeblebi Vch54 antifaguri Sratis gamoyenebiT.

3.5.5. V. cholerae O1–is mimarT specifikuri baqteriofagebis zegavlena Stamebis mier qoleris toqsinis produqciaze GM1 gangliozid damokidebuli enzimdakavSirebuli imunosorbentuli analizis (GM1-ELIZA) gamoyenebiT nanaxi iyo V. cholerae O1–is mimarT specifikuri baqteriofagebis garkveuli zegavlena Stamebis mier qolera toqsinis produqciaze fagisa da Stamis sxvadasxva TanafardobiT urTierTqmedebisas drois sxvadasxva intervalis SemTxvevaSi. qolera toqsinis (CT) produqciis daTvla xdeboda standartul mrudTan Sedarebis mixedviT, romelic asaxavda V. cholerae O1–is standartuli Stamis (V.chCIP62.13, V. cholerae 4805-aziatikusi) filtratis sxvadasxva ganzavebebis SemTxvevaSi producirebul toqsinis raodenobas (OD-optikuri simkvrive).cdaSi gamoyenebuli iyo V. cholerae O1–is mimarT specifikuri baqteriofagi Vch20K-4805, romlis zemoqmedebas CT-is produqciaze vswavlobdiT standartuli Stamis-V.ch-4805 ( CIP-is koleqciidan) magaliTze. drois intervalis gazrdasTan erTad SeiniSneboda qolera toqsinis produqciis Semcireba (R2=0.5. p<0.03) (sur.31).

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sur.31. CT-is produqciis Semcireba drois intervalTan damokidebulebiT miRebuli Sedegebidan gamomdinare Semdgomi kvleva, baqteriofagebis biologiuri Tvisebebis daxasiaTebiT gagrZelda 3 -V. cholerae ara-O1-is, 6 -V. cholerae O1-is mimarT specifikuri da 3 -V. mimicus-is mimarT specifikuri baqteriofagiT.

3.6. fiziko-qimiuri faqtorebis zegavlena baqteriofagebis sicocxlisunarianobaze

fagebis stabilobis Seswavla sxvadasxva areSi xorcieldeboda 6 Tvis ganmavlobaSi, periodulad maTi titris dadgeniT.Bbaqteriofagebis praqtikuli gamoyenebisTvis didi mniSvneloba aqvs maT stabilobas sakvlev garemoSi, SemdgomSi fagebis samkurnalo-profilaqtikuri mizniT da ekologiuri kvlevebisTvis, sxvadasxva wyalsatevebSi trasirebis eqsperimentebis Casatareblad. baqteriofagebis stabiloba aseve Seiswavleba Senaxvisa da transportirebis pirobebSi maTi aqtivobis gaTvaliswinebis mizniT. fagebis stabilobas vswavlobdiT sxvadasxva mikrobiologiur areebsa da xsnarebSi, aseve bunebriv wylebSi, risTvisac viyenebdiT tbisa da zRvis wylebs (cxr.13). miRebulma Sedegebma gamoavlina V. cholerae O1-is mimarT specifikuri baqteriofagebis maRali stabiloba sxvadasxva areSi, 1-dan 6 Tvemde Catarebul kvlevaSi. umniSvnelo cvlilebebs qonda adgili sacdeli areebis umravlesobis SemTxvevaSi. yvelaze nakleb stabiluri V. cholerae ara-O1-is

80

mimarT specifikuri fagi-Vch105 aRmoCnda, romlis titris dacemac 1 Tvis Semdeg Semowmebul cdebSi aRiniSna. V. cholerae O1-is mimarT specifikuri baqteriofagebis titris dacema ZiriTadad SeiniSneboda eqsperimentis dawyebidan 6 Tvis gasvlis Semdeg. V. cholerae O1-is mimarT specifikurma fagebma: Vch16-O1 da Vch20-4805 gamoavlines naklebi stabiloba fiziologiur xsnarSi. 6 Tvis Semdeg sicocxlisunariani faguri nawilakebis ricxvi 2 logiT ecemoda. orive tipis fagebisTvis araxelsayreli aRmoCnda marilis maRali procentobis Semcveli are. M miRebuli Sedegebidan dadginda, rom V. cholerae O1-is mimarT specifikur baqteriofagebs aqvT unari metad SeinarCunon mdgradoba V. cholerae ara-O1-is mimarT specifikur baqteriofagebTan SedarebiT.

cxr.13. baqteriofagebis stabiloba sxvadasxva areSi

*moniSnuli ujrebi miuTiTebs aqtiuri faguri nawilakebis 1-dan 2 log.-mde dacemas F

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fagebis Termoinaqtivaciis eqsperimenti tardeboda 10,20,30 wT-is intervaliT 550C, 600C, 650C da 700C temperaturaze. siCqaris mudmiva (K) ganisazRvra V. cholerae ara-O1-is mimarT specifikuri 3 fagis, V. cholerae O1-is mimarT specifikuri 6 fagis da V. mimicus-is mimarT specifikuri 3 fagis SemTxvevaSi. siCqaris mudmivas sidide gansxvavdeboda TiToeuli fagisTvis temperaturidan da drois intervalidan gamomdinare. V. cholerae-s mimarT specifikuri 9 arsebuli fagidan 8 fagi (Vch105-is gamoklebiT) da V. mimicus-is mimarT specifikuri yvela fagi inaqtivirebuli iyo 550C-ze 10 wT-is eqspoziciis pirobebSi. naqtivirebuli

faguri nawilakebis ricxvi aRemateboda 70%-s (sur.32-34).

sur.32. V. cholerae O1-is mimarT specifikuri fagebis Termoinaqtivacia

mxolod Vch105 fagis SemTxvevaSi sicocxliunariani faguri nawilakebis ricxvis mniSvnelovani dacema (90%-ze meti) SeiniSna 700C t-ze 10 wT-is intervalSi da siCqaris mudmivas maqximumi miRweuli iyo 700C t-ze 30 wT-is

eqspoziciis dros (sur.35). 600C-dan 700C-mde Catarebulma cdebma aCvena, rom xuTi fagi srulad inaqtivirda 30 wT-ze nakleb droSi. maT Soris: V. cholerae ara-O1-is mimarT specifikuri fagi Vch10CH-600C-ze, V. cholerae O1-is mimarT specifikuri fagebi: Vch20K-4805, Vch20K-O1, Vch16K-O1- 650-ze da V. cholerae ara-O1-is mimarT specifikuri fagi-VchBS3 700C-ze.

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sur.33. V. cholerae ara-O1-is mimarT specifikuri fagebis Termoinaqtivacia

sur.34. V. mimicus-is mimarT specifikuri fagebis Termoinaqtivacia

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gamokvleuli baqteriofagebis erTmaneTisgan gansxvavebuli TermomgrZnobeloba miuTiTebs maTi cilovani struqturebis gansxvavebaze. V. cholerae O1-is mimarT specifikuri fagebis TermomgrZnobeloba gansxvavdeboda, rogorc V. cholerae ara-O1-is mimarT specifikuri fagebis, aseve erTmaneTis TermomgrZnobelobisgan. V. choleraeara-O1-is mimarT specifikuri samive fagi am parametris mxriv erTmaneTisgan gansxvavebuli aRmoCnda. amrigad fagi Vch105-is cilovani struqtura sxva fagebis cilovan struqturaze metad Termostabiluri aRmoCnda. V. mimicus-is mimarT specifikuri fagebis SemTxvevaSi reaqciis siCqaris piki dafiqsirda 700C temperaturaze 30 wT-is

eqspoziciis Semdeg (sur.36). amrigad, es ukanasknelni, V. cholerae-s mimarT specifikur fagebisgan gansxvavebiT metad Termostabilurebi aRmoCndnen.

sur.35. V. cholerae ara-O1 da O1-is mimarT specifikuri fagebis Termoinaqtivacis siCqare

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sur.36. V. mimicus-is mimarT specifikuri fagebis Termoinaqtivaciis siCqare wyalbad-ionTa koncentraciis zegavlenas fagebze vswavlobdiT pH-is sxvadasxva koncentraciis pirobebSi: 1-dan 12-mde, 30 da 60 wT-is intervalze, ris Semdegac vsazRvravdiT sicocxlisunariani faguri nawilakebis ricxvs. baqteriofagebis umravlesoba sakmaod stabiluria pH-is Sualedur diapazonSi, romelic standartul pirobebSi (320C-370C) Seadgens pH-5-9-s. sakvebi aris gatutovneba iwvevs virionebis TandaTanobiT degradacias, Tavdapirvelad

xdeba morCebis daSla, Semdeg ki iwyeba dnm-is gamosvla. კafsidis struqturuli elementebis daSlas iwvevs, rogorc maRali tutianobis pirobebi (pH-12), aseve pH-is eqstremalurad dabali maCvenebeli. (<pH-2). pH-is zomierad dabali maCveneblis dros baqteriofagebis gamoleqva SesaZloa infeqciurobis dakargvis gareSec moxdes. pH-is gamoxatulad maRali an dabali mniSvnelobis dros gamovlenili faguri nawilakebi SesaZloa mutantur formebs warmoadgenen, romelTa gadarCevac cvalebad pirobebSi mimdinareobs. Cvens mier Catarebulma cdebma aCvena, rom fagebisTvis kritikuli aRmoCnda pH-is eqstremalurad dabali (pH-1) maCvenebeli, romlis drosac gadarCenili faguri nawilakebis ricxvi mxolod 1%-s utoldeboda. pH-4-9-s Soris intervalSi V. cholerae ara-O1-is mimarT specifikur fagebSi (3 fagi) SeiniSneboda titris mcire dacema V. cholerae O1-is mimarT specifikur fagebTan (6 fagi) SedarebiT. mxolod ori V. cholerae ara-O1-is mimarT specifikuri fagi:Vch105 da Vch10CH inarCunebda aqtivobas pH 12-ze 30 da 60 wT-is

85

intervalze. V. cholerae O1-is mimarT specifikuri yvela fagi inaqtivirda pH-12-ze. V. mimicus-is mimarT specifikuri fagebisTvis (3 fagi) iseve, rogorc danarCenebisTvis kritikuli aRmoCnda pH-1-is maCvenebeli, V. cholerae ara-O1-is mimarT specifikuri fagebis msgavsad inarCunebdnen srul aqtivobas pH-4-9 maCvenebelze. pH-12-is SemTxvevaSi fagi VmimL4-is sicocxlisunariani faguri nawilakebis maCvenebeli 40%-ze mets Seadgenda, gansvavebiT danarCeni ori fagisgan (Vmim8.07K da V.mim29.08K), romlebic V. cholerae O1-is mimarT specifikuri fagebis msgavsad naklebad rezistentuli aRmoCndnen maRali tutianobis pirobebSi (cxr.14). dadginda, rom V. cholerae O1-is mimarT specifikuri fagebi iCendnen nakleb rezizstentobas pH-is sxvadasxva koncentraciis mimarT V. cholerae ara-O1-is da V. mimicus-is mimarT specifikur fagebTan SedarebiT.

cxr.14. wyalbad-ionTa koncentraciis mainaqtivirebeli zemoqmedeba fagebze

baqteriofagebis mgrZnobeloba UV sxivebis mimarT. bunebriv pirobebSi fagebis populaciis SenarCunebisaTvis didi mniSvneloba aqvs baqteriofagebis ultraiisferi sxivebisadmi gamZleobas. UV gamosxivebas damazianebeli efeqti aqvs fagebze, amcirebs ra maT litiur aqtivobas. gamokvleuli 12 fagidan (6- V. cholerae O1-is da 3 ara-O1-is mimarT specifikuri, 3-V. mimicus-is mimarT specifikuri) yvelaze naklebad rezistentuli UV gamosxivebis mimarT aRmoCnda V. cholerae ara-O1-is mimarT specifikuri fagi-Vch10CH, romlis SemTxvevaSic aqtiuri faguri nawilakebis ricxvi 30 wamis eqspoziciis dros aRwevda 0.8%-s (sur.37). V. cholerae O1-is mimarT specifikuri 6 fagidan yvelaze

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naklebi medegobiT gamoirCeoda 2 fagi: Vch20K-4805 da Vch16K-O1, romelTa aqtiuri faguri nawilakebis sixSirec 30 wamis intervalze utoldeboda 19 da 28%-s, gansxvavebiT sxva V. cholerae O1-is mimarT specifikuri fagebisa, romelTa SemTxvevaSic aqtiuri faguri nawilakebis procentuli maCvenebeli 30 wamis intervalze aRwevda 70%-s (sur.38).rac Seexeba V. mimicus-is mimarT specifikur fagebs, maRali medegobiT gamoirCeoda fagi- VmimL4, gansxvavebiT danarCeni 2 V. mimicus-is mimarT specifikur fagisa. cdebma gamovalina, rom fagebis litiuri aqtivoba mcirdeboda UV gamosxivebis zemoqmedebis drois gazrdasTan erTad. V. cholerae ara-O1-is mimarT specifikuri fagebi nakleb medegobas iCendnen UV gamosxivebis mimarT, V. cholerae O1-is mimarT specifikur fagebiTan SedarebiT. V. mimicus-is mimarT specifikuri fagebi V. cholerae ara-O1-is mimarT specifikuri fagebis msgavsad naklebad rezistentuli aRmoCndnen UV gamosxivebis damazianebeli efeqtis mimarT.

sur.37. UV gamosxivebis zemoqmedeba V. cholerae ara-O1-is mimarT specifikuri fagebis sicocxlisunarianobaze

87

sur.38. UV gamosxivebis zemoqmedeba V. cholerae O1-is mimarT

specifikuri fagebis sicocxlisunarianobaze

fagebis mgrZnobeloba natriumis citratis mimarT gamokvleuli iyo 12 fagze (6-V. cholerae O1-is da 3 ara-O1-is mimarT specifikuri, 3-V. mimicus-is mimarT specifikuri). natriumis citratis zemoqmedeba maRali iyo V. cholerae ara-O1-is mimarT specifikuri fagis -VchBS3 da V. cholerae O1-is mimarT specifikuri fagis -Vch20K-4805 mimarT. sicocxlisunariani faguri nawilakebis raodenoba utoldeboda 1 da 24%-s. saerTod ar SeiniSneboda titris cvlileba V. cholerae ara O1-is mimarT specifikuri fagis -Vch105-is SemTxvevaSi da titris mcire dacema aRiniSna V. cholerae ara O1-is mimarT specifikuri fagisTvis-Vch10CH (aqtiuri faguri nawilakebis ricxvi utoldeboda 83%-s). V. mimicus-is mimarT specifikur fagebis gamokvlevam ki cxadyo, rom maRali medegobiT gamoirCeoda fagi-VmimL4, gansxvavebiT danarCeni ori-V. mimicus-is mimarT specifikuri fagisa, romelTa gadarCenili faguri nawilakebis procentuli maCvenebeli dabali iyo, msgavsad V. cholerae ara-O1-is mimarT specifikuri fagisa-VchBS3 da utoldeboda 11 da 13%-s (sur.39). umniSvnelo xarisxis inaqtivacia arapirdapirad miuTiTebs imze, rom baqteriofagebi ar arian damokidebulni Ca++-is orvalentian ionebze, rogorc adsorbciis kofaqtorebze.

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sur.39. sicocxlisunariani faguri nawilakebis procentuli ganawileba natriumis citratis zemoqmedebidan 15 wT-iseqspoziciis Semdeg

*gavlebuli zRvari miuTiTebs aqtiuri faguri nawilakebis 1 log.-iT dacemas

fagebis mgrZnobeloba osmosuri Sokis mimarT aseve SeviswavleT aRniSnul 12 fagze (6-V. cholerae O1-is da 3 ara-O1-is mimarT specifikuri, 3-V. mimicus-is mimarT specifikuri). kvlevam aCvena, rom V. cholerae ara-O1-is mimarT specifikuri fagebi iCendnen met medegobas osmosuri Sokis zemoqmedebis mimarT V. cholerae O1-is mimarT specifikur fagebTan SedarebiT. V. cholerae ara-O1-is mimarT specifikuri fagebis: VchBS3 da Vch10CH-is aqtiuri faguri nawilakebis procentuli raodenoba 15 wuTiani eqspoziciis Semdeg 83%-mde aRwevda gansxvavebiT fagi-Vch105-sgan (42.5%), romlis medegobis maCvenebelic sxvadasxva faqtorebis mimarT ufro maRali iyo zemoT xsenebul or fagTan SedarebiT. V. cholerae O1-is mimarT specifikuri fagebis: Vch16K-4805, Vch9K, Vch20K-O1-is SemTxvevaSi gadarCenili faguri nawilakebis procentuli maCvenebeli utoldeboda mxolod 19.5, 26 da 31.5%-s. unda aRiniSnos, rom xsenebuli 3 fagi meti medegboT gamoirCeoda natriumis citratis mimarT. V. mimicus-is mimarT specifikur fagebis kvlevam, iseve, rogorc sxva parametrebis mxriv gamoavlina VmimL4 fagis maRali medegobis unari mainaqtivirebeli faqtoris mimarT. osmosuri Sokis zemoqmedebis Semdeg, 15wT-iani eqspoziciisas gadarCenili faguri nawilakebis ricxvi 83%-s aRwevda. aseve maRali iyo fagis-V.mim29.08K–is medegobis maCvenebeli (75%). yvelaze naklebi rezistentobiT, rogorc V.

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mimicus-is aseve V. cholerae-s mimarT specifikur fagebTan SedarebiT gamoirCeoda fagi Vmim8.07K (1%) (sur.40).

sur.40. sicocxlisunariani faguri nawilakebis procentuli ganawileba osmosuri Sokis zemoqmedebidan 15 wT-is eqspoziciis Semdeg

*gavlebuli zRvari miuTiTebs aqtiuri faguri nawilakebis 1 log.-iT dacemas

3.7. baqteriofagebis genetikuri maxasiaTeblebi V. cholerae-s mimarT specifikuri fagebis dnm-is restriqciuli analizisTvis SevarCieT ramodenime endonukleaza, kerZod: EcoR I, EkoR V, Hind III, BamH I, Pst I,Not I (sur.41-43). Cvens mier Seswavlili baqteriofagebis dnm orjaWviania da mgrZnobiare aRmoCnda Sewavlili restriqciuli endonukleazebis umravlesobis mimarT. fagebis dnm-ebis molekuluri wonis dasaTvlelad gamoviyeneT Hind III endonukleaziT daWrili lambda(λ) dnm, rogorc markeri da Sesabamisad vixelmZRvaneleT amave dnm-is standartuli mrudiT. Seswavlili fagebis dnm-ebis yvelaze msxvili fragmentebi 22.5 kb-s aRwevda. endonukleazebiT faguri dnm-ebis restriqcia ZiriTadad wvril fragmentebad moxda da umetesoba fragmentebisa dabali molekuluri wonis iyo (1.1 kb). V. mimicus-is mimarT specifikuri fagebis restriqciulma profilma gamoavlina maTi SedarebiT dabali mgrZnobeloba endonukleazebis mimarT. mxolod 2 fagi

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aRmoCnda mgrZnobiare EkoR V da BamH I fermentebis mimarT (VmimL4 da V.mimK5).restriqciuli profili saSualebas gvaZlevs vimsjeloT fagebis genetikur mravalferovnebaze. genetikuri maxasiaTeblis mixedviT fagebis dajgufebisTvis avageT filogenezuri xe (sur.44).

sur.41. EcoR I fermentiT daWrili V. cholerae-s mimarT specifikuri fagebis restriqciuli profili

sur.42. Hind III fermentiT daWrili V. cholerae-s mimarT specifikuri fagebis restriqciuli profili

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sur.43. EcoR V fermentiT daWrili V. cholerae-s mimarT specifikuri fagebis restriqciuli profili

1. λ -DNA+Hind III 2. Vch16K-O1 3. Vch16K-4805 4. Vch20K-O1 5. Vch20K-4805 6. VchLLVIII 7. VchXKL 8. Vch25MT 9. Vch83-6 10. VchK24 11. Vch9K

12. VchK1 13. VchK3 14. VchB1 15. VchB4 16. Vch54 17. Vch105 18. VchBS3 19. Vch105S

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sur.44. UPGMA (Nei-Li daSorebuli analizi) xe V. cholerae-s da V. mimicus-is mimarT specifikuri fagebis dajgufeba restriqciuli profilis mixedviT

93

IV Sedegebis ganxilva

sasmeli wylis da sarekreacio mizniT gamoyenebuli wyalsatevebis mikrobuli dabinZureba sazogadoebrivi jandacvis mTavar sakiTxad ganixileba. ukanasknel wlebSi klimatis cvlilebasTan dakavSirebiT gansakuTrebul interess iwvevs adamianis paTogeni arahalofiluri, qoleras jgufis vibrionebis V. cholerae-s da V. mimicus-is ekologiasa da biomravalferovnebasTan dakavSirebuli sakiTxebi. V. cholerae-s epidemiuri serotipebi mwvave infeqciuri daavadebis-qoleras gamomwvevi agentebia. saqarTvelos Tbili, subtropikuli klimati xelsayrel pirobas qmnis vibironebis gamravlebisa da ganviTarebisTvis. dabali da saSualo marilianoba, aseve temperatura, romelic 18°C-s aRemateba, qmnis maTTvis sasurvel zrdis pirobebs. garemo-klimaturi pirobebi da maTi sezonuri cvlileba did zegavlenas axdens paTogenuri vibrionebis, aseve maTi mimarT specifikuri

baqteriofagebis gavrcelebaze. baqteriofagebs did roli eniWebaT baqteriuli populaciis regulaciasa da genetikur mravalferovnebaSi. baqteriofagebs gamorCeuli antibaqteriuli moqmedeba axasiaTebT. aRwerilia maTi indikatorebad da traserebad, bunebrivi Ria wyalsatevebis dabinZurebis xarisxis, wyarosa da gavrcelebis gzebis dadgenis mizniT gamoyeneba. V. cholerae O1-is mimarT specifikuri baqteriofagebi odiTganve interesis sferos warmoadgenda-infeqciebis sadiagnostikod. misi baqteriofagebis specifikuri nakrebebi dRemde gamoiyeneba V. cholerae O1-is serotipis “klasikuri” da “El Tor” biotipebis gansasxvaveblad. Camdinare wylebSi paTogenuri Stamebisa da Sesabamisi fagebis deteqcia SesaZloa iyos specifikuri saSualeba qoleras afeTqebebis winaswar ganWvretisa. daavadeba qolera misi aRmocenebis adgilidan sakmaod swrafad vrceldeba. epidemiologiuri TvalsazrisiT mniSvnelovania daavadebis gavrcelebis Tvalyuris devneba. V. cholerae–s mimarT specifikuri baqteriofagebis intensiuri daxasiaTeba saSualebas iZleva dagrovdes informacia paTogenuri vibrionebis populaciis struqturis cvlilebaze da paTogenurobis meqanizmebis gagebaze. fagebis profili da siuxve wylian garemoSi zemoqmedebs wylismieri infeqciebis eTiologiur struqturaze da informacias iZleva paTogenebis Sesaxeb, am ukanasknelTa ararsebobis SemTxvevaSic ki. saqarTvelos wylis aqvatoriis regularuli monitoringis Sedegad dagrovili informacia saSualebas iZleva daigegmos prevenciuli zomebi wylismieri infeqciuri daavadebebis gavrcelebis Tavidan asacileblad. Cveni kvlevis ZiriTad mizans warmoadgenda saqarTvelos wylian garemoSi arahalofiluri (qoleris jgufis) vibrionebis mimarT specifikuri baqteriofagebis gamoyofa da daxasiaTeba, aseve qoleras jgufis vibrionebis, rogorc baqteriofagebis maspinZeli Stamebis winaswari koleqciis Seqmna. 2006-2009 wlebSi Savi zRvis sanapiro zolSi da Tbilisis SemogarenSi arsebul mtknar wyalsatevebSi Catarebulma monitoringma aCvena, rom saqarTvelos wyliani garemo da klimati xelsayrel pirobebs qmnis arahalofiluri, qoleras jgufis baqtriebis da Sesabamisi baqteriofagebis

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cxovelmyofelobisTvis. baqteriebisa da Sesabamisi baqteriofagebis gamoyofa sezonur xasiaTs atarebda da damokidebuli iyo gamoyofis wyaroze. V. cholerae-s Stamebis intensiuri gamoyofa moxda zafxulis da adreuli Semodgomis TveebSi (ivlisi-seqtemberi). swored am periodSi gamoiyo V. cholerae-s izolatebis umravlesoba-56.1%. mniSvnelovnad mcirdeboda V. cholerae-s Stamebis gamoyofis sixSire zamTaris da adreuli gazafxulis periodSi (ianvari-marti). mxolod 2.5% iyo gamoyofili aRniSnul TveebSi. Savi zRvis sxvadasxva saitidan V. cholerae-s Stamebis sul 16.7% gamoiyo. V. cholerae-s Stamebis umravlesoba-83.3% gamoiyo Tbilisis SemogarenSi arsebuli mtknari wyalsatevebidan, sadac Sav zRvasTan SedarebiT marilianoba dabalia da Sesabamisad Savi zRva SesaZloa warmoadgendes araxelsayrel garemos arahalofiluri vibrionebis gamravlebisTvis. araxelsayrel garemo pirobebSi V. cholerae inarCunebs ra sasicocxlo Tvisebebs gadadis arakultivirebad mdgomareobaSi. es movlena literaturaSi sicocxlisunariani, magram arakultivirebadi mdgomareobis (VBNC) saxelwodebiT aris cnobilia. V. cholerae-s baqteriebis arakultivirebad mdgomareobaSi gadasvla dakavSirebulia, rogorc maRal marilianobasTan aseve wylis dabal temperaturastan. eqsperimentulad Catarebulma kvlevebma, sadac VBNC-mdgomareobaSi V. cholerae-s baqteriebis gadasvla iyo naCvenebi cxadyo, rom araxelsayreli pirobebi, rogoricaa: maRali marilianoba, dabali temperatura da sakvebi nivTierebebis nakleboba, sasicocxlo procesebis daqveiTebasa da kultivirebis unaris dakargvas iwvevs. Alami da Tanaavtorebi (2007) swavlobdnen cocxal da arakultivirebad V. cholerae Ol-is Stamebs wylis garemos biofilmebSi da maT rols qoleras gadacemaSi. am eqsperimentis Sedegad daginda, rom arsebuli Stamebi inarCunebdnen sicocxlisunarianobas 495 dRis ganmavlobaSi, matbarias (bangladeSi) wyalsatevSi. mniSvnelovania garemodan gamoyofili V. cholerae-s epidemiuri serotipebis zusti identifikacia rac ganxorcielda polimerazuli jaWvuri reaqciis sxvadasxva saxis saSualebiTac (PCR): 1.polimerazuli jaWvuri reaqcia (ITS – PCR, Internal Transcribed Spacer PCR)specifikuri praimerebiT. 2. multipleqsuri polimerazuli jaWvuri reaqcia (Multiplex-PCR) V. cholerae O1-is, V. cholerae O139-is, toqsigenuri genebis aRmosaCenad (ctxA).Ggamovlinda ctxA+ 8 Stami, maT Soris 6 Stami iyo V. cholerae ara-O1-is da 2 -V. cholerae O1-is. kvlevebma aCvena bioqimiuri identifikaciis maRali sizuste. izolatebis 98.5%-Tvis bioqimiuri identifikacia aRmoCnda zusti. garemoSi V. cholerae-s epidemiri serotipebis pirdapiri deteqcia mniSvnelovania am paTogenis mier gamowveuli daavadebis kontrolisa da Tavidan aridebisTvis. pirdapiri fluorescentuli meTodis (DFA) gamoyenebiT saqarTvelos wylis aqvatoriaSi aRmoCenil iqna V. cholerae-s epidemiuri serotipebi. wylis pirdapirma fluorescentulma analizma gamoavlina V. cholerae O1–is arseboba rogorc, zRvis, aseve tbebis wylis nimuSebSi (kumisisa da lisis tbebi, Tb.zRva). gamokvleuli sinjebidan 31% dadebiTi iyo. dadebiTi nimuSebidan 90% nimuSi pozitiuri iyo V. cholerae O1-is SemTxvevaSi, 10%_V.

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cholerae O139-is (savaraudod VBNC-sicocxlisunarian, magram arakultivirebad formaSi) SemTxvevaSi. V. cholerae-s antiSrati -“poli” warmoadgens serologiuri testis erT-erT magaliTs, romelic dafuZnebulia aglutinaciis reaqciaze da romlis meSveobiTac moxda garemoSi V. cholerae-s toqsigenuri izolatebis pirdapiri detqcia. aRsaniSnavia, rom slaid-aglutinaciis Sedegebi dadebiT korelaciaSi modioda polimerazuli jaWvuri reaqciiT da pirdapiri fluorescentuli analiziT Catarebuli kvlevis Sedegad miRebul SedegebTan. V. cholerae O1-is epidemiuri serotipebis gadamowmebis mizniT gamoyenebuli serologiuri testis_mgrZnobiare, membranuli antigenis swrafi testis (Cholera O1 SMARTTM) Sedegebi aseve dadebiT korelaciaSi modioda sxva, Catarebuli serologiui testebis SedegebTan. V. cholerae O1-is Stamebis mier toqsinis produqcia Semowmda GM1 gangliozid damokidebuli enzimdakavSirebuli imunosorbentuli analiziT (GM1-ELIZA).Semowmda, rogorc standartuli Stamebis (kulturaTa sxvadasxva koleqcia, NCDC,CIP) mier aseve garemodan gamoyofili epidemiuri Stamebis mier toqsinis gamoyofa. Sedegad ramoednime (6), garemodan gamoyofilma Stamma dadebiTi Sedegi anaxa toqsinis produqciaze. V. cholerae-s 81 (135-dan) izolati da V. mimicus-is 7 (7-dan) izolati mgrZnobiare aRmoCnda maT mimarT specifikuri sakvlevi baqteriofagebis mimarT.Ffagotipirebis Sedegebi gaTvaliswinebuli unda iqnas am paTogenebiT gamowveuli infeqciebis samkurnalo preparatis Seqmnis procesSi. 2006-2009 wlebSi saqarTvelos wylian aqvatoriaSi Catarebuli monitoringis Sedegad gamoyofili baqteriofagebis gamoyofis sixSire maRali iyo aprili-

ivnisis periodSi (49.3%). oqtomberi–dekembris periodSi fagebis yvelaze mcire

(19.7%) raodenoba iyo gamoyofili. fagebis gamoyofam cxadyo, rom maTi

umravlesoba (51.2%) gamoiyo Tbilisis SemogarenSi arsebuli mtknari wyalsatevebidan. fagebis gamoyofis siuxviT xasiaTdeboda kumisis tba,

saidanac yvelaze didi raodenobiT (24.4%) faguri izolatis gamoyofa moxda. fagebi naklebi siuxviT gamoirCeodnen Sav zRvaSi (22%). fagebis umciresi raodenoba gamoiyo kulturaTa supernatantebidan (13.4%). V. mimicus-is mimarT specifikuri 7 faguri izolatidan mxolod 1-is gamoyofa moxda Savi zRvis wylis sinjebidan. Sul V.choelrae O1-is mimarT specifikuri 17 pirveladi izolati gamoiyo. gamoyofis sixSire V.choelrae O1-is mimarT specifikuri

fagebis SemTxvevSi maRali iyo (58.8%) ivlisi-seqtembris periodSi. umetesoba fagebisa (76.5%) gamoiyo Tbilisis SemogarenSi arsebuli wyalsatevebidan aRebuli wylis sinjebidan. kvlevebma cxadyo, rom V. cholerae-s aqvs dabali faguri potenciali. mag.: 2009 wlis ivlisSi aRebuli sinjebidan 24 gamididrebuli wylis nimuSidan mxolod 5 pirveladi faguri izolatis gamoyofa moxerxda. fagis gamoyofis sixSirem aCvena sezonuri gamoxatuleba. fagebis gamoyofa win uswrebda maspinZeli baqteriuli Stamebis gamoyofis piks. Sedegebma gamoavlina V. cholerae O1-is da V. mimicus-is mimarT specifikuri fagebis farTo lizisuri speqtri, gansxvavebiT V. cholerae ara-O1-is mimarT

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specifikuri fagebisa, romlebic ZiriTadad Stam-specifikurobiT xasiaTdebodnen. fagebis 36%–maaCvena viwro speqtri, aqtiurebi iyvnen mxolod maspinZeli Stamis mimarT. V. cholerae O1-is mimarT specifikuri yvela sxva fagi (71%) gamoirCeoda lizisuri aqtivobis saSualo da farTo speqtriT. fagebis garkveulma jgufebma gamoavlina msgavseba lizisuri speqtris mixedviT. lizisuri aqtivobis Sedegebidan gamomdinare agebul filogennezuri xis suraTSi naCvenebia, rom V. cholerae ara-O1-is da V. cholerae O1-is mimarT sepcifikuri fagebi damoukidebel did klasterebSi erTiandebian. unda aRiniSnos, rom sxvadasxva patron-ujredis mqone V. cholerae O1-is mimarT sepcifikuri fagebi axlomdgom, Tumca gansxvavebul mcire klasterTa jgufebSi gaerTianda. V. mimicus-is mimarT specifikuri fagebis klasterTa jgufebi V. cholerae O1-is mimarT sepcifikuri fagebis klasterebis momijnavea. gamomdinare zedapiruli cilovani struqturebis-fagospecifikuri receptorebis mravalferovnebidan, gamokvleulma baqteriofagebma meti mravalferovneba anaxes moqmedebis diapazonis mixedviT, gansxvavebiT sxva maxasiaTeblebisgan. lizisuri aqtivobis farTo speqtri Terapiuli fagebisTvis sasurveli maxasiaTebelia, Tumca maspinZeli Stamis mimarT maRali specifikurobis mqone fagebi warmatebiT gamoiyeneba Stamebis fagotipirebisTvis. fagebis eleqtronuli mikroskopiT Seswavlam gviCvena, rom Cvens mier gamoyofili V. cholerae-s da V. mimicus-is mimarT specifikuri baqteriofagebi xasiaTdebian morfologiuri mravalferovnebiT. gamovlinda kudiani fagebis samive morfotipis arseboba. V. cholerae-s mimarT specifikuri baqteriofagebis SemTxvevaSi Warbobda mioviride (izometruli Tavi da grZeli kumSvadi kudi) da sifovirides (arakumSvadi, grZeli kudi) ojaxis warmomadgenlebi, aseve gamovlinda mcire raodenobiT podovirides (mokle arakumSvadi kudi) morfotipis fagebi. V. mimicus-is mimarT specifikur baqteriofagebSi Warbobda sifovirides morfotipis fagebi. gansxvavdeboda aseve sakvlevi baqteriofagebis nukleokapsidisa da morCis zomebi. samkurnalo preparatebis SemadgenlobaSi arsebuli baqteriofagebis umravlesoba kudiani fagebia da, ZiriTadad, miovirides da sifovirides ojaxebs miekuTvneba.Aamdenad, Cvensmier Seswavlili baqteriofagebis umetesoba savaraudod atarebs Terapiul potencials. baqteriofagsa da maspinZel baqteriul ujreds Soris urTierTqmedebis meqanizmebis gageba saSualebas iZleva ukeT iqnes Secnobili molekuluri meqnizmebi, ramac SesaZloa migviyvanos baqteriuli infeqciebis sawinaaRmdego mkurnalobis meTodebis gaumjobesebamde. Terapiuli mizniT baqteriofagebis gamoyenebisTvis mniSvnelovania maTi swrafi da efeqturi gamravlebis unari. elisma da delbrukma [69] moaxdines postulireba, rom gamosavlianobis sidide izrdeba infeqciis drosTan mimarTebaSi. gamosavali, romelic Sedegia mokle inkubaciuri periodis iqneba mcire did inkubaciur periodTan SedarebiT. Tumca kvlevbma, romlebic Catarebulia erTsa da imave periodis xangrZlivobiT, cxadyo, rom fagis gamosavali SesaZloa sxvadasxva iyos. amave avtorebma gamoTqves hipoTeza, rom faguri nawilakebis gamoTavisufleba ar xdeboda erTdroulad, rac mizezi iyo gansxvavebuli gamosavlianobis

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sididisa. Coi da Tanaavtorebi [52] swavlobdnen ra ujredis sididis gavlenas gamosavalze, gamoTqves mosazreba, romlis mixedviTac arsebobs ujredis zomis kritikuli maCvenebeli, romlis iqiTac korelacia ujredis sididesa da gamosavals Soris wydeba. gamosavlianobis sxvadasxvaoba dRemde srulyofilad Seswavlili ar aris. Cvens mier Catarebulma eqsperimentebma aCvena, rom latenturi periodis xangrZlivoba dadebiT korelaciaSi iyo faguri nawilakebis gamosavalTan, gansakuTrebiT ki ramodenime fagis SemTxvevaSi sadac erT baqteriul ujredze 200-mde faguri nawilaki aRiniSna (Vch105 da V.mim8.07K). aqve unda aRiniSnos, rom V. cholerae O1-is mimarT specifikuri 3 fagis SemTxvevaSi aRiniSneboda latenturi periodis mokle monakveTi, romlis Semdegac faguri gamosavali erT baqteriul ujredze utoldeboda xangrZlivi latenturi periodis mqone fagebis gamosavals. baqteriofagebis daxasiaTebisa da diferencirebisTvis metad mniSvnelovan kriteriums warmoadgens maT Soris antagonisturi naTesaobis dadgena, rasac safuZvlad udevs jvaredini neitralizaciis reaqciebi. miRebulma Sedegebma saSualeba mogvca gvemsjela am fagebis serologiur naTesaobaze, kerZod, moxda baqteriofagebis dajgufeba 5 sxvadasxva jgufSi. V. cholerae O1-is mimarT specifikuri fagebis erT jgufSi gaerTianeba miuTiTebs maT serologiur naTesaobaze, rac Seesabameba lizisuri speqtris monacemebsac .iseve, rogorc lizisuri speqtris mixedviT, serologiuri analizidan gamomdinare erTmaneTisgan gansxvavebuli fagebi aRmoCnda V. cholerae ara-O1-is mimarT specifikuri fagebi. serologiuri TvalsazrisiT gansxvavebuli fagebi aRmoCnda aseve V. mimicus-is mimarT aqtiuri fagebi, iCenden, ra, serologiur naTesaobas erTmaneTTan, Tumca gansxvavdebodnen V. cholerae-s mimarT specifikuri fagebisagan.serologiurma analizma anaxa, rom V. cholerae O1-is mimarT specifikuri fagebi ar iCendnen naTesaobas V. cholerae ara-O1-is mimarT specifikur fagebTan. GM1-gangliozid damokidebuli enzimdakavSirebuli imunosorbentuli analiziT (GM1-ELIZA) nanaxi iyo V. cholerae O1–is mimarT specifikuri baqteriofagebis antibaqteriuli zegavlena Stamebis mier qolera toqsinis produqciaze fagisa da Stamis sxvadasxva TanafardabiT urTierTqmedebisas drois sxvadasxva intervalis SemTxvevaSi. cdebma gamoavlina qoleris toqsinis produqciis Semcireba drois intervalis gazrdasTan erTad. baqteriofagebis struqturuli komponentebis Senebisa da funqcionirebis SefasebisaTvis Seiswavleba maTze fiziko-qimiuri faqtorebis mainaqtivirebeli moqmedeba. Seswavlil iqna sakvlevi baqteriofagebis mdgradoba sxvadasxva mikrobiologiur areebsa da xsnarebSi, aseve bunebriv wylebSi (tbisa da zRvis wylebi), aseve damokidebuleba iseTi faqtorebis mimarT, rogoricaa ultraiisferi gamosxiveba, temperatura, wyalbad ionTa maRali an dabali maCveneblebi, maRali ionuri Zala da a.S. Bbaqteriofagebis praqtikuli gamoyenebisTvis didi mniSvneloba aqvs maT stabilobas sakvlev garemoSi, SemdgomSi fagebis samkurnalo-profilaqtikuri mizniT da ekologiuri kvlevebisTvis, sxvadasxva wyalsatevebSi trasirebis eqsperimentebis Casatareblad. baqteriofagebis stabiloba aseve Seiswavleba Senaxvisa da transportirebis pirobebSi maTi aqtivobis gaTvaliswinebis mizniT.

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miRebulma Sedegebma gamoavlina V. cholerae O1-is mimarT specifikuri baqteriofagebis maRali stabiloba sxvadasxva areSi, 1-dan 6 Tvemde Catarebul kvlevaSi. umniSvnelo cvlilebebs qonda adgili sacdeli areebis umravlesobis SemTxvevaSi. miRebuli Sedegebidan dadginda, rom V. cholerae O1-is mimarT specifikur baqteriofagebs aqvT unari, metad, SeinarCunon mdgradoba V. cholerae ara-O1-is mimarT specifikur baqteriofagebTan SedarebiT. V. cholerae-s mimarT specifikuri baqteriofagebis mdgradoba iseTi organuli gamxsnelis mimarT, rogoricaa qloroformi, SesaZloa miuTiTebdes virionebSi lipiduri komponentis arsebobaze. Termoinaqtivaciis Seswavlam gamoavlina gamokvleuli baqteriofagebis erTmaneTisgan gansxvavebuli TermomgrZnobeloba, rac miuTiTebs maTi cilovani struqturebis gansxvavebaze. V. cholerae O1-is mimarT specifikuri fagebis TermomgrZnobeloba gansxvavdeboda, rogorc V. cholerae ara-O1-is mimarT specifikuri fagebis, aseve erTmaneTis TermomgrZnobelobisgan. V. cholerae ara-O1-is mimarT specifikuri samive fagi erTmaneTisgan gansxvavebuli aRmoCnda am parametris mxriv, maTi cilovani struqtura sxva fagebis cilovan struqturaze metad Termostabiluri aRmoCnda. 550C-ze 10 wT-is eqspoziciis pirobebSi Catarebuli cdebis Sedegad V. cholerae-s mimarT specifikuri 9 fagidan 8 fagis (fagi -Vch105-is garda) inaqtivirebuli faguri nawilakebis ricxvi Seadgenda 70%-s. 600C-dan 700C-mde Catarebulma cdebma aCvena, rom xuTi fagi srulad inaqtivirda 30 wT-ze nakleb droSi. V. mimicus-is mimarT specifikuri fagebi V. cholerae-s mimarT specifikuri fagebisgan gansxvavebiT metad Termostabilurebi aRmoCndnen, inarCunebdnen ra sicocxlisunarianobas 700C temperaturis pirobebSic. wyalbad-ionTa koncentraciis zemoqmedebis Seswavlam gamoavlina, rom baqteriofagebis umravlesoba sakmaod stabiluria pH-is Sualedur (pH-4-9) diapazonSi. Cvens mier gamokvleuli fagebisTvis kritikuli aRmoCnda pH-is eqstremalurad dabali (pH-1) maCvenebeli, romlis drosac gadarCenili faguri nawilakebis ricxvi mxolod 1%-s utoldeboda. pH-4-9-s Soris intervalSi SeiniSneboda titris mcire dacema. unda aRiniSnos, rom V. mimicus-is mimarT specifikuri fagebi V. cholerae ara O1-is mimarT specifikuri fagebis msgavsad inarCunebdnen srul aqtivobas pH-4-9 maCvenebelze. mxolod ori V. cholerae ara-O1-is mimarT specifikuri fagi (Vch105 da Vch10CH) inarCunebda aqtivobas pH 12-ze. V. mimicus-is iseve, rogorc V. cholerae O1-is mimarT specifikuri fagebi naklebad rezistentuli aRmoCndnen maRali tutianobis pirobebSi. kvlevma aCvena, rom V. cholerae O1-is mimarT specifikuri fagebi iCendnen nakleb rezizstentobas pH-is sxvadasxva koncentraciis mimarT V. cholerae ara-O1-is da V. mimicus-is mimarT specifikur fagebTan SedarebiT. pH-is eqstremalurad maRali an dabali mniSvnelobisas gadarCenili faguri nawilakebi SesaZloa mutantur formebs warmoadgenen. sakvebi aris amgvari pH-is maCveneblebi iwvevs virionis degradacias da infeqciurobis dakargvas. bunebriv pirobebSi fagebis populaciis SenarCunebisaTvis didi mniSvneloba aqvs baqteriofagebis ultraiisferi sxivebisadmi gamZleobas. UV gamosxivebas damazianebeli efeqti aqvs fagebze, amcirebs ra maT litiur aqtivobas. cdebma

99

gamoavlina, rom fagebis litiuri aqtivoba mcirdeboda UV gamosxivebis zemoqmedebis drois gazrdasTan erTad. V. cholerae ara-O1-is mimarT specifikuri fagebi nakleb medegobas iCendnen UV gamosxivebis mimarT, V. cholerae O1-is mimarT specifikur fagebTan SedarebiT. V. mimicus-is mimarT specifikuri fagebi V. cholerae ara-O1-is mimarT specifikuri fagebis msgavsad naklebad rezistentuli aRmoCndnen UV gamosxivebis damazianebeli efeqtis mimarT. baqteriofagebis urTierTqmedebis Seswavlam adsorbciis kofaqtorebTan, magaliTad, 2%-iani natriumis citratTan, gamoavlina, rom natriumis citratis zemoqmedeba maRali iyo mxolod erTeuli fagebis SemTxvevaSi (VchBS3,Vch20K-4805,Vmim8.07K daV.mim29.08K). umniSvnelo xarisxis inaqtivacia arapirdapirad miuTiTebs imze, rom baqteriofagebi ar arian damokidebulni orvalentian ionebze (Ca++), rogorc adsorbciis kofaqtorebze. kvlevam gamovalina, V. cholerae ara-O1-is mimarT specifikuri fagebis medegoba osmosuri Sokis zemoqmedebis mimarT rac miuTiTebs, imaze, rom maTi cilovani garsi advilad atarebs wylis molekulebs da ionebs. V. cholerae O1-is iseve, rogorc V. mimicus-is mimarT specifikuri (Vmim8.07K, V.mim29.08K) baqteriofagebi naklebad mdgradi aRmoCnda osmosuri Sokis mimarT.A osmosuri Sokisadmi mgrZnobeloba SesaZloa damokidebuli iyos fagebis

cilovani kafsidis simkvriveze, ანუ kafsomerebis SefuTvis kompaqturobaze. andersonis mier, T-fagebi swored am niSnis mixedviT iqna dajgufebuli mgrZnobiare (T-wyvili fagebi) da rezistentul (T3,T5,T7) jgufebad. aRsaniSnavia, rom Cvenma kvlevam gamoavlina podovirides ojaxis warmomadgeneli fagebis naklebi rezistentoba osmosuri Sokis mimarT, mioviride da sifovirides ojaxis warmomadgenel fagebTan SedarebiT. fagebis genetikuri mravalferovneba Seswavlili iyo maTi restriqciuli profilis saSualebiT. genetikuri maxasiaTeblis mixedviT fagebis dajgufebisTvis agebulma filogenezurma xem V. cholerae ara-O1-is mimarT specifikuri fagebi, rogorc erTmaniTisgan, aseve V. cholerae O1-is mimarT specifikuri fagebisgan gansxvavebul klasterebSi moaqcia. ramodenime monaTesave klasterSi gadanawilda V. cholerae O1-is mimarT specifikuri fagebi. V. mimicus-is mimarT specifikuri fagebi gansxvavebuli aRmoCnda V. cholerae-s mimarT specifikuri fagebisgan restriqciuli profilis mixedviT. miRebuli Sedegebi msgavsi iyo lizisuri speqtris da serologiis mixedviT Catarebuli eqsperimentebiT miRebul SedegebTan. K klimatis temperaturuli maCveneblis momatebasTan dakavSirebiT SesaZloa gafarTovdes qoleras afeTqebebis geografiuli areali, im regionebis CaTvliT, romlebic ar warmoadgenen endemur garemos qolerasTvis.Bbaqteriofagebi baqteriebis arsebobis efeqtur indikatorebad gvevlinebian. warmoadgenen potenciur masalas paTogenuri vibrionebis deteqciisa da biokontrolisaTvis. SesaZlebelia maTi gamoyeneba rogorc profilaqtikuri da samkurnalo, aseve msgavsi serotipis baqteriuli izolatebis diferencirebis mizniT.

100

V daskvnebi

1. V. cholerae–s da V. mimicus–is mimarT specifikuri baqteriofagebi farTod

aris gavrcelebuli rogorc saqarTvelos Sida, mtknar da mciremarilian

wyalsatevebSi, aseve Savi zRvis sanapiro zolSi.

2. baqteriofagebis gavrceleba da sezonuroba ZiriTadad korelirebs

patroni baqteriebis sivrcul-droiT ganawilebasTan.

3. V. cholerae–s da V. mimicus–is fagebis wylian garemoSi gavrcelebisaTvis

mniSvnelovan garemo faqtors warmoadgens temperatura, xolo

marilianobis cvlileba arsebul gavlenas ar axdens.

4. gamovlinda specifikuri baqteriofagebis gavlena V. cholerae-s toqsigenuri

Stamebis mier qoleris toqsinis produqciaze, rac gamoixata toqsinis

raodenobrivi maCveneblis SemcirebiT.

5. SeirCa V. cholerae–s da V. mimicus–is mimarT specifikuri, maRali Terapiuli

potencialis mqone baqteriofagebi maTi biologiuri Tvisebebis

safuZvelze.

6. V. cholerae–s da V. mimicus–is fagebis SerCeuli nakrebebi SeiZleba maRali

efeqturobiT iqnas gamoyenebuli V. cholerae–s epidemiuri da araepidemiuri

Stamebis diferencirebisaTvis.

101

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Ppublikaciebi

1. Elbakidze T., Kokashvili T., Janelidze N., Porchkhidze K., Koberidze T., Tediashvili M. „Biological characterization of V.cholerae -specific bacteriophages isolated from water sources in Georgia“. Journal of Georgian Med News. 2015 Mar; (240):65-72

2. Kokashvili T, Elbakidze T, Jaiani E, Janelidze N, Kamkamidze G, Whitehouse C, Huq A, Tediashvili M. “Comparative phenotypic characterization of Vibrio cholerae isolates collected from aquatic environments of Georgia” Journal of Georgian Med News. 2013 Nov; (224):55-62.

3. E.Jaiani, T.Kokashvili, N.Mitaishvili, T.Elbakidze, N.Janelidze, N. Lashkhi, R.Kalandadze, E.Mikashavidze, G.Natroshvili, C.A.Whitehouse, A.Huq, M.Tediashvili. “Microbial water quality of recreational lakes near Tbilisi, Georgia “Journal of Water and Health. 2013 Jun. Vol 11 No2, pp 333-345.

4. Mitaishvili N., Tskhvediani A., Elbakidze T., Kokashvili T., Natroshvili G., Kajaia G., Tediashvili M. 2011. “Biological series B” Proceedings of the Georgian Academy of Sciences. “The abundance and diversity of Vibrio species not pathogenic to humans in Georgian aquatic environment”. Tbilisi. Georgia. v.9 No.1-4.pp43-49.

5. T.Elbakidze, N.Janelidze, E.Jaiani, T.kokashvili, N.Mitaishvili, T. Koberidze, K. Porchkhidze, M.Tediashvili. 2010. I international conference-“The main ecological problems of modernity and Caucasus”. Chemical journal. “Occurrence of epidemic strains of Vibrio cholerae in recreational and drinking water reservoirs in Georgia”. Tbilisi, Georgia. v.10, pp.120-122, N4.

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saerTaSoriso samecniero konferenciebze warmodgenili Tezisebi :

1. Tinatin Elbakidze, Tamar kokashvili, Nino Janelidze, Marina Tediashvili, Anwar Huq. July 14-18, 2014. Swiss Federal Institute of Technology (ETH). Zurich, Switzerland. Embo Conference on Viruses of Microbs III: Structure and Function-from Molecules to Communities, Abstract book, Poster N209. “Specific phages with potential for subtyping and biocontrol of V. cholerae”

2. Tinatin Elbakidze, Ekaterine Jaiani, Tamar Kokashvili, Tamar Koberidze,Ketevan Porchkhidze,Marina Tediashvili. Abstract of VII International Scientific Conference for Graduate and PhD Students YOUTH AND THE PROGRESS OF BIOLOGY, Abstract book, p.252. April 5 – 8, 2011. Ivan Franko National University of Lviv. Faculty of Biology.”Isolation and characterization of environmental Vibrio cholerae corresponding bacteriophages by use of immunologycal method”. Lviv, Ukraine

3. Kokashvili T., Jainai E., Janelidze N., ElbakidzeT., Tediashvili M. Abstract of VII International Scientific Conference for Graduate and PhD StudentsYOUTH AND THE PROGRESS OF BIOLOGY , Abstract book, p.256. April 5 – 8, 2011. Ivan Franko National University of Lviv.Faculty of Biology. “Ecology and diversity of V. cholerae in Georgia water environment”. Lviv, Ukraine.

4. Tinatin Elbakidze, Ekaterina Jaiani, Tamar Kokashvili, Marina Tediashvili .Georgian Congress of Allergology and Immunology, IV International Congress “Health and Drug”. Europian Board for Accreditation in Pneumology (EBAP), Oct 1-3, 2010. Abstarct book, p203. “Application of immunological methods for detection, identification and characterization of environmental Vibrio cholerae “.

5. T.Elbakidze, T.kokashvili, E.Jaiani, N.Janelidze, T.Koberidze, G.Tsertsvadze, C.A. Whitehouse, A.Huq, M.Tediashvili. Jun21-25, 2010. Abstract book, p.172.Conference:“Virusis of microbes”,“ Bacteriophages specific to environmental isolates of V. cholerae-ecology and diversity.” Pasteur Institute, Paris, France.

6. M.Tediashvili, E. Jaiani, N. Janelidze, T. Kokashvili, T.Elbakidze, C. A. Whitehouse, A. Huq, R.Colwell. ”8th ASM Biodefense and Emerging Diseases Research Meeting”. Feb 21-24, 2010. Abstract book, p.83.”Epidemic Strains of Toxigenic Vibrio cholerae in Recreational and Drinking Water Reservoire in Georgia”. Baltimore, MD. USA.

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