saqarTvelos wylian garemoSi gavrcelebuli Vibrio ... thesis... · saqarTvelos wylian garemoSi gavrcelebuli Vibrio parahaemolyticus-is da mis mimarT specifikuri baqteriofagebis gamoyofa,

1

……………………………………………………………………………………………………………………………………………………………………………………………………………………………

………………………………………………………………………………………………………………………………………………………………………………

saqarTvelos wylian garemoSi gavrcelebuli Vibrio parahaemolyticus -

is da mis mimarT specifikuri baqteriofagebis gamoyofa,

daxasiaTeba da biologiuri Tvisebebis Seswavla

ana cxvediani

ivane javaxiSvilis saxelobis Tbilisis saxelmwifo universiteti

zust da sabunebismetyvelo mecnierebaTa fakulteti

mimarTuleba: imunologia, mikrobiologia

sadisertacio naSromi Sesrulebulia g.eliavas saxelobis baqteriofagiis,

mikrobiologiisa da virusologiis institutSi sabunebismetyvelo

mecnierebaTa doqtoris akademiuri xarisxis mosapoveblad

samecniero xelmZRvaneli: marine TediaSvili, bmd

nino gaCeCilaZe, bmd

Tbilisi 2014

2

anotacia

bolo aTwleulSi aRiniSna halofiluri vibrionebiT gamowveuliinfeqciebis sixSiris zrda. Vibrio parahaemolyticus ganixileba rogorc mZimegastroenteritebis da WrilobiT infeqciebis gamomwvevi yvelazemniSvnelovani saxeoba (araqolerul vibrionebs Soris). Ees baqteria aseveasocirdeba zRvis organizmebis infeqciebTan. saqarTvelos Savi zRvissanapiro zolis klimati da saSualod mariliani wyali halofilurivibrioebisTvis xelsayrel garemos warmoadgens. halofiluri vibrionebisda Sesabamisi baqteriofagebis Seswavla did interess iwvevs maTipaTogenobis meqanizmebis dasadgenad da Stamebis diferenciaciisaTvis. gardaamisa, baqteriofagebi SeiZleba ganvixiloT, rogorc alternatiulisaSualeba, am paTogenebiT gamowveuli daavadebebis profilaqtikisa damkurnalobisTvis.

mocemuli kvlevis ZiriTad mizans warmoadgenda saqarTvelo wyliangaremoSi V.parahaemolyticus –is gamovlena, gamoyofa da daxasiaTeba mis miergamowveuli infeqciebis riskis Sefasebisa da biomravalferovnebis dadgenismizniT. agreTve V.parahaemolyticus –is mimarT specifikuri baqteriofagebisgamoyofa da daxasiaTeba gamomwvevis identifikaciisa da subtipirebis, asevesamkurnalo da prevenciuli mizniT maTi Semdgomi gamoyenebisTvis.

Kkvlevis farglebSi saqarTvelos Savi zRvis sanapiro zolis wylebidangamoiyo V.parahaemolyticus-is 200-mde izolati, moxda maTi daxasiaTebaserotipebisa da virulentobis faqtorebis Semcvelobis mixedviT.gamovlinda V.parahaemolyticus-is Savi zRvis izolatebis mniSvnelovanimravalferovneba, maT Soris pandemuri klonebis arseboba.

Ggamoyofili da daxasiaTebuli iyo V.parahaemolyticus-is mimarTspecifikuri 70 –mde baqteriofagi, maTgan 12 baqteriofagi SerCeul iqnaserotipisa da genetikuri struqturis mixedviT axlos mdgomiV.parahaemolyticus-is baqteriuli Stamebis diferencirebisTvis. V.parahaemolyticus–is ramdenime baqteriofagma gamoavlina maRali Terapiuli potenciali.

3

Abstract

The prevalence of infections caused by halophilic Vibrio species appears to be increased in thelast decade. V. parahaemolyticus is the most important non-cholerae Vibrio causing seriousgastroenteritis and wound infections. Bacteria may be also pathogenic for marine organisms. Climateand water of Black See costal zone in Georgia provide favorable conditions for survival andpropagation of halophilic vibrios. Research about halofilic vibrios and corresponding bacteriophages ofV.parahaemolyticusis of special importance for study pathogenicity mechanisms and straindifferenciation. Besides lytic bacteriophages can be discussed as an alternative approach for theprevention and treatment of infections caused by these pathogens.

The aim of the study was to detect, isolate and characterize V.parahaemolyticus and itsbacteriophages in order to asess infection risk in this region and to study the strain biodiversity, also toselect specific bacteriophages for diagnostic, subtyping and therapeutic purpose.

Up to 200 bacterial strains were collected from Black Sea coastal waters in Georgia.Characterization by serological properties and virulence factors was performed. High diversity ofBlack Sea isolates, also presence of pandemic strains was revealed.

Up to 70 V.parahaemolyticus-specific bacteriophages were isolated and characterized. Twelveout of them were selected for subtyping of strains of the same serotype and genetic structure. Numbersof bacteriopages with high therapeutic potential have been selected.

4

Sinaarsi

Tavfurceli------------------------------------------------------------------------------------------------------------ 1

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

Sinaarsi------------------------------------------------------------------------------------------------------------------- 3

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

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

1. Vibrio parahaemoliticus–is zogadi daxasiaTeba----------------------------------------------------- 9

1.1. istoriuli cnobebi-------------------------------------------------------------------------------------------- 9

1.2. Vibrio parahaemolyticus-is klasifikacia da taqsonomia-------------------------------------- 10

1.3. V. parahaemolyticus –is ujredisa da koloniis morfologia-------------------------- 11

1.4. V.parahaemolyticus-is kultivireba da bioqimiuri identifikacia------------------- 13

1.5. Vibrio parahaemolyticus-is genomi-------------------------------------------------------------------------- 13

1.6. V. parahaemolyticus-is virulentobis faqtorebi----------------------------------------------- 14

1.7. V. parahaemolyticus-is izolatebis serologiuri Tvisebebis Seswavla---------- 16

1.8. Vibrio parahaemolyticus-iT gamowveuli infeqciebi da maTi epidemiologia--- 17

1.9. Vibrio parahaemolyticus -is ekologia------------------------------------------------------------------- 17

2. baqteriofagebi----------------------------------------------------------------------------------------------- 20

2.1. klasifikacia da taqsonomia------------------------------------------------------------------------- 20

2.2. baqteriofagebis biologia--------------------------------------------------------------------------- 22

2.2.1. virulenturi baqteriofagebis sasicocxlo cikli------------------------------------ 22

2.2.2. zomieri baqteriofagebis sacicoxlo cikli-------------------------------------------- 24

2.3. baqteriofagebis gamoyeneba Terapiuli mizniT (fagoTerapia)----------------- 25

2.4. baqteriofagebis daxasiaTebisa da identifikaciis meTodebi------------------ 26

2.5. Vibrio parahaemolyticus-is mimarT specifikuri baqteriofagebi---------------------- 27

II.Mmasalebi da meTodebi--------------------------------------------------------------------------------------- 31

1. sakvlevi masala--------------------------------------------------------------------------------------------------- 31

2. kvlevaSi gamoyenebuli meTodebi----------------------------------------------------------------------- 35

2.1. Ria wyalsatevebSi wylis sinjebis aReba da damuSaveba--------------------------- 35

2.2. VIbrio-s gvaris baqteriebis gamoyofa--------------------------------------------------------------- 35

5

2.3 baqteriuli Stamebis biologiuri Tvisebebis Seswavla------------------------------ 37

2.4. mikrokosmebis eqsperimenti VBNC-ujredebze------------------------------------------------ 39

2.5. baqteriofagebis gamoyofa------------------------------------------------------------------------------- 39

2.6. baqteriofagebis biologiuri Tvisebebis Seswavla-------------------------------------- 39

2.7. fagebis mgrZnobeloba fizikuri da qimiuri faqtorebis mimarT---------------- 41

2.8. fagis gamravleba da koncentrireba--------------------------------------------------------------- 42

2.9. baqteriofagebis genetikuri Tvisebebis Seswavla--------------------------------------- 43

III. miRebuli Sedegebi--------------------------------------------------------------------------------------------- 44

1. Vibrio parahaemolyticus-is gamoyofa da ekologia------------------------------------------------ 44

2. Vibrio parahaemolyticus-is baqteriebis gamovlena pirdapiri deteqciis meTodiT--

---------------------------------------------------------------------------------------------------------------------------------- 48

3. baqteriuli Stamebis biologiuri Tvisebebis Seswavla--------------------------------- 49

4. arakultivirebadi, magram sicocxlisunariani mdgomareobaSi ( VBNC)-------- 61

5. Vibrio parahaemolyticus-is miarT specifikuri baqteriofagebis gamoyofa da

ekologia------------------------------------------------------------------------------------------------------------------ 63

6. Vibrio parahaemolyticus-is miarT specifikuri baqteriofagebis daxasiaTeba------ 64

7. fagebis mgrZnobeloba fizikuri da qimiuri faqtorebis------------------------------ 71

8. proteolituri fermentebis moqmedebis Seswavla baqteriofagebze-------------- 78

9. baqteriofagebis genetikuri Tvisebebis Seswavla---------------------------------------- 78

IV. Sedegebis ganxilva------------------------------------------------------------------------------------------ 81

V. daskvnebi------------------------------------------------------------------------------------------------------------ 89

VI. gamoyenebuli literatura------------------------------------------------------------------------------- 90

6

S e s a v a l i

saqarTveloSi, iseve rogorc msoflioSi, infeqciuri daavadebebi, maTSoris wylismieri infeqciebi, erT-erTi aqtualuri problemaa. Ees daavadebebidabinZurebul wyalTan sxvadaxva saxis kontaqtiTaa gamowveuli da maTiriski dRiTi-dRe matulobs. bolo periodSi Catarebuli gamokvlevebismixedviT gamovlinda saqarTveloSi rekreaciul wyalsatevebSi, kerZod, SavzRvasa da Sida tbebSi, wylis xarisxis TandaTanobiTi gauareseba [90]. mzardanTropogenur dabinZurebasTan erTad mniSvnelovania globaluri daTbobisprocesTan dakavSirebuli klimatis cvlileba. Kklimaturi pirobebigansazRvravs wyalSi avtoqtonuri organizmebis arsebobas, romlebicinfeqciuri agentebis gadatanas uzrunvelyofen. amdenad, sasmeli wylis darekreaciuli wyalsatevebis dabinZureba da araadekvaturi dacva SesaZloainfeqciuri afeTqebebis mizezi gaxdes. dabinZurebul wylebSi SesaZloagavrceldes wylismieri infeqciebis iseTi gamomwvevebi, rogoricaa: Salmonellaspp., Shigella spp., Vibrio spp., enteropaTogenuri E. coli da sxva baqteriebi, gardaamisa 100-ze meti tipis paTogenuri virusi, umartivesebi da sxva [47, 118].

sayovelTaod miRebulia, rom wylismieri infeqciuri daavadebebisgavrcelebis Tavidan acilebisTvis metad mniSvnelovania rekreaciulwyalsatevebSi da zRvis sanapiro zolSi regularuli monitoringisCatareba. Des RonisZiebebi saSualebas gvaZlevs gamovlindes wylismieripaTogenebi da dadgindes infeqciis wyaro da misi gavrcelebis gzebi.

bolo periodSi gansakuTrebiT imata halofiluri vibrionebiTgamowveuli infeqciebis sixSirem. es daavadebebi ZiriTadad asocirebuliaumi da cudad damuSavebuli zRvis produqtebis moxmarebasTan an warmoadgensdabinZurebul wylebTan kontaqtiT gamowveul WrilobiT infeqciebs.halofiluri vibrionebi aseve paTogenuria zRvis organizmebisTvis damniSvnelovan zarals ayenebs aqvakulturis meurneobebs. araqolerulvibrionebs Soris V.parahaemolyticus yvelaze mniSvnelovani saxeobaa, romelicxSirad iwvevs seriozul gastroenteritebs. Bbaqteriis gamoyofa gamoyofaCveulebriv xdeba zRvisa da estuariebis wylebidan, mtknari wylebisekosistemebSi misi aRmoCena ki droebiTia da damokidebulia biologiurmaspinZelze. saSualo da maRali marilianoba, agreTve 18°C-ze maRalitemperatura, optimalur pirobebs warmoadgens araqoleruli vibrionebiszrdisa da gamravlebisTvis. am TvalsazrisiT, saqarTveloSi arsebulisubtropikuli klimati da wyliani garemo, gansakuTrebiT Savi zRvissanapiro, halofiluri vibrioebisTvis xelsayrel garemos warmoadgens.garemo-klimaturi pirobebi da maTi sezonuri cvlilebebi mniSvnelovanwiladaisaxeba rogorc paTogenuri vibrioebis, aseve maTi specifiuribaqteriofagebis gavrcelebaze.

baqteriuli virusebi (baqteriofagebi) sicocxlis yvelazegavrcelebuli formebia dedamiwaze. Bbunebriv pirobebSi baqteriofagebimonawileoben baqteriuli populaciis regulaciaSi [173] da populaciebis

7

genetikur mravalferovnebaSi, rac genetikuri gacvlis gziT xorcieldebabaqteriul Stamebs da saxeobebs Soris [173].

baqteriofagebs didi mniSvneloba eniWeba, rogorc anti-infeqciuragentebs medicinasa da veterinariaSi. d’erellis mier fagis aRmoCenidanmaleve moxda misi gamoyeneba dizenteriis samkurnalod [36]. dReisTvisvirulenturi baqteriofagebi ganixileba, rogorc antibiotikebis erT-erTialternatiuli saSualeba sxvadasxva eTiologiis mqone infeqciebisprevenciisaTvis da samkurnalod. fagebis mimarT maRali interesiganpirobebulia, erTis mxriv, antibiotikebis mimarT multirezistentulibaqteriuli Stamebis gavrcelebiT, meores mxriv ki fagebis moqmedebis maRalispecifikurobis, efeqturobisa da uvneblobis faqtoriT [32, 36, 111, 162, 163].

infeqciur paTologiaSi baqteriofagis gamoyenebis rekomendaciebidafuZnebulia im gamocdilebaze, rac dagrovda mravali wlis manZilze.dReisaTvis baqteriofagebi warmatebiT gamoiyeneba Cirqovan-anTebiTiinfeqciebis, nawlavuri infeqciebis, Sard-sasqeso gzebis anTebiTi procesebissamkurnalod.

Bbaqteriofagebi gansxvavdeba lizisuri speqtris mixedviT: specifikuribaqteriofagebi, viwro speqtris baqteriofagebi da moqmedebis farTo speqtrismqone baqteriofagebi. TiToeul maTgans praqtikuli gamoyeneebis Tavisiareali aqvs. mTel rig SromebSi aRwerilia baqteriofagebis gamoyenebaindikatorebad da traserebad bunebrivi Ria wyalsatevebis dabinZurebisxarisxis, wyarosa da gavrcelebis gzebis dadgenis mizniT. xangrZlivipraqtikuli gamocdilebiT naCvenebia, rom faguri preparatebi xasiaTdebianuvneblobiT da gamomwvevisadmi maRali specifikurobiT. maTi miRebis datiraJirebis SedarebiT dabali ekonomikuri danaxarjebis gaTvaliswinebiTSesaZlebelia maRaltitriani, koncentrirebuli preparatebis martivi miReba[93].

halofiluri vibrionebis baqteriofagebi did interess iwvevs, rogorcpaTogenobis meqanizmebis Sesaswavlad da Stamebis diferenciaciisaTvis, aseverogorc alternatiuli saSualeba, am paTogenebiT gamowveuli daavadebebisprofilaqtikisa da mkurnalobisTvis.

mocemuli kvlevis ZiriTad mizans warmoadgenda saqarTvelo wyliangaremoSi V.parahaemolyticus –isa gamoyofa da daxasiaTeba mis mier gamowveuliinfeqciebis riskis Sesfasebisa da biomravalferovnebis Seswavlis mizniT.agreTve V.parahaemolyticus –is mimarT specifikuri baqteriofagebis daxasiaTebamaTi Semdgomi gamoyenebisTvis samkurnalo da prevenciuli mizniT.

aqedan gamomdinare warmodgenili kvlevis ZiriTadi amocanebi iyo:1. saqarTvelos wylian garemoSi ( Savi zRva, lisis tba, kumisis tba,

Tbilisis zRva) V.parahaemolyticus –is gamovlena da Sesabamisi baqteriuliStamebis Segroveba;

2. V.parahaemolyticus –is Stamebis identifikacia, daxasiaTeba dabiomravalferovnebis Seswavla;

3. V.parahaemolyticus –is mimarT specifikuri baqteriofagebis gamoyofada seleqcia;

8

4. fagebis morfologiisa da biologiuri Tvisebebis Seswavla(moqmedebis diapazoni, gamravlebis erTjeradi ciklis parametrebi,serologiuri maxasiaTeblebi);

5. fagebis stabilurobis Seswavla garemos sxvadasxva pirobebSi(temperatura, pH, qloroformi, proteolituri fermentebi);

6. V. parahaemolyticus –is mimarT specifikuri baqteriofagebis genetikuridaxasiaTeba da mravalferovnebis Seswavla.

7. V.parahaemolyticus –is mimarT specifikuri baqteriofagebis SeswavlamaT mier gamowveuli infeqciebis biokontrolis mizniT.

9

I. literaturuli mimoxilva

1. Vibrio parahaemoliticus–is zogadi daxasiaTeba

1.1. istoriuli cnobebi

1950 wels fujinom da misma TanamSromlebma iaponiaSi, osakaSi,arasaTanadod damuSavebeli zRvis produqtisgan pirvelebma gamoyves Vibrioparahaemolyticus, rogorc kvebiTi warmoSobis gastroenteritebis gamomwvevi. am

incidentis Sedegad dazaralda 272 adamiani, 20- daiRupa. baqteria gamoyves

agreTve 1953 wels Proteus morganii-Tan Sereuli infeqciis dros pacientebisfekaliebidan da nawlavuri masidan. am droidan baqterias ewoda Pasteurellaparahaemolyticus. 1958 wels V. parahaemolyticus gamoyves iokohamas hospitalSikvebiTi infeqciis mqone pacientebidan [165]. 1960 wels halofiluri, glukozisfermentatori baqteria - Oceanomonas parahaemolyticus, gamoyves pacientebidan daagreTve zRvis wylis nimuSidan. iaponiis jandacvis saministros kvlevebismixedviT, morfologiuri, kulturaluri da qimiuri maxasiaTeblebiT P.parahaemolyticus da O. parahaemolyticus identificirda, rogorc erTi da igiveorganizmi. 1974 wels iaponiaSi, tokios saerTaSoriso simpoziumze baqteriamiakuTvnes Vibrio-s gvars rogorc saxeoba Vibrio parahaemoliticus [65].

mas Semdeg Vibrio parahaemolyticus gamoyofa araerTxel daukavSirdamsoflios mraval qveyanaSi gastroenteritebis afeTqebebsa da calkeulsporadul SemTxvevebs [81, 100]. amJamad Vibrio parahaemolyticus miiCnevamsoflioSi gastroenteritebis erT- erT mTavar gamomwvevad [81, 94, 100].

gansxvavebiT Vibrio cholerae-gan, romlis mxolod ori serotipia dakavSirebuli

epidemiebsa da pandemiebTan, Vibrio parahaemolyticus-is mier gamowveuli

gastroenteritebi asocirdeba O და K-serotipebis 76 sxvadasxva kombinaciasTan.

ukanasknel aTwleulSi Vibrio parahaemolyticus-is ramdenime specifikuriserotipis Stamma, romlebic savaraudod warmoiSva saerTo winapariklonisgan, gamoiwvia gastroenteritebis pandemia [10, 15, 21, 40, 42, 43, 44, 45,52, 54, 68, 73, 88, 89, 104, 137, 138, 143, 144, 171, 174].

10

1.2. Vibrio parahaemolyticus-is klasifikacia da taqsonomia

Vibrio parahaemolyticus miekuTvneba Proteobacteria-s tips, Gamma Proteobacteria-sklass, Vibrionales rigs, Vibrionaceae-s ojaxs da Vibrio-s gvars [62] (suraTi 1).

ojaxi Vibrionaceae pirvelad aRweril iqna 1965 wels [94]. mocemul ojaxiaerTianebs adamianisa da cxovelebis oportunistul paTogenebs [52, 166] daaseve Tavisuflad mcxovreb qemoheterotrofebs da zRvis faunis komensalebs[69, 70]. es aris gram uaryofiTi halofiluri baqteriebi, Cveulebriv moZraviCxirebi, mezofilurebi, qemoorganotrofebi da axasiaTebT fakultaturifermentuli metabolizmi.

gvari Vibrio 65 saxeobas aerTianebs [19]. maTgan 12 saxeoba, Cveulebriv,gamoiyofa klinikuri nimuSebidan da maT mier gamowveuli daavadebebi moicavsdiareebs, septicemiebs da WrilobiT infeqciebs [80, 94]. 12 klinikurisaxeobidan 11 adamianis diareul da sxva nawlavur infeqciebTan arisasocirebuli. adamianis vibriozebis umetesoba dakavSirebulia gvaris sammniSvnelovan warmomadgenelTan: V.cholerae, V.parahaemolyticus da V.vulnificus,romelTaganc ori saxeoba (V.parahaemolyticus da V.vulnificus ) halofilebsmiekuTvneba.

sur.1. V. parahaemolyticus-is taqsonomia

samefo- Bacteria

tipi- Proteobacteria

klasi- Gamma Proteobacteria

rigi- Vibrionales

ojaxi- Vibrionaceae

gvari- Vibrio

saxeoba- Vibrio parahaemolyticus

11

1.3. V. parahaemolyticus –is ujredisa da koloniis morfologia

V. parahaemolyticus gram (-), Cxiris formis zoma 0.5-0.8/1.4-2.6 mkm-ia. zrdisoptimaluri pirobebia 35-370C, pH 7.5-8.0 da marilis Semcveloba daaxloebiT 0.5M NaCl [100]. arsebobs V.parahaemolyticus –is sxvadasxva fenotipuri variaciebi,kerZod, koloniebis morfologiuri tipebi. erTi koloniisgan miRebulujredul kulturaSi SesaZloa iyos gamWvirvalobis mixedviT gansxvavebuliori tipis-gaumWvirvali da gamWvirvale koloniebi (OP da TR). opaR-geniseqspresia korelaciaSia gaumWvirvale koloniebis warmoqmnasTan. Ees genebitranskribirdeba gaumWvirvale koloniebSi (OP), magram ar gvxvdeba TR-gamWvirvale ujredebSi [124].

V.parahaemolyticus Seguebulia sicocxlis sxvadasxvagvar stilTan,rogoricaa: planqtonTan asocirebuli, Tavisufal mdgomareobaSi, sxvadasxvamikrobul TanasazogadoebebTan asocirebuli (biofilmebi),mikroorganizmebTan komensalur urTierTobaSi an patron organizmTanpaTogenur damokidebulebaSi. V. parahaemolyticus-is ujredebi xasiaTdebabiofilmebis warmoqmnis maRali unariT. biofilmebis warmoqmnis unari qitin-damokidebuli polusiTa (ChiRP) da manozis mimarT mgrZnobiare hemaglutininiT(MSHA) regulirdeba [159]. V. parahaemolyticus-is gamWvirvale da gaumWvirvalikoloniebi sxvadasxvagvar biofilmebs warmoqmnian [59]. gamWvirvale koloniebismier warmoqmnili biofilmebi metad erTgvarovania da ujredebi mWidrobaaganlagebuli, gansxvavebiT gaumWvirvali koloniebisgan warmoqmnilibiofilmisa, sadac ujredebs Soris ufro didi napralebia da, Sesabamisad,biofilmi ufro gamWvirvalea.

imisaTvis, rom gadarCes cvalebad pirobebSi, V.parahaemolyticus–s gaaCniasxvadasxva adaptoruli meqanizmebi-Secvalos qceviTi da genetikuri pasuxebigaremos da intraujreduli faqtorebis sapasuxod.

V.parahaemolyticus –is Tavisuflad mcxovrebi forma kargadaa SeguebuliTxier garemoSi moZraobasTan. Cxiris formis baqteria SeiaraRebulia erTi

polaruli SoltiT. Eenergiis wyari Soltis samoZraod Na- damokidebulimeqanizmia [14]. Aam tipis energiis wyaros upiratesoba aqvs zRvis wyalSiarsebobisTvis, radgan wylis pH daaxloebiT 8.0-is tolia [108]. am tipis

moZraoba Zalze swrafia. 300 milimoli NaCl –is Semcvel areSi baqteriamoZraobs 60 mkm/wm siCqariT.

Ppolaruli Solti rTuli agebulebisaa da or lokusSi organizebuliflagelinis eqvsi geniT kodirdeba [124]. igi dafarulia SaliTiT, romelicujredis gareTa membranis gagrZelebas warmoadgens [7]. SaliTiani flagelinismoZraobis zusti meqanizmi ar aris cnobili. savaraudod Soltis filamentebimoZraoben SaliTis SigniT an SaliTa da filamentebi moZraoben, rogorcerTi mTliani warmonaqmni [64]. Solti gadamwyvet rols TamaSobs baqteriiszedapirze mimagrebaSi. mTelma rigma kvlevebma sxvadasxva baqteriebze aCvena,

12

rom moZraobis unari mniSvnelovania ara mxolod adsorbciis, aramedpaTogenezis TvalsazrisiTac [148].

Mmyar zedapirze an blant garemoSi zrda iwvevs V.parahaemolyticus –isujredebis diferencirebas koloniebis saxiT. ujredis gayofa wydeba daizrdeba sigrZeSi daaxloebiT 30 nm-mde. iwyeba meoradi samoZrao aparatis -lateraluri sistemis formireba, romelic peritriqulad ganlagebulimravali Soltisgan Sedgeba. Aam tipis ujredebi adaptirebulebi arian myarzedapirebze da blant areSi moZraobasTan. Ppolaruli Soltebi myar arezesustad viTardeba, maSin rodesac, lateraluri Soltebi efeqturadmoqmedeben am tipis garemoSi. Llateraluri aparatis mqone ujredebismoZraobis siCqare mudmivia da 25 mkm/wm-s Seadgens polivinilpirolidonis(10% PVP-360) damatebisas. Ees aris grZeli, datotvili polimeri, romeliczedapiris simyares zrdis. Aamgvar pirobebSi polaruli Soltis mqoneujredebis moZraobis sixSire klebulobs [14].

Llateraluri Soltebi araa dafaruli SaliTiT da filamentflagelinis erTi suberTeulisgan Sedgeba [125]. isini absoluturadgansxvavdebian polaruli Soltebisgan. Ggenetikurma kvlevebma aCvena, rom maTsamoZrao aparatebSi ar gaaCniaT saerTo struqturuli komponentebi. SoltiTmoZraobis sistemas, Cveulebriv, akontrolebs 40 geni mainc. amasTan, es genebior sakmaod dacilebul genebis nakrebs ekuTvnis.

polaruli da lateraluri Soltebi ara marto struqturulad,energiis wyaros mixedviTac gansxvavdebian erTmaneTisgan. LpolaruliSoltebisgan gansxvavebiT lateraluri Soltebi samoZraod iyenebenprotonebis wyaros [14]. Ggarda imisa, rom zedapirze mimagrebaSi monawileobs,polaruli Soltebi moqmedebs, rogorc sensorebi. igi warmoiqmneba yvelaSemTxvevaSi-rogorc Txier areSi, aseve zedapirTan asocirebuli zrdispirobebSi.

garemoSi raime saxis stres-faqtoris arsebobis sapasuxod (mag.araxelsayreli temperatura, Jangbadis nakleboba, osmosuri stresi) baqteriis

koloniis warmomqmneli erTeulebis raodenoba klebulobs- V.parahaemolyticus –is ujredebi gadadis sicocxlisunarian, magram arakultivirebad- VBNC-mdgomareobaSi. VBNC-mdgomareobaSi gadasvlisas adgili aqvs mTel rigmetabolur cvlilebebs-mcirdeba sakvebi nivTierebebze moTxovnileba,sunTqvis intensivoba, makromolekulebis sinTezi. VBNC-mdgomareobaSi myofibaqteriebi ar izrdebian mikrobul sakveb areze, sadac Cveulebriv warmoqmniankoloniebs, magram ujredebi cocxalia da aqvT metaboluri aqtivobisganaxlebis unari [145].

13

1.4. V.parahaemolyticus-is kultivireba da bioqimiuri identifikacia

Vibrio-ebis gamoyofisa da kultivirebisTvis gamoiyeneba specifikuriTCBS-agari (Thiosulfate-Citrate-Bile-Sucrose). sxva niadagebs Soris is maRaliSerCeviTobiT xasiaTdeba. safuaris eqstraqti da peptoni warmoadgens azotisda vitaminebis wyaros; natriumis citrati, natriumis Tiosulfati da naRvlismarilebi ganapirobeben tute pH –s gram-dadebiTi organizmebis dasaTrgunadda koliformebis ricxvis Sesamcireblad. saqaroza ganicdis fermentacias,xolo natriumis qloridi astimulirebs mikrobTa zrdas. saqaroza -dadebiTiStamebi, Cveulebriv iZleva yviTeli feris, xolo saqaroza-uaryofiTebi-mwvaneferis koloniebs.

Bsaxeobis doneze Vibrio-ebis bioqimiuri identifikaciis yvelazegavrcelebuli sqema [8] gankuTvnilia gram-uaryofiTi, fakultaturi anaerobibaqteriebisTvis, romlebic TCBS-agarze izrdebian. igi moicavs 28 tests,romelTagan maqsimum 10 sakmarisia yvelaze rTuli identifikaciisTvisac ki.maT Soris aris lizin- da ornitin- dekarboqsilazebis da arginindehidrolazas, sxvadasxva naxSirwylebis fermentaciis da marilis sxvadasxvakoncentraciaze zrdis unaris testebi.

V.parahaemolyticus-is bioqimiuri identifikacia SedarebiT rTulia wylidanda zRvis produqtebidan gamoyofili Stamebis SemTxvevaSi. aseT SemTxvevebSi

nimuSebi Seicavs Vibrio-s monaTesave saxeobebs da sxva analogiuri bioqimiuriTvisebebis mqone baqteriebs [98].

1.5. Vibrio parahaemolyticus-is genomi

erT-erTi klinikuri Stamis sekvenirebis magaliTze Catarebul iqna

Vibrio parahaemolyticus-is genomis analizi [117]. dadginda, rom genomiwarmodgenilia ori wriuli qromosomiT [176]. sicocxlisa da zrdisTvisaucilebeli genebis umetesoba, aseve ribosomuli cilebis makodirebeligenebi da t-rnm-is genis minimum 1 asli moTavsebulia pirvel qromosomaSi,romelic zomiT aRemateba meore qromosomas. Tumca meore qromosomaSimoTavsebulia metabolizmis procesis makodirebeli genebi da, amdenad, vervityviT, rom is ar aris CarTuli sasicocxlo Tvisebebis da zrdisprocesebis kontrolSi. rogorc aRmoCnda, meore qromosoma pirvelqromosomaze metadaa CarTuli transkrifciul da nivTierebaTa

transportis regulaciis procesebSi. V.cholerae-sa da V.parahaemolyticus-isgenomebis Sedarebam aCvena, rom pirveli qromosomebi TiTqmis ar gansxvavdeba

zomaSi, maSin rodesac, meore qromosoma V.parahaemolyticus-is SemTxvevaSi arisgacilebiT didi. es faqti SesaZloa dakavSirebuli iyos evoluciis manZilze

14

V.parahaemolyticus-is genomSi zogierTi genis ufro met dublikaciasTan an

genebis horizontalur transferTan an, piriqiT, V.cholerae-s SemTxvevaSi

deleciis meti procesiT. V.parahaemolyticus-is meore qromosomaSi gvxvdebaramdenime saxis mobiluri elementi, rogoricaa magaliTad, transpozonebi,baqteriofagebi an g+c Semcveli Tanmimdevrobebi (rac genebis lateralurtransferze migvaniSnebs.). amiT SegviZlia davaskvnaT, rom ucxo dnm-is

horizontaluri gadatana ar aris V.cholerae-sa da V.parahaemolyticus-is genomebiszomaSi gansxvavebis ZiriTadi faqtori [66].

1.6. V. parahaemolyticus-is virulentobis faqtorebi

V. parahaemolyticus-is virulentobis faqtorebi moicavs: hemolizinebs,virulentobis genebs, mesame tipis sekreciul sistemas (TTSSs), kolonizaciisfaqtorebs da membranul proteinebs.

hemolizinebi. 1968 wels vagacumam Seqmna axali sisxliani niadagi,

romliTac gansazRvravdnen Vibrio parahaemolyticus-is hemolizur Tvisebebs daamgvarad, Stamebi daiyo or jgufad: hemolizur da arahemolizur Stamebad

[100, 134]. diareiani pacientebis fekaluri masalidan gamoyofili Stamebi,Cveulebriv, hemolizuria, xolo sakvebidan gamoyofili Stamebi-arahemolizuri. sakazakis mier mravalricxovan izolatebze Catarebulmaeqsperimentebma (1968) aCvena, rom am organizmebis unari, moaxdinon hemolizivagacuma-agarze- rac cnobilia kanagavas fenomenis saxelwodebiT-mWidrodaadakavSirebuli gastrointestinaluri daavadebebis gamowvevasTan [121].

kanagavas fenomenis asaxsnelad Catarebulma mravalricxovanmaeqsperimentebma saboloo jamSi mecnierebi miiyvana pirdapiri Termostabiluri

hemolizinis ( pTh TDH) aRmoCenamde [81]. gvian 1970-ian wlebSi hemolizinigamoiyo, gasufTavda da dadginda, rom igi warmoadgens homodimerulistruqturis cilas. hemolizini warmoadgens enterotoqsins da amJRavnebssxvadasxva biologiur aqtivobas [60, 81, 86, 155] , mag. iwvevs sxvadasxvacxoveluri warmoSobis sisxlis wiTeli ujredebis liziss. is agreTve

citotoqsiuria sxvadasxva kultivirebuli ujredebis mimarT [81].1988 wels hondam da Tanaavtorebma aRmoaCines, rom kanagava-uaryofiTi

klinikuri izolatebi, romlebic gamoyofil iqna maldiveli mogzaurebisgan,Seicavda sxva toqsins da mas uwodes Termostabilur hemolizinTan

monaTesave hemolizini(TRH) [83]. mocemuli toqsinis aminomJavuri Tanmimdevroba67%-iT emTvxeva Termostabilur hemolizinis aminomJavur Tanmimdevrobas da

axasiaTebs msgavsi biologiuri Tvisebebi [81]. zemoTxsenebuli toqsinebismakodirebeli genebis klonebis miRebam SesaZlo gaxada maTi arsebobisSemowmeba klinikur da garemodan gamoyofil izolatebSi [53, 81, 139, 147, 164 ].

dadginda, rom umravles klinikur izolatebs aqvT tdh-geni, Tumca mocemuli

geni gvxvdeba zogierT garemodan gamoyofil izolatSic. trh-dadebiTiStamebis umravlesoba garemodanaa gamoyofili ( zRvis wylidan da zRvis

15

produqtebidan) da umravlesoba paTogenuri Stamebia. damtkicebulia, rom es

ori geni Vibrio parahaemolyticus-is ZiriTadi virulentobis faqtorebia, radganmaT armqone mutantebSi enterotoqsiuri Tvisebebi daqveiTebulia.

mesame tipis sekreciuli sistema. V.parahaemolyticus-is paTogenobasTandakavSirebuli yvelaze mniSvnelovani aRmoCena iyo mis genomSi mesame tipissekreciis sistemasTan dakavSirebuli genTa ori nakrebis aRmoCena [117]. es iyopirveli SemTxveva, rodesac Vibrio-s gvaris warmomadgenelSi aRmoCnda am tipissekreciuli sistema. mesame tipis sekreciuli sistema aris gram uaryofiTibaqteriebisTvis damaxasiaTebeli aparati, romelic monawileobsvirulentobis faqtori cilebis gamoyofasa da translokaciaSi eukariotuliujredis citozolSi [85]. msgavsi tipis sekreciuli sistema damaxasiaTebeliaim baqteriuli paTogenebisTvis, romlebic iwveven daavadebebs eukariotulujredebTan pirdapiri kontaqtiT, aseTebia: Salmonella, Shigella, Yersinia, mcenareTa

paTogenebi.imis gansasazRvrad, aris Tu ara V.parahaemolyticus-is mesame tipis

sekreciuli sistemis genebis ori nakrebi funqcionirebadi, Seiqmna mutanturi

Stamebi, romelTac tdh-is orive geni hqondaT amoWrili genomidan. Cveulebriv,

V.parahaemolyticus- s SeuZlia eukariotuli ujredebis swrafad mokvla(daaxloebiT 5 saaTSi). mocemuli kvleva ki mimarTuli iyo imis dasadgenad,

axdens Tu ara gavlenas TTSS1 da TTSS2 - genebis amoWra mocemuli baqteriis

citotoqsiur efeqtze. dadginda, rom TTSS1 monawileobs citotoqsiuraqtivobebSi, maSin roca TTSS2 ar monawileobs am procesebSi.

meore Zalzed mniSvnelovani kvleva dakavSirebuli iyo mesame tipis

sekreciuli sistemis genebis- TTSS1 da TTSS2 -is enerotoqsiurobasTan kavSirisdadgenasTan, romelic agreTve mutanturi Stamebis gamoyenebiT Catardabocvris qsovilebis magaliTze [168]. aRmoCnda, rom paTogenobisgan

gansxvavebiT enterotoqsiurobis Tviseba dakavSirebulia TTSS2-genTan da ara-

TTSS1-Tan. damtkicda agreTve isic, rom TTSS1 da TTSS2- genebi akontrolebs

sxvadasxva, aramonaTesave cilebis sekrecias. TTSS2- sisitema specifikuraddakavSirebulia kanagava dadebiT klinikur izolatebTan da ara garemodangamoyofil izolatebTan [117], rac kidev erTxel adasturebs mis monawileobas

Stamebis paTogenuri Tvisebebis CamoyalibebaSi. sxvadasxva StamebSi TTSS2-genebis Tanaarseboba tdh-genebTan, romlebic virulentobis mTavar faqtors

warmoadgenen [81], kidev erTi sabuTia imis, rom mocemuli sistema paTogenobis

meqanizmebSia CarTuli. gansxvavebiT TTSS2-gan TTSS1 gvxvdeba nebismierizolatSi, ganurCevlad imisa klinikuri warmoSobisaa Tu garemodan arisgamoyofili. aqedan gamomdinare zusti pasuxi imaze, aris Tu ara igipaTogenobaze pasuxismgebeli ar arsebobs [149]. N Tumca arsebobs versia, rom

SesaZloa igi aqtiuria mxolod TTSS2-Tan Tanaarsebobis pirobebSi [29].

is faqti, rom V.parahaemolyticus Seicavs TTSS- sisitemas, romelic araa

damaxasiaTebeli V.cholerae-Tvis, metyvelebs imaze, rom infeqciis meqanizmebi am

ori paTogenisTvis aris aramonaTesave. V.parahaemolyticus iwvevs anTebiT

16

diareasTan asocirebul gastroenteritebs, maSin rodesac, V.cholerae-s infeqciasekreciul diareasTanaa dakavSirebuli [23, 52].

1.7. V. parahaemolyticus-is izolatebis serologiuri Tvisebebis Seswavla

adreul serologiur kvlevebSi [165, 169] vibrios izolatebi dayofiliiyo serotipebad, magram vibrio-specifikuri antigenebi ar iyoidentificirebuli. mogvianebiT, sakazakim [157]. aRwera Termostabilurisomaturi O- antigeni, Termolabiluri kafsularuli K-antigeni daflagelaruli H-antigeni. omorim da Tanaavtorebma [146]. gasufTavebuli K-antigenis imunoqimiur kvlevaSi aCvenes, rom antigenuri masala Zlebda 1000C-zegaxurebisas 2 saaTis ganmavlobaSi, magram Termuli damuSavebis Sedegad

scildeboda ujredis zedapirs. sakazakim moaxdina V. parahaemolyticus-ისStamebis klasificireba 10 O- antigenis da 32 K-antigenis mixedviT. Mmisi

azriT rutinulad mxolod K-antigenis tipireba xdeba, radgan O- antigenis

gansazRvra gaxangrZlivebuli procesia da K-antigeni specifikuria TiToeuli

O- jgufisTvis. Tumca baqteriuli izolatebis mniSvnelovani nawili,gamoyofili Tevzebidan da zRvis wylidan [6, 12, 140, 158 ], iseve rogorcklinikuri wyaroebidan [75, , 133, 161] ar aglutinirdeboda K-antigeniT.Sedegad mravali axali O- da K-antigeni iqna aRmoCenili [75, 158 ], zogierTiki gamoiricxa [110 ].Aantigenuri sqema, romelic iTvaliswinebs am cvlilebebs,aRweril iqna zen-iojis da TanaavtorTa mier da moicavs 12 O- antigens da 48K-antigens. Tanamedrove sqemis mixedviT arsebobs 75 serovarianti 13 O-antigenis da 71 K-antigenis sxvadasxva kombinaciebis mixedviT. Tumcavibrionebis sakmaod mzard ricxvs mocemuli sqema zustad ar miesadageba, rackvlevis am mimarTulebiT gagrZelebis aucileblobaze mianiSnebs [168].

1.8. Vibrio parahaemolyticus-iT gamowveuli infeqciebi da maTi epidemiologia

Vibrio parahaemolyticus-is gastroenteritebisaTvis damaxasiaTebeliZiriTadi simptomebia diarea (wyliani, lorwovani, sisxliani an sisxlian-lorwovani) da abdominaluri tkivili; agreTve cxeleba, pirRebineba dazogadi sisuste, SesaZloa Tan sdevdes gauwyloveba da kolafsi. infeqciaTviTdasrulebadia da, rogorc wesi, grZeldeba 2-10 dRe. Ddaavadebis erT-erTi garTuleba ukavSirdeba kardio-vaskularul sistemas. imunodeficiturpirebSi SesaZloa sistemuri infeqciebis, mag. sefsisis ganviTareba.

infeqciis ZiriTadad wyaros warmoadgens daumuSavebeli an cudad

damuSavebuli zRvis produqtebi. Vibrio cholerae-s mier gamowveuli infeqciisgan

17

gansxvavebiT, romelic Cveulebriv siRaribesa da cud sanitarul pirobebs

ukavSirdeba, Vibrio parahaemolyticus-is infeqcia xSiria agreTve kargi socio-ekonomikuri pirobebis mqone qveynebSi, sadac zRvis produqtebi kvebismrewvelobis wamyvani komponentia [52 ].

Vibrio parahaemolyticus farTodaa gavrcelebuli estuariebSi, zRvebSi dasanapiro wylebSi mTel msoflioSi [16, 52, 100, 170] da warmoadgensinfeqciebisa da garemos dabinZurebis wyaros CrdiloeT amerikaSi, afrikaSida xmelTaSua zRvis sanapiroze. wylis temperatura, marilianoba,zooplanqtonis Semcveloba da gaxsnili Jangbadis raodenoba gavlenas axdenspaTogenis sivrcesa da droSi gavrcelebaze [102]. wylis temperaturismniSvnelobaze infeqciis epidemiologiaSi miuTiTebs is faqti, rom umetesiafeTqebebi swored Tbili Tveebis dros iCens Tavs [167].

daavadebebs, romelTa gamomwvevia Vibrio-s gvaris erTi romelimekonkretuli saxeoba an ramdenime saxeobis gamomwvevTa erToblioba,moixsenieben vibriozebad. zogadad, vibriozebis samkurnalodrekomendirebulia didi raodenobiT siTxis miReba an intravenurad gadasxma,aseve damatebiT SesaZlebelia antibiotikebiT (tetraciklini, doqsiciklini,agreTve ceftazidini) mkurnaloba /profilaqtika.

1.9. Vibrio parahaemolyticus -is ekologia

Hhalofiluri vibrionebi umetesad gvxvdeba wylian garemoSi-estuariebSi, zRvis sanapiro wylebSi, agreTve sxva wyalsatevebSi, romlebicsairigacio wylebiT, Camdinare wylebiT da niadagis sasuqebis CanarecxebiTbinZurdeba. halofiluri saxeobebi gavrcelebulia ZiriTadad zRvebSi damarilian wyalsacavebSi, Tumca maTi naxva SeiZleba agreTve mtknar wylebSi.

wylian garemoSi V.parahaemolyticus –is gavrcelebis sixSireze gavlenasaxdens sxvadasxva ekologiuri faqtorebi. aseTi faqtorebia temperaturulicvlilebebi, wylis marilianoba, gaxsnili nawilakebis raodenoba dasimRvrive, qlorofili a –s raodenoba da a.S.

wylis temperaturasa da V.parahaemolyticus-s Soris urTierTkavSirisarseboba gansazRvravs baqteriis gavrcelebis geografiul areals amerikaSi.evropasa da aziaSi [87]. temperatura mniSvnelovan gavlenas axdens, zogadad,vibrio-ebis sixSireze, Tumca pirdapiri kavSiri temperaturasa da paTogenuriV.parahaemolyticus-is sixSires Soris ar arsebobs. kvlevebma aCvena, rompaTogenuri V.parahaemolyticus-is raodenoba saerTo raodenobasTan SedarebiTmaRalia dabali temperaturis pirobebSi. amgvarad, paTogenuri izolatebitemperaturis cvlilebaze gansxvavebulad reagireben. DdadasturebuliaagreTve garemo faqtorebis gavlena sedimentebSi vibrio-ebis gavrcelebaze.kerZod, kaneko da kolveli [102] aRniSnaven zamTris TveebSi sedimentebSi vibriospp.-baqteriebis sasicocxlo ciklze temperaturis gavlenas. erT-erTi axalikvlevis Sedegebis mixedviT [56], mocemuli baqteria sedimentebSi gvxvdeba

18

xangrZlivi drois ganmavlobaSi, zamTris TveebSic ki, Tumca wlisganmavlobaSi maTi gamoyofa sedimentebidan ar xdeba.

wylis simRvrive, Tumca naklebad mniSvnelovan ekologiur faqtoradmiiCneva, xSirad dakavSirebulia sakvebi nivTierebebis (organuli substratis)didi raodenobiT SemcvelobasTan, rac Tavis mxriv baqteriebis zrdas dasedimentebze maT mimagrebas astimulirebs da, amasTan, mzis gamosxivebisganawilebas uSlis xels [22, 99]. bleqvelma da oliverma gamoavlinestemperaturisa da simRvrivis gavlena V.parahaemolyticus-is baqteriebis sixSirezeda gamoricxes marilianobis, rogorc ekologiuri faqtoris, roli mocemulimaCveneblis formirebaSi.

sxva kvlevebis mixedviT damokidebuleba marilianobasa da vibrioebissixSires Soris araxazobrivia da marilianobis maCvenebeli cvalebadobssakmaod did farglebSi. UTumca arsebobs sawinaaRmdego mosazrebac,ukanaskneli kvlevebis mixedviT, marilianoba gacilebiT did gavlenas axdens,vidre temperatura. arsebiTi kavSiri wylis marilianobasa da V.parahaemolyticus-is raodenobas Soris sedimentebSi, SeiZleba davukavSiroT marilianobispirdapir gavlenas an mtknar da sxvadasxva wylebis Cadinebas. rac ufromaRalia wylis marilianoba, miT naklebad mniSvnelovania mtknari wylisdinebebi da miT naklebi iqneba maTi gavlena. amasTan maRali marilianobaganapirobebs baqteriebis ukeTes sedimentacias. wylis marilianobis dadebiTigavlena vibrionebis gavrcelebaze vlindeba, ZiriTadad, maSin, rocamarilianobis maCveneblebi varirebs farTo sazRvrebSi da nimuSebisraodenoba aris sakmarisad didi [54, 120].

qlorofili a , temperaturis msgavsad, sxvadasxvagvar damokidebulebaSia

paTogenuri da, zogadad, V. parahaemolyticus –is izolatebis saerTo sixSiresTan.qlorofilis momatebuli mniSvneloba miCneulia qoleris epidemiis

winapirobad [46]. SesaZloa arsebobdes kavSiri qlorofil a –s, fitoplanqtons

da zooplanqtons Soris. Vibrio–s saxeobebze zooplanqtonis arsebobisa da

sixSiris gavlena naCvenebi iyo ufro adrindel SromebSi. qlorofili a– sgavlena ar ukavSirdeba fitoplanqtonis romelime saxeobis yvavilobas,aramed mTlianobaSi fitoplanqtons an mas zooplanqtonTan erTobliaobaSi.

kvlevebma aCvena, rom V. parahaemolyticus –is raodenoba wylian garemoSizRvis sxvadasxva organizmebis, Tevzebis, moluskebis, Rrubelebis, sxvadasxvazooplanqtonis raodenobazea damokidebuli [48]. sedimentebSi da wylissxvadasxva organizmebSi [97], Vibrio spp. baqteriebi ZiriTadad gvxvdeba zomierda subtropikul wylebSi, maTi raodenoba wyalSi korelaciaSi modis wylistemperaturasTan, zamTris TveebSi isini umeteswilad gvxvdeba sedimentebSi[13].Ggazafxulis dadgomisas, roca temperatura aris 13-150 C, sedimentebSigamozamTrebuli vibrionebi iwyeben gamoTavisuflebas da zooplanqtonzemimagrebas, temperaturis matebasTan erTad ki gamravlebasac [95]. planqtonTanasocirebuli vibrionebi gamovlenil iqna wylis nimuSebSi, romelTatemperatura iyo 190 C. zooplanqtonze arsebuli baqteriebis raodenobadamokidebulia agreTve temperaturaze, ZiriTadad Tavmoyrilia zedapirebzeda aris specifikuri, gansxvavebulia sedimentebSi napovni populaciisagan

19

[102]. garemo stresebis sapasuxod vibrionebi xSirad warmoqmnian biofilmebskibosnairTa da zRvis sxva organizmTa egzoskeletonTan asociaciaSi. amgvarasociaciaSi baqteriebs SeuZliaT rezistentulni iyvnen antibiotikebismimarT da xelsayreli urTierToba daamyaron sxva baqteriebTan anmaspinZelTan [48].

sicocxlisunariani, magram arakultivirebadi mdgomareoba (VBNC).daaxloebiT 30 sxvadasxva gvaris baqterias aqvs unari araxelsayrelipirobebis dros (temperaturis, Jangbadis Semcvelobis an osmosuri wneviscvlileba, sakvebis deficiti) gadavides sicocxlisunarian, magramarakultivirebad (VBNC) mdgomareobaSi. am organizmebs miekuTvnebian iseTibaqteriebi, rogoricaa: Escherichia coli, Pseudomonas fluorescens, Salmonella typhimurium daVibrio spp. zogadad, VBNC ujredebi xasiaTdeba ujredis zomis SemcirebiT, dnm-is , rnm-is da cilis sinTezis SemcirebiT [145]. amasTan icvleba ujredebislipiduri profilic-savaraudod membranis Txevadi struqturis SenarCunebisgamo temperaturis klebis dros [120; 145]. imisaTvis, rom mocemuli mdgomareobaCaiTvalos fiziologiur, „Tavis gadamrCen“ meqanizmad, VBNC ujredebs undaSeeZloT am mdgomareobidan ukan dabruneba ise, rom SesaZlo iyosstandartuli mikrobiologiuri meqanizmebiT maTi xelaxali gamovlena.

V.parahaemolyticus-isTvis damaxasiTebeli sezonuri variabeloba- is romufro xSirad iTeseba zafxulis TveebSi, vidre civ TveebSi [44; 102], agreTveV.parahaemolyticus -is infeqciebis maRali sixSire aprili-oqtombris periodSi[52]. sxvadasxva avtorTa mier aixsneba temperaturisa da sakvebis naklebobisdros V. parahaemolyticus -is VBNC-mdgomareobaSi gadasvliT [96]. am mdgomareobaSigadasvlisas kultivirebadi ujredebis raodenoba mcirdeba manam, sanam argaxdeba arakultivirebadi standartuli mikrobiologiuri meTodebisgamoyenebiT. Tumca am arakultivirebad ujredTa mniSvnelovani populaciaibrunebs cxovelmyofelobis unars [145].

VBNC- mdgomareobaSi gadasvla sxvadasxva SemzRudvel faqtors SeiZlebaukavSirdebodes, magram ujreduli mdgomareobis dinamika damokidebuliaTiToeulis intensivobaze. dabali temperaturis an dabali marilianobisgavlena V. parahaemolyticus-ze kultivirebis unariis dakargvas iwvevs. ujredebisfiziologiurma mdgomareobam aCvena, rom arakultivirebadi ujredebiinarCuneben respiraciul da membranul mTlianobas. maT miiCneven sicocxlisunarian, magram arakultivirebad ujredebad (VBNC) [47; 145].

marilianobis deficitiT gamowveuli stresi zamTris periodSi iwvevs V.parahaemolyticus-is VBNC-mdgomareobaSi gadasvlas, maSin rodesac, zafxulisTveebSi mcire raodenobiT ujredebi kultivirebis unars inarCuneben.sicivisgan gamowveulma stresma, nawilobriv, SesaZloa ujredebi daicvasmarilianobis deficitiT ganpirobebuli stresisgan, saSualeba misces ra,gadavidnen VBNC mdgomareobaSi. is rom erTi saxis stresi damcvelobiTrols TamaSobs meorenairi stresisgan ujredebis gadasarCenad, ZvelkvlevebSic iqna naCvenebi [52].

20

2. baqteriofagebi

2.1. klasifikacia da taqsonomia

Bbaqteriuli virusebi anu baqteriofagebi cocxali samyaros erT-erTyvelaze mravalferovan jgufs warmoadgenen. maTi gavrcelebis areali farToa.maTi naxva SesaZlebelia haerSi, wyalsa da niadagSi, agreTve cxoveluriorganizmebis, maT Soris adamianis, sxeulis sxvadasxva Rruebsa da nawlavebSi.baqteriofagebis aRmoCena me-20 saukunis erT-erTi umniSvnelovanesi movlena iyo,romelic ori mecnieris, britaneli f.tuortis (1915 w.) da kanadeli mecnierisf.derelis (1917w.) saxelebs ukavSirdeba. saxelwodeba baqteriofagi pirveladf.derelma uwoda dizenteriis gamomwvevi mikrobis sawinaaRmdego viruss.aRsaniSnavia agreTve qarTveli mecnieris, giorgi eliavas, damsaxureba, romelmac1917 wels mtkvris wyalSi vibrionis aRmoCenasTan erTad aRwera baqteriofagisfenomeni [33].

Bbaqteriofagebis pirveli klasifikacia [112] efuZneboda patronibaqteriuli ujredebis tipebs da maT mier gamowveuli daavadebebis simptomebs.1962 wels lvofis, hornis da tunieris klasifikacias safuZvlad daedovirionis morfologia, kerZod, nukleinis mJavis tipi, kafsidis forma dakafsomerebis raodenoba [5, 33, 112].Ees sqema mogvianebiT cnobili iyo LHT-sistemis saxelwodebiT, romlis SemadgenlobaSic Sedioda ФX174 da orikudiani fagi.

mogvianebiT, praqtikulma aucileblobam mecnierebi miiyvana im daskvnamde,rom baqteriofagebis klasifikacias safuZvlad unda daedos morfotipi dagenetikuri Tvisebebi.

morfotipebis mixedviT cnobilia bredlis da a. tixonenkosklasifikaciis sqemebi. Bbredlis klasifikaciis mixedviT fagebi daiyo 6jgufad: fagebi kumSvadi wanazardiT, mokle wanazardiani fagebi, kuburiformis fagebi, erTZafiani dnm-is Semcveli, rnm-is Semcveli da Zafiseburi(filamenturi) fagebi.

akermanma klasifikaciis dazustebisaTvis SemoiRo morfologiuriqvetipebi, sadac gaTvaliswinebulia fagis Tavisa da kudis SefardebiTizomebi, Tavis forma da masze specifikuri warmonaqmnebis arseboba. 10 ojaxidayofilia 4 morfologiur tipad: 1) kudiani fagebi 2) kuburi ukudo fagebi 3)Zafisebri fagebi 4) pleomorfuli ukafsido (lipidebiani) fagebi.

1) kudiani (2 jaWviani dnm-iT) fagebis tipi Sedgeba 3 ojaxisagan:I - mioviride; II- sifoviride; III- podoviride [5, 28]

morfologiuri kriteriumi aucilebelia, magram arasakmarisia, radganarseboben morfologiurad identuri fagebi, romelTac maspinZlisgansxvavebuli speqtri aqvT da piriqiT.

Tanamedrove klasifikaciis mixedviT (1992 ICTV) baqteriofagebigaerTianebulia Caudovirales klasis 13 ojaxSi da 31 gvarSi. klasifikaciadamyarebulia Semdeg kriteriumebze:1) virionis ultrastruqtura: binaluri simetriis (wanazardiani), kuburi,helikaluri (spiraluri), filamenturi an pleiomorfuli

21

2) nukleinis mJavis tipi3) Semadgenloba da antigenuri Tvisebebi4) molekuluri masa5) patron-ujredebis wre

eleqtronuli mikroskopiiT gamokvleul 5100 baqteriofags Soris 4950(96%) wanazardiani( kudiani) fagebia. isini miekuTvnebian 3 did filogenetikurojaxs.1) Myoviridae- fagebi grZeli kumSvadi wanazardiT. Aam ojaxSi Sediswanazardiani fagebis 25%.2) Siphoviridae- fagebi grZeli, moqnili kudiT, romlis SaliTas ar gaaCniaSekumSvis unari. Aam ojaxs miekuTvneba kudiani fagebis 61% [5]. tipiuriwarmomadgenlebia: T1, T5, L5, λ fagebi da sxva. Oojaxi moicavs 6 gvars.3) Podoviridae- mokle wanazardiani fagebia. tipiuri warmomadgenlebi arian: P22,T7 fagebi da sxva. Aam ojaxSi gaerTianebulia 3 gvaris, kudiani fagebis 14 %.

wanazardian fagebs gaaCniaT ikosaedris an oqtaedris formis Tavi. FagiSedgeba Tavis, kudis da fakultaturi struqturebisgan, rogoricaa Tavis ankudis fibrilebi, sayelo, bazaluri firfita, terminaluri boloebi.kafsidSi moTavsebulia superspiralizebuli orZafiani dnm. wanazardianifagebi SesaZloa iyos virulenturi an- zomieri (50%-50%) [33].

zemoT CamoTvlili sami ojaxidan didi zomis kafsidiT da dnm-ismaRali SemcvelobiT xasiaTdeba Myoviridae, xolo Podoviridae da Siphoviridae, ammaxasiaTeblebiT ufro axlo msgavsebas avlenen [33].

ojaxi Myoviridae warmodgenilia fagebis mravalferovani jgufiT, romlisZiriTadi morfologiuri maxasiaTebelia kumSvadi wanazardi. aRniSnuliojaxis warmomadgenlebs unari aqvT daainficiron rogorc gram-dadebiTi,aseve gram-uaryofiTi baqteriebi da arqebaqteriebi. virusebis taqsonomiissaerTaSoriso komitetis klasifikaciis mixedviT, winazardiani fagebis samiveojaxi aerTianebs 15 gvars, aqedan Myoviridae-s ojaxi dayofilia Semdeggvarebad: T4-is msgavsi virusebi, P1-msgavsi virusebi, P2-msgavsi virusebi, Mu-msgavsi virusebi, SPO1-msgavsi virusebi, PBS1-msgavsi virusebi, H-msgavsivirusebi. aRniSnuli gvarebi mkveTrad gansxvavdebian erTmaneTisgansasicocxlo ciklis da genomis struqturiT. mag.: P1-jgufi lizogenur fagebsaerTianebs, Mu-transpozonebs warmoadgens da ar eqvemdebareba P2-is msgavsassaitspecifikur rekombinaciebs, xolo T4 liTiuri baqteriuli virusia. maTigenomis zomebi varirebs 30000 nw-dan 17000 nw-mde. Myoviridae-s ojaxSi fagebiZiriTadad gaerTianebulia morfologiuri niSnebis mixedviT.

brusovis da Tanaavtorebis mier 1996 wels Catarebulma kvlevam cxadyo,rom fagis erTi morfotipis meored gardaqmna mutaciiT ar xdeba. erTiojaxis fagebs Soris SemCneulia mxolod mcire struqturuli gansxvavebebi.mag. maRali dnm-dnm homologiis mqone rZemJavuri streptokokuri fagebigansxvavdebodnen mxolod sayelos morfologiuri SenebiT.

Aamgvarad, aucilebelia axlad gamoyofili fagis identifikacia da imisgarkveva, romel ojaxs miekuTvneba igi, radgan erTi ojaxis fagebs gaaCniaTmsgavsi genetikuri organiazacia, maTi genomebi SegviZlia warmovidginoT,rogorc genebis blokebis modulebis Semadgenloba, romelTagan TiToeuli

22

akontrolebs garkveul funqcias da dakavSirebulia sxva modulebTangansazRvruli TanmimdevrobiT (romelic damaxasiaTebelia mocemuli ojaxisfagebisaTvis) [25].

2.2. baqteriofagebis biologia

baqteriofagis urTierTqmedeba baqteriul ujredTan mkacradspecifikuria, anu baqteriul virusebs aqvT unari moaxdinon mxolodgarkveuli saxeobis baqteriebis inficireba [33, 76, 112]. baqteriofagis daujredis urTierTobis mixedviT fagebi SeiZleba daiyos 3 tipad, kerZod:WeSmaritad virulenturi fagebi, zomieri fagebi da fagebi, romlebic ujredisinficirebis Semdeg ar iwveven mis daSlas. ujredi, romelic dasnebovnebuliaaseTi fagiT, imyofeba ganuwyveteli (permanentuli) infeqciis mdgomareobaSi.fagis aseTi ganuwyveteli (producirebis) gamo ujredis gayofis siCqare

baqteriofagebi, romlebic iwveven produqciul infeqcias da fagissicocxlisunarian STamomavlobas gvaZleven, iwodebian aradefeqtur fagebad.yvela aradefeqturi fagisaTvis cnobilia 2 an 3 mdgomareoba: 1) araujreduli(Tavisufali, momwifebuli fagis mdgomareoba); 2) vegetaciuri fagi da 3)zomieri fagisaTvis arsebobs kidev profagis mdgomareoba [113].

2.2.1. virulenturi baqteriofagebis sasicocxlo cikli

ganviTarebis lizisuri cikli iwyeba fagis baqteriul ujredzeadsorbciiT [33, 76]. baqteriofagis adsorbcia patron ujderze mkacradspecifikuria da zedapiruli struqturebis- receptorebis saSualebiTxorcieldeba. gram-uaryofiT baqteriaSi nebismieri cila, oligosaqaridi,lipopolisaqaridi SeiZleba warmoadgendes receptors fagisaTvis.morfologiurad msgavsi fagebis receptoruli ubnebi SeiZleba gansxvavebuliiyos da piriqiT. aris fagebi, romlebic adsorbciisaTvis iyeneben baqteriisspecifiur warmonaqmnebs e.w. pilebs. adsorbciis procesi, misi kinetikasxvadasxva fagis SemTxvevaSi gansxvavebulia. adsorbciis sixSire xSiraddamokidebulia mraval garemo faqtorze da maspinZeli mikrobis

fiziologiur mdgomareobaze. magaliTad, λ receptorebis eqspresia xdebamxolod maltozis Saqris Tanaarsebobisas [113]. mravali fagisTvis sxvadasxvakofaqtorebis arsebobaa aucilebeli, magaliTad Ca2+ Mg2+ ionebis, an sxvamartivi kaTionebis. mutaciebis dros, romelsac Tan axlavs baqteriulireceptorebis cvlileba, ujredi kargavs fagis adsorbciis unars da iZensrezistentobas mis mimarT. fagebis umetesobisTvis adsorbciis procesiSeuqcevadia, romelic ujredSi fagis genomis eJeqciiT mTavrdeba.

baqteriofagis genomi kudis gavliT Sedis ujedSi. es procesi arganixileba rogorc dnm-is martivi ineqcia, aramed am procesSi CarTulia dnm-is transferis ramdenime meqanizmi [113]. fagis adsorbciis Semdeg, ujrediskedlis SemadgenlobaSi arsebuli TuTiis ionebi (Zn2+ ) iwvevs fagis kudis

23

distaluri nawilis boWkoebis gaglejas. gamoTavisuflebuli bazalurifirfita kudis distalur nawilSi moTavsebuli fermentis endolizinis(lizocimis) gamoyofiT axdens baqteriuli ujredis kedlis mimdebarefragmentis lizirebas. amavdroulad SaliTaSi gamoTavisuflebuli Ca2+ionebi iwvevs atf-azas aqtivirebas, rac Tavis mxriv ganapirobebs SaliTisSekumSvas da ujredSi citoplazmaturi membranis gavliT kudis RerZisSeWras. amis Semdgom adgili aqvs virusuli dnm-is ujredis citoplazmaSiswraf gadasvlas [33, 113].

naCvenebia, rom SesaZlebelia zogierTi fagisaTvis eJeqciis procesisstimulireba. am dros mimdinareobs gravitaciuli urTierTqmedeba fagis kudisgamonazardsa da citoplazmur membranis cilovan komponentebs Soris, amsaxis stimulacia xels uwyobs ujredSi fagis genomis droze adre SeWras [91,92].

xSirad fagis adsorbciuli aparatis moqmedebiT xdeba ujredismembranuli SeRwevadobis potencialis cvlileba. am dros SeiZleba dairRvesujredis bioqimiuri aqtivoba, romlis Tavidan asacileblad fags gaaCnia genebi,romlebic axorcieleben inficirebuli ujredis membranis mTlianobis dadaurRvevlobis kontrols [105].

ujredSi fagis dnm-is inJeqciis Semdeg sawyis safexurs warmoadgensmaspinZeli ujredis rnm-polimerazas mier fagis promotoris Secnoba daadreuli genebis transkrifcia [105]. am genebis produqtebi icaven fagur dnm-sda axdenen maspinZeli ujredis restruqturizacias: inaqtivireben maspinZeliujredis proteazebs, restriqciul enzimebs, Slian sxvadasxva cilebs.Sualeduri genebis transkripcia mimarTulia fagis axali dnm-issinTezisTvis. Sualedur genebs moyveba gviani genebi, romlebic ukvekodireben faguri nawilakebis asawyob struqturebs. axali fagebis TavmoyrasujredSi ewodeba fagis momwifeba. fagis dnm-is inJeqciidan fagebis axaliSTamomavlobis gamoTavisuflebamde drois intervals ewodeba latenturiperiodi [112]. latenturi periodi damokidebulia maspinZlis fiziologiaze dameryeobs 20 wT-dan 30-40 wT-mde.

fagis sasicocxlo ciklis saboloo safexuri, romelic mohyvebabaqteriofagiT infeqcias, aris fagis axali Taobis gamoTavisufleba damaspinZeli ujredis lizisi [113, 123 ]. kudian baqteriofagebs amgvar procesSisWirdebaT fagiT kodirebuli ori cilis moqmedeba. pirveli aris e.w. holini,patara transmembranuli proteini, romelic warmoSobs mcire zomisdazianebebs citoplazmur membranaSi. es dazianebebi forebis funqcias asrulebsda aZleven saSualebas ganTavisufldes meore cila _ enzimi-lizini,romelsac aqvs peptidoglikanis hidrolizis unari. sxvadasxva fagis SvileulpopulaciebSi virionebis raodenoba met-naklebad mudmivia da iwodeba,rogorc fagis gamosavlianoba, anu fagis gamosavali.

virulenturi fagi SesaZlebelia aRmoCndes e.w. fsevdolizogenurmdgomareobaSi, romelsac zogjer matareblobas uwodeben [113]. cnobiliafsevdolizogeniis ramdenime meqanizmi. zogjer is viTardeba baqteriebisfagisagan droebiTi izolaciis Sedegad. es gamoyofa SesaZlebelia sufTameqanikur xasiaTs atarebdes, magaliTad, romelime webovani substratiujredebs gars ekvris da xels uSlis fagebiT maT swraf inficirebas. xSir

24

SemTxvevaSi fsevdolizogenias gansazRvravs sakvebi nivTierebebis nakleboba.aseT dros ar aris sakmarisi energia imisTvis, rom fagma ganaxorcielosTavisi sasicocxlo cikli da fagis genomi maspinZeli ujderis SigniT arisaraaqtiur mdgomareobaSi.

2.2.2. 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 mdgomareobisganmsazRvrel mraval faqtorze [33, 112]. lizisuri gzis SemTxvevaSi,lizogenuri fagebi gadian ganviTarebis igive etapebs, rasac litiuri fagebi.zomieri sasicocxlo ciklis SemTxvevaSi, faguri dnm integrirdebabaqteriul dnm-Si profagis saxiT da replicirdeba sinqronulad baqteriulgenomTan erTad. swored am movlenas lizogenia ewodeba, xolo baqteriulkulturas lizogenuri. lizogenia sakmaod stabiluri Tvisebaa da mxolodgarkveuli garemo faqtorebis gavleniT aRiniSneba fagis virulentobisganmsazRvreli genebis stimulacia da fagis induqcia [33, 112].

lizogeniis koncefcias gaaCnia cvalebadi istoria. fagis pirvelmkvlevarebs 1920-1930 wlebSi sjerodaT, rom fagi dakavSirebuli iyobaqteriasTan da Tavad baqterias hqonda fagebis spontanuri generaciis unari,romelic principSi am dros ufro fermentad miiCneoda da ara cocxalvirusad [33, 112]. SemdgomSi naxes, rom es ujredebi mravldebodnen fagisproduqciis gareSe, Tumca garkveul SemTxvevaSi maT SeamCnies ujredisspontanuri lizisi da areSi Tavisufali fagi. lvofma fagis ujderSidamdgomareobas profagi uwoda da mogvianebiT aCvena, rom lizogenuri ujredisultraiisferi dasxivebiT adgili hqonda profagis litiuri zrdisiniciacias da Sedegad fagis induqcias [112].

lizogeniis movlenas xSirad Tan axlavs e.w. lizogenuri konversia,romlis drosac aRiniSneba virusuli dnm-iT gamowveuli baqteriuli ujredisgenetikuri Tvisebebis cvlileba [38, 61]. lizogenur fagebs unari aqvTgadaitanon maspinZeli ujredis genebi erTi baqteriidan meoreSi(transduqcia) [34, 112]. mraval lizogenur fags gaaCnia integrirebisTvisspecifikuri saitebi. am SemTxvevaSi transduqcia moixsenieba, rogorcspecializirebuli transduqcia. sxva fagebisTvis damxaxasiaTebeli ar arisintegrirebisTvis specialuri ubnebis arseboba da isini integrirdebianbaqteriuli dnm-is nebismier ubanSi, SemTxveviTad. aseTi saxis transduqciaukve moiazreba, rogorc generalizirebuli transduqcia [112].

fagebis roli bunebaSi, rogorc genebis horizontalurad gadamtanebisa,sakmaod didia, gansakuTrebiT baqteriebis evoluciis TvalsazrisiT [34].mikrobTa virusebi miiCnevian baqteriebs Soris genebis, maT Sorisantibiotikorezistentuli genebis horizontalur gadatanaSi monawileyvelaze metad gavrcelebul veqtorebad. Tanamedrove kvlevebi mowmobs, romfagebi rezistentuli genebis rezervuars warmoadgens da monawileobs maTgavrcelebaSi, filogenetikurad aramonaTesave mikrobebs Sorisac ki. fagebiT

25

genebis horizontaluri gadatana sakmaod xSiri movlena unda iyos, racaxsnis antibiotikorezistentuli genebis aseT mravlobiTobas da gavrcelebisfarTo areals [96].

2.3. Bbaqteriofagebis gamoyeneba Terapiuli mizniT (fagoTerapia)

Dd’Herelle-s mier fagis aRmoCenidan maleve daiwyo misi gamoyenebadizenteriis samkurnalod. swored es iyo fagis pirveladi Terapiuligamoyeneba [36]. mocemuli kvleva mimdinareobda parizSi 1919 wels. Ppirvelieqsperimentebi sakmaod efeqturi aRmoCnda, Tumca warmatebuli SedegebimaSinve ar gamoqveynebula, ufro metic, pirveli angariSi, sadac miTiTebuliafagoTerapiis warmatebuli SemTxveva, gamoica 1921 wels, roca fagi gamoiyeneskanis stafilokokuri daavadebis samkurnalod [36]. maleve Catarda mravalimsgavsi kvleva, romlis Sedegebic xazs usvamda fagebis did potencialssxvadasxva infeqciebis samkurnalod, ramac xeli Seuwyo ramdenime faguripreparatis warmoebas sxvadasxva kompaniebis mier.

Dd’Herelle-s laboratoria uSvebda 5 samkuralo komerciul preparatssxvadasxva baqteriuli infeqciebis samkurnalod [4]. samkurnalo fagebiswarmoeba aseve mimdinareobda amerikis SeerTebul StatebSic. es preparatebigamoiyeneboda iseTi infeqciebis samkurnalod, rogoricaa abscesebi,Wrilobebi, vaginitebi, qronikuli infeqciebi, zeda respiratoruli traqtisinfeqciebi da a.S. magram antibiotikebis eram advilad Caanacvla fagoTerapia,gansakuTrebiT dasavleT evropaSi. Tumca fagebis gamoyeneba samkurnalomizniT gagrZelda aRmosavleT evropaSi da yofil sabWoTa kavSiris qveynebSi[36, 55, 84, 111, 114, 123]. erT-erTi mniSvnelovani instituti, romelic aqtiuradiyo CarTuli fagebis kvlevasa da warmoebaSi, iyo g. eliavas saxelobisbaqteriofagiis, mikrobiologiisa da virusologiis instituti.

infeqciur paTologiaSi baqteriofagis gamoyenebis rekomendaciebidafuZnebulia im gamocdilebaze, rac dagrovda mravali wlis manZilze.dReisTvis baqteriofagebi warmatebiT gamoiyeneba Cirqovan-anTebiTi,infeqciebis, nawlavuri da Sard-sasqeso gzebis infeqciuri anTebiTiprocesebis samkurnalod [32, 84, 114, 128, 162, 163].

26

2.4. baqteriofagebis daxasiaTebisa da identifikaciis meTodebi

baqteriofagebis kvlevaSi maTi daxasiaTebisaTvis gamoiyeneba Semdegistandartuli 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.

fagis erTjeradi gamravlebis cikli. fagis erTjeradi gamravlebisdros unda iqnes uzrunvelyofili fagis ganviTarebis dawyeba erTdrouladinficirebuli baqteriebis ujredebis umravlesobaSi. amis miRweva SeiZlebasxvadasxva meTodiT. fagebi, romlebic kargad adsorbirdeba, Txevad areSimaqsimalur adsorbcias aRweven daaxloebiT 5wT-is ganmavlobaSi.

erTjeradi gamravlebis cikli moicavs ganviTarebis TiToeuli periodisxangrZlivobas. fagis ujredSida gamravlebis process yofen 3 periodad:eklipsi (faruli periodi), latenturi periodi da lizisis dro. farulperiodSi (dro infeqciis dawyebidan anu fagis genomis baqteriaSi SeRwevidanpirveli momwifebuli faguri nawilakis warmoqmnamde) xorcieldebaujredSida ganviTarebis yvela etapi, romelic fagis momwifebisaTvis arisaucilebeli. faruli da latenturi periodebis xangrZlivoba erT-erTiSedarebiT mdgradi damaxasiaTebeli Tvisebaa ama Tu im fagisaTvis.standartuli cdebis pirobebSi sakontrolo fagis Tanaobisas SeiZlebamoxdes Sedareba kontrolTan. lizisis da latenturi periodis xangrZlivobasxvadasxvaa. lizisis dro da infeqciis mravlobiToba ar iZleva fagisidentifikaciis saSualebas, radgan maTze moqmedebs garemo aris bevrifaqtori da sxva.

negatiuri koloniis morfologia (nk). es maxasiaTebeli axlad gamoyofilifagis pirveladi identifikaciis saSualebaa. fagis negatiuri kolonia myar annaxevradTxevad agarze warmoiqmneba fagis mier baqteriuli ujredisinficirebis Sedegad. fagis identifikaciisas Tundac naTesauri fagebisSemTxvevaSi negatiuri koloniis forma da zoma aris sakmaod specifikuriTviseba. negatiuri koloniiT SeiZleba erTi fagis meorisagan gansxvaveba:centraluri lizisis zonis sidide, simRvrive, oreolis arseboba, misi formada zoma, opalesenciis unari da sxva. Tumca, mxolod negatiuri koloniebisSedarebiT ar SeiZleba Sefasdes fagebis msgavseba, magram Tu ori fagisnegatiuri kolonia erTmaneTisagan gansxvavdeba, es fagebi ar SeiZlebaidenturni iyvnen.

lizisuri aqtivobis speqtri. lizisuri aqtivobis speqtri, anu maspinZelbaqteriaTa speqtri warmoadgens baqteriofagebis daxasiaTebisa daidentifikaciis erT-erT saSualebas da mas saklasifikacio mniSvneloba aqvs.imisaTvis, rom lizisuri aqtivoba gamoyenebuli iqnes fagis saidentifikaciodaucilebelia:

a) aRebul iqnes potenciuri maspinZlebis farTo jgufi da agreTvemaTi sxva NnaTesauri saxeobebis baqteriebi;

b) aucileblad unda Semowmdes fagis aqtivoba sxvadasxva mutanturStamebze, romlebic sxva fagebis mimarT rezistentulni arian;

27

g) sadac SesaZlebelia, dadgindes, Tu ratom ar aris fagi aqtiuri amaTu im Stamis mimarT; misi ganviTarebis romeli periodis daTrgunva xdebamaspinZel ujredSi (adsorbcia, ujredSida gamravleba) [5, 41].

antifaguri Sratis gamoyeneba baqteriofagebis identifikaciisaTvis.specifikuri antifaguri Sratebi erT-erTi umTavresi saSualebaa fagebisidentifikaciisa da klasifikiciisaTvis. antifaguri Sratebis momzadebismeTodi martivia, Tumca sakmaod Sromatevadi, xolo neitralizaciisjvaredini reaqcia _ sakmarisad zusti, romlis Sedegad SeiZleba dadgindesaris Tu ara ori fagi erTmaneTis msgavsi. Tu erTi da igive wyarodan mciredrois periodSi gamoyofil iqna 2 fagi, romelTac aqvT erTnairimgrZnobeloba antifaguri Sratis mimarT, magram gansxvavdebian lizisuriaqtivobis speqtriT, miuTiTebs, rom gvian aRmoCenili fagi, warmoadgenspirveli fagis mutants (gafarTovebuli lizisuri speqtriT). sxvadasxvamaCveneblebis mixedviT msgavsi fagebi SeiZleba aRmoCndnen sxvadasxvaserotipebSic.

faguri nawilakebis fiziko-qimiuri Tvisebebi. fagis virionismorfologiuri parametrebi, romlebic Seiswavleba eleqtronulimikroskopiis saSualebiT, aris fagis erT-erTi mniSvnelovani damaxasiaTebeliTavisebureba, magram ara gadamwyveti.

baqteriofagebis daxasiaTebisaTvis metad mniSvnelovan maCveneblebswarmoadgens sxvadasxva fizikur-qimiuri faqtorebis moqmedeba, rogoricaa:qloroformis da sxva organuli gamxsnelebis moqmedeba, Termoinaqtivacia, UVgamosxiveba, aris sxvadasxva pH da mravali sxva.

2.5. Vibrio parahaemolyticus-is mimarT specifikuri baqteriofagebi

Vibrio-s gvaris mimarT specifikuri baqteriofagebi pirveladidentificirebul iqna derelis mier 1926 wels da 1950 wlisTvis aRweril iqna

V.cholerae-s mimarT specifikuri ramdenime baqteriofagi. V.cholerae-sbaqteriofagebis Sesaxeb kvlevebs gadaxeda politzerma. adreuli Sromebi

V.cholerae-s fagebis Sesaxeb Seexeboda ZiriTadad maT gamoyenebas mkurnalobisda 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 fagotipirebissqemis Seqmna liTiuri fagebis da meores mxriv, klasifikaciis- lizogenuri

fagebis gamoyenebiT. aRmoCenilia Vibrio-s gvaris mimarT specifikuri 85 fagi,84 maTgani miekuTvneba kudian fagebs, mxolod erTi aris filamenturi.kudiani fagebi mikuTvnebian 6 mTavar morfotips da Myoviridae-s, Siphoviridae-s,da Podoviridae-s ojaxebs. 63 fagi klasificirda 18 saxeobad. filamenturi fagi

aris Inovirus-is gvaris da Inoviridae-s ojaxidan. Vibrio-s fagebi Zalzed

28

heterogenulia da moicavs morfologiurad saintereso virusebs. zogierTifagi axlos dgas sxva gram (-) baqteriaTa fagebTan, rac SesaZloa maTfilogenezur naTesaobazec mianiSnebdes.

Bbaqteriofagebi, romlebic gvxvdeba okeaneebSi, moqmedeben maspinZelibaqteriis winaaRmdeg da mniSvnelovan gavlenas axdenen zRvis baqteriebis

sixSiresa da mravalferovnebis Camoyalibebaze [30, 173]. baqteriofagebigarkveul rols TamaSobs diareul afeTqebebSi, romlis mizezic aris zRvis

sxvadasxva mikroorganizmi, maT Soris Vibrio parahaemolyticus-ic. baqteriofagebisyvelaze mniSvnelovani rolia baqteriebis koncentraciis Semcireba maTi

pirdapiri mokvlis gziT [26]. garda amisa isini SesaZloa monawileobdnenlizogenur gardaqmnebSi da genebis lateralur gadatanaSi [31].baqteriofagebs, romlebic wamyvan rols TamaSoben baqteriuli genomismravalferovnebaSi, SeuZliaT warmoqmnan mocemuli Stamebis sxvadasxva

variantebi.zRvis baqteriofagebis genomis sekvenirebam aCvena, rom maTi

umravlesobis aTvlis CarCo naklebad gavs genebis bankSi arsebul ukveSeswavlil sxva genomebs. Uunikaluri aTvlis CarCoebis aseTi maRaliprocentuli maCvenebeli ganasxvavebs maT kargad Seswavlili fagebisgan(kolifagebi da streptokokuri fagebi), romelTa genebis 80% adregamoyofili fagebis genebis homologiuria. Tumca vibriofagi HSIC (maspinZelibaqteria Listonella pelagia) Seicavs aTvlis CarCos, romelic sxvadasxvabaqteriebis mimarT specifikuri fagebis aTvlis CarCoebis msgavsia: mag.Streptococcus pyogenes-is da Yersinia pestis-is profagebis da Bordetella pertussis-is,Bordetella bronchioseptica-s da Cyanobacter-is baqteriofagebis.

zRvis baqteriofagebis TiTqmis 1/3 , romelTa sekvenirebac dRemdegakeTebula, vibriofagebia. Mmravali maTgani gansazRvravs maspinZlispaTogenur Tvisebebs, mag. rogorc toqsin-asocirebuli genebi. zogierTimaTgani lizogenur an fsevdolizogenur urTierTobaSia Tavis maspinZelTan.

xSir SemTxvevaSi morfologiurad gansxvavebuli fagebi genomisorganizaciiT msgavsia da emsgavsebian Siphoviridae-s zomier warmomadgenlebs.vibriofagebis 4 sxvadasxva Myoviridae-s warmomadgenlis genomis SedarebiTmaanalizma aCvena [150], rom genomis struqturiT yvela maTgani gavda Siphoviridae-szomier fagebs. maT gaaCniaT garkveulwilad konservatiuli organizaciisgenomi, Tundac mxolod im klasteris mixedviT, romelic virionis Semadgenelproteinebs akodirebs [150]. aTvlis CarCoTa garkveuli nawili mocemulifagebisTvis erTnairia. Ggarda amisa genomSi daculia Semdegi Tanmimdevroba:terminaza, struqturuli, kafsidis da kudis cilebis genebi. SeswavlilioTxive fagi ufro metad gavda Siphoviridae-s zomier fagebs, vidre virulenturMyoviridae-s.

V. parahaemolyticus-is mimarT specifikuri baqteriofagebi wylidanpirvelad gamoyves 1996 wels nakaniSim da Tanaavtorebma. es iyo samigansxvavebuli baqteriofagi, romlebic gamoyofil iqna zRvis wylidan,adamianTa fekaliebidan da lizogenuri Stamidan, romelic aRmoCnda Zalzedgansxvavebuli negatiuri koloniis morfologiis mixedviT. amas mohyva

29

baroSisa da Tanaavtorebis mier zRvis nimuSebidan sxva baqteriofagebisgamoyofa. dReisaTvis cnobilia baqteriofagebi, romlebic ainficireben daklaven V. parahaemolyticus sxvadasxva Stamebs, magram litiuri fagi pandemuri

Stamis winaaRmdeg ar aris cnobili. cnobilia V. parahaemolyticus-is fagebis

sixSire zRvis cxovelebSi [17], da genetikuri mravalferovneba oisterebsa da

zRvis wyalSi [49, 50].vibriofagebi zooplanqtonisa da wylis nimuSebSi gvxvdeba

koncentraciiT 104 virusi sm3 -Si. V. parahaemolyticus-is fagebis didi nawilisakmaod detaluradaa daxasiaTebuli. maTi umravlesoba miekuTvnebasifovirideebs, raodenobrivad maT mosdevT podovirides ojaxis

warmomadgenlebi. VpV262 baqteriofagi, romlis genomi Sedgeba 46012

nukleotiduri wyvilisagan, savaraudod, aris T7-baqteriofagis Soreuli

naTesavi [74]. sruladaa gansazRvruli agreTve T4-msgavsi, farTo speqtris

mqone vibriofagis KVP40-is genomi [132]. V. parahaemolyticus-is yvela baqteriofagi,garda inovirides ojaxis ramdenime filamenturi fagisa, miekuTvneba kudianfagebs: miovirides, sifovirides an podovirides ojaxidan. es fagebi,ZiriTadad, saxeoba-specifikuri, xandaxan ki Stam- specifikuria, TumcazogierTi maTgani amJRavnebs moqmedebis SedarebiT farTo speqtrs vibrios da

monaTesave gvaris fotobaqteriumis winaaRmdeg [122]. V. parahaemolyticus-is mimarTspecifikuri baqteriofagebis gamoyeneba SesaZloa agreTve Stamebis

tipirebisTvis; Tumca Cveulebriv maTi gamoyeneba am mizniT arc ise xSiria

naklebi specifikurobis gamo. zomieri fagebi V. parahaemolyticus-is winaaRmdeggamoyofilia mitomicin C -s da ultraiisferi sxivebis saSualebiT [106, 142].erT-erTi kvlevis Tanaxmad V. parahaemolyticus-is izolatebis 10% Seicavs

lizogenur fags [136]. aRmoCnda, rom zomieri fagi inovirides ojaxidan- f237CarTulia umravlesi pandemuri Stamebis genomSi orf8-aTvlis CarCos saxiT daiTvleba pandemuri Stamebis markerad [88, 138]. ori sxva, mogvianebiTgamoyofili zomieri fagi miovirides ojaxidan miakuTvnes fagebis sruliad

axal - telomerul jgufs. am fagTagan erT-erTi aZlierebs lizogenurimaspirZlis ultraiisferi sxivebis mimarT mgrZnobelobas da SesaZloa amiTmniSvnelovnad amcirebdes pandemuri Stamis okeaneSi gadarCenisa da

gamravlebis unars. bolo wlebSi Catarebuli erT-erTi kvlevis Sedegad,

aRmoCnda, rom arsebobs V.parahaemolyticus-is mimarT specifikuri fagebisgarkveuli jgufi, romlebic mravldebian pandemur StamebSi yovelgvarizianis motanis gareSe da SesaZloa maTi ko-kultivireba maspinZelTan erTad,lizogenizaciis gamowvevis gareSe [150].

mniSvnelovania V.parahaemolyticus-is mimarT specifikuri fagebisgansakuTrebuli jgufi- es aris filamenturi fagebi- baqteriofagebis jgufi,romelic xasiaTdeba Cxiris an filamentis formiT, Cveulebriv erTjaWviandnm-s Seicavs da ZiriTadad, gram-uaryofiT baqteriebs ainficirebs.

filamenturi fagebi or klasad iyofa. Ppirvel klass miekuTvnebaEscherichia coli –is fagebi M13, fd, f1, If1 da Ike. Mmeore klasSi ki Sedian Pf1 da Pf3,

30

romlebic ainficirebs Pseudomonas aeruginosa-s, agreTve Xanthomonas oryzae-is fagiXf. rogorc wesi maTi dnm erTjaWviani da wriulia, magram SeuZliaT iarseboninficirebul ujredSi orjaWviani dnm-is msgavsad, romelic SeiZlebagamoiyos eqstraqromosomuli plazmidis saxiT. zogierT maTgans DSeuZliaintegrireba qromosomul dnm-Si. inficirebuli baqteria sakmaod xangrZliviperiodis ganmavlobaSi agrZelebs faguri nawilakebis warmoqmnas lizisisgareSe. Aamgvarad, logikuria davaskvnaT, rom isini mniSvnelovan rolsTamaSoben genebis horizontalur gadatanaSi msgavsad lambda fagisa,romelic verocitotoqsinis gens atarebs.

Vibrio cholerae –s filamenturi baqteriofagi CTX iTvleba qolera-toqsinisgenis klasteris (ctxAB) gadatanis meqanizmad. Mmocemuli fagi integrirdebaqromosomul dnm-Si attRS- mimagrebis saitis saSualebiT , xolo StamebSi,romelTac es saiti ar gaaCniaT, replicirebs plazmidis saxiT. vinaidan ctxABCTX fagis struqturis nawilia, mas SeuZlia ctxAB-s horizontalurad gadatanaV. cholera-s toqsigenuridan aratoqsigenur Stamebze. filamenturibaqteriofagebi VSK, fs1 da fs2 gamoyofilia V. cholerae O139-dan. VSK-s SeuZliaqromosomaSi integrireba da lizogenis warmoqmna.

V. parahaemolyticus-ის filamenturi fagebis Vf12-is da Vf33-is magaliTzeswavlobdnen kavSirs kanagava fenomensa da filamenturi fagis SemcvelobasSoris. es aris daaxloebiT 1,400 nm sigrZis da 7 nm siganis fagebi, erTjaWviani,wriuli dnm-iT, romlis zoma daaxloebiT 8.4 kb-ia. Tumca eqstraqromosomulielementebis qonasa da kanagava fenomens Soris pirdapiri kavSiri ver moinaxa,dadginda, rom Vf12 da Vf33 fagebis genomebs SeuZliaT urTierTqmedebamaspinZeli ujredis plazmiduri da qromosomuli warmoSobis dnm-ebTan daSesaZloa garkveuli roli iTamaSon V. parahaemolyticus- is filamenturi fagispaTogenobis genebis dinamikur mobilizaciaSi [40].

MmogvianebiT dadginda, rom V. parahaemolyticus-ის O3:K6 serotopis

StamebTan asocirebulia f237-baqteriofagi, romelsac garkveuli genebi dagenomuri struqtura V. cholera-s CTX-filamenturi fagis analogiuri aqvs. ctxAB-is sanacvlod f237-baqteriofags aqvs unikaluri aTvlis CarCo - ORF8, romelsacar gaaCnia homologi dnm-is sxva TanmimdevrobebTan.

Kkvlevam aCvena, rom mocemuli f237-baqteriofagi ara mxolod O3:K6serotipis StamebTan aris asocirebuli, aramed V. parahaemolyticus –is sxvaserovarebTanac, rogoricaa magaliTad, O4:K68 da O1:KUT. O3:K6 –monaTesaveStamebis umravlesoba aRmoCnda ORF8-dadebiTi. Hhibridizaciiuli kvlevebiTdadasturda, rom f237 integrirebulia baqteriul qromosomaSi. Aamgvarifenotipi SesaZloa, organizms icavs garkveuli garemo stresisgan, sanam isdaainficirebs adamians. Mmeores mxriv ki- f237–is, rogorc zrdadi pandemuripotencialis mqone komponentis roli V. parahaemolyticus –is StamebSi, SemdgomSeswavlas saWiroebs [88].

31

II.Mmasalebi da meTodebi

1. sakvlevi masala

P wylis sinjebi: pirvelad sakvlev masalad baqteriebisa dabaqteriofagebis gamoyofis mizniT viyenebdiT wylis sinjebs. sinjebsviRebdiT saqarTvelos mtknari wyalsatevebidan (lisis da kumisis tbebi,Tbilisis zRva) da Savi zRvis sxvadasxva wertilebidan (md.sufsis SesarTavi,md.Woroxis SesarTavi, mwvane koncxis sanapiro zoli da baTumis bulvari).

baqteriuli Stamebi: standartuli Stami ATCC # 17802kvlevis procesSi saqarTvelos wyliani garemodan 2006-2008 wlebSi

gamoyofili Vibrio parahaemolyticus-is 199Bbaqteriuli Stami. mocemuli Stamebiinaxeba g. eliavas saxelobis baqteriofagiis, mikrobiologiisa davirusologiis institutSi.

baqteriofagebi: kvlevis farglebSi gamoyofili V. parahaemolyticus–ismimarT specifikuri 30 baqteriofagi .

antibiotikebi: ampicilini, cefazolini, sulfamidi, furazolidoni,kanamicini, polimiqsini B, doqsiciklini, trimetoprimi, tetraciklini,qloramfenikoli, eriTromicini, imipenemi, gentamicini, streptomicini,ciprofloqsacini ( mwarmoebvelebi: Sigma, Liophilchem )

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) 1mM EDTA buferuli xsnari pH 7,4 5M NaCl- xsnari 10mM Tris 1% SDS

mikrobiologiuri areebi:baqteriebisa da baqteriofagebis kultivirebisTvis da Sesanaxad:

LB (Luria-Bertani) luria-bertanis agari da bulioni (Difco) soios agari da bulioni (“DiFco”, “Biomereux”); peptoniani tute wyali

baqteriebis saidentifikaciod:

TCBS- Tiosulfat-citrat-naRvlis marilebis- saqarozas Semcvelimyari are Vibrio spp. bateriebisaTvis.

32

Bbaqteriebis fiziologiur- bioqimiuri Tvisebebis Sesaswavlad:

T1N1 agari – tripton mariliani agari

GSA agari _3% natriumis qloridis Semcveli agari

GA agari _Jelatiniani, umarilo agari

hiu-leivsonis (HF) glukoziani are

naxSirwylebi: saqaroza, manoza, arabinoza, laqtoza

aminomJavebi: arginini, lizini, orniTini

antigenebis komerciuli nakrebi (Denka Seiken co., Niigata-ken, Japan)V. parahaemolyticus baqteriebis serotipirebisTvis;

specialuri komerciuli nakrebi (Bio-Rad Laboratories, Hercules, CA da Gen Elute,Sigma ) baqteriuli dnm-is gamosayofad;

polimeraza jaWvuri reaqciis praimerebi V. parahaemolyticus baqteriebisgenetikuri identifikaciisTvis gamoyenebuli (cxrili 1.)

cxrili 1.

samizne geni Tanmimdevroba (5’-3’) amplikonis zoma(nukleotiduri wyvili)

wyaro

VA-F F'-5’-CGAGTACAGTCACTTGAAAGC-3’737 Di Pinto et al.,

2006VA-R R-5’- CACAACAGAACTCGCGTTACC-3’

Pbaqteriebis genetikuri mravalferovnebis Sesaswavlad pulsirebadvelSi gel-eleqtroforezisTvis viyenebdiT Not I-restriqciul endonukleazas.(cxrili 2.)

cxrili 2.

restriqciuliendonukleaza

amosacnobiTanmimdevroba

gaWris adgili

Not I 5' GCGGCCGC 3' GCGGCC-- GC

3' CGCCGGCG 5' CG-- CCGGCG

33

baqteriebis virulentobis faqtorebis Sesaswavlad gamoyenebulipolimeraza jaWvuri reaqciis ( PCR) praimerebi: toxR, tdh,trh, tlh,gs,orf8 (cxrili 3.)

cxrili 3. virulentobis faqtorebis genetikuri maxasiaTeblebi

samizne geni Tanmimdevroba (5’-3’)

amplikonis zoma(nukleotiduri

wyvili) wyaro

toxR toxR-F: GTC TTC TGA CGC AAT CGT TG 368Kim et.al.(1999)

toxR-R: ATA CGA GTG GTT GCT GTC ATG

Tdh tdh-F:GTA AAG GTC TCT GAC TTT TGGAC 269Bej et.al.(1999)

tdh-R: TGG AAT AGA ACC TTC ATC TTCACC

Trh trh-F: TTG GCT TCG ATA TTT TCA GTA TCT 500Bej et.al.(1999)

trh-R: CAT AAC AAA CAT ATG CCC ATTTCC G

Tlhtlh-F: AAA GCG GAT TAT GCA GAA GCACTG 450

Bej et.al.(1999)

tlh-R: GCT ACT TTC TAG CAT TTT CTC TGC

GS gs-F:TAA TGA GGT AGA AAC A 651Catherine Cet.al. (2006)

gs-R:ACG TAA CGG GCC TAC A

Orf8 orf8-F: GTT CGC ATA CAG TTG AGG ~700Nasu et.al.,(2000)

orf8-R: AAG TAC AGC AGG AGT GAG

baqteriofagebis serologiuri Tviseebebis Sesaswavlad:

laboratoriuli cxovelebi: bocvrebi

adiuvanti (Freund’s adjuvant, Difco) antifaguri Sratebi: anti-Vpa 19KP da anti-Vpa A11

proteolizuri fermentebi:

pepsinis 1%-iani xsnari

qemotrifsinis 1%-iani xsnari

restriqciuli endonukleazebi baqteriofagebis restriqciuliprofilis Sesaswavlad (cxrili 4):

34

cxrili 4.

restriqciuliendonukleaza

amosacnobiTanmimdevroba gaWris adgili

EcoR I5'GAATTC 3' 5'--G AATTC--3'3'CTTAAG 5' 3'--CTTAA G--5'

EcoR V5'GATATC 3' 5'--GAT ATC--3'3'CTATAG 5' 3'--CTA TAG--5'

Hind III5'AAGCTT 3' 5'--A AGCTT--3'3'TTCGAA 5' 3'--TTCGA A--5'

BamH I5'GGATCC 3' 5'--G GATCC--3'3'CCTAGG 5' 3'--CCTAG G--5'

Sau3A5'GATC 3' 5'-- GATC---3'3'CTAG 5' 3'-- CTAG --5'

Pst I5' CTGCAG 3' 5'--CTGCA G--3'3' GACGTC 5' 3'--G ACGTC--5'

Sal I5' GTCGAC 3' 5'--G TCGAC--3'3' CAGCTG 5' 3'--CAGCT G--5'

Xba I5'TCTAGA 3' 5'--T CTAGA--3'3'AGATCT 5' 3'--AGATC T--5'

35

2. kvlevaSi gamoyenebuli meTodebi

2.1. Ria wyalsatevebidan wylis sinjebis aReba da damuSaveba

wylis sinjebis aReba warmoebda erTi ZiriTadi sqemis mixedviT, Tumcagamokvlevebis miznisa da specifikis gaTvaliswinebiT, sinjebis aRebissixSire da damuSavebis procedura icvleboda. sinjebs viRebdiT dRis pirvelnaxevarSi 11-15 saaTamde intervalSi. sinjebs viRebdiT wylis zedapiridan 15-30sm siRrmeze sterilur WurWelSi 1 litris moculobiT. paraleluradviniSnavdiT: aminds, temperaturas da sxva klimatur pirobebs. wylis nimuSebisanalizs vanxorcielebdiT sinjis aRebidan araumetes 4 sT-is ganmavlobaSi.

wylis fiziko-qimiuri parametrebis gansazRvra. wylis zogierT fizikur-qimiur parametrs, rogoricaa marilianoba, gamtaroba, wyalbad ionTakoncentracia, wyalSi gaxsnili Jangbadis raodenoba da myari nawilakebisraodenoba, vsazRvravdiT multiparametruli portatuli sazomi xelsawyos(YSI 556 MPS) saSualebiT.

mikrobuli dabinZurebis xarisxis gansazRvra. sakvlev wyalSi saerTomikrobuli ricxvis gansazRvrisaTvis viyenebdiT 1,8% -iani LB agaris finjnebs.sakvlevi wylis sawyisi da aTjeradi ganzavebebi 1ml raodenobiT dagvqondapetris finjanze da vamatebdiT 15-20ml gamlRval 45ºC gagrilebul LB agars.vaxdendiT inkubacias 37oC-ze 24-48 saaTis ganmavlobaSi da viTvlidiTwarmoqmnili baqteriuli koloniebis raodenobas. Aerobic Count plates (3M, aSS)petrifilmebis meSveobiT vsazRvravdiT wylis nimuSSi aerobebis saerToraodenobas. pertifilmze dagvqonda sakvlevi wylis sawyisi da aTjeradiganzaveba 1ml raodenobiT. vaxdendiT inkubacias 37oC-ze 24 saaTisganmavlobaSi. Sedegs viwerdiT Sesabamisi sqemis meSveobiT.

2.2. Vibrio-s gvaris baqteriebis gamoyofa

Vibrio-s gvaris baqteriebis gamosayofad 100 litri moculobis wyalsvatarebdiT 200mkm-ian da 64mkm-ian badeSi. 100ml gafiltrul (planqtonisganTavisufal) wyals (PFW-plankton free water) vfiltravdiT 0,45mkm-ian filtrSi,romelsac filtraciis Semdeg vaTavsebdiT 100 ml peptonian wyalSi.paralelurad 10-50 ml planqtonmocilebul wyals vfiltravdiT 0,45mkm-ianfiltrSi da filtrs vaTavsebdiT TCBS-is niadagze. Oorive SemTxvevaSiinkubacias vaxdendiT TermostatSi 370C-ze 24 saaTis ganmavlobaSi.

planqtonis fraqciidan baqteriebis gamoyofis mizniT 9ml peptonianwyals vumatebdiTa 1 ml planqtons (64mkm da 200 mkm-ian badeSi gafiltruli)da vaxdendiT inkubacias TermostatSi 370C-ze 24 saaTis ganmavlobaSi.

36

gamdidrebis meTodivibrionebis momravleba 1% peptonur wyalSi

sakvlevi masala

seleqtiuri niadagebis gamoyeneba (TCBS agari)

kultivireba 1% NaCl-is SemcvelJelatinian agarze

bioqimiurisaidentifikaciotestebiAPI 20 NE

oqsidaza testi

zrda 0%-10% NaCl-is Semcvel TxevadniadagSi

bioqimiuri testebi

Saqrebis fermentaciasaqaroza, manoza,arabinoza, laqtoza

aminomJavebisfermentaciaargininiliziniorniTini

polimerazuli jaWvuri reaqcia (pjr)

Bwylis sinjebidan vibrioebs gamoyofis da identifikaciis sqema

sur.2. Vibrio-s gvaris baqteriebis identifikaciis sqema

37

2.3. baqteriuli Stamebis biologiuri Tvisebebis Seswavla

sakvlevi Stamebis kulturalur-morfologiur Tvisebebs vswavlobdiTbaqteriebis zrdis xasiaTiT myar, Txevad da naxevradTxevad areebze.

Bbioqimiuri Tvisebebis Seswavla. izolatebi mowmdeboda marilismoxmarebaze, glukozis Jangvasa da fermentaciaze (hiu-leivsonis testi),naxSirwylebis moxmarebaze (saqaroza, laqtoza, manoza, arabinoza), citoqromoqsidazas, Jelatin dehidrolazas, lizin- da orniTin- dekarboqsilazasa daarginin-dehidrolazas Semcvelobaze. Bbioqimiur testebze dayrdnobiTidentificirebuli Vibrio parahaemolyticus-is Stamebis genetikur identifikaciaspolimeraza jaWvuri reaqciiT.

G genetikuri identifikacia. genomuri dnm-s gamosayofad viyenebdiT dnm-isgamosayof specialur komerciul nakrebs (Bio-Rad Laboratories, Hercules, CA).bioqimiurad identificirebuli V. parahaemolyticus –is izolatebis identifikaciaxdeboda polimeraza jaWvuri reaqciis (pjr) saSualebiT saxeoba-specifikuripraimerebis gamoyenebiT (cxrili 1). Ppolimeraza jaWvur reaqciaSigamoyenebul iqna baqteriuli kulturebis dnm, Taq-dnm polimeraza,Tavisufali nukleotidebis narevi (10 mmoli TiToeuli koncentracia) da oripraimeri (pirdapiri da uku- mimarTulebis). Ppjr-produqtebisgasaanalizeblad viyenebdiT eleqtroforezs 1-1,2% agarozasgelSi.Bbioqimiurad araidentificirebuli Stamebis genetikuriidentifikaciisTvis vaxdendiT 16S ribosomuli rnm-s amplificirebas,seqvenirebas da vadarebdiT NCBI da RDPII bazebs.

PFGE-analizi. baqteriuli Stamebis qromosomuli dnm-is restriqciulifragmentebis polimorfuli analizi ganxorcielda pulsirebad velSi gel-eleqtroforezis saSualebiT (PFGE - Pulsed-Field Gel Electrophoresis). genomuri dnm-is eqstraqcias vaxdendiT 1% agarozas (SeaKem-Gold Agarose. Lonza) gelSi,romelic muSavdeboda 1 mg/ml lizocimisa da 100 mkg/ml proteinaza K-sSemcveli malizirebeli buferiT (6mM Tris-acetati pH-8,0; 1M NaCL; 100mM EDTApH8,0; 0,2% deoxycholate; 0,5% N-Lauroylsarcosine).

intaqturi dnm-is fragmentirebas vaxdendiT restriqciuliendonukleaziT NotI (Neb. BioLabs), mwarmoeblis mier mowodebuli anotaciismixedviT. fermentiT damuSavebuli dnm-is fragmentebis dayofas vaxdendiTpulsirebadi eleqtroforeziT 1,2% SeaKem-Gold agarozas gelSi, Sesabamisieleqtroforezis buferiT (0,5xTris-Borate-EDTA), Gene Navigator™ System (Amersham)aparatis gamoyenebiT. eleqtroforezisaTvis gamoyenebuli iqna molekulurimasis markeri (Pulse Marker ™ 50-1,000kb, Sigma). eleqtroforezis pulsirebisparametrebi Seesabameboda: 10-50 wami, 200 volti (6 V/sm), 20 saaTi, 140C.

gelis vRebavdiT eTidiumis bromidis xsnarSi (0,5mg/ml) 20 wuTisganmavlobaSi. dnm-is fragmentebis Sefasebas vaxdendiT UV-transiluminatoris meSveobiT.

restriqciuli fragmentebis analizisaTvis gamoyenebuli iqna Freetree-threeview kompiuteruli programa. genetikuri msgavsebis amsaxveli UPGMA

38

(unweighted pair group method with arithmetic means) dendrograma konstruirebuli iqnarestriqciuli fragmentebis msgavsebis koeficientis mixedviT.

Vibrio –s gvaris sxvadasxva saxeobebis gamovlena PCR/ESI- MS –issaSualebiT. Vibrio PCR/ESI-MS gamoyenebul iqna garemos nimuSebiidan Vibrio-spaTogenuri saxeobebis gamovlenisa da identifikaciisaTvis [172]. Ddnm-isgamoyofa xdeboda wylis nimuSidan da gamoyofili dnm gamoiyeneboda 8sxvadasxva saxeobis mimarT specifikur pjr-reaqciaSi. imis gamo, romsistemis SemadgenlobaSi Sedioda 8 sxvadasxva praimeri, genomis sxvadasxvanawilis TiToeuli nimuSisTvis aRebul iqna mravalricxovani anaTvlebi.nimuSis Semadgeneli nawilebis komponentebi emateboda bazaSi Vibrio-sgaSifruli Stamebis genomebTan Sesadareblad.

virulentobis faqtorebis gansazRvra. Ggamoyofili izolatebispandemurobis markerebis da virulentobis faqtorebis gansazRvra xdebodapolimeraza-jaWvuri reaqciiT. PCR- iT Stamebi Semowmda tdh-ze, trh-ze, tlh-ze,agreTve f237-is ORF8 –Tanmimdevrobaze, pandemur jguf-specifikur toxRS -Tanmimdevrobaze(GS-PCR ), aseve TTSS1-is VP1680-sa da VP1686 –ze. 25 nanogramidnm-s Sereva xdeboda 2.5 mM dNTP –Tan, 15 mM PCR-buferTan , emateboda 5U-1 µlTAQ dnm-polimeraza da 20 µm Sesabamisi praimeri. virulentobis faqtorebisa dapandemurobis markerebis vizualizacia xdeboda 1.5%-ian agarozis gelSi,romelsac vRebavdiT eTidium bromidis xsnarSi da vnaxulobdiT UV-transiluminatorSi.

kanagava-fenomenis analizi xdeboda vagacuma agarze. sakvlev StamebsTavdapirvelad vzrdidiT myar 7% NaCl-ian sakveb agarze. 18-24 sT-ianiinkubaciis Semdeg 370C-ze calkeuli koloniebi iTeseboda Tevad areSi,romlis marilianobac aseve Seadgenda 7%-s. 370C-ze 18-24 sT-iani inkubaciisSemdeg miRebuli kulturis 10 mkl zolebis saxiT iTeseboda vagacumasagarze. Sedegebis aRricxva xdeboda 370C-ze 18-24 sT-iani inkubaciis Semdeg.Stamebi, romlebic iwvevdnen sisxlis wiTeli ujredebis β-hemolizs,iTvleboda kanagava-dadebiTad.

molekuluri mravalferovnebis gansazRvra xdeboda rep-PCR-issaSualebiT, Chokesajjawatee-s meTodis mixedviT. PCR-produqtebis gancalkevebaxdeboda 1%-ian agarozis gelSi, TAE-buferSi. Ffingerprintis Sedegebisgaanalizebas vaxdendiT GelDoc-ItTM -sistemiT( Ultra-Violet Products, Upland, CA).

serotipis gansazRvra. sakvlev Stamebs vzrdidiT 370C-ze 3% NaCl-ian LB-agarze. kulturis 24 sT-iani nazrdidan 10 mkl-iani maryuJiT xdebodakulturis resuspendireba 1 ml 0.9%-ian NaCl-is xsnarSi. miRebuli xsnarinawildeboda 2 sinjaraSi: 500-500 mkl raodenobiT. erTi sinjara 2 saaTisganmavlobaSi ixarSeboda. moxarSuli ujredebis 10 mkl-is Sereva xdeboda O-antigenis SratebTan, xolo mouxarSavis 10 mkl ereoda K-antigenis SratebTan.reaqciis Sedegad xiluli slaid-aglutinaciiT ganisazRvreboda antigeni.(Denka Seiken co., Niigata-ken, Japan).

Aantibiotikebis mimarT mgrZnobeloba SeviswavleT SerCeuli Stamebzediskis difuziis meTodiT. baqteriul kulturas vzrdidiT myar- Muller-Hinton-is agarze. gamoyenebul iqna 12 antibiotiki. Sedegs aRvricxavdiT meTodikis

39

klasikuri Sefasebis Sesabamisad: mgrZnobiare, saSualod mgrZnobiare darezistentuli baqteriuli Stamebi (S, I da R).

2.4. mikrokosmebis eqsperimenti VBNC-ujredebze

Tavdapirvelad kulturebs vzrdidiT dacerebul 3%-ian LB-agarze. 24saaTiani inkubaciis Semdeg 370C-ze, nazrds vrecxavdiT 5 ml fiziologiurixsnariT da Segvqonda sxvadasxva areebSi: LB-bulionSi, lisis tbis da zRviswylebSi. TiToeuls vaTavsebdiT macivris (40C) da oTaxis (250C)temperaturebze. mikrokosmebis TiToeuli sinjaridan drois garkveuliintervaliT vaxdendiT baqteriebis amoTesvas. Uujredebi iTvlebodaarakultivirebad mdgomareobaSi, rodesac xdeboda 0.1 uj/ml -ze naklebikultivirebadi ujredis gamovlena. Gkultivirebadi ujredebis gamovlenislimiti dgindeboda mikrokosmis 10 ml-is gafiltvriT 0.22 mkm forovnebisfiltrSi da Semdeg filtris moTavsebiT 3%-ian LB-agarze.

arakultivirebad VBNC-mdgomareobaSi gadasuli ujredebiskultivirebadSi gadayvanis mizniT mikrokosmis SigTavsis 1 ml-is inkubaciasvaxdendiT 220C-ze 24 saaTiT, meore dRes ki vTesavdiT 3%-ian LB-agarze.

2.5. baqteriofagebis gamoyofa

baqteriuli virusebis gamoyofas da kultivirebas vaxdendiT orimeTodiT:

gamdidrebis standartuli meTodiT (1) gafiltrul wylis sinjsvumatebdiT koncentrirebul bulions da baqteriuli kulturis narevs,proporciiT 10:1:1 da vaTavsebdiT TermostatSi 18-24sT 37ºC-ze. 5000 br/wTcentrifugirebis Semdeg suspenzias vfiltravdiT 0,2 mkm zomis filtrSi davawveTebdiT indikatori baqteriis gazonze. fagis lizisuri zonebisaRmosaCenad finjnebs vamowmebdiT 18-24 saaTSi.

kulturaTa supernatantebidan (fiskas meTodi)-bulionSi gazrdilkulturebs vaZvelebdiT 48-72 saaTis ganmavlobaSi. vacentrifugirebdiT 5000br/wT siCqareze. supernatantebs vawveTebdiT indikatori baqteriis gazonze.lizisur zonebs vnaxulobdiT 18-24 saaTis Semdeg.

2.6. baqteriofagebis biologiuri Tvisebebis Seswavla

fagebis sufTa xazebis misaRebad viyenebdiT orSriani agaris meTods,risTvisac Tanmimdevruli pasaJebiT vaxdendiT morfologiurad erTgvarovanikoloniebidan 5-10 koloniis SerCevas.

40

fagebis lizisuri speqtris saorientacio gansazRvras vawarmoebdiTmyar sakveb areze kreijis meTodis modifikaciis mixedviT. sacdeli fagissamuSao titri Seadgenda 1x108 naw/ml. fagis suspenziis wveTebi dagvqonda4sT-iani bulioniani kulturis gamSral zolze da 280C-ze 4-12 sT inkubaciisSemdeg aRvricxavdiT Sedegebs.

fagebis lizisis stabilobis Seswavlas vaxdendiT Txier areSi apelmanismeTodis gamoyenebiT. 4,5ml LB bulionis Semcvel sinjarebSi Segvqonda2X109ujr/ml koncentraciis baqteriuli kulturis 10mkl da vumatebdiT 0,2mkm-ian membranul filtrSi gafiltrul 0,5ml sakvlev baqteriofags iseTiganzavebebiT, rom faguri nawilakebis raodenoba pirveli sinjaridan bolosinjarisken TanmimdevrobiT Semcirebuliyo 108naw/ml-101naw/ml. vaxdendiTinkubacias 370C-ze 4-6-24-48 saaTis ganmavlobaSi. sakvlevi fagis lizisisstabilobis maxasiaTeblad aRvricxavdiT inkubaciis dros da im bologanzavebas, romel sinjaraSic ar aRiniSneboda baqteriuli kulturis zrda.

fagis zusti titris dadgenas test-Stamze vaxendiT orSriani agarismeTodiT [2].

fagis daTesvis efeqturobas (d.e.) sakvlev Stamze vsazRvravdiT rogorcfagis titris Sefardebas mocemul Stamze da sawyis, standartul baqteriulStamebze, romelzedac gamravlebuli iyo mocemuli baqteriofagi [2]

fagis negatiuri koloniis formas vadgendiT standartuliSemadgenlobis niadagze (LB niadagi) da sxvadasva temperaturaze erTi daigive test-baqteriebis gamoyenebiT orSriani agaris meTodis saSualebiT.negatiuri koloniebis analizs vaxdendiT maTi formirebis damTavrebisSemdeg (18-24sT) [2].

fagis virionis morfologias vswavlobdiT eleqtronuli mikroskopissaSualebiT. preparatebs vamzadebdiT negatiuri konstrastirebis meTodiTuranil-acetacis 2%-iani wyalxsnaris gamoyenebiT. fagis baqteriis suspenziaan koncentrati dagvqonda sakontrasto nimuSebTan erTad kolodiumis apkebze– badeebze. struqturul kvlevas vawarmoebdiT IEM-1200CX (Jeol, iaponia) da M10(Opton-Zeiss, germania) eleqtronuli mikroskopebis saSualebiT.

patron - ujredTan urTierTqmedebis fazebis Seswavla xdebodaTiToeuli parametris mixedviT, kerZod:

a) adsorbciis siCqares da intensivobas maspinZeli baqteriuli Stamisujredebze vsazRvravdiT araadsorbcirebuli fagis raodenobis mixedviTqloroformirebis meTodiT [69, 70].

b) latenturi periodis xangrZlivobas da saSualo gamosavlissidides vadgendiT Eliss-delbruck-is klasikuri meTodiT [2].

baqteriofagis mimarT rezistentuli baqteriuli mutantebis misaRebadLB – agaris Semcveli finjnebze Segvqonda 106 baqteriuli ujredi da 108

naw/ml fagi. 24sT-iani inkubaciis Semdeg gazrdili calkeuli koloniebigadagvqonda LB-agarian da fagis Semcvel finjnebze, vaxdendiT ra Semdgomiklonirebis ramdenime cikls gasufTavebis mizniT. fagisadmi gamZle TiToeulklons vamowmebdiT sxva fagebisadmi mgrZnobelobaze.

fagis efeqturi mravlobiTobis gansazRvrisaTvis vawarmoebdiTganzavebebis or serias: 1) 4,5ml bulionian sinjarebSi Segvqonda myar areze

41

gazrdili baqteriuli kulturis Camonarecxi iseTi ganzavebiT, rom mikrobTaricxvi TanmimdevrobiT Semcirebuliyo 108ujr/ml-101ujr/ml. sinjarebsvamatebdiT 0,2 mkm forovnebis membranul filtrSi gafiltrul sakvlevibaqteriofagis 0,5ml-s (108naw/ml). 2) igive moculobis bulionian sinjarebSiSegvqona 0,2mkm forovnebis membranul filtrSi gafiltruli sakvlevibaqteriofagis iseTi ganzaveba, rom faguri nawilakebis ricxviTanmimdevrobiT Semcirebuliyo 108naw/ml-101naw/ml da TiToeul sinjarasvamatebdiT dacerebul agarze gazrdili baqteriuli kulturis Camonarecxis10mkl-s. vaxdendiT inkubacias TermostatSi 18 saaTis ganmavlobaSi 370C-ze, risSemdegac yvela sinjaraSi vamowmebdiT sakvlevi fagis titrs graciasmeTodiT. efeqtur mravlobiTobad miviCnebdiT sakvlevi baqteriofagis dabaqteriuli kulturis im Sefardebas, romelSic baqteriofagi maqsimaluradgamravlda. (mag. 1 faguri nawilaki : 100 baqteriuli ujredi).

fagis serologiuri Tvisebebs vswavlobdiT neitralizaciis cdebSispecifiuri antifaguri Sratebis meSveobiT. antifaguri Sratis misaRebad 2-3kg wonis bocvrebis kanqveS Segvyavda sakvlevi baqteriofagis (1-5X1011 naw/ml)da froindis adiuvandtis narevi (1:1). 1 Tvis Semdeg vimeorebdiT aRniSnulproceduras adiuvantis gareSe. 10 dRiani intervalis Semdeg bocversvuRebdiT sisxls gulidan; gamovyofdiT sisxlis Srats, vfiltravdiT davinaxavdiT – 200C. vsazRvravdiT neitralizaciis konstantas homologiursistemaSi.

fagis serologiur naTesaobas vswavlobdiT fagis neitralizaciisjvaredina reaqciis Catarebis gziT sxvadasxva specifiurobis antifaguriSratis gamoyenebiT. fagi – antifaguri Sratis TiToeuli wyvilis SemTxvevaSivsazRvravdiT neitralizaciis procents da konstantas [2].

2.7. fagebis mgrZnobeloba fizikuri da qimiuri faqtorebis mimarT

baqteriofagebis inaqtivacia UV sxivebiT 1-51010 naw/ml titris mqonefagis suspenzias 1:100 vanzavebdiT 0,1 M fosfatur buferSi, vasxamdiT petrisTasSi 5ml-is odenobiT da vasxivebdiT ultraiisferi naTuriT 35sm-isdistanciaze. sinjebs viRebdiT 10 wm-is intervaliT da vsazRravdiTsicocxlisunariani faguri nawilakebis ricxvs orSriani agaris meTodiT.

baqteriofagebis Termostabilobis Seswavla. sakvlev fagebs vanzavebdiT103 naw/ml Semcvelobamde da vaTavsebdiT wylis abazanaSi sxvadasxvatemperaturaze (400C, 500C, 600C, 700C, 800C) 30 wuTiT. kontrolad viRebdiT fagiansinjaras igive ganzavebiTa da moculobiT, romelsac vaTavsebdiT 370C-ze. 30wuTis Semdeg sakvlevi da sakontrolo sinjarebidan gadmogvqonda fagis 0,1ml da vtitravdiT gracias meTodiT. kritikuli temperaturis dadgenisSemdeg eqsperiments vimeorebdiT 5 wuTis intervaliT kritikultemperaturaze.

mgrZnobeloba pH-is sxvadasxva mniSvnelobebis mimarT. fagze garemoaris pH-is gavlenas vswavlobdiT Semdegnairad: 1X108 naw/ml titris mqone

42

fagis suspenzia Segvqonda specialur fagur buferSi (SM-buferi) (pH 2,0 -10,0)da vaxdendiT inkubacias 1 sT-is ganmavlobaSi. vsazRvravdiTsicocxlisunariani nawilakebis raodenobas.

baqteriofagis stabilobas Senaxvisas vsazRvravdiT sxvadasxva areSi:LB-bulioni pH7,4; LB-bulioni qloroformiT pH7,4; onkanis wyali; gamoxdiliwyali; fiziologiuri xsnari; 0,05M fosfaturi buferi pH7,4; zRvis wyali(Savi zRvis wyali); mtknari wyali (kus da lisis tbis wyali); dabuferebulipeptonuri wyali pH7,4; dabuferebuli peptonuri wyali pH8,0; soiosbulioni pH7,4; λ bulioni pH7,4; faguri buferi pH7,4. sakvlevi fagebi titriT109naw/ml gavfiltreT 0,2mkm-ian membranul filtrSi da SevinaxeT macivarSi40C-ze erTi wlis manZilze. fagis stabilobas vamowmebdiT 1 kviris, 2 kviris,da Semdeg yovelTviurad gracias meTodiT.

fagebis stabilobis Sesaswavlad -800C –ze sakvlevi fagebi titriT 108

naw/ml gavfiltreT 0,2mkm-ian filtrSi, TanafardobiT 1:1 SevurieT 50%glicerins da SevinaxeT -800C-ze macivarSi erTi wliT. fagis stabilobasvamowmebdiT yovelTviurad gracias meTodiT.

osmosuri Sokis moqmedebas vswavlobdiT andersonis meTodiT dabali damaRali ionuri Zalis mqone xsnarebSi baqteriofagebis inokulaciis gziT [9].

2.8. fagis gamravleba da koncentrireba

fagis koncentratebis miReba xdeboda Semdegi meTodebis gamoyenebiT:myar areze koncentrats vRebulobdiT standartuli meTodiT [2],

romelic gulisxmobs Txeli agaris fenaSi maRalkoncentrirebulibaqteriuli suspenziis fagoliziss da minimaluri moculobis Txevad areSifagis Semdgom eqstragirebas. fagur koncentrants vamzadebdiT 10-20 finjanze:0,7% LB agarSi Segvqonda baqteriebi 1-5X108 uj/ml da baqteriofagbi 1X105

naw/ml saboloo koncentraciiT. inkubacias vaxdendiT 280C-ze 18-24 sT,vxsnidiT agaris zeda fenas SpateliT, vacentrifugirebdiT 5000 br/wT 30 wTda vamowmebdiT titrs gracias meTodiT. miRebuli fagolizatis titrisaSualod Seadgenda 1-5X1010-1X1011 naw/ml.

Txier areSi fagolizatis miRebisas kulturas vzrdidiT bulionTankolbebSi sanjRrevelaze 370C-ze logariTmuli fazamde (1-5X108 uj/ml). Semdegxdeboda fagis suspenziis damateba sxvadasxva mravlobiTobiT (1:10, 1:25, 1:50,1:100) da vagrZelebdiT kultivirebas Semcirebuli aeraciiT. 280C-ze 1,5-2,0 sT-is Semdeg xdeboda baqteriebis sruli lizisi. qloroformiT (1:5) damuSavebisSemdeg, lizats vtovebdiT 14-16 sT macivarSi sruli lizisisTvis. lizatsvacentrifugirebdiT 3000 br/wT 20 wT (45 wT) da vadgendiT titrs graciasmeTodiT. miRebuli fagolizatis titri aRwevda 1-5X1010 naw/ml koncentracias[2].

koncentrirebisa da baqteriofagis Semdgomi gawmendis mizniT vaxdenTdiferencialur centrifugirebas 20000br/wT (35000g) +40C-ze, 1,5-3sT-isganmavlobaSi, preparatis moculobidan gamomdinare (centrifuga Bekman Allegra64R). naleqs vxsnidiT buferul xsnarSi 1 SSC (pH 7,0) an PBS (pH 7,2), vdgamdiT

43

magnitur sarevelaze +40C-ze 3-4 sT-is ganmavlobaSi da SemdegvacentrifugirebdiT 3500g baqteriuli narCenebis mocilebis mizniT [2].

2.9. baqteriofagebis genetikuri Tvisebebis Seswavla

faguri dnm-is gamoyofas vaxdendiT fenol/qloroformiTdeproteinizaciis meTodiT. baqteriofagis gawmendil koncentrats(5x1011naw/ml) vumatebdiT sawyisi moculobis 1/10 lizisur bufers ( 1% SDS,10mM EDTA), vayovnebdiT oTaxis temperaturaze 10 wuTis ganmavlobaSi. SemdegvaxdendiT sametapian deproteinizacias fenol/qloroformiT. narevsvumatebdiT fenols (pH 8.0) TanafardobiT 1:1, vanjRrevdiT davacentrifugebdiT 15000br/wT 10wT. Semdgom etapze vumatebdiT fenol-qloroforms 1:1 SefardebiT, kvlav vanjRrevdiT da vacentrifugirebdiT 15000br/wT 10wT. Bbolos ki- izoamil-qloroforms 1:24 TanafardobiT, vanjRrevdiTda vatrialebdiT igive reJimze. dnm-s gamoleqvas vaxdendiT 5M NaCl daizopropanoliT. narevs vacentrifugebdiT igive reJimze, supernatantsvamatebdiT 70% eTanols (2:1). narevis centrifugirebis Semdeg daleqil dnm-ski vaSrobdiT oTaxis temperaturaze, vukeTebdiT resuspendirebas TE-buferSida vinaxavdiT macivarSi Semdgomi procedurebisaTvis [2].

faguri denm-is restriqciuli analizis mizniT gamoyofil fagur dnm-svamuSavebdiTY sxvadasxva restriqciuli enzimebiT (EcoRI, EcoRV, HindIII, BamHI, XhoIda sxva) mwarmoeblis mier mowodebul pirobebSi. Semdeg sinjebi Segvqonda0,8% agarozis gelSi da vawarmoebdiT eleqtroforezs 1,5-2 sT-isganmavlobaSi. markerul preparatad viyenebdiT HindIII fermentiT daWril λfagis dnm-s (Sigma). gels vRebavdiT eTidium bromidiT (0,5mkg/ml)sanjRrevelaze 45 wuTis ganmavlobaSi. Sedegs vnaxulobdiT UV-iluminatorze.miRebuli restriqiciuli fragmentebis molekulur masas vsazRvravdiTsakalibracio mrudis saSualebiT.

44

III. miRebuli Sedegebi

1. Vibrio parahaemolyticus-is gamoyofa da ekologia

V.parahaemolyticus–is da Sesabamisi baqteriofagebis monitoringixorcieldeboda 2006-2008 wlebSi, saqarTveloSi, Savi zRvis sanapiros oTxsxvadasxva wertilSi da Tbilisis SemogarenSi arsebul wyalsatevebSi(Tbilisis zRva, lisis tba, kumisis tba), rogorc wylidan, aseve fito- dazooplanqtonidan (sur.3).

Cveni gamokvlevis farglebSi saqarTvelos wyalsatevebidan gamoiyo

V.parahaemolyticus –is 183 izolati. V.parahaemolyticus –is gamoyofis sixSire Vibrio-s gvaris sxva halofilur saxeobebTan SedarebiT maRali iyo da gamoyofiliizolatebis daaxloebiT 50% warmoadgenda V.parahaemolyticus –s. (sur.4)

sur. 3. Mmonitoringis saitebi

45

sur. 4. halofiluri vibrionebis gamoyofis sixSire

wylis sinjebSi V.parahaemolyticus –is gamovlena sxvadasxva biotur daabiotur ekologiur faqtorzea damokidebuli, rogoricaa wylis temperatura,marilianoba, gaxsnili Jangbadis raodenoba, planqtonis Semcveloba da a.S.[71] (cxrili 5).

cxrili 5. Eekologiuri faqtorebis gavlena V.parahaemolyticus-is gamoyofaze

Eekologiuri faqtorimarilianoba

0/00

wylistemperatura

0C pH

gaxsniliJangbadisraodenoba(mg/l)

sinjSi V.parahaemolyticus-isSemcveloba + - + - + - + -

PplanqtonisaganTavisufali wyali 12,9 12 22,8 16,5 7,7 7,8 4,4 4,3

Ffitoplanqtoni 13,6 12,3 25,4 17,3 7,9 7,8 4,1 4,4

zooplanqtoni 12,8 12,3 24,6 17 7,6 7,8 4,4 4,3

46

Catarebulma monitoringma aCvena, rom wylis temperaturaV.parahaemolyticus-is gamoyofis mniSvnelovani ekologiuri faqtoria, rogorcplanqtonmocilebuli wylis, aseve fito- da zooplanqtonebis SemTxvevaSi.mocemuli Sedegi SeiZleba imiT aixsnas, rom Cvens mier Catarebulimonitoringis manZilze temperaturisagan gansxvavebiT, wylis marilianoba,masSi gaxsnili Jangbadis raodenoba da mJavianoba, wylis ekosistemis,metnaklebad, ucvlel maxasiaTeblebs warmoadgenda. temperaturazedamokidebuleba gansazRvravs V.parahaemolyticus –is gamoyofis sezonur xasiaTs.V.parahaemolyticus-is Stamebis umravlesoba 100 izolati ( 54.7%) gamoiyoSemodgomis TveebSi, 69 (37.7%) –zafxulSi, xolo 14 izolati (7.6%)-gazafxulze. V.parahaemolyticus-is gamoyofis piki dafiqsirda seqtemberSi - 55izolati (30.1%) ( sur. 3).

sur. 5. V.parahaemolyticus-is da specifikuri baqteriofagebis gamoyofisdinamika

garda temperaturisa mniSvnelovani maxasiTebelia wylis marilianoba,ramdenadac V.parahaemolyticus –is Stamebis umravlesoba- 180 izolati (98.3%)gamoiyo Savi zRvis sxvadasxva wertilidan, sadac mtknar wyalsacavebTanSedarebiT marilianoba maRalia. Mmxolod 3 izolati ( 1.7%) gamoiyo mtknariwyalsatevebidan, maTgan 2- mdinare mtkvris, 1 ki- kus tbis wylidan. amasTan

47

Savi zRvis TiToeul saitSi gamoyofis sixSire iyo gansxvavebuli (sur. 6).yvelaze maRali sixSire dafiqsirda mwvane koncxSi, xolo yvelaze dabali-Woroxis estuariaSi. Stamebis gamoyofis es kanonzomierebac dakavSirebuliamocemul wertilebSi marilis gansxvavebul SemcvelobasTan. V.parahaemolyticus,rogorc halofiluri saxeoba, Warbobs swored im adgilebSi, sadacmarilianoba ufro maRalia.

sur. 6. V.parahaemolyticus-is izolatebis gamoyofa

rac Seexeba planqtonis Semcvelobis gavlenas, V.parahaemolyticus –isizolatebis sakmaod didi nawili- 50 baqteriuli Stami (27%) gamoiyoplanqtonidan, maT Soris 30 ( 16.4%) zooplanqtonidan, xolo 20 ( 10.9%) –fitoplanqtonidan.

48

2.Vibrio parahaemolyticus-is baqteriebis gamovlena pirdapiri deteqciis meTodiT

PCR/ESI-MS gamoyenebiT wylis nimuSebidan Vibrio-s sxva paTogenursaxeobebTan erTad moxda Vibrio parahaemolyticus-is gamovlena da identifikacia[172]. V.parahaemolyticus –is dnm aRmoCnda rogorc zRvis aseve tbis wylebissinjebSi, maSin rodesac V.parahaemolyticus ar amoTesila tbis wylis arc erTinimuSidan. sainteresoa, rom es monacemebi dadebiT korelaciaSiaV.parahaemolyticus –is mimarT specifikuri baqteriofagebis gamoyofis

SedegebTan ( სურ. 7.). es SeiZleba aixsnas, erTis mxriv, mtknari wylishidrobiontebiT zRvis baqteriis gadatanis unariT, da aseve, am baqteriispersistirebiT sicocxlisunarian, magram arakultivirebad formaSi (ix. TaviIII.4 ).

sur. 7. V.parahaemolyticus-is da Sesabamisi baqteriofagebis deteqcia

49

3. baqteriuli Stamebis biologiuri Tvisebebis Seswavla

sakvlevi Stamebis kulturalur-morfologiur Tvisebebs vswavlobdiTbaqteriebis zrdis xasiaTiT rogorc zogad, aseve specifikur( TCBS); myar,Txevad da naxevradTxevad areebze (sur. 8).

myar LB-niadagze Vibrio parahaemolyticus-is izolatebi warmoqmnian 2-5 mmzomis lorwovan koloniebs. Ppeptonian wyalSi izrdebian zedapirulad, apkissaxiT. TCBS-agarze Vibrio parahaemolyticus-is izolatebi, rogorc wesi,warmoqmnian mwvane feris koloniebs, Tumca aRmoCnda, rom izolatebisdaaxloebiT 13% TCBS-agarze izrdeboda yviTlad, rac miuTiTebs ambaqteriebis saqarozis fermentaciis unarze.

a) b)

g) d)

sur. 8. sxvadasxva baqteriul areze gazrdili V.parahaemolyticus-is Stamebia-b) TCBS-agari g) sisxliani agari d) LB-agari

50

SeviswavleT Vibrio parahaemolyticus-is zogierTi izolatis morfologiaM10 (Opton-Zeiss, germania) eleqtronuli mikroskopiis saSualebiT (sur. 9).

Vibrio parahaemolyticus-is baqteriebi moxrili Cxiris formisaa, zomiTsigrZeSi daaxloebiT 1-1,5 mkm-ia, xolo siganeSi 0,4-0,8 mkm.

a) b)

sur. 9. V.parahaemolyticus-is Stamebis eleqtronuli mikroskopiaa) V.parahaemolyticus 372 Bb) V.parahaemolyticus 548

bioqimiuri Tvisebebis Seswavlas vawarmoebdiT specializirebul sakvebareebze: kerZod: proteolizur aqtivobas vswavlobdiT Jelatinis gajirjvebismixedviT GSA da GA agarze. Vibrio-s gvaris baqteriebs aqvT rogorc glukozisfermentaciis, aseve misi daJangvis unari. vswavlobdiT agreTve sxvadasxvakoncentraciis natriumis qloridis Semcvel Txevad niadagSi mikrobTazrdas. V.parahaemolyticus-is izolatebi danarCeni saxeobebis msgavsadaucileblad izrdebian 1%-ian marilianobis niadagSi. Tumca, rogorchalofilur saxeobebs, ar axasiaTebT umarilo areSi zrda. samagierodizrdebian 6%-ian da zogierT SemTxvevaSi 8%-ian marilianobis areSic ki.SeviswavleT agreTve V.parahaemolyticus-is izolatebis mier naxSirwylebisa daaminomJavebis fermentacia. (cxrili 6.)

51

cxrili 6. V.parahaemolyticus-is zogierTi Stamis bioqimiuri Tvisebebi

Stami

Stamebis maxasiaTeblebi

TCB

S

citoqr

om oqs

idaz

a

Jelat

in hid

rolaz

a

citrat

is moxmareb

a

zrda umarilo areS

i

zrda 6%

mar

iliano

bis areS

i

zrda 8%

mar

iliano

bis

areS

i

gluko

zis

Jangva

gluko

zis

fer

mentac

ia

saqar

ozis

fer

ment

acia

mano

zis

fer

ment

acia

laqtozis

fer

ment

acia

arab

inozis

fer

mentac

ia

liz

in d

ekar

boqs

ilaz

a

argin

in d

ehid

rolaz

a

orni

Tin

dekar

boqs

ilaz

a

V.parahaemolyticus S.W.7 z + + - - + + + + - + - [+] + - +

V.parahaemolyticus 315 z + + - - + + + + - + - - + - +

V.parahaemolyticus 328 z + + - - + - + + - + - + + - +V.parahaemolyticus 372 z + + - - + + + + - [+] [+] [+] + - +

V.parahaemolyticus 401 Ym + + [+] - + + + + + + + + + - +

V.parahaemolyticus 428 z + + - - + + + + - + - + + - +V.parahaemolyticus 957 z + + - - + + + + - + - [+] + - +

V.parahaemolyticus 970 z + + - - + + + + - + - [+] + - +

V.parahaemolyticus 974 Yy + + - - + + + + + [+] - - + - [+]V.parahaemolyticus 641 Ym + + - - + + + + + + - + + - +

V.parahaemolyticus 1025 z + + - - + + + + - + - + + - +

V.parahaemolyticus 3025 z + + - - + + + + - + - + + - +

V.parahaemolyticus 2335 Mm + + - - + [+] + + - + - + + - +

V.parahaemolyticus 2344 Mm + + - + - [+] + + + + - + + - +

V.parahaemolyticus 2358 Mm + + - + - + + + + + - + + - +

z - zeTisxilisferim - mwvaney - yviTeli+ - dadebiTi_ - uaryofiTi[+] – arazusti reaqcia

52

genetikuri identifikacia. Bbaqtriuli izolatebis bioqimiuriidentificiis sizustis gadasamowmeblad genetikur doneze viyenebdiTpolimeraza jaWvur reaqcias (PCR) da saxeoba-specifikur praimerebs(cxrili 1). Stamebis 98%-Tvis bioqimiuri identifikacia aRmoCnda zusti.(sur.10)

sur. 10. Pkolagenaza specifikuri polimeraza jaWvuri reaqcia

1. PCR-markeri; 2. V.para SW7; 3.V.para SW8; 4.V.para 315; 5.V.para 552; 6. V.para 548; 7.V.para1025; 8. V.para 1025; 9. V.para 528; 10. V.para 563; 11. V.para 651; 12.V.para 652; 13.V.para 653;14.V.para 654; 15.V.para 328; 16.V.para 372; 17.V.para 932; 18. V.para 950; 19.Vpara 905; 20.V.para 957;21.V.para 966; 22.V.para 1010; 23. V.para 970; 24.V.para1019; 25.V.para1023; 26. PCR-markeri.

serotipis gansazRvra. serotipireba antigenis gansazRvris mizniTganxorcielda slaid-aglutinaciis meTodiT V.parahemolyticus–is 81 izolatze(cxrili 7). aRniSnul Stamebs Soris gamovlinda 9 serojgufis da 27sxvadasxva serotipis warmomadgenlebi aratipirebuli izolatebis CaTvliT.maTi sakmaod didi nawilis (24 izolati) serotipireba ver moxerxda.Semowmebuli Stamebis 25% da tipirebuli Stamebis 35% miekuTvneboda O3-serotips. foTsa da qobuleTSi mocemuli serotipis arc erTi izolati argamoyofila, maSin rodesac aq aRiniSneboda O8-serotipis dominantoba(gamoyofili Stamebis 60%). O3-serotipi kvebiTi infeqciebis gamomwveviyvelaze xSiri warmomadgenelia. Mmas miekuTvneba pandemuri kloni- O3:K6 [137],romlis mier araerTi globaluri infeqcia iqna gamowveuli ukanasknelwlebSi. Cvens kvlevaSi arcerTi Stami ar aRmoCnda mocemuli serotipis mqone,Tumca mwvane koncxidan gamoyofili erT-erTi Stami tipirebul iqna, rogorc01:KUT , rac O3:K6-is pandemuri klonis serovariants warmoadgens. Mmocemulibaqteriuli izolati tdh- da trh-uaryofiTi iyo, Tumca gaaCnda O3:K-is GS-PCR

53

markeri. pandemuri Stamis markerTan erTad mocemuli serotipis arseboba SavzRvaSi pandemuri Stamebis an maTi variantebis arsebobaze miuTiTebs. Uufrometic, klinikurad mniSvnelovani O2:K28, O3:K31, O3:KUT, O4:KUT-serotipebisgamoyofis sixSre regionSi klinikurad mniSvnelovani Stamebis arsebobasadasturebs.

virulentobis faqtorebis Seswavla ganxorcielda V.parahemolyticus-isgenetikurad identificirebul 81 Stamze. PCR-is saSualebiT moxda mocemuliStamebis Semowmeba tdh, trh, tlh, filamenturi fagis f237-is ORF8, pandemuri jguf-specifikuri toxRS-is da TTSS1-is VP1680 da VP1686-genebis mixedviT. A

arc erTi Stami ar aRmoCnda tdh-genis matarebeli. Seswavlili StamebiagreTve ar aeqspresirebda tdh –is monaTesave trh-is gens. rac SeexebaTermolabilur hemolizins, 3 Stamis garda, yvela Seicavda mis makodirebelgens. am Stamebidan ori iyo O2:K28-serotipis, erTi ki aratipirebadi. arcerTiStami ar Seicavda filamenturi fagis f237-is ORF8- gens. 7 izolati Seicavdapandemuri jguf-specifikur toxRS-markers, risi dadasturebac moxda agarozisgelze 651 nukleotiduri wyvilis sididis bendis vizualizaciiT. TiToeuliStami sxvadasxva serotips miekuTvneboda, maT Soris iyo: O1:KUT, O3:KUT,O3:K31, O3:K33, O3:K65, UT. gansakuTrebiT mniSvnelovania O1:KUT- is arseboba,romelic erT-erTi pandemiis dros iqna dafiqsirebuli. 6 maTgani UT-serotipsmiekuTvneboda, 5- O3:K31-s, 1 ki- O1:K58-s.

78 Stami (96%) dadebiTi aRmoCnda TTSS1-mesame tipis sekreciuli sistemisefeqtoruli proteinebis- VP1680 da VP1686-is arsebobis mixedviT. erTi StamiVP1686-is mixedviT aRoCnda dadebiTi, Tumca uaryofiTi iyo VP1680-is da sxvavirulentobis faqtorebisa da pandemuri markerebis mixedviT da verganxorcielda maTi tipireba. 2 Stami, romlebic rogorc VP1680-is, aseveVP1686-is arsebobis mixedviT, uaryofebi aRmoCndnen, da agreTve ardafiqsirda maTSi arc erTi virulentobis genis eqspresia, tipirebul iqnarogorc O2:K28 da O10:K61.

Termolabiluri hemolizinis da mesame tipis sekretoruli sistemisefeqtoruli proteinebis arseboba regionSi paTogenuri Stamebis arsebobisSesaZleblobaze miuTiTebs. zemoTnaxsenebi TvisebebiT xasiaTdebodaSemowmebuli Stamebis 96%, rac SesaZleblobas iZleva davaskvnaT, rom SavizRvis V.parahaemolyticus-is populaciaSi virulentobis faqtorebi farTodaagavrcelebuli. Ggarda amisa, tlh- da TTSS1-dadebiT Stamebs Soris O3:K6-pandemuri klonis serovariantis arseboba saqarTveloSi Savi zRvissanapiroze sazogadoebrivi jandacvisTvis sayuradRebo movlenaa, ramdenadacaseTi tipis izolatebi infeqciis wyaroa daziaebuli kanis (WrilobiTiinfeqciebi) an zRvis produqtebis dabinZurebis (kvebiTi infeqciebi)SemTxvevaSi.

hemolizis unaris-igive kanagava-fenomenis analizi xdeboda vagacumasisxlian-agarze. ACvens mier gamoyofili arc erTi Stami ar aRmoCnda kanagava-dadebiTi. Mmocemuli Sedegi dadasturebul iqna genetikuradac, virulentobisfaqtorebis Seswavlisas arc erT maTgans ar aRmoaCnda tdh-geni.

54

cxrili 7. V.parahaemolyticus-is Stamebis serologiuri da genetikurimaxasiaTeblebi

Ser

otipi

izolat

ebis

rao

denoba

Ter

mostab

iluri

pirdapir

i hemo

liz

ini

(tdh

)

Ter

molab

iluri

hemo

liz

ini

(tlh

)

Ter

mostab

iluri

pirdapir

i hemo

liz

inis

monaTes

ave hemo

liz

ini

(trh

)

mesame tipis

sekrec

iuli

sis

temis

cil

aVP

1680

mesame tipis

sekrec

iuli

sis

temis

cil

aVP

1686

fil

amenturi

fag

is

ORF

8-geni

pand

emuri

kloni

s j

guf-

specif

ikuri

pjr-mar

keri

O1:K32 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0O1:K58 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0O1:KUT 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 1 (100%)O2:K28 8 0 7(87.5%) 0 7 (87.5%) 7 (87.5%) 0 0O2:KUT 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0O3:K5 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0

O3:K31 7 0 7( 100%) 0 7( 100%) 7( 100%) 0 1 (1)O3:K33 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 1 (100%)O3:K51 2 0 2 (100%) 0 2 (100%) 2 (100%) 0 0O3:K65 2 0 2 (100%) 0 2 (100%) 2 (100%) 0 1 (50%)O3:KUT 7 0 7( 100%) 0 7( 100%) 7( 100%) 0 1 (14%)O4:K12 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0O4:K34 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0O4:K37 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0O4:KUT 7 0 7( 100%) 0 7( 100%) 7( 100%) 0 0O5:K68 2 0 2 (100%) 0 2 (100%) 2 (100%) 0 0O5:KUT 2 0 2 (100%) 0 2 (100%) 2 (100%) 0 0O6:KUT 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0O8:KUT 2 0 2 (100%) 0 2 (100%) 2 (100%) 0 0O10:K61 1 0 0 0 0 0 0 0O10:K60 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0O10:KUT 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0O11:KUT 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0OUT:K27 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0OUT:K33 2 0 2 (100%) 0 2 (100%) 2 (100%) 0 1 (50%)OUT:K52 1 0 1( 100%) 0 1( 100%) 1( 100%) 0 0

UT 24 0 23(95.8%) 0 23 (95.8%) 24 (100%) 0 1 (4%)sul: 81 0 78 0 78 79 0 7

55

molekuluri mravalferovnebis Seswavla. 45 SemTxveviTad SerCeulbaqteriul Stamze gakeTda rep-PCR (sur. 11). Sedegebis gaanalizeba moxdabendebis ganawilebis mixedviT GelDoc-ItTM –sistemiT (Ultra-Violet Products, Upland,CA). arsebuli suraTis safuZvelze aigo dendrograma (sur. 12)GgamovlindaSavi zRvidan gamoyofili Stamebis klonuri mravalferovneba, racSesabamisobaSia serotipebisa da serojgufebis mravalferovnebasTan.

sur. 11. V.parahaemolyticus-is izolatebis Rep-PCR.

1. Rep-PCR-markeri; 2. V.para SW7; 3.V.para SW8; 4.V.para 315; 5.V.para 552; 6. V.para 548;7. V.para1025; 8. V.para 1025; 9. V.para 528; 10. V.para 563; 11. V.para 651; 12.V.para 652; 13.V.para 653;14.V.para 654; 15.V.para 328; 16.V.para 372; 17.V.para 932; 18. V.para 950; 19.Vpara 905; 20.V.para 957;21.V.para 966; 22.V.para 1010; 23. V.para 970; 24.V.para1019; 25.V.para1023; 26. Rep-PCR-markeri.

56

sur. 12 V.parahaemolyticus-is SemTxveviTad SerCeuli zogierTi Stamis Rep-PCR-is dendrograma

57

Stamebis genotipirebisaTvis da molekuluri mravalferovnebisSesaswavlad alternatiul meTodad gamoviyeneT aseve pulsirebad gelSi gel-eleqtroforezis meTodi (PFGE). Ees meTodi farTod aris aprobirebulimravali baqteriuli agentis diferencirebisaTvis.

SemTxveviTad SerCeul 73 baqteriul Stamze gakeTebulma analizma aCvenaStamebis sakmaod didi mravalferovneba, rac zogadad damaxasiaTebeliagaremodan gamoyofili StamebisTvis. (sur. 13).

0.1

V.p. 1025

V.p. 970

V.p. 328

V.p. 1010

V.p. 548

V.p. 315

V.p. 654

V.p. 552

V.p.372

Sw 7

V.p. 1019

V.p. 653

Sw 8

V.p. 651

V.p. 652

V.p. 528

V.p. 563

V.p. 905

V.p. 950

V.p. 1023

V.p. 957

V.p. 966

V.p. 932

V.p. 428

V.p.1013

sur. 13. V.parahaempolytricus -is izolatebis PFGE- dendrograma

58

antibiotikebisadmi mgrZnobeloba. SerCeuli Stamebi Semowmda agreTveantibiotikebis mimarT mgrZnobelobaze diskebis difuziis meTodiT myar areze(Muller-Hinton-s agari). gamoyenebul iqneba 12 antibiotiki. Sedegi Caiwera ammeTodis klasikuri Sefasebis Sesabamisad: mgrZnobiare, saSualod mgrZnobiareda rezistentuli (S, I da R). Seswavlili Stamebis daaxloebiT 40%rezistentuli aRmoCnda yvela antibiotikis mimarT. antibiotikorezistentulibaqteriebis maRali procentuli maCvenebeli dakavSirebulia imasTan, romCvens mier Seswavlili Stamebi garemodan gamoyofili, arapaTogenuriizolatebia, romlebic, rogorc wesi, antibiotikebis mimarT naklebimgrZnobelobiT xasiaTdebian. yvelaze maRali mgrZnobeloba dafiqsirdaciprofloqsacinis, streptomicinisa da gentamicinis mimarT. Aamave drosStamebis umravlesobam gamoavlina rezistentoba ampicilinis mimarT. (sur. 14)

sur.14. V.parahaemolyticus-is antibiotikebis mimarT mgrZnobiareizolatebis %-uli raodenoba

59

Bbaqteriofagebis mimarT mgrZnobeloba. Ggamoyofili baqteriuliizolatebi Semowmda kvlevis farglebSi gamoyofili baqteriofagebis mimarTmgrZnobelobaze. 87 Stami (47.5%) mgrZnobiare aRmoCnda mocemuli fagebismimarT. fagomgrZnobelobis profilis mixediT baqteriuli Stamebis Sedarebisda maTi dajgufebis mizniT avageT dendrograma ( sur. 14).

sainteresoa agreTve fagomgrZnobelobisa da Stamebis serologiurimaxasiaTeblebis urTierTkavSiri (cxrili 8). fagebis mimarT yvelaze maRalimgrZnobelobiT xasiaTdebian O3:K51, O3:K65 da O10:KUT serotipebis Stamebi.rac Seexeba O3:K6-is pandemuri klonis serovariants- 01:KUT ( V.parahaemolyticus1019) - is, mis mimarT aqtiuri aRmoCnda mxolod erTi baqteriofagi- Vpa A11.

cxrili. 8. V.parahaemolyticus-is sxvadasxva serotipis StamebisfagomgrZnobeloba

aqtiu

ri

baqter

iofag

ebi

ser

otipi

O1:

K58

O1:

KUT

O2:

K28

O2:

KUT

O3:

K51

O3:

K65

O3:

KUT

O4:

K12

O5:

K68

O5:

KUT

O8:

KUT

O10

:K60

O10

:KU

T

UT

OU

T:K2

7

OU

T:K3

3

rao

denoba

1 1 3 2 19 8 3 1 2 2 2 5 14 3 4 1

%

2, 43 2, 43 7, 32 4, 88 46,3 19,5 7, 32 2, 43 4, 88 4, 88 4, 88 12, 2 34,2 7, 32 9, 76 2, 43

60

sur. 15. V.parahaemolyticus-is Stamebis dendrograma baqteriofagebis mimarTmgrZnobelobis mixedviT

61

4. arakultivirebadi, magram sicocxlisunariani mdgomareoba ( VBNC)

V.parahaempolytricus –is baqteriebis gadasvlis unars vswavlobdiTarasakmarisi sakvebis Semcvelobis da araxelsayreli temperaturuli reJimisSeqmniT. viyenebdiT lisisa da Savi zRvis wylebs, Sesadareblad ki mdidarsakveb ares LB-niadags. araxelsayreli temperaturis magaliTad viyenebdiT40C-s, kontrolad ki optimalur temperaturas- 250C-s.Uujredebi iTvlebodaarakultivirebad mdgomareobaSi, rodesac xdeboda 0.1 uj/ml -ze naklebikultivirebadi ujredis gamovlena.

kvlevam aCvena, rom 40C-ze tbis wyalSi V.parahaempolytricus –is baqteriebiufro swrafad (21 dReSi) gadadian arakultivirebad mdomareobaSi, vidrezRvis wyalSi (1-2 Tve) (sur.15. a, b). mocemuli maCvenebeli dakavSirebuliaV.parahaempolytricus –is, rogorc halofiluri saxeobis mier marilismTxovnilebasTan. rac Seexeba mdidar sakveb ares, aq 2 Tviani periodisganmavlobaSi kultivirebadi baqteriebis raodenoba mxolod 2 log- iTSemcirda (sur. 15.… g) da, Sesabamisad, ar moxda arakultivirebadmdgomareobaSi gadasvla. es faqti SesaZloa aixsnas areSi sakmarisiraodenobiT sakvebi nivTierebebis arsebobiT.

rac Seexeba optimalur 250C temperaturaze Senaxul baqteriebs, arc erTareSi ar moxda arakultivirebad mdgomareobaSi gadasvla, 2 Tviani dayovnebisSemdeg baqteriuli ujredebis raodenoba 103-104 iyo. Tumca sakvebinivTierebebiT Rarib wyalSi, sicocxlisunarian baqteriul ujredTaraodenoba ufro swrafad mcirdeboda, vidre LB-niadagSi.

62

a)

b)

g)sur. 16. V.parahaempolytricus –is baqteriebis sicocxlisunarianobis

Seswavla 40C-ze sxvadasxva areSi a) lisis tbis wyali b) zRvis wyali g) LB-niadagi

63

5. Vibrio parahaemolyticus-is miarT specifikuri baqteriofagebis gamoyofa daekologia

Cveni gamokvlevis farglebSi saqarTvelos mtknari wyalsatevebidan dazRvidan, agreTve kulturaTa supernatantebidan gamoiyo V.parahaemolyticus –ismimarT specifikuri baqteriofagebis- 69 izolati. baqteriofagebis gamoyofissixSire Seadgenda 6,5%-s. V.pa fagebi yvelaze xSirad iyofoda zRvis wylissinjebidan ( 39 izolati). 16 fagi gamoiyo tbis wylebidan, 14 ki- kulturaTasupernatantebidan (sur.17). zRvidan V.pa-fagebis gamoyofis piki aRiniSnaSemodgomis TveebSi, rac Tan sdevda maspinZeli baqteriis gamoyofis piks(sur.5).

sur. 17. Vibrio parahaemolyticus-is miarT specifikuri baqteriofagebis gamoyofa

64

6.B Vibrio parahaemolyticus-is miarT specifikuri baqteriofagebis daxasiaTeba

fagis negatiuri koloniis formas vnaxulobdiT baqteriuli Stamebisgazonebze maTi formirebis damTavrebis Semdeg. sxvadasxva baqteriofagebsaRmoaCndaT morfologiurad gansxvavebuli negatiuri koloniebi. romelTazomebi varirebda 1-3 mm diametris farglebSi. TiToeuli sakvlevi fagisaTvis,morfologiurad erTgvarovani koloniebis 5-10 Tanmimdevruli pasaJis SedegadmiviReT sufTa faguri klonebi Semowmebuli el. mikroskopiT da agarozisgelSi eleqtroforeziT. Ggarkveul SemTxvevebSi fagebi kvlav xasiaTdebodnenpolimorfizmiT, rac negatiuri koloniebis araerTgvarovnebaSigamoixateboda.

fagis virionis morfologias vswavlobdiT eleqtronuli mikroskopissaSualebiT. struqturul kvlevas vawarmoebdiT M10 (Opton-Zeiss, germania)eleqtronuli mikroskopis saSualebiT. fagebis eleqtronul-mikroskopiurmaSeswavlam gviCvena, rom yvela maTgani miekuTvneba Myoviridae-s ojaxs,heqsagonaluri TaviTa da kumSvadi kudiT. aRiniSna mcire sxvaoba sakvlevifagebis nukleokapsidisa da morCis zomebSi (cxrili 9), ( sur 18).

cxrili 9. V.parahaemolyticus-is mimarT specifikuri baqteriofagebismorfologia

baqteriofagebi O ojaxi Tavi kudissigane

kudissigrZe

Vpa ch2s Myoviridae 60±2 16±2 120±2

Vpa lab Myoviridae 60±2 20±2 140±2

Vpa 555 Myoviridae 80±2 15±2 175±2

Vpa 5/II(372) Myoviridae 60±2 20±2 140±2

Vpa 19KP Myoviridae 60±2 20±2 135±2

Vpa 549 Myoviridae 55±2 13±2 125±2

Vpa 26 Myoviridae 50±2 17±2 112±2

Vpa 18 Myoviridae 70±2 15±2 85±2

Vpa 37 Myoviridae 60±2 14±2 88±2

Vpa G40 Myoviridae 66±2 20±2 92±2

Vpa 80 Myoviridae 60±2 20±2 100±2

Vpa A11 Myoviridae 80±2 12±2 97±2

65

a) b)

g) d)

e)

fagebis lizisuri speqtrissaorientacio gansazRvras

sur. 18. V.parahaemolyticus-is

mimarT specifikuri

baqteriofagebis morfologia

a) Vpa 26 b )Vpa 19KP g)Vpa A11

d) Vpa ch2s e) Vpa G40

66

vawarmoebdiT myar sakveb areze [2]. sacdeli fagis samuSao titri Seadgenda107-108naw/ml. skriningi ganxorcielda V.parahaemolyticus-is 158 izolatze.

kvlevebis Sedegad dadginda, rom baqteriofagebis umravlesobaxasiaTdeboda maspinZeli Stamis mimarT maRali specifikurobiT, maSinrodesac zogierT fags SeeZlo 8-24 izolatis lizisi. xolo Stamebis 30% ariyo mgrZnobiare mocemuli fagebis mimarT.

cxrili 10.

V.pa

raha

emol

ytic

us-

baqter

iofag

i

mgrZnobiare Stameb

is

ric

xvi

მგrZnobiareStameb

is

%

Vpa ch2s 19 12.03Vpa 555 5 3.16Vpa lab 3 1.9Vpa 5/II(372) 8 5.06Vpa 19KP 2 1.27Vpa 549 7 4.43Vpa 26 33 20.89Vpa 18 4 2.53Vpa 37 4 2.53Vpa G40 13 8.23Vpa 80 11 6.96Vpa A11 22 13.92

Ffagebis garkveulma jgufebma gamoavlina msgavseba lizisuri speqtrismixedviT ( sur. 19). Ees monacemebi korelaciaSia serologiuri da genetikurimaxasiaTeblebis SedegebTan. Tumca unda aRiniSnos, rom Seswavlilibaqteriofagebi moqmedebis diapazonis mixedviT ufro mravalferovania, vidresxva maxasiaTeblebis mixedviT. es aixsneba zedapiruli cilovanistruqturebis-fagospecifikuri receptorebis mravalferovnebiT.

67

sur. 19. Bbaqteriofagebis dajgufeba lizisuri speqtris mixedviT

68

baqteriofagis mimarT rezistentuli baqteriuli mutantebis analizmagviCvena, rom baqteriofagebs Zalian msgavsi, receptoruli aparati gaaCniaT,radgan jvaredina skriningis eqsperimentebma baqteriofagebisfagorezistentul mutantebze gviCvena, rom es fagebi ar arian mgrZnobiarenierTmeneTis fagorezistentuli baqteriuli mutantebis mimarT.

patron -ujredTan urTierTqmedebis fazebis Seswavlisas TavdapirveladSevarCieT adsorbciisTvis optimaluri temperatura, romelic ganisazRvra290C-iT, gansxvavebiT V.parahaemolyticus-is baqteriebisTvis optimaluri 370C-sa.

Semdgom etapze SeviswavleT adsorbciis siCqare, latenturi periodi dasaSualo gamosavli TiToeul ujredze. sakvlev baqteriofagebs SorisumniSvnelo gansxvaveba dafiqsirda maTi ujredSida gamravlebis ciklismaxasiaTeblebis mixedviT, rogoricaa adsorbciis efeqturoba, latenturiperiodis xangrZlivoba da fagur nawilakTa gamosavali (sur. 20, cxrili 11).

sur. 20. V. parahaemolyticus-is baqteriofagebis adsorbcia

69

cxrili 11.

ფაგები K(ადს) მლ/წთ ადს % ლატ.პერიოდი გამოსავალი( 1უჯრ)

Vpach2s 7,5X 10-9 80% 40-45 193

Vpa19KP 4,5X10-11 69% 40-45 100

VpaG40 6X 10-7 40% 55-60 74

Vpa26cl 8,0X 10-9 57% 50-55 108

VpaA11 2,2X10-10 60% 35-40 184

Catarebuli eqsperimentebis Sedegad dadginda, rom baqteriofagebixasiaTdeba sakmaod Senelebuli, Tumca maRalprocentuli adsorbciis unariT,xangrZlivi latenturi periodiT da virusuli nawilakebis maRaligamosavaliT, gansakuTrebiT zogierTi fagis (Vpach2, VpaA11) SemTxvevaSi.

fagebis serologiur naTesaobas vswavlobdiT fagis jvaredinaneitralizaciis reaqciis Catarebis gziT sxvadasxva baqteriofagebisa daspecifikuri antifaguri Sratebis gamoyenebiT. vsazRvravdiT neitralizaciismaqsimalur procents (sur. 21) da neitralizaciis konstantas [1, 2].specifikuri Vpa 19KP da Vpa A11 antifaguri Sratebis gamoyenebiT Catarebulijvaredina neitralizaciis reaqciebis Sedegebis mixedviT movaxdineT fagebisdayofa 4 jgufad. serologiuri monacemebi korelaciaSi aRmoCnda fagebislizisuri speqtris (cxrili 12) da restriqciuli profilis SedegebTan(ixileT qvemoT)

sur. 21. Bbaqteriofagebis neitralizacia antifaguri SratebiT

70

cxrili 12.

baqteriofagebi

dajgufebaneitralizaciis Sedegebis

(%) mixedviT

dajgugebalizisuri speqtris

msgavsebis (%) mixedviT

Vpa 19 KP Vpa A11 jgufi Vpa 19 KP Vpa A11 jgufi

Vpa lab 99 28

I

78 44

I

Vpa 555 99 45 78 44

Vpa 5/II(372) 98 30 79 45

Vpa 19KP 100 23 100 45

Vpa 549 99 29 70 45

Vpa ch2s 99 63 64 40

IIVpa 37 55 20

III

68 46

Vpa 26 22 24 44 34 III

Vpa 18 82 71

II

66 46

II

Vpa 80 83 80 63 44

Vpa G40 56 90

III

63 44

Vpa 7cl 58 38 65 41

Vpa A11 32 100 IV 45 100 IV

71

6. fagebis mgrZnobeloba fizikuri da qimiuri faqtorebis mimarT

cnobilia, rom baqteriofagebis struqturuli komponentebis Senebisa dafunqcionirebis SefasebisaTvis didi mniSvneloba aqvs maTze mTeli rigifizikuri da qimiuri faqtorebis mainaqtivirebeli moqmedebis Seswavlas.amitomac Seswavlil iqna sakvlevi baqteriofagebis damokidebuleba iseTifaqtorebis mimarT, rogoricaa ultraiisferi gamosxiveba, temperatura,wyalbad ionTa maRali an dabali maCveneblebi, maRali ionuri Zala da a.S.

baqteriofagebis inaqtivacia UV sxivebiT. Bbunebriv pirobebSi fagebispopulaciis SenarCunebisaTvis didi mniSvneloba aqvs baqteriofagebisultraiisferi sxivebisadmi gamZleobas.Ggarda amisa, es niSan-TvisebadakavSirebulia fagebis nukleinis mJavebis struqturis TaviseburebebTan damniSvnelovnad ganapirobebs fagebis cxovelmyofelobis SenarCunebas bunebrivpirobebSi.

Uultraiisferi sxivebis mimarT mgrZnobelobas vswavlobdiT dinamikaSi(30; 60 da 90 wm-is ganmavlobaSi) 254 nm talRis sigrZeze. miRebuli SedegebismixedviT Seswavlili baqteriofagebi gansxvavdebian erTmaneTisganultraiisferi gamosxivebis mimarT gamZleobis mixedviT. Vpa ch2s da Vpa 19KPbaqteriofagebisTvis 100 %-iani inaqtivaciis dro 60 wm-ia, xolo Vpa G40 , Vpa26 da Vpa A11 baqteriofagebis SemTxvevaSi- 90 wm.

sur. 22. V.parahaemolyticus-is mimarT specifikuri baqteriofagebisultraiisferi sxivebis mimarT mgrZnobeloba

72

baqteriofagebis Termostabilobis Seswavlas vaxdendiT sxvadasxvatemperaturuli reJimis (550C, 600C, 650C, 700C) da eqspoziciis pirobebSi. miRebulSedegebze dayrdnobiT SeiZleba iTqvas rom, Vpa 40 , Vpa 26 da Vpa A11baqteriofagebi meti TermostabilobiT xasiaTdeba, vidre Vpa ch2s da Vpa 19KPbaqteriofagebi, romelTa Termuli inaqtivacia 600C-ze xdeboda 30 wT-Si.Tumca Vpa A11 Termuli inaqtivaciis kritikul temperaturad SesaZloaCavTvaloT 700C-c, rac am fagis cilebis Termostabilurobaze migviTiTebs darac ganasxvavebs mas danarCeni fagebisgan (sur. 23), ( cxrili 13).

sakvlevi baqteriofagebis sxvadasxva TermomgrZnobeloba miuTiTebs maTicilovani struqturebis gansxvavebaze da amdenad, fagebis taqsonomiurimikuTvnebisa da arsebuli koleqciis farglebSi dajgufebisaTvis garkveulimniSvneloba aqvs.

cxrili 13.B V.parahaemolyticus-is mimarT specifikuri FbaqteriofagebisTermoinaqtivacia

ბაქტერიოფაგები საწყისი ტიტრი

550C 600C 650C 700C

საბოლოო ტიტრი (30wT)

Vpa ch2s 2,5X108 3X105 3X104 120 28

Vpa19KP 3X108 6,8X105 3X104 90 2

Vpa G40 4X108 8,4X107 8X106 5X106 1

Vpa 26 3,4X108 1,5X107 1,4X106 1X106 16

Vpa A11 5X108 1X108 3X107 1,5X107 1,1X105

73

a)

b)Nsur. 23 V.parahaemolyticus-is mimarT specifikuri Fbaqteriofagebis

mgrZnobeloba sxvadasxva temperaturis mimarT a) 550C b) 700C

74

SeviswavleT baqteriofagebis osmosuri Sokisadmi damokidebuleba(cxrili 14). NaCl- is 4M xsnarSi inkubaciisa da gamoxdil wyalSi Semdgomi 100-jeradi ganzavebis Semdeg fagis infeqciuri nawilakebis raodenobis mkveTriSemcireba ar aRiniSneboda. Mmocemuli Sedegi imaze miuTiTebs, rom maTicilovani garsi advilad atarebs wylis molekulebs da ionebs. Aarsebobsmosazreba, rom osmosuri Sokisadmi mgrZnobeloba damokidebulia fagebiscilovani kafsidis simkvriveze, kerZod, kafsomerebis SefuTviskompaqturobaze. andersonis mier T-fagebi swored am niSnis mixedviT iqnadayofili mgrZnobiare (T-wyvili fagebi) da rezistentul (T3,T5,T7) jgufebad.

cxrili 14. V.para-fagebis damokidebuleba ionuri Zalis mimarT

Fbaqteriofagebiaqtiuri faguri nawilakebisprocentuli Semcveloba (%)

Vpa ch2s 81.25

Vpa 19KP 56.30

Vpa 26 61.50

Vpa G40 45.80

Vpa A11 99.90

Seswavlili iqna agreTve baqteriofagebis mgrZnobeloba wyalbad-ionTasxvadasxva mniSvnelobebis mimarT. cnobilia, rom baqteriofagebisumravlesoba sakmaod stabiluria pH-is Sualedur diapazonSi pH-5-9. sakvebiaris gatutovneba iwvevs virionebis TandaTanobiT degradacias, amasTanTavdapirvelad iSleba morCebi, Semdeg ki iwyeba dnm-is gamosvla, pH-12

tutianobis pirobebSi kafsidi iSleba calkeul suberTeulebad. Kკafsidisstruqturuli elementebis daSlas aseve iwvevs eqstremalurad dabalimaCveneblebi (<pH-2). Tumca pH-is zomierad dabali maCveneblis drosbaqteriofagebis gamoleqva xdeba infeqciurobis dakargvis gareSe. arsebobsmosazreba, rom pH-is gamoxatulad maRali an dabali mniSvnelobis drosgamovlenili faguri nawilakebi mutantur formebs warmoadgenen, romelTagadarCevac cvalebad pirobebSi mimdinareobs.

sabolood, baqteriofagebis mJavur-tutovani denaturaciis cdebmagviCvena V.parahaemolyticus-is fagebis sakmaod maRali stabiluroba pH-4-7-ispirobebSi. E pH-is eqstremalurad dabali mniSvnelobis dros nawilakebisraodenoba mkveTrad mcirdeba. igives ver vityviT eqstremalurad tute areze

75

pH-10-12, rigi fagebis SemTxvevaSi (Vpa 19KP da Vpa ch2s) maRali pH aramarto aramcirebs, aramed xels uwyobs faguri nawilakebis sicocxlisunarianobisSenarCunebas (sur.24).

ა)

ბ)

76

გ)

sur. 24. baqteriofagebis mgrZnobeloba wyalbad-ionTa sxvadasxva

mniSvnelobebis mimarT. ა) Vpa 19KP ბ) Vpa G40 გ) Vpa A11

Seswavlil iqna sakvlevi baqteriofagebis sxva fiziologiuriTvisebebic. kerZod, natriumis citratisadmi damokidebuleba (cxrili 15). 2%-iani natriumis citratis mimarT fagebi avlenen sakmaod maRal mgrZnobelobasrac SesaZloa miuTiTebdes imaze, rom es fagebi damokidebulni arianorvalentian ionebze, rogorc adsorbciis kofaqtorebze.

cxrili 15. V.para-fagebis damokidebuleba natriumis citratze

baqteriofagebiaqtiuri faguri nawilakebisprocentuli Semcveloba (%)

Vpa ch2s 0.06

Vpa 19KP 0.15

Vpa 26 0.01

Vpa G40 0.06

Vpa A11 0.08

77

Catarebul eqsperimentebSi gamovlinda V.parahaemolyticus-is fagebismdgradoba organuli gamxsnelebis (spirti, qloroformi) mimarT. racarapirdapirad miuTiTebs maT virionebSi lipiduri komponentis ararsebobaze.

baqteriofagebis stabiluroba sxvadasxva areSi Senaxvisas.Bbaqteriofagebis praqtikuli gamoyenebisTvis rogorc samkurnalo-profilaqtikuri mizniT, aseve ekologiuri kvlevebis, sxvadasxva wyalsatevebSitrasirebis eqsperimentebis Casatareblad, didi mniSvneloba aqvs maTstabilobas sakvlev garemoSi da aseve Senaxvisa da transportirebis pirobebSi.

amitom Catarebul iqna V.parahaemolyticus-is fagebis stabilobiseqsperimentebi sxvadasxva mikrobiologiur areebsa da xsnarebSi, asevebunebriv tbisa da zRvis wylebSi (cxrili 16).Kkvlevebma gviCvena, rom fagebisaqtivoba umniSvnelod ecemoda 6 Tvis ganmavlobaSi. Ffagebis Sesanaxad

SedarebiT naklebefefqtur ared SeiZleba CaiTvalos lisis tbis, rogorcmtknari wyalsatevis, wyali. am SemTxvevaSi dabali marilianobaV.parahaemolyticus –is baqteriofagebisTvis, iseve rogorc TviT baqteriebisTvis,araxelsayrel fizikur faqtors warmoadgens.

cxrili 16. baqteriofagebis stabiluroba sxvadasxva areebSi 40C-ze Senaxvisas

Ddro Ffagebi PBSSM- faguri

buferizRviswyali

lisis tbiswyali

24 saaTi

Vpa ch2s 2,5x108 2,5x108 2,5x108 2,5x108

Vpa A11 2x108 2x108 2x108 2x108

10 dRe

Vpa ch2s 2,5x108 2,5x108 2,5x108 2,5x108

Vpa A11 2x108 2x108 2x108 2x108

2 Tve

Vpa ch2s 2,5x108 2,5x108 2,5x108 6x107

Vpa A11 8x107 2x108 6,6x107 7,2x107

6 Tve

Vpa ch2s 5x107 4,5x107 4,4x107 1,5x107

Vpa A11 6,5x107 7,2x107 6,5x107 4,2x107

P

78

7. proteolituri fermentebis moqmedebis Seswavla baqteriofagebze

P proteolituri fermentebis moqmedebis efeqti Vibrio parahaemolyticus –ismimarT specifikur baqteriofagebze, Seswavlil iqna pepsinisa daqemotrifsinis magaliTze.

pepsini, qemotrifsinTan da trifsinTan erTad saWmlis momnelebelisistemis ZiriTadi fermentia. Ppepsinis moqmedebis efeqti yvelaze maRalianormaluri kuWis wvenis mJavianobis pirobebSi (pH 1.5-2.5) [178].Qqemotrifsinipankreasis wvenis erT-erTi komponentia, romelic moqmedebs TormetgojanawlavSi da Slis cilebs da polipeptidebs. Ppraqtikul medicinaSi isgamoiyeneba per-oralurad moxmarebisTvis an zedapirebis dasamuSaveblad,abscesebTan, travmebTan da qirurgiul operaciebTan asocirebuli siwiTlisada SeSupebis Sesamcireblad [151].

Eeqsperimentebma fermentebis gavlenis SeswavlasTan dakavSirebiT, aCvena,rom Cvens mier gamoyofili Vibrio parahaemolyticus –is mimarT specifikuribaqteriofagebi mgrZnobiarea pepsinis 1% -iani xsnaris mimarT. Ffagebis titripepsinis xsnarSi Zalian male, TiTqmis SetanisTanave Semcirda >2 log –iT. esmonacemi SesabamisobaSia Cvens gamokvlevebTan, mocemuli fagis pH-issxvadasxva mniSvnelobebze damokidebulebasTan dakavSirebiT. imdenadramdenadac, pepsinis xsnaris pH 1-1.5-ia, xolo Cvenma kvlevebma aCvenasicocxlisunariani faguri nawilakebis raodenobis mniSvnelovani kleba pH 2-ze, mocemuli Sedegi savsebiT mosalodneli unda yofiliyo. miRebulinegatiuri efeqtebis gaTvaliswinebiT, kuWis wvenis mJave pH–isgasaneitraleblad umjobesi iqneba fagebis miReba tute bunebis xsnarebTanerTad.

rac Seexeba qemotrifsinis gavlenas fagebis sicocxlisunarianobaze, ariyo TvalSisacemi. Qqemotrifsinis sxvadasxva koncentraciebma (0.1%; 0.25%; 0.5%;1%) da drois sxvadasxva eqspoziciebma (10 wT, 30 wT, 60 wT) ar gamoiwvia fagistitris mniSvnelovani cvlilebebi ( < 1 log). Mmocemuli Sedegi SesaZleblobasiZleva vifiqroT V.parahaemolyticus –is mimarT specifikuri baqteriofagebisgamoyenebaze iqnes WrilobiTi infeqciebis samkurnalod qemotrifsinTanerTobliobaSi.

79

8. baqteriofagebis genetikuri Tvisebebis Seswavla

faguri denm-is restriqciuli analizi Cvens mier Seswavlili yvelabaqteriofagis dnm aris orjaWviani da mgrZnobiarea Sewavlili restriqciuliendonukleazebis umravlesobis mimarT. dnm-ebis molekuluri wonisdasaTvlelad SevarCieT ori endonukleaza (Sal I da PstI), romlebicSeswavlili fagebis dnm-s SedarebiT msxvil fragmentebad Wridnen (didizomis fragmentebiT molekuluri wona SedarebiT zustad iangariSeba).

fagebis umravlesobis dnm 43-44 kb-ia, SedarebiT maRali molekuluriwona aqvs Vpa A11-is dnm-s- 60 kb.

cxrili 17. faguri dnm-is mgrZnobeloba restriqciuli endonukleazebismimarT

baqteriofagebi

restriqciuli endonukleaza

EcoR I EcoR V Hind III BamH I Sau3A Pst I Sal I Xba I

Vpa ch2s + + + - - - + +

Vpa lab + + + - - - + +

Vpa 555 + + + - - - + +

Vpa 5/II(372) + + + - - - + +

Vpa 19KP + + + - - - + +

Vpa 549 + + + - - - + +

Vpa 26 + + + + + + + +

Vpa 18 + + + + + + + +

Vpa 37 + + + + + + + +

Vpa G40 + + + + + + + +

Vpa 80 + + + + + + + +

Vpa A11 + + + + + + - +

80

restriqciuli profili saSualebas gvaZlevs vimsjeloT fagebisgenetikur mravalferovnebaze. amisaTvis avageT filogenetikuri xe, sadc Cans,rom fagebi sam klasterul erTeulSia gaerTianebuli. aRsaniSnavia, rom erTi

da imave serotipis maspinZeli ujredis mqone baqteriofagebi (mag. Vpa ch2s da

Vpa 19KP, Vpa 26 da Vpa G40) erT klasterSia gaerTianebuli. amasTan, calkejgufSia Vpa A11, romelic genomis zomiT, serologiuri da fiziologiurimaCveneblebiT gansxvavebulia danarCeni fagebisgan.

Aa) b)

sur. 25. V.para-baqteriofagebis denm-is restriqciuli analizi

Aa)1.Hind III+ Lambda-dnm;2. Pst I+Vpa ch2s-dnm;3. Pst I+ Vpa19KP-dnm4. Pst I+Vpa G40-dnm5. Pst I+Vpa 26-dnm6. Pst I+Vpa A11-dnm

b)1.Hind III+ Lambda-dnm;2. Sal I+Vpa ch2s-dnm;3. Sal I+ Vpa19KP-dnm4. Sal I+Vpa G40-dnm5. Sal I+Vpa 26-dnm6.Sal I+Vpa A11-dnm

81

sur. 26. V.para-baqteriofagebis dajgufeba restriqciuli profilismixedviT.

82

IV.Sedegebis ganxilva

wyliani garemos dabinZureba Tanamedrove msoflios da maT SorissaqarTvelos erT-erTi umniSvnelovanesi prolemaa. Ggazrdili anTropogenulizemoqmedeba klimaturi pirobebis cvlilebasTan erTad sakmaod mokle droSiaisaxeba wylis dabinZurebis xarisxze. sasmeli wylisa da sarekreaciowyalsatevebis regularuli monitoringi da dabinZurebis wyaroebisgamovlena, konkretuli RonisZiebebis dagegmvisa da CatarebisTvis metadmiSvnelovania.

wyliani garemos dabinZureba ganapirobebs sxvadasxva baqteriuli Tuvirusuli wylismieri infeqciis gavrcelebas, rac ZiriTadad, dabinZurebulwyalTan sxvadaxva saxis kontaqtiTaa gamowveuli. mikrobul paTogenebaddabinZurebul wylebSi gvxvdeba: Salmonella spp., Shigella spp., Vibrio spp.,enteropaTogenuri E. coli da sxva baqteriebi, garda amisa 100-ze meti tipispaTogenuri virusi, umartivesebi da sxva.Dsxvadasxva daavadebebis Tavidanacilebis mizniT aucilebelia infeqciis wyaros da misi gavrcelebis gzebisdadgena.

Bbolo or aTwleulSi mniSvnelovnad imata halofiluri vibrionebiT

gamowveuli infeqciebis sixSirem. maT Soris, Vibrio parahaemolyticus yvelazexSirad iwvevs seriozul gastroenteritebs, ZiriTadad asocirebulia umi dacudad damuSavebuli zRvis produqtebis moxmarebasTan. Ggarda amisa,V.parahaemolyticus-iT dabinZurebul wylebTan kontaqtis Sedegad viTardebaWrilobiTi infeqciebi. halofiluri vibrionebi aseve paTogenuria zRvisorganizmebisTvis (Tevzebi, xamanwkebi) da zarals ayenebs aqvakulturismeurneobebs.

V.parahaemolyticus-is gamoyofa Cveulebriv xdeba zRvis rogorc gaSliliubnebidan, aseve mdinareTa SesarTavebis wylebidan. saSualo da maRalimarilianoba, agreTve 18°C-ze maRali temperatura optimalur pirobebswarmoadgens araqoleruli vibrionebis zrdisa da gamravlebisTvis. amTvalsazrisiT, saqarTveloSi Savi zRvis sanapiroze arsebuli subtropikuliklimati da wyliani garemo, halofiluri vibrioebisTvis xelsayrel garemoswarmoadgens. garemo-klimaturi pirobebi da maTi sezonuri cvlilebebimniSvnelovnad aisaxeba rogorc paTogenuri vibrionebis, agreTve maTispecifiuri baqteriofagebis gavrcelebaze.

halofiluri vibrionebis baqteriofagebi did interess iwvevs, wyalSiSesabamisi baqteriuli saxeobebis Semcvelobis gamosavlenad (fagi-indikatorebi), aseve paTogenobis meqanizmebis Sesaswavlad da StamebisdiferenciaciisaTvis, dabolos rogorc alternatiuli saSualeba, ampaTogenebiT gamowveuli daavadebebis profilaqtikisa da mkurnalobisTvis.

kvlevis ZiriTad mizans warmoadgenda saqarTvelos wylian garemoSiV. parahaemolyticus –is da Sesabamisi baqteriofagebis gavrcelebis dabiomravalferovnebis Seswavla. amisaTvis Savi zRvidan da mtknariwyalsatevebidan gamovyaviT V. parahaemolyticus –is baqteriebi da Sesabamisibaqteriofagebi. SevisawvleT maTi biologiuri Tvisebebi.

83

Cveni kvlevis farglebSi 2006-2008 wlebSi Savi zRvis sanapiro zolSi dazogierT mtknar wyalsacavSi Catarebulma monitoringma aCvena, romsaqarTvelos wyliani garemo da klimati xelsayrel pirobebs qmnisV.parahaemolyticus-is baqtriebis da Sesabamisi baqteriofagebiscxovelmyofelobisTvis. baqteriis, iseve rogorc specifikuribaqteriofagebis, gamoyofa sezonuri xasiaTisaa da damokidebulia gamoyofisadgilze. V.parahaemolyticus-i halofiluri baqteriaa, Sesabamisad izolatebisgamoyofa ZiriTadad Savi zRvidan moxda, erTeuli izolatebi gamovyaviTmtknari wyalsacavidanac, Tumca literaturis Tanaxmad, baqteriis arsebobanaklebadmarilian garemoSi droebiTia. aRsaniSnavia, rom mtknariwyalsacavebidan gamoyofil iqna V.parahaemoliticus-is mimarT specifikuribaqteriofagebi, rac aseve SesaZloa mianiSnebdes mocemul garemoSi patronibaqteriis arsebobaze. Aamaze metyvelebs agreTve pirdapiri deteqciis meTodiT(PCR-ESI/MS) tbebSi V.parahaemolyticus-is genomis arsebobis dadgena. MmocemuliSedegebidan gamomdinare SegviZlia vivaraudoT, rom mtknar wyalsatevebSi,sadac garemo pirobebi araxelsayrelia halofiluri baqteriebisTvisgamravlebisTvis, V.parahaemolyticus gadadis arakultivirebad mdgomareobaSi,Tumca inarCunebs sasicocxlo Tvisebebs. Mmocemuli mdgomareobaliteraturaSi cnobilia sicocxlisunariani, magram arakultivirebadimdgomareobis (VBNC) saxelwodebiT. V.parahaemolyticus-is baqteriebisarakultivirebad mdgomareobaSi gadasvla aramxolod dabal marilianobasTanaris dakavSirebuli. is faqti, rom V.parahaemolyticus-is mimarT specifikuribaqteriofagebis gamoyofa xdeboda gvian Semodgomis TveebSi, maSin rodesacSesabamisi baqteriebi mcire raodenobiT an saerTod ar iTeseboda, agreTvemetyvelebs imaze, rom wylis dabali temperatura dabal marilianobasTanerTad aris faqtori, romelic ujredebis arakultivirebad mdgomareobaSigadasvlas ganapirobebs. es mosazreba eqsperimentulad dadasturebul iqnaCveni kvlevis SedegebiT, sadac VBNC-mdgomareobaSi V.parahaemolyticus-isbaqteriebis gadasvla iyo naCvenebi. sxvadasxva tipis araxelsayreli pirobebiszegavleniT, kerZod, arasakmarisma marilianobam, dabalma temperaturam dasakvebi nivTierebebis naklebobam, sasicocxlo procesebis daqveiTeba dakultivirebis unaris dakargva gamoiwvia. D

daavadebis riskis ganviTarebaTan mimarTebaSi didi mniSvneloba aqvsV.parahaemolyticus-is gamoyofili izolatebis serologiur maCveneblebs.V.parahaemolyticus-is serotipirebis Tanamedrove sqemis mixedviT

identificirebulia 75 serovarianti 13 O- antigenis da 71 K-antigenissxvadasxva kombinaciebis mixedviT. maTgan O3-serotipi kvebiTi infeqciebisgamomwvevi yvelaze xSiri warmomadgenelia. Mmas miekuTvneba pandemuri kloni-O3:K6, romelmac ukanasknel wlebSi araerTi afeTqeba gamoiwvia mravalsxvadasxva regionSi. Cveni kvlevis farglebSi Seswavlil 81 izolats Sorisdadgenil iqna 9 serojgufis da 27 sxvadasxva serotipis izolatebisSemcveloba. O3:K6-serotipis arc erTi Stami ar gamovlinda, Tumca mwvanekoncxidan gamoyofili erT-erTi Stami tipirebul iqna, rogorc 01:KUT , racO3:K6-is pandemuri klonis serovariants warmoadgens. Mmocemuli baqteriuli

84

izolati virulentobis faqtorebis mixedviT- tdh- da trh-uaryofiTi iyo, TumcagaaCnda O3:K-is GS-PCR markeri.

pandemuri Stamis markerTan erTad mocemuli serotipis arseboba SavzRvaSi pandemuri Stamebis an maTi variantebis SesaZlo arsebobaze miuTiTebs.amis SesaZleblobaze miuTiTebs agrTve Termolabiluri hemolizinis damesame tipis sekretoruli sistemis efeqtoruli proteinebis deteqciagamoyofil izolatebSi. zemoTnaxsenebi virulenturi TvisebebiTxasiaTdeboda Semowmebuli Stamebis 96%, rac SesaZleblobas iZlevadavaskvnaT, rom Savi zRvis V.parahaemolyticus-is populaciaSi virulentobisfaqtorebi farTodaa gavrcelebuli. aRsaniSnavia is faqti, rom literaturisTanaxmad garemodan gamoyofili izolatebis mxolod 1% Seicavsvirulentobis faqtorebs.AgansakuTrebiT sayuradReboa mocemul StamebsSoris O3:K6-pandemuri klonis serovariantis arseboba. saqarTveloSi, SavizRvis sanapiro zolSi, wylidan gamoyofili baqteriuli izolatebSivirulenturi maxasiaTeblebis arseboba unda ganvixiloT, rogorcsazogadoebrivi jandacvisTvis sayuradRebo movlena, ramdenadac aseTi tipis(tdh, trh, TTSS2-dadebiTi) izolatebi infeqciis wyaros warmoadgens dazianebulkanTan kontaqtis (WrilobiTi infeqciebi) an dabinZurebuli zRvisproduqtebis moxmarebis (kvebiTi infeqciebi) SemTxvevaSi.

V.parahaemolyticus-is izolatebis genetikuri mravalferovneba Seswavliliqna ori meTodiT, pulsirebad velSi gel eleqtroforeziT (PFGE) da Rep-PCR-iT. Oorive meTodma gviCvena V.parahaemolyticus-is izolatebis didimravalferovneba, Tumca serologiur maxasiaTeblebTan mniSvnelovanikorelacia ar gamovlenila. Ees aixsneba serotipirebis SedegebTan SedarebiTgenetikuri mravalferovnebis maRali mravalferovnebiT. es faqti kideverTxel adasturebs biomravalferovnebis Sesaswavlad sxvadasxva meTodebisgamoyenebis aucileblobas.

V.parahaemolyticus-is Stamebis fagomgrZnobelobis Seswavla baqteriebistipirebis kidev erTi alternativaa. Ffagotipirebam aCvena, rom erTi da igiveserotipisa da genetikuri struqturis mqone baqteriuli izolatebifagomgrZnobelobis mixedviT kidev gansxvavdebian erTmaneTisgan. mocemuliSedegi SeiZleba aixsnas imiT, rom fagotipireba asaxavs zedapirulistruqturebis-fagis saadsorbcio receptorebis mravalferovnebas.

V.parahaemolyticus-is Stamebis antibiotikebis mimarT mgrZnobelobisSeswavlam aCvena, rom izolatebis daaxloebiT 40% multirezistentulia.rogorc cnobilia, garemodan gamoyofili, arapaTogenuri izolatebi,antibiotikebis mimarT naklebi mgrZnobelobiT xasiaTdebian. Cvens kvlevaSiyvelaze maRali mgrZnobeloba dafiqsirda ciprofloqsacinis,streptomicinisa da gentamicinis mimarT. Aamave dros Stamebis umravlesobamgamoavlina rezistentoba ampicilinis mimarT, rac agreTve dasturdebasxvadasxva avtorTa kvlevebSi.

G V.parahaemolyticus-is izolatebis 47.5% mgrZnobiare aRmoCnda kvlevisfarglebSi gamoyofili baqteriofagebis mimarT. sainteresoa sxvadasxvaserotipis Stamebis baqteriofagebis mimarT mgrZnobelobis Sedegebi. KkvlevamaCvena, rom infeqciebis gamomwvevi O3-serotipi (O3:K51, O3:K65) yvelaze maRali

85

mgrZnobelobiT xasiaTdeba. rac Seexeba O3:K6-is pandemuri klonisserovariants- 01:KUT -s, naSromis farglebSi gamoyofil iqna mocemuliserotopis mimarT aqtiuri baqteriofagi- VpaA11. Ffagotipirebis SedegebigaTvaliswinebuli unda iqnas V.parahaemolyticus-is mier gamowveuli infeqciebissamkurnalo preparatis Seqmnis procesSi.

fagebis eleqtronul-mikroskopiurma Seswavlam gviCvena, rom Cvens miergamoyofili V.parahaemolyticus-is baqteriofagebi ar xasiaTdebianmorfologiuri mravalferovnebiT- yvela maTgani miekuTvneba Myoviridae-sojaxs- heqsagonaluri TaviTa da kumSvadi kudiT, Tumca gansxvaveba aRiniSnebavirionis zomebSi. literaturaSi MMioviridae-s garda aRwerilia Siphoviridae-sojaxis sxvadasxva gvari warmomadgenlebic [107]. cnobilia, rom samkurnalopreparatebis SemadgenlobaSi arsebuli baqteriofagebis umravlesoba kudianifagebia da, ZiriTadad, Myoviridae-s da Siphoviridae-s ojaxebs miekuTvneba.amdenad, V.parahaemolyticus-is mimarT specifikuri baqteriofagebi savaraudodatareben Terapiul potencials.

Terapiuli mizniT baqteriofagebis gamoyenebisTvis mniSvnelovaniaagreTve maTi swrafi da efeqturi gamravlebis unari. Catarebulmaeqsperimentebma aCvena, rom Cvens mier gamoyofili fagebi xasiaTdeba efeqturiadsorbciis unariT (maRali procentuli maCvenebliT) da virusulinawilakebis maRali gamosavaliT. SeviswavleT baqteriofagebisurTierTqmedeba adsorbciis kofaqtorebTan, magaliTad, 2%-iani natriumiscitratTan, fagebi avlenen sakmaod maRal mgrZnobelobas rac SesaZloamiuTiTebdes imaze, rom es fagebi damokidebulni arian orvalentian ionebze,rogorc adsorbciis kofaqtorebze.

baqteriofagebi daxasiaTebisas didi mniSvneloba eniWeba maTserologiur maCveneblebs. antifaguri Sratebis gamoyenebiT Catarebulijvaredina neitralizaciis reaqciebis Sedegebis mixedviT movaxdineTsakvlevi fagebis dayofa 4 serojgufad. aRsaniSnavia, rom serologiurimonacemebi korelaciaSi aRmoCnda fagebis lizisuri speqtris darestriqciuli profilis SedegebTan.

kvlevebis Sedegad aRmoCnda, rom baqteriofagebis 70% aqtiuriagamoyofili Stamebis mimarT. Tumca fagebis umravlesoba xasiaTdebodamaspinZeli Stamis mimarT maRali specifikurobiT. mocemuli SedegiTerapiuli fagebisTvis arasasurveli maxasiaTebelia, Tumca meores mxriv,moqmedebis viwro speqtris mqone fagebi warmatebiT gamoiyeneba StamebisfagotipirebisTvis.

baqteriofagebis dajgufeba garkveul korelaciaSi modis genetikurmaxasiaTeblebTan, kerZod, faguri dnm-is restriqciul profilTan. Tumcaunda aRiniSnos, rom Seswavlili baqteriofagebi moqmedebis diapazonismixedviT ufro mravalferovania, vidre sxva maxasiaTeblebis mixedviT. iseverogorc baqteriebis SemTxvevaSi, baqteriofagebis mravalferovneba lizisurispeqris mixedviT aixsneba zedapiruli cilovani struqturebis- saadsorbcioreceptorebis maRali mravalferovnebiT.

meores mxriv, fagorezistentuli (meoradi) baqteriuli mutantebisanalizma gviCvena, rom Cvens mier gamoyofil baqteriofagebs msgavsi

86

receptoruli aparati gaaCniaT, radgan jvaredina skriningis eqsperimentebmasxvadasxva fagorezistentul mutantebze gviCvena, rom fagebi ar avlenenaqtivobas fagorezistentuli baqteriuli mutantebis mimarT.

restriqciuli profili saSualebas gvaZlevs vimsjeloT fagebisgenetikur mravalferovnebaze. aRsaniSnavia, rom erTi da imave serotipismaspinZeli ujredis mqone baqteriofagebi erT klasterSia gaerTianebuli.amasTan, calke jgufSia Vpa A11, romelic genomis zomiT, serologiuri dafiziologiuri maCveneblebiT gansxvavebulia danarCeni fagebisgan.

cnobilia, rom baqteriofagebze fizikuri da qimiuri faqtorebismainaqtivirebeli moqmedebis Seswavlas didi mniSvneloba aqvs struqturulikomponentebis Senebisa da funqcionirebis SefasebisaTvis. Seswavlil iqnasakvlevi baqteriofagebis damokidebuleba iseTi faqtorebis mimarT,rogoricaa ultraiisferi gamosxiveba, temperatura, wyalbad ionTa maRali andabali maCveneblebi, maRali ionuri Zala da a.S.

Catarebul eqsperimentebSi gamovlinda V.parahaemolyticus-is fagebismdgradoba organuli gamxsnelebis (spirti, qloroformi) mimarT. racarapirdapirad miuTiTebs maT virionebSi lipiduri komponentis ararsebobaze.imis gaTvaliswinebiT, rom literaturaSi aRwerili V.parahaemolyticus-isfagebis umravlesobaMmgrZnobiarea organuli gamxsnelebis mimarT, Cvens miergamoyofili faguri izolatebi gansxvavdeba aqamde Seswavlili

baqteriofagebi gansxvavdebian erTmaneTisgan ultraiisferi gamosxivebismimarT gamZleobis mixedviT. Tumca yvelaze aramdgradi baqteriofagebisSemTxvevaSic inaqtivaciis dro 60 wm-ia, zogierT SemTxvevaSi ki 90 wm.

TermomgrZnobelobis Seswavlam aCvena, rom gamoyofili baqteriofagebisakmaod mdgradia maRali temperaturis mimarT (mag E.coli-is daV.parahaemolyticus-is sxva fagebTan SedarebiT), amasTan isini gansxvavdebianerTmaneTisgan temperaturaze damokidebulebis mixedviT. sakvlevibaqteriofagebis sxvadasxva TermomgrZnobeloba miuTiTebs maTi cilovanistruqturebis gansxvavebaze da amdenad, fagebis taqsonomiuri mikuTvnebisa daarsebuli koleqciis farglebSi dajgufebisaTvis garkveuli mniSvneloba aqvs.

Seswavlili baqteriofagebi mdgradi aRmoCnda osmosuri Sokis mimarT.mocemuli Sedegi imaze miuTiTebs, rom maTi cilovani garsi advilad atarebswylis molekulebs da ionebs. Aarsebobs mosazreba, rom osmosuri SokisadmimgrZnobeloba damokidebulia fagebis cilovani kafsidis simkvriveze, kerZod,kafsomerebis SefuTvis kompaqturobaze. andersonis mier T-fagebi swored amniSnis mixedviT iqna dayofili mgrZnobiare (T-wyvili fagebi) da rezistentul(T3,T5,T7) jgufebad.

cnobilia, rom baqteriofagebis umravlesoba sakmaod stabiluriaSualedur pH-5-9 diapazonSi. sakvebi aris pH-is eqstremalurad maRali andabali maCveneblebi iwvevs virionis degradacias da infeqciurobis dakargvas.

Seswavlili V.parahaemolyticus-is baqteriofagebi sakmaod maRalistabilurobiT xasiaTdeba pH-4-7-is pirobebSi. E pH-is eqstremalurad dabalimniSvnelobis dros nawilakebis raodenoba mkveTrad mcirdeba. igives vervityviT eqstremalurad tute areze pH-10-12, rigi fagebis SemTxvevaSi maRalipH aramarto ar amcirebs, aramed xels uwyobs faguri nawilakebis

87

sicocxlisunarianobis SenarCunebas. Ees SesaZloa aixsnas patroni baqteriebisalkalofiluri TvisebebiTac.

baqteriofagebis praqtikuli gamoyenebisTvis rogorc samkurnalo-profilaqtikuri mizniT, aseve ekologiuri kvlevebis, magaliTad sxvadasxvawyalsatevebSi trasirebis eqsperimentebis Casatareblad, didi mniSvneloba aqvsmaT stabilobas sakvlev garemoSi da aseve Senaxvisa da transportirebispirobebSi.

Aam mizniT Catarebul iqna V.parahaemolyticus-is fagebis stabilobisSeswavla sxvadasxva mikrobiologiur areebsa da xsnarebSi, aseve bunebrivtbisa da zRvis wylebSi.Kkvlevebma gviCvena, rom V.parahaemolyticus-isbaqteriofagebi gamoirCevian stabilurobiT. maTi aqtivoba umniSvnelodecemoda 6 Tvis ganmavlobaSi.P baqteriofagebis, rogorc Terapiuli preparatebis, dasaxasiaTebladmniSvnelovania masze proteolituri fermentebis moqmedebis Seswavla. amtipis eqsperimentebi V.parahaemolyticus –is baqteriofagebze, rogorc potenciurTerapiul saSualebaze aqamde ar gamoqveynebula.

ECvens mier Catarebulma eqsperimentebma aCvena, rom gamoyofiliV.parahaemolyticus –is mimarT specifikuri baqteriofagebi mgrZnobiarea pepsinis1% -iani xsnaris mimarT. imdenad ramdenadac, pepsinis xsnaris pH 1-1.5-ia, xoloCvenma kvlevebma aCvena sicocxlisunariani faguri nawilakebis raodenobismniSvnelovani kleba pH 2-ze, mocemuli Sedegi savsebiT mosalodneli undayofiliyo. miRebuli negatiuri efeqtebis gaTvaliswinebiT, kuWis wvenis mJavepH–is gasaneitraleblad umjobesi iqneba fagebis miReba tute bunebisxsnarebTan erTad.

rac Seexeba qemotrifsinis gavlenas fagebis sicocxlisunarianobaze, ariyo TvalSisacemi. Mmocemuli Sedegi SesaZleblobas iZleva vifiqroTV.parahaemolyticus –is mimarT specifikuri baqteriofagebis gamoyenebaze iqnesWrilobiTi infeqciebis samkurnalod qemotrifsinTan erTobliobaSi.

momdevno etapze saWiroa in vivo- eqsperimentebis Catarebabaqteriofagebis gamoyenebasTan dakavSirebuli uaryofiTi Sedegebisgamoricxvis mizniT.

Cvens mier Catarebuli kvlevis Sedegad SegviZlia davaskvnaT, romsaqarTvelos Savi zRvis sanapiros wylebi xelsayrel garemos warmoadgensVibrio parahaemolyticus-is da Sesabamisi baqteriofagebis sicocxlisunarianobisada gavrcelebisTvis. sayuradReboa baqteriebis serotipirebisa davirulentobis faqtorebis Seswavlis Sedegebi, romlis Tanaxmad gamovlindaSav zRvaSi V. parahaemolyticus-is pandemuri klonebis arseboba.

mtknar wyalsatevebidan baqteriebis gamoyofis dabal sixSire daSesabamisi baqteriofagebis gamoyofis SesaZlebloba mianiSnebs, romaraxelsayrel pirobebSi (mag. Ddabali marilianoba) Vibrio parahaemolyticus-isbaqteriebi gadadian arakultivirebad mdgomareobaSi.

Cvens mier gamoyofili V.parahaemolyticus-is mimarT specifikuribaqteriofagebis biologiur maxasiaTeblebi mianiSnebs maT maRal Terapiulpotencialze.

88

garda Terapiuli daniSnulebisa Seswavlili baqteriofagebis gamoyenebaSesaZlebelia fagotipirebis mizniT erTnairi serotipisa da genetikuristruqturis V.parahaemolyticus-is baqteriuli Stamebis diferencirebisTvis.

89

V. Ddaskvnebi

1. saqarTvelos Savi zRvis sanapiros wylebi xelsayrel pirobebs qmnisVibrio parahaemolyticus-is da Sesabamisi baqteriofagebissicocxlisunarianobisa da gavrcelebisTvis. maTi SemcvelobakorelaciaSia temperaturis matebasTan.

2. mtknari da momlaSo Sida wyalsatevebi araxelsayrel garemoswarmoadgens Vibrio parahaemolyticus-is baqteriebis sicocxlisunarianobisTvis,rac aisaxeba mtknar wyalsatevebSi baqteriebis gamoyofis dabal sixSireze.

3. eqsperimentuli kvleviT dadasturda, rom araxelsayrel pirobebTanSeguebis mizniT Vibrio parahaemolyticus gadadis arakultivirebadmdgomareobaSi.

4. V. parahaemolyticus–is saqarTveloSi gamoyofili Stamebi xasiaTdebamaRali fenotipuri da genetikuri mravalferovnebiT. Ddadgenilia SavzRvaSi Vibrio parahaemolyticus-is pandemuri klonebis arseboba.

5. saqarTveloSi gamoyofili V. parahaemolyticus –is mimarT specifikuribaqteriofagebi xasiaTdeba maRali patron-specifikurobiT, SedarebiTdabali morfologiuri da genetikuri mravalferovnebiT.

6. V. parahaemolyticus –is mimarT specifikuri baqteriofagebi avlenen garemofaqtorebisadmi ufro maRal gamZleobas, vidre Sesabamisi baqteriuliStamebi, rac maT indikatorul Tvisebebs ganapirobebs

7. SerCeulia Vibrio parahaemolyticus-is mimarT specifikuri ramdenimebaqteriofagi, romelTa Terapiuli potenciali efuZneba maT biologiurmaxasiaTeblebs.

8. V. parahaemolyticus –is mimarT specifikuri fagebis nakrebis gamoyenebaSedegs iZleva ganxorcieldes erTnairi serotipisa da genetikuristruqturis mqone V. parahaemolyticus-is izolatebis subtipireba.

90

VI. gamoyenebuli literatura:

1. Адамс М. Бактериофаги. Москва, изд-во «Иностранная Литература», 1961г.

2. Габрилович И.М. Основы бактериофагии. Минск, «Вимеймая школа», 1973.

3. Покровский В.И. Медицинская Микробиология. Учебная литература для студентовмедицинских вузов и врачей. ГЭОТАР МЕДИЦИНА. Москва, 1999, 1184 стр.

4. Abedon ST, Kuhl SJ, Blasdel BG, Kutter EM. 2011. Phage treatment of human infectionsBacteriophage. 1:66-85.

5. Ackermann H.W. Bacteriophage taxonomy. J. Microbiol. Sci. 1987, v.4, №7, p. 214-218.

6. Akiyama, S., K. Takizawa, A. Matsushima, and Y. Miyamoto. 1966. Contamination of Vibrioparahaemolyticus in circulation of sea fish and serotypes of the vibrios isolated from them. Jap. J. Pub.Health 13:159.

7. Allen, R., and Baumann, P. 1971. Structure and arrangement of flagella in species of the genusBeneckea and Photobacterium fischeri. J. Bacteriol. 107: 295-302.

8. Alsina, M., and Blanch, A.R. (1994). Improvement and update of a set of keys for biochemicalidentification of Vibrio species. Journal of Applied Bacteriology, 77: 719-721

9. Anderson T.F., Rapport C., Muscarite N.A. On the structure and osmotic properties of phageparticles. Ann. Inst. Pasteur. 1953, v. 84, pp. 5-14.

10. Arakawa, E.; Murase, T.; Shimada, T.; Okitsu, T.; Yamai, S. and Watanabe, H.; 1999;Emergence and Prevalence of a Novel Vibrio parahaemolyticus O3:K6 Clone in Japan; J. Infect. Dis.,52 (6), 246-247

11. Arbedon S.T., Heschler T.D., Stopar D. Bacteriophage Latent-Period Evolution as a Responseto Resource Aviability. J. Appl. Enviro. Micriobiol, 2001, vol. 67, №9, p. 4233-4241.

12. Asakawa, Y., S. Akabane, and M. Noguchi. 1966. Studies on Vibrio parahaemolyticus. 5.Distribution of the vibrios in Hamana Lake. Jap. J. Pub. Health 13:158.

13. Asplund ME, et al. 2011. Water column dynamics of Vibrio in relation to phytoplanktoncommunity composition and environmental conditions in a tropical coastal area. Environ. Microbiol.13:2738–2751.

14. Atsumi, T., McCarter, L., and Imae, Y. 1992. Polar and lateral flagellar motors of marine Vibrioare driven by different ion membrane forces. Nature. 355: 182-184

91

15. Bag, P. K., S. Nandi, R. K. Bhadra, T. Ramamurthy, S. K. Bhattacharya, M. Nishibuchi, T.Hamabata, S. Yamasaki, Y. Takeda, and G. B. Nair. 1999. Clonal diversity among recently emergedstrains of Vibrio parahaemolyticus O3:K6 associated with pandemic spread. J. Clin. Microbiol.37:2354–2357.

16. Barbieri, E., Falzano, L., Fiorentini, C ., Pianetti, A., Baffone, W., Fabbri, A., Matarrese P.,Casiere, A., Katouli, M., Kuhn, 1., Mollby, R., Bruscolini, F., Donelli, G.(1999). Occurrence, diversity,and pathogenicity of halophilic Vibrio spp. And non-O1 Vibrio cholerae from estuarine waters alongthe Italian Adriatic coast. Applied arid Environmental Microbiology, 65 :2748-2753.

17. Baross, J. A., J. Liston, and R. Y. Morita. 1978. Incidence of Vibrio parahaemolyticusbacteriophages and other Vibrio bacteriophages in marine samples. Appl. Environ. Microbiol. 36:492-499.

18. Bastias, R., Higuera, G., Sierralta, W., Espejo, R.T., 2010. A new group of cosmopolitanbacteriophages induce a carrier state in the pandemic strain of Vibrio parahaemolyticus.

19. Bergey’s Manual of Systematic Bacteriology (2005). Brenner, D.J., Krieg, N .R. and Staley,J.T. Eds. 2nd ed. Volume 2, Part B, Springer, New York, USA.

20. Bergh, O., K. Y. Borsheim, G. Bratbak, and M. Heldal. 1989. High abundance of viruses foundin aquatic environments. Nature 340:467-8.

21. Bhuiyan, N.A, Ansaruzzaman, M., Kamruzzaman, M., Alam, K., Chowdhury, N.R., Nishibuchi,M., Faruque, S.M., Sack, D.A., Takeda, Y., and Nair, G.B., (2002), Prevalence of the pandemicgenotype of Vibrio parahaemolyticus in Dhaka, Bangladesh, and significanceof its distribution acrossdifferent serotypes. J.Clin.Microbiol. 40, 284-286

22. Blackwell KD, Oliver JD., The ecology of Vibrio vulnificus, Vibrio cholerae, and Vibrioparahaemolyticus in North Carolina estuaries, J Microbiol. 2008 Apr;46(2):146-53.

23. Blake, and Weaver, R.W. (1980). Diseases of humans (other than cholera) caused by vibrios.Annual Revieu of microbiology, 34:341-367.

24. Borsheim, K. Y., G. Brantbak, and M. Heldal. 1990. Enumeration and biomass estimation ofplanktonic bacteria and viruses by transmission electron microscopy, Appl. Environ. Microbiol.56:352-356.

25. Botstein D.A. Theory of modular evolution for bacteriophages. Ann. №4. Acad. Sci. 1980. v.354. p. 484-490.

26. Bouvier T, del Giorgio PA, 2007,Key role of selective viral-induced mortality in determiningmarine bacterial community composition. Environ Microbiol. Feb;9(2):287-97.

27. Bratbak, G., M. Heldal, S. Norland, and T. F. Thingstad. 1990. Viruses as partners in springbloom microbial trophodynamics, Appl. Environ. Microbiol. 56:1400-1405.

92

28. Bredley D. E. Ultrastructure of bacteriophages and bacteriocins. J. Bacteriol. Rev.1967,v.31.pp.230-314.

29. Broberg CA, Calder TJ, Orth K. 2011. Vibrio parahaemolyticus cell biology and pathogenicitydeterminants. Microbes Infect. 13:992–1001.

30. Brussaard, C.P.D.; Timmermans, K. R.; Uitz ,J.; Veldhuis, M. J. W.; 2008; Virioplanktondynamics in the waters southeast of the Kerguelen (Southern Ocean). Deep-Sea Res. Pt. II 55:752-765.

31. Brüssow, H., Canchaya, C. & Hardt, W. D. (2004). Phages and the evolution of bacterialpathogens: from genomic rearrangements to lysogenic conversion. Microbiol Mol Biol Rev 68, 560–602.

32. Burrowes B, Harper DR, Anderson J, McConville M, Enright MC. 2011. Bacteriophagetherapy: potential uses in the control of antibiotic-resistant pathogens Expert. Rev. Anti. Infect. Ther.9:775-85.

33. Calendar, R. 2006, The Bacteriophages. OXFORD, University Press.

34. Canchaya C, Fournous G, Chibani-Chennoufi S, Dillmann ML, Brussow H. 2003. Phage asagents of lateral gene transfer Curr. Opin. Microbiol. 6:417-24.

35. Ceccarelli D., Hasan N.A, Huq A., Colwell1 R.R., Distribution and dynamics of epidemic andpandemic Vibrio parahaemolyticus virulence factors, 2013; Front Cell Infect Microbiol. 2013; 3: 97.

36. Chanishvili N. 2012. Phage therapy--history from Twort and d'Herelle through Sovietexperience to current approaches Adv. Virus Res. 83:3-40.

37. Caruso, G., Zaccone, R.. Genovese, L., and Crisafi, E. (I998). Microbiological monitoring ofCastellamare Gulf (TP) waters for their suitability in marine aquaculture. New Microbiology, 21:169-182.

38. Casas V., 2011. Role of bacteriophage-encoded exotoxins in the evolution of bacterialpathogens.

39. Chakraboity, S., Nair, G.B., and Shimada, S. (1997). Pathogenic vibrios in the natural aquaticenvironment. Reviews on Environmental Health, 12:63-80

40. Chang, B., S. Yoshida, H. Miyamoto, M. Ogawa, K. Horikawa, M. Nishibuchi, and H.Taniguchi. 2000. A unique and common restriction fragment pattern of the nucleotide sequenceshomologous to the genome of Vf33, a filamentous bacteriophage, in pandemic strains of Vibrioparahaemolyticus

41. Chanishvili N., Chanishvili T., Tediashvili M., Barrow P.A. Phages and their applicationagainst drug- resistant bacteria .Review. J.Che. Technol. Biotechnol, 2001, 76, p. 689-699.

93

42. Chiou, C., S. Hsu, S. Chiu, T. Wang, and C. Chao. 2000. Vibrio parahaemolyticus serovarO3:K6 as cause of unusually high incidence of food-borne disease outbreaks in Taiwan from 1996 to1999. J. Clin. Microbiol. 38:4621–4625.

43. Chowdhury A, Ishibashi M, Thiem VD, Tuyet DT, Tung TV, Chien BT, Seidlein Lv L, CanhDG, Clemens J, Trach DD, Nishibuchi M. Emergence and serovar transition of Vibrioparahaemolyticuspandemic strains isolated during a diarrhea outbreak in Vietnam between 1997 and1999. Microbiol Immunol 2004a; 48(4): 319–327.

44. Chowdhury N. R., Chakraborty S., Ramamurthy T., Nishibuchi M., Yamasaki S., Takeda Y., etal. (2000). Molecular evidence of clonal Vibrio parahaemolyticus pandemic strains. Emerg. Infect.Dis. 6, 631–636.

45. Chowdhury N. R., Stine O. C., Morris J. G., Nair G. B. (2004b). Assessment of evolution ofpandemic Vibrio parahaemolyticus by multilocus sequence typing. J. Clin. Microbiol. 42, 1280–1282.10.1128/JCM.42.3.1280-1282.

46. Colwell. R.R (1996). Global climate and infectious disease: the cholera paradigm. Science, 274:2025—2031.

47. Colwell R., Huq A. Vibrios in the environment:viable but nonculturable vibrio cholerae.p.117-133. InI.K. Wachmuth, O.Olsvik , P.Blakke (ed.). Vibrio cholerae and cholera: molecular andglobal perspectives.1994. ASM, Wahington, D.C.

48. Colwell R.R., West P. A., Maneval D., Remmers, E.F., Elliot, E.L., and Carlson, NE.,1984;Ecology of pathogenic vibrios in Chesapeake Bay. pp: 367-387. In R.R.Colwell-(ed.), Vibrios in theenvironment, John Wiley & Sons, lnc., New York.

49. Comeau, A. M., A. M. Chan, and C. A. Suttle. 2006. Genetic richness of vibriophages isolatedin a coastal environment. Environ. Microbiol. 8:1164–1176.

50. Comeau, A. M., Buenaventura E., C. A. Suttle., A Persistent, Productive, and SeasonallyDynamic Vibriophage Population within Pacific Oysters (Crassostrea gigas) Appl. Environ.Microbiol., Sept. 2005, 71: 5324–5331.

51. Curtis A. Suttle, Viruses in the sea, 2005, Nature 437, 356-361.

52. Daniels NA, Ray B, Easton A, Marano N, Kahn E, McShan AL 2nd, et al. Emergence of a newVibrio parahaemolyticus serotype in raw oysters: a prevention quandary. JAMA. 2000; 284:1541-5.

53. DePaola, A., C. A. Kaysner, J. Bowers, and D. W. Cook. 2000. Environmental investigations ofVibrio parahaemolyticus in oyster after outbreaks in Washington, Texas, and New York (1997 and1998). Appl. Environ. Microbiol. 66:4649–4654.

94

54. DePaola, A., Ulaszek J.,, Kaysner, C.A., Tenge, B.J., Nordstrom. J.L.. Wells. 1.. Puhr, N.,Gendel. S.M. (2003). Molecular serological and virulence characteristics of Vibrio parahaemolyticusisolated from environmental food and clinical sources in North America and Asia. Applied andEnvironmental Microbiology 69:3999-4005.

55. Deresinski S. 2009. Bacteriophage therapy: exploiting smaller fleas Clin. Infect. Dis. 48:1096-1101.

56. Deter J., Lozach S.,, Véron Antoine., Chollet J., Derrien A., Hervio-Heath D., 2010,Environmental microbiology Volume 12, Issue 4, Pages 929 - 937

57. Diez, B., J. Anton, N. Guixa-Boixereu, C. Pedros-Alio, and R. Rodriguez-Valera.2000. Pulse-field gel electrophoresis analysis of virus assemblages present in a hypersaline environment. Internatl.Microbiol. 3:159-164.

58. Dinh Thi Tuyet,Vu Dinh Thiem, Lorenz von Seidlein, Ashrafazzuman Chowdhury,Eunsik Park,Do Gia Canh, Bui Trong Chien,Tran Van Tung, Abdollah Naficy, Malla Raghav Rao, Mohammad Ali,Hyejon Lee,Trinh Hung Sy, Mitsuaki Nichibuchi, John Clemens and Dang Duc Trach, Clinical,Epidemiological, and Socioeconomic Analysis of an Outbreak of Vibrio parahaemolyticus in KhanhHoa Province, Vietnam, The Journal of Infectious Diseases 2002;186:1615–1620.

59. Enos-berlage, J. L., Guvener, Z. T., Keenan, C. E. & Mccarter, L. L. (2005). Geneticdeterminants of biofilm development of opaque and translucent Vibrio parahaemolyticus. MolMicrobiol 55, 1160–1182.

60. Fabbri, A., Falzano, L., Frank, C ., 1999, Vibrio parahaemolyticus thermostable directhemolysin modulates cytoskeletal organisation and calcium homeostasis in intestinal culturedcells. Infection and Immunity, 67, 1139- 1148.

61. Faruque SM, Mekalanos JJ. 2012. Phage-bacterial interactions in the evolution of toxigenicVibrio cholerae Virulence. 3.

62. Farmer I., James J., Janda J.M, Brenner FW, Cameron DN, Birkhead KM. Genus I. VibrioPacini 1854, 411AL. In: Brenner DJ, Krieg NR, Staley JT, editors. Bergey’s manual of systematicBacteriology. The Proteobacteria. Part B. The Gammaproteobacteria. 2nd ed. New York: Springer;2005. v. 2, p. 494-546.

63. Freifelder D. Molecular biology. Jones and Bartlett Inc., Boston, 1987.

64. Fuerst, J.A. 1980. Bacterial sheathed flagella and the rotary motor model for the mechanism ofbacterial motility. J. Theor. Biol. 84: 761-774.

65. Fujino, T., Sakaguchi, G., Sakazaki, R. & Takeda, Y. (2002). Discovery of Vibrioparahaemolyticus. J Jpn Soc Intern Med 91, 2903–2906.

66. Gavilan RG, Zamudio ML, Martinez-Urtaza J (2013) Molecular Epidemiology and GeneticVariation of Pathogenic Vibrio parahaemolyticus in Peru. PLoS Negl Trop Dis 7(5) .

95

67. George, M.R., John, K.R., lyappan, T., and Jeyaseelan, M.J.P (2005). Genetic heterogenecityamong Vibrlo alginolyticus isolated from shrimp farms by PCR fingerprinting. Letters in AppliedMicrohiology40:369-372.

68. Gonzalez-Escalona, N., Cachicas, V., Acevedo, C., Rioseco, M. L., Vergara, J. A., Cabello, F.,Romero, J. & Espejo, R. T. (2005). Vibrio parahaemolyticus diarrhea, Chile, 1998 and 2004. EmergInfect Dis 11, 129–131.

69. Grimes, D.J., Brayton, P., Colwell, R.R., and Gmber, S.H. (1985). Vibrios as autochthonousflora of neritic sharks. Systematic and Applied .Microbiology, 6:221-226.

70. Guerinot M.L. and Patriquin, D.G.(1981). N2-fixing vibrios isolated from the gastrointestinaltract of sea urchins. Canadian Journal of Microbiology, 27:311-317.

71. Haley BJ, Kokashvili T, Tskshvediani A, Janelidze N, Mitaishvili N, Grim CJ, Constantin deMagny G, Chen AJ, Taviani E, Eliashvili T, Tediashvili M, Whitehouse CA, Colwell RR, HuqA.;Molecular diversity and predictability of Vibrio parahaemolyticus along the Georgian coastal zoneof the Black Sea.; Front Microbiol. 2014; 5:45.

72. Hara, S., K. Terauchi, and I. Koike. 1991. Abundance of viruses in marine water: Assessmentby epifluorescence and transmission electron microscopy. Appl. Enviorn. Microbiol. 57:2731-2734.

73. Hara-Kudo, Y., Kasuga, Y., Kiuchi, A., Horisaka, T., Kawasumi, T., and Kumagai, S. (2003).Increased sensitivity in PCR detection of tdh-positive Vibrio parahaemolyticus in seafood with purifiedtemplate DNA. Journal of Food Protection, 66: 1675-7680.

74. Hardies, S. C., A. M. Comeau, P. Serwer, and C. A. Suttle. 2003. The complete sequence ofmarine bacteriophage VpV262 infecting Vibrio parahaemolyticus indicates that an ancestralcomponent of a T7 viral supergroup is widespread in the marine environment. Virology 310:359–371.

75. Hashimoto, H., S. Baba, T. Terada, H. Zen-Yoji, S.Sakai, Y. Kudo, and T. Ito. 1966. Isolationof new types of Vibrio parahaemolyticus and its etiological distribution. Jap. J. Bacteriol. 21:409-410.

76. Hatfull GF, Hendrix RW. 2011. Bacteriophages and their genomes Curr. Opin. Virol. 1:298-303.

77. Hervio-Heath, D., Colwell, R.R., Derrien, A., Robert-Pillot, A., Fournier, J., and Pommepuy,M. (2003). Occurrence of pathogenic vibrios in coastal areas of France. Journal of AppliedMicrobiology, 92:1123-1135.

78. Hlady, and Klontz, KC. (1996). The epidemiology of Vibrio infections in Florida, 1981 -1993.Journal of Infectious Diseases, 173:1176-1183.

79. Hollis, D.G., Weaver, R.E., Baker, C.N., and Thomsbeny, C, (1976). Halophilic Vibrio spp.isolated from blood cultures. Journal of Clinical Microbiology, 3:425.

96

80. Holmberg .S.D. (1992). Vibrio. In: Gorbach, S.L., Bartlett, J.G., Blacklow,N.R., (eds.),Infectious Diseases, Philadelphia, WB Saunders Company.

81. Honda T, Iida T. The pathogenicity of Vibrio parahaemolyticus and the role of the thermostabledirect hemolysin and related hemolysins. Rev Med Microbiol. 1993;4:106–113.

82. Honda, S., Matsumoto, S., Miwatani, T. & Honda, T. (1992). A survey of urease-positiveVibrio parahaemolyticus strains isolated from traveller’s diarrhea, sea water and imported frozen seafoods. Eur J Epidemiol8, 861-864.

83. Honda, T., Ni, X.Y., and Miwatani, T. (1988). Purification and characterization of a hemolysinproduced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and relatedto the thermostable direct hemolysin. Infection and Immunity, 56: 961-965.

84. Housby JN, Mann NH. 2009. Phage therapy Drug Discov. Today 14:536-40.

85. Hueck CJ., Type III protein secretion systems in bacterial pathogens of animals and plants,1998, Microbiol Mol Biol Rev. Jun;62(2):379-433.

86. Huntley JS, Hall AC, Sathyamoorthy V, Hall RH. Cation flux studies of the lesion induced inhuman erythrocyte membranes by the thermostable direct hemolysin of Vibrio parahaemolyticus. InfectImmun. 1993 Oct;61(10):4326–4332.

87. Igbinosa E.O., Okoh A.I. Emerging Vibrio species: an unending threat to public health indeveloping countries. Res Microbiol. 2008; 159:495–506.

88. Iida, T., A. Hattori, K. Tagomori, H. Nasu, R. Naim, and T. Honda. 2001. Filamentous phageassociated with recent pandemic strains of Vibrio parahaemolyticus. Emerg. Infect. Dis. 7:477–478.Infectious Disease. Philadelphia, WB Saunders Company

89. Islam, M.S.. Tasmin, R., Khan, S.1.._ Bakht, H.B.M., Mahmood, Z.H., Rahman, M.Z.,Bhuiyan, N., Nishibuchi, M., Nair, G.B., Sack, R.B., Huq, A.._ Colwell, R.R., and Sack, D.A. (2004).Pandemic strains of 031146 Vibrio parahaemolyticus in the aquatic environment of Bangladesh.Canadian. Journal of Microbiology, 50:827-834.

90. Jaiani E, Kokashvili T, Mitaishvili N, Elbakidze T, Janelidze N, Lashkhi N, Kalandadze R,Mikashavidze E, Natroshvili G, Whitehouse CA, Huq A, Tediashvili M.; Microbial water quality ofrecreational lakes near Tbilisi, Georgia.; J Water Health. 2013 Jun;11(2):333-45.

91. Jaiani E., Tediashvili M., Janelidze N., Chanishvili N., Ivanova A., Mdzinarashvili T.,Mrevlishvili G., Tushisvili D. Study of the Peculiarities of the Phage-Host Cell Interactions and DNAejection processes in Different Conditions on the model of Cb and Un Phages. (2001) EvergreenInternational phage biology Meeting Evergreen,USA, (2001) p.42.

97

92. Jaiani E., Tediashvili M., Janelidze N., Chanishvili N., Tsertsvadze G., Mdzinarashvili T.,Mrevlishvili G. Peculiarities of the Interactions of Cb and Un Phages with the Host Cell in DifferentConditions. Interact. Microb.World. Amsterdam, (2001) p.395.

93. Jaiani E., Natroshvili G.,Tediashvili M.,Giorgadze N.,Lashkhi N. Investigation of microbialpollution in aquatic reservoires of Tbilisi by biological methods. I -Caucasian Symposium on medical-Biological Sciences, Thesis, September 21-25, Tbilisi,1999, p73-74.

94. Janda, J.M., Powers, Bryant, R.G.. and Abbott, S. (1988). Current perspectives on theepidemiology and pathogenesis of clinically significant Vibrio spp. Clinical Microbiology Reviews, 1:245-267.

95. Jeffrey W. Turner, Leena Malayil, Dominic Guadagnoli, D. Cole, Erin K. Lipp, Detection ofVibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae with respect to seasonal fluctuations intemperature and plankton abundance, Environmental Microbiology, 2014, 16, 3

96. Jiang, S. C., and J. H. Paul. 1998. Gene transfer by transduction in the marine environment.Appl. Environ. Microbiol. 64:2780-7.

97. Johnson C.N., Bowers J.C., Griffitt K.J., Molina V., Clostio R.W., Pei S., Laws E., ParanjpyeR.N., Strom M.S., Chen A., Hasan N.A., Huq A.,. Noriea N.F, Grimes D.J., Colwelle R.R., Ecology ofVibrio parahaemolyticus and Vibrio vulnificus in the Coastal and Estuarine Waters of Louisiana,Maryland, Mississippi, and Washington (United States) Appl Environ Microbiol., 2012; 78(20): 7249–7257.

98. Jones J. L., Lüdeke C.H. M., Bowersc J. C., Fischerb N.G, M., Parsonsd M.B.,. Boppd C.A andDePaolaa A., Biochemical, Serological, and Virulence Characterization of Clinical and Oyster Vibrioparahaemolyticus Isolates, April 2012, J. Clin. Microbiol. vol. 50 no. 7 ,2343-2352

99. Jones, S.H. and B. Summer-Brason. 1998. Incidence and detection of pathogenic Vibrio sp. in anorthern New England estuary, USA. J. of Shellfish Research 17:1665-1669.

100. Joseph SW, Colwell RR, Kaper JB. Vibrio parahaemolyticus and related halophilic vibrios. CritRev Microbiol. 1982;10:77-124.

101. Kampelmacher E.H.. van Noorle, Jansen L.M., Mossel. D.A.A.. Groen, F.J. (1972). A surveyon the occurrence of Vibrio parahaemolyticus and V. alginolyticus on mussels and oysters and inestuarine waters in the Netherlands. Journal of Applied Bacteriology, 35, 431-438.

102. Kaneko, T., and Colwell, R.R. (1978). The annual cycle of Vibrio parahaemolyticus inChesapeake Bay. Microb. Ecol. 4:135-156.

103. Kawagishi, I., Imagawa, M., Imae, Y., McCarter, L., and Homma, M. 1996. The sodium-drivenpolar flagellar motor of marine Vibrio as the mechanosensor that regulates lateral flagellar geneexpression. Mol Microbiol. 20: 693-699.

98

104. Khan, A. A., S. McCarthy, R. F. Wang, and C. E. Cerniglia. 2002. Characterization of UnitedStates outbreak isolates of Vibrio parahaemolyticus using enterobacterial repetitive intergenicconsensus (ERIC) PCR and development of a rapid PCR method for detection of O3:K6 isolates.FEMS Microbiol. Lett. 206:209–214.

105. Khvedelidze M., Mdzinarashvili T., Ivanova A., Tediashvili M., Tushishvili D., MrevlishviliG. The influence of pH on thermal and hydrodynamic properties of DDVI phage and on DNA ejectionfrom phage induced by bacterial membrane fragments. J. Biol. Phys. Chem. 2004, 4, pp.209-213.

106. Koga, T., and T. Kawata. 1991. Comparative characterization of inducible and virulent Vibrioparahaemolyticus bacteriophages having unique head projections. Microbiol. Immunol. 35:49–58.

107. Koga T., Toyoshima S., Kawata T. ; Morphological Varieties and Host Ranges of Vibrioparahaemolyticus Bacteriophages Isolated from Seawater; Appl. And Env. Microbio., 1982, Vol. 44,No. p. 466-470

108. Kogure, K. 1998. Bioenergetics of marine bacteria. Curr. Opin. Biotechnol. 9: 278-82.

109. Kristensen, K.K. (1974). The occurrence of Vibrio paraliaemolyrieus and Vibrio alginolyticusin the Sound. Nord Veterinary Medicine, 26:188-196.

110. Kudoh, Y. 1969. Studies on K antigens of Vibrio parahaemolyticus. 2. Immunochemicalspecificity of K antigens and their sugar constituents. Jap. J. Bacteriol. 24:331-337.

111. Kutateladze M, Adamia R. 2010. Bacteriophages as potential new therapeutics to replace orsupplement antibiotics Trends Biotechnol. 28:591-5.

112. Kutter Elizabeth, Alexander sulakvelidze. Bacteriophages: biology and applications. BurtonGuttman, Raul Raua Elizabeth Kutter. Basic Phage Biology. 2005.

113. Kutter Elizabeth Alexander Sulakvelidze. Bacteriophages: biology and applications. Hans-W.Ackermann. Bacteriophage classification. 2005.

114. Kutter E, De VD, Gvasalia G, Alavidze Z, Gogokhia L et al. 2010. Phage therapy in clinicalpractice: treatment of human infections Curr. Pharm. Biotechnol. 11:69-86.

115. MacRae, J., and M. Srivastava. 1998. Detection of viruses by electron microscopy: An efficientapproach. J. Virol. Meth. 72:105-108.

116. Magny G, Murtugudde R, Sapiano MR, Nizam A, Brown CW, et al. (2008) From the Cover:Environmental signatures associated with cholera epidemics. Proc Natl Acad Sci U S A 105: 17676–17681

117. Makino, K., Oshima, K., Kurokawa, K., Yokoyama, K.. Uda, T., Tagomori, K., Iijima, Y.,Najima M., Nakano, M., Yamashita, A., Kubota, Y., Kimura, S._, Yasunaga, T., Honda, T., Shinagawa,H., Hattori, M., and Iida, T. (2003). Genome sequence of Vibrio parahaemolyticus: a pathogenicmechanism distinct from that of V. cholerae. Lancer, 361 :743-749.

99

118. Manz W., Amann R., Ludwig W., Wagner M., Schleifer K-H. Phylogeneticoligodeoxynucleotide probes for the major subclasses of proteobacteria: problems and solutions. Syst.Appl. Microbiol. 1992, 15, 593–600.

119. Marie, D., C. P. D. Brussard, R. Thyrhaug, G. Bratbak, and D. Voulot. 1999. Enumeration ofmarine viruses in culture and natural samples by flow cytometry, Appl. Enviorn. Microbiol. 65:45-52.

120. Martinez-Urtaza, J.; Lozano-Leon, A.; Varela-Pet, J.; Trinanes, J.; Pazos, Y.; Garcia-Martin, O.;(2008), Environmental determinants of the occurrence and distribution of Vibrio parahaemolyticus inthe rias of Galicia, Spain, Appl. Environ. Microbiol,74, 265-274

121. Matsuda S, et al. 2010. Association of Vibrio parahaemolyticus thermostable direct hemolysinwith lipid rafts is essential for cytotoxicity but not hemolytic activity. Infect. Immun. 78:603–610

122. Matsuzaki, S., T. Inoue, S. Tanaka, T. Koga, M. Kuroda, S. Kimura, and S. Imai. 2000.Characterization of a novel Vibrio parahaemolyticus phage, KVP241, and its relatives frequentlyisolated from seawater. Microbiol. Immunol. 44:953–956.

123. Matsuzaki S, Rashel M, Uchiyama J, Sakurai S, Ujihara T et al. 2005. Bacteriophage therapy: arevitalized therapy against bacterial infectious diseases J. Infect. Chemother. 11:211-9.

124. McCarter, L. 1995. Genetic and molecular characterization of the polar flagellum of Vibrioparahaemolyticus. J. Bacteriol. 177: 1595-1609.

125. McCarter, L.L., and Wright, M.E. 1993. Identification of genes encoding components of theswarmer cell flagellar motor and propeller and a sigma factor controlling differentiation of Vibrioparahaemolyticus. J. Bacteriol. 175: 3361-3371.

126. Mdzinarashvili T.J., Mrevlishvili G.M., Al-Zaza M., Tushishvili D.G., Tediashvili M.I., KiziriaE.L. Thermodynamics of temperature- Induced Destruction of Phages, J. Georg. Enginer. News (GEN),2000, №2 p. 89-92.

127. Merril C.R., Biswas B., Carlton R. Long circulating bacteriophages as antibacterial agents.Proc. Nat. Acad. Sci. USA. 1996. V. 93. P. 3188-3192.

128. Miedzybrodzki R, Borysowski J, Weber-Dabrowska B, Fortuna W, Letkiewicz S et al. 2012.Clinical aspects of phage therapy Adv. Virus Res. 83:73-121.

129. Miller, R. V. 1998. Bacterial gene swapping in nature. Scientific American 278: 66-71.

130. Miller, R. V., S. Ripp, J. Replicon, O. A. Ogunseitan, and T. A. Kokjohn. 1992. Virus-mediatedgene transfer in freshwater environments. In Gauthier, M.J. (ed.), Gene Transfers and Environment,Springer-Verlag, Berlin, German, pp. 50-62.

100

131. Miller, R. V. 1988. Potential for transfer and establishment of engineered genetic sequences inthe environment. In Hodgson, J., and Sugden, A.M. (eds.), Planned Release of Genetically EngineeredOrganisms (Trends Biotech. and Trends Ecol. Evolution Special Publication), Elsevier, Cambridge, pp.S23-7.

132. Miller V. R. Marine Phages, The bacteriophages. 2th edition, R. Calendar (ed), OxfordUniversity Press.

133. Miwatani, T., S. Shinoda, T. Tamura, H. Nishimune, A. Tomaru, A. Yoshihara, and T. Fujino.1969. Antigens of Vibrio parahaemolyticus. I. Preparation of specific antisera to somatic (0) antigenand their application in antigen analysis of Vibrio parahaemolyticus.

134. Miyamoto, Y., T. Kato, Y. Obara, S. Akiyama, K. Takizana, and S. Yamai. 1969. In vitrohemolytic characterization of Vibrio parahaemolyticus: its close correlation with human pathogenicity.J. Bacteriol. 100, 1147-1149.

135. Morris, 1. and Black, R. (1985). Cholera and other vibrioses in the United States. New EnglandJournal of Medicine, 312: 343-350.

136. Muramatsu, K., and H. Matsumoto. 1991. Two generalized transducing phages in Vibrioparahaemolyticus and Vibrio alginolyticus. Microbiol. Immunol. 35:1073–1084.

137. Myers M.L., Panicker G., Bej, A.K.(2003). PCR Detection of a newly emerged pandemicVibrio parahaemolyricus O3:K6 pathogen in pure cultures and seeded waters from the Gulf of Mexico.Applied and Envirorinmental Microbiology, 69: 1194-2200.

138. Nasu, H., T. Iida, T. Sugahara, Y. Yamaichi, K. S. Park, K. Yokoyama, K. Makino, H.Shinagawa, and T. Honda. 2000. A filamentous phage associated with recent pandemic Vibrioparahaemolyticus O3:K6 strains. J. Clin. Microbiol. 38:2156–2161.

139. Nishibuchi. M.. and Kaper. J.B. (1995). Thermostable direct hemolysin gene of Vibrioparahaemolyticus : A virulence gene acquired by a marine bacterium. Infection and Immunity,63:2093-2099.;

140. Nishio, T. 1967. Ecological studies on Vibrio parahaemolyticus.3. Comparison of K antigentype and hemolytic activity between cultures isolated from human patients and from sea water. Jap. J.Bacteriol. 22:446-447.

141. Noble, R. T., and J. A. Fuhrman. 1998. Use of SYBR Green I for rapid epifluorescence countsof marine viruses and bacteria. Aquatic Microb. Ecol. 14:113-118.

142. Ohnishi, T., and K. Nozu. 1986. Induction of phage-like particles from a pathogenic strain ofVibrio parahaemolyticus by mitomycin C. Biochem. Biophys. Res. Commun. 141:1249–1253.

101

143. Okuda J, Ishibashi M, Hayakawa E, Nishino T, Takeda Y, Mukhopadhyay AK, Garg S,Bhattacharya SK, Nair GB, Nishibuchi M. Emergence of a unique O3:K6 clone of Vibrioparahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from SoutheastAsian travelers arriving in Japan. J Clin Microbiol 1997; 35: 3150–3155.

144. Okura, M., Osawa, R., Iguchi, A., Arakawa, E., Terajima, J., and Watanabe, H. (2003).Genotypic analyses of Vibrio parahaemolyticus and development. of a pandemic group specificmultiplex PCR assay. Journal of Clinical Microbiology, 41: 4676-4682

145. Oliver, J.D. 2000. The public health significance of viable but nonculturable bacteria, p. 277-299. In R.R. Colwell and D.J.Grimes (eds.), Nonculturable Microorganisms in the Environment.American Society for Microbiology Press, Washington, D.C.

146. Omori, G., M. Iwao, S. lida, and K. Kuroda. 1966. Studies on K antigen of Vibrioparahaemolyticus. I. Isolation and purification of K antigen from Vibrio parahaemolyticus A55 andsome of its biological properties. Biken J. 9:33-43.

147. Osawa, R., T. Okitsu, H. Morozumi, and S. Yamai. 1996. Occurrence of urease-positive Vibrioparahaemolyticus in Kanagawa, Japan, with specific reference to presence of thermostable directhemolysin (TDH) and the TDH-related-hemolysin genes. Appl. Environ. Microbiol. 62:725–727.

148. Ottemann, K.M., and Miller, J.F. 1997. Roles for motility in bacterial-host interactions. Mol.Microbiol. 24: 1109-1117.

149. Paranjpye R. N., Myers M. S., Yount E. C., Thompson J. L., Zebrafish as a model for Vibrioparahaemolyticus virulence, Microbiology, 2013, 159, Pt_12, 2605

150. Paul and Sulivan, 2005,Marine phage genomics,Current Opinion in Biotechnology, 16:299–307

151. Petsko, Gregory; Ringe, Dagmar (2009). Protein Structure and Function. Oxford: OxfordUniversity Press. pp. 78–79.

152. Pollitzer 1959. History of the disease, pp.11-50. In R. Pollitzer,(ed.), Cholera World HealthOrganization, Geneva, Switzerland.

153. Proctor, L. M., A. Okubo, and J. A. Fuhrman. 1993. Calibrating estimates of phage-inducedmortality in marine bacteria: Ultrastructural studies of marine bacteriophage development from one-step growth experiments. Microb. Ecol. 25:161-182.

154. Proctor, L. M., and J. A. Fuhrman. 1990. Viral mortality of marine bacteria and cyanobacteria.Nature 343:60-62.

155. Raimondi F., Kao J. P., Fiorentini C., Fabbri A., Donelli G., Gasparini N., et al.(2000).Enterotoxicity and cytotoxicity of Vibrio parahaemolyticus thermostable direct hemolysin in invitro systems. Infect. Immun. 68, 3180–3185.

102

156. Reali, D., Caroli, G., Filippi, S., Simonetti, S. (1977). Isolation of halophilic vibrios as possibleagents of food poisoning , Allergy and Immunology Communicable Diseases, 19:455-463.

157. Sakazaki, R. 1965. Vibrio parahaemolyticus a non-choleragenic enteropathogenic vibrio. InProceedings of the cholera research symposium. U.S. Dept. of Health, Education, and Welfare,Washington, D.C.

158. Sakazaki, R., S. Iwanami, and K. Tamura. 1968. Studies on the enteropathogenic, facultativelyhalophilic bacteria, Vibrio parahaemolyticus. I. Serological characteristics. Jap. J. Med. Sci. Biol.21:313-324.

159. Shime-Hattori, A., Iida, T., Arita, M., Park, K. S., Kodama, T. & Honda, T. (2006). Two typeIV pili of Vibrio parahaemolyticus play different roles in biofilm formation. FEMS Microbiol Lett 264,89–97.

160. Shimouchi, H. 1968. Studies on new serological types of Vibrio parahaemolyticus. 2. New K-antigens of new 0 group strains. Bull. Pub. Health Inst. Hyogo Pref. 3:7- 11.

161. Shimouchi, H. 1968. Vibrio parahaemolyticus. 3. New K antigens of 0-3, 0-4, 0-8, and 0-9strains. Bull. Publ. Health Inst. Hyogo Pref. 3:12-16.

162. Sulakvelidze A. 2005. Phage therapy: an attractive option for dealing with antibiotic-resistantbacterial infections Drug Discov. Today 10:807-9.

163. Sulakvelidze A, Alavidze Z, Morris JG, Jr. 2001. Bacteriophage therapy Antimicrob. AgentsChemother. 45:649-59.

164. Suthieukul, O., lshibashi, M., lida, T., Nettip, N., Supavej, S., Eampokalap, B., Makino, M., andHonda, T. (1995). Urease production correlates with possession of trh’-gene in Vibrioparahaemolyticus strains isolated from Thailand. Journal of infectious Diseases, 172: 1405- 1408.

165. Takikawa, L. 1958. Studies on pathogenic halophilic bacteria. Yokohama Med. Bull. 2:313-322.

166. Thompson FL, Iida T, Swings J. Biodiversity of vibrios. Microbiol Mol Biol Rev. 2004; 68:403-31.

167. Turner JW, Paranjpye RN, Landis ED, et al. Population structure of clinical and environmentalVibrio parahaemolyticus from the Pacific Northwest coast of the United States. PLoS One 2013;8:726-736.

168. Twedt R.M., et al, 1972 Antigenic Relationships Among Strains of Vibrio parahaemolyticusApplied mMicrobiology, p.966-971.

169. Twedt, R. M., P. L. Spaulding, and H. E. Hall. 1969. Morphological, cultural, biochemical, andserological comparison of Japanese strains of Vibrio parahaemolyticus with related cultures isolated inthe United States. J. Bacteriol. 98:511-518.

103

170. Vasconcelos G.J., Stang, W.l., and Laidlaw, R.H. (1975). lsolation of Vibrio parahaemolyticusand Vibrio alginolyticus from estuarine areas of southeastern Alaska. Applied Microbiology, 29: 557-559.

171. Vuddhakul, V., Chowdhury, A., Loahaprertthisan, V., Pungrasamee, P., Patararungrong, N.,Thianmontri, P., Ishibashi, M., Matsumoto, C. & Nishibuchi, M. ,2000., Isolation of a pandemicO3 : K6 clone of a Vibrio parahaemolyticus strain from environmental and clinical sources inThailand. Appl Environ Microbiol 66, 2685–2689.

172. Whitehouse C. A, Rangarajan Sampath C.B., Lawrence B. B., Melton R, Li F., Hall T. A,Matthews V.H., Tediashvili M., , Jaiani E., Kokashvili T., Janelidze N., Grim C., Colwell R.R., HuqA.; Identification of Pathogenic Vibrio Species by Multilocus PCR-Electrospray Ionization MassSpectrometry and Its Application to Aquatic Environments of the Former Soviet Republic of Georgia ;Appl Environ Microbiol. 2010; 76-86.

173. Wommack E.K., Colwell R.R., 2000, Virioplankton: Viruses in Aquatic EcosystemsMicrobiology and Molecular Biology Reviews, Vol. 64, No. 1 pp. 69–114.

174. Wong, H. C., S. H. Liu, T. K. Wang, C. L. Lee, C. S. Chiou, D. P. Liu, M. Nishibuchi, and B.K. Lee. 2000. Characteristics of Vibrio parahaemolyticus O3:K6 from Asia. Appl. Environ. Microbiol.66:3981–3986.

175. Xie, Z.Y., Hu. C.Q., Chen, C., Zhang, L.P. and Ren, CH. (2005) investigation of seven Vibriovirulence genes among Vibrio alginolyticus and Vibrio parahaemolyticusstrains from the coastalmariculture systems in Guangdong, China. Letters in Applied Microbiology, 41:202-207.

176. Yamaichi Y, Iida T, Park KS, Yamamoto K, Honda T. Physical and genetic map of the genomeof Vibrio parahaemolyticus: presence of two chromosomes in Vibrio species. MolMicrobiol. 1999(5):1513-21

177. Zen-Yoji H, Le Clair RA, Ota K, Montague TS. Comparison of Vibrio parahaemolyticuscultures isolated in the United States with those isolated in Japan. J Infect Dis. 1973 Mar;127(3):237–241.

178. http://www.britannica.com/EBchecked/topic/450873/pepsin

104

publikaciebi:

Haley, Kokashvili, Tskhvediani, Janelidze, Mitaishvili, Grim, Constantin de Magny, Chen, Taviani,Eliashvili, Tediashvili, Whitehouse, Colwell, Huq, "Molecular Diversity and Predictability of Vibrioparahaemolyticus along the Georgian coastal zone of the Black Sea", 2014, Frontiers in Microbiology,AquaticMicrobiology, Volume5, Article 45, 1-9.

Tskhvediani A., Khukhunashvili T., Eliashvili T., Tsertsvadze G., Gachechiladze N., Tediashvili M.,“The possible use of V. parahaemolyticus –specific bacteriophages for prevention and therapy ofinfections caused by V. parahaemolyticus”, 2014, Georgian Medical News, No 6 (231), pp. 82-88.

N. Janelidze, E. Jaiani, N. Lashkhi, A. Tskhvediani ,T. Kokashvili,T. Gvarishvili , D. Jgenti ,E.Mikashavidze, R. Diasamidze, S. Narodny, R. Obiso, C.A. Whitehouse, A. Huq, M. Tediashvili“Microbial water quality of the Georgian coastal zone of the Black Sea” 2011, Mar. Pollut. Bull., vol.62, pp. 573-580.

A.Tskhvediani, T.Kokashvili, N.Janelidze, N.Lashkhi, T.Khukhunashvili, T.Eliashvili, G.Tsertsvadze,M.Tediashvili. Tbilisi, 2010. “V.parahaemolyticus-specific phages in relation to bacterial host ecologyand diversity”. Georgia Chemical Journal, V.10, N4 (special issue „The main ecological problems ofmodernity and Caucasus“), pp.123-125.

105

sakonferencio moxsenebebi:

2014- Diversity of bacteriophages specific to Vibrio parahaemolyticus (Poster Presentation), ASMgeneral meeting 2014, Boston, USA

2011- V.parahaemolyticus Bacteriophages- Characterisation and Biodiversity, VII internationalscientific conference for students an PhD students "Youth and Progress of Biology", Ivan FrankoNational University of Lviv

2010- V.parahaemolyticus bacteriophages: Relationship to Bacterial Ecology and Diversity, «Virusesof Microbes", Pasteur Institute, Paris, France

Documents

saqarTvelos wylian garemoSi gavrcelebuli Vibrio ... thesis... · saqarTvelos wylian garemoSi gavrcelebuli Vibrio parahaemolyticus-is da mis mimarT specifikuri baqteriofagebis gamoyofa,