STUDIES ON THE SCREENING FORKLEBSIELLA PENEU MONIAE VBNCMUTANTS AND ITSTRANSFOR MATI ON CHARACTERISTICS

VBNC (viable but non-culturable state) bacteria are considered to represent a sub-population of cells which are not able to grow in the usual culturemedia but yet remainmetabolically active, and can be resuscitatedwhen conditions be-come favorable So far, themolecular basis for entry into and resuscitation from theVBNC state are still poorly known, and all these obscurationsmustmake clear, becauseVBNC bacteria is concerned about public health, evaluatingwater quality, epide m ic prevention To better understand the molecular mechanism of bacterial VBNC state, th is study used transposon Tn5 to induce the gram negative bacteria-K lebsiella peneumoniae by parental con jugation method Exconjugations were iso-lated from culturemed ium with 50 ug/mL km and 15 ug/mL Tc and detected by PCR using prmiers IS1 and IS-which had been designed usingTn5 as the template?PCR productswere visualized on a1% agarose gel After that, exconjugations were incubated in sterilized distilled waterwith 3% sodium chloride at4 for screeningmutantswhichwas the fastest and the easiest entered theVBNC state?The results showed that transposon Tn5 were inserted in chromosome ofK?peneumoniae successfully, about 2.3*104cell/mL exconjugationswere obtained, and its transposon frequencywas high at 7.6*10-5. Exconjugations cou ld be detected by PCR using prmiers IS. and IS-Comparing with the other stains, a mutantKPQT-7 was foundmuch faster and easier to enterVBNC state, throughout the incubation, total direct cell counts re mained near the original inoculum level of 106to 107cell/mL, and the plate cell counts decreased day by day, more than 30% ofKPQT-7 popu lation had been into VBNC state after 9d stress treatment, wh ile the control stain entered the VBNC state after-1d stress treat ment It can be concluded thatK peneumoniae entered the VBNC statem ight dealwith the sequence flanking the Tn5 insertion site The genetic analysis on themutantKPQT-7 are ongoing, this expermient has laid a solid foundation for the further research on molecularmechanism of the VBNC transformation in aquatic bacteria.