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0301 SELECTION OF ENTEROHAEMORRHAGIC ESCHERICHIA COLI PHAGES FOR THEIR USE IN PHAGE THERAPY IN CATTLE C Dini1 2, P de Urraza1 21 Cátedra de Microbiología General, Departamento de Ciencias Biologicas, Facultad de Ciencias Exactas, UNLP, La Plata, Argentina. 2 Centro de investigación y desarrollo en Criotecnología de Alimentos (CIDCA-CCT-CONICET), La Plata, Argentina Purpose of the study: Selection of the best cocktail of phages from eleven previously isolated coliphages for reducing Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 population in calves. Phages are analyzed for its pH resistance, bacterial cell damage and presence of shiga toxins (stx) by PCR. Phage genomes are characterized by RAPD and REP-PCR. Methods: PH resistance: Phages were exposed to different buffer solutions ranged from pH 1 to 7 for 20 hs at 37ºC, and PFUs were determined by the double agar layer method after treatment.Evaluation of bacterial membrane damage by flow cytometry: Bacterial damage using one selected coliphage was first measured by comparing fluorescence intensity at different incubation times after carboxyfluorescein diacetate (CFDA) staining and compared with non-phage treated EHEC EDL 933 cells. Bacterial lysis was also measured by events reduction against controls. Exponential phase culture was mixed with phage suspension in SM buffer at multiplicity of infection of 1 or with sterile SM buffer (controls) and incubated at 37ºC. Samples were taken at 0, 30 and 60 min of incubation. In a second assay cell damage was compared between all phages in the same conditions as previously with an incubation time of 75min. Flow cytometric analysis was performed as mentioned above.Phage genome analysis by PCR: DNA was obtained from DNAse treated phage lysates by standard phenol-chloroform extraction and isopropanol precipitation. Phage DNA quantity was mesured by optical density at 260/280 nm and standarized at 30 ng/ul for PCR assays. Stx1 and stx2 presence was screened by multiplex PCR. Phage genomes were compared by REP-PCR and RAPD-PCR amplification profiles using three different RAPD primers.Results and conclusions: All phages showed total resistance from pH 7 to 5. A decrease of less than 1 logarithmic order on PFUs of some phages was observed at pH 4,4. At pH 3,4 three phages showed the highest acid resistance by a PFU reduction of less than 4 log. while the others showed higher reductions or total inactivation. No PFUs were detected from pH 3 to 1. Flow cytometric analysis at different incubation times showed a reduction of 60% on cell population at 60 min and a progressive increase of fluorescence intensity in time on treated samples. Controls remained constant, indicating cell damage and lysis as a result of phage infection. On the second assay a reduction of 1 log on total events was observed for almost all phages except for two that didnt show significant increase on fluorescence intensity either, suggesting that those two phages arent as good infecting EDL 933 as they are on their original host. All other phages produced a significant increase on fluorescence intensity, associated to a higher bacterial membrane permeability compared with the control.None phage showed amplification of any stx genes by PCR. All RAPD profiles were similar for all phages, but REP profiles were different for the same two phages with different flow cytometric results than those of the other phages.