Local bacteriophage isolates showed anti- Escherichia coli O157:H7 potency in an experimental ligated rabbit ileal loop model Muntasir Alam, Marufa Zerin Akhter, Mahmuda Yasmin, Chowdhury Rafiqul Ahsan, and Jamalun Nessa Abstract: Escherichia coli O157:H7 is considered among the most important recently emerged food-borne bacteria causing severe hemorrhagic diarrhea. Antibiotic treatment is not recommended as a prospective curative agent against this pathogen. Therefore, potency assessment of the local lytic phage isolates infecting E. coli O157:H7 as an alternate remedy to antibiot- ics was the principal concern of this study. Phage isolates against E. coli O157:H7 were checked by polymerase chain reac- tion for the presence of the virulence genes stx1 and stx2, and the safe phages were further screened in vitro for their capacity as biocontrol agents. Two bacteriophage strains, namely PAH6 and P2BH2, that had expressed potential antibacte- rial activity (P < 0.05) in vitro were selected for in vivo testing in ligated rabbit ileal loop models. Both phage isolates were capable of decreasing fluid accumulation in rabbit ileal loops along with reducing bacterial growth (r = 0.992). Combined application of the phages was found most satisfactory, reducing seven log cycles of bacterial growth. Consistent results in both in vivo and in vitro experiments demonstrate the applicability of bacteriophages as a rapid response tool against E. coli O157:H7. To our knowledge, this is the first successful application of the rabbit ileal loop test for therapeutic evaluation of bacteriophages. Key words: Escherichia coli O157:H7, bacteriophage, phage therapy, ligated rabbit ileal loop assay. Résumé : Escherichia coli O157 :H7 fait partie des bactéries émergentes d’ origine alimentaire causant des diarrhées hémor- ragiques sévères les plus importantes. Le traitement antibiotique comme thérapie curative prospective n’est pas recommandé contre ce pathogène. C’est pourquoi l’évaluation du potentiel d’isolats de phages lytiques locaux infectant E. coli O157 :H7 comme remède alternatif aux antibiotiques était la principale préoccupation de cette étude. Des isolats de phages visant E. coli O157 :H7 ont été examinés par PCR afin de déterminer la présence des gènes de virulence stx1 et stx2, et les phages sécuritaires ont été davantage caractérisés in vitro quant à leur potentiel d’action comme agent de contrôle biologique. Deux souches de bactériophages, PAH6 et P2BH2, qui manifestaient une activité antibactérienne potentielle (P < 0,05) in vitro ont été sélectionnées pour des tests in vivo dans un modèle de ligature de l’anse iléale chez le lapin. Les deux isolats de phages étaient capables de diminuer l’accumulation de liquide dans les anses iléales de lapin, parallèlement à la réduction de la croissance bactérienne (r = 0,992). L’application combinée des phages s’est avérée la plus satisfaisante en réduisant de sept log les cycles de croissance bactérienne. La cohérence des résultats des expériences réalisées in vitro et in vivo dé- montre l’applicabilité de l’utilisation de bactériophages comme outil rapide de réponse envers E. coli O157 :H7. À notre connaissance, il s’agit de la première application fructueuse d’un test utilisant l’anse iléale de lapin dans l’évaluation théra- peutique des bactériophages. Mots‐clés : Escherichia coli O157 :H7, bactériophage, thérapie par phages, test sur l’anse iléale de lapin ligaturée. [Traduit par la Rédaction] Introduction Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga-toxin-producing E. coli, is an important enteropathogen associated with severe gastrointestinal diseases. Although several EHEC serotypes have been associated with human in- fections, one particular serotype, EHEC O157:H7, is recog- nized as the most important recently emerged food-borne pathogen that is predominantly associated with the develop- ment of hemorrhagic colitis and more severe complications, like hemolytic uremic syndrome (Karmali et al. 1983). In spite of its relatively low incidence compared with other en- teric pathogens, E. coli O157:H7 has the potential to produce severe, life-threatening illnesses. Unfortunately, natural infec- tions with E. coli O157:H7 do not confer immunity and no vaccine is available yet to provide protection against this pathogen and other Shiga toxin (Stx)-producing strains Received 28 July 2010. Revision received 26 January 2011. Accepted 15 February 2011. Published at www.nrcresearchpress.com/cjm on XX May 2011. M. Alam, M.Z. Akhter, M. Yasmin, C.R. Ahsan, and J. Nessa. Department of Microbiology, University of Dhaka, Dhaka 1000, Bangladesh. Corresponding author: J. Nessa (e-mail: jamalun_nessa@hotmail.com). 408 Can. J. Microbiol. 57: 408–415 (2011) doi:10.1139/W11-020 Published by NRC Research Press Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by 116.212.107.98 on 05/06/11 For personal use only.