In this era of global rapid spread of multiple antibiotic resistance in clinically relevant bacterial species, alternative or adjuvant therapies to antibiotics are urgently needed to both combat refractory infections and limit dissemination of problematic opportunistic pathogens [1]. Bacteriophage therapy may provide a valid clinical alternative, as obligately lytic bacteriophages can be readily isolated and have the potential to be effective against multidrug resistant bacteria [2]. However, routine implementation of bacteriophage treatment in the clinic is hindered by poor understanding of therapeutic applicability, penetration, and resistance issues [3]. We are currently investigating a rational design approach to the development of bacteriophage therapeutic cocktails to be used against pathogenic bacterial clones. The targets chosen for this work are ST131 E. coli, with bla_CTX-M-15, and cc258 K. pneumoniae, carrying bla_KPC, both globally disseminated highly virulent clones dominating the epidemiology of life-threatening multidrug resistant nosocomial infections [4]. These species are also common gut colonizers able to persist asymptomatically for up to 12 months, thus increasing the risk of transmission to vulnerable individuals [5]. We have fully characterized sets of target bacteria (60 ST131 E. coli; 20 cc258 K. pneumoniae), and tested the infectivity of >30 bacteriophages (AmpliPhi Biosciences Corporation) from our extensive libraries. Phages specific to each target population (n=8 for E. coli; n=12 for K. pneumoniae) were selected for detailed characterization. Using a combined genomic and molecular microbiology approach, we found that the genetic diversity of each bacterial population was associated with specific phage susceptibility profiles reflecting the structural specificity of the bacterial outer cell envelope. This represents the first step in our workflow towards the establishment of a robust scientific protocol for the selection of phages for the development of optimal therapeutic cocktails.