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<p>Abstract</p> <p>Background</p> <p><it>Burkholderia pseudomallei</it> and <it>B</it>. <it>mallei</it> are closely related Category B Select Agents of bioterrorism and the causative agents of the diseases melioidosis and glanders, respectively. Rapid phage-based diagnostic tools would greatly benefit early recognition and treatment of these diseases. There is extensive strain-to-strain variation in <it>B</it>. <it>pseudomallei</it> genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from <it>B</it>. <it>pseudomallei</it> lysogens, for example φK96243, φ1026b and φ52237.</p> <p>Results</p> <p>We have isolated a P2-like bacteriophage, φX216, which infects 78% of all <it>B</it>. <it>pseudomallei</it> strains tested. φX216 also infects <it>B</it>. <it>mallei</it>, but not other <it>Burkholderia</it> species, including the closely related <it>B</it>. <it>thailandensis</it> and <it>B</it>. <it>oklahomensis</it>. The nature of the φX216 host receptor remains unclear but evidence indicates that in <it>B</it>. <it>mallei</it> φX216 uses lipopolysaccharide O-antigen but a different receptor in <it>B</it>. <it>pseudomallei</it>. The 37,637 bp genome of φX216 encodes 47 predicted open reading frames and shares 99.8% pairwise identity and an identical strain host range with bacteriophage φ52237. Closely related P2-like prophages appear to be widely distributed among <it>B</it>. <it>pseudomallei</it> strains but both φX216 and φ52237 readily infect prophage carrying strains.</p> <p>Conclusions</p> <p>The broad strain infectivity and high specificity for <it>B</it>. <it>pseudomallei</it> and <it>B</it>. <it>mallei</it> indicate that φX216 will provide a good platform for the development of phage-based diagnostics for these bacteria.</p>