Figure 3 Multiplex PCR for detection of φX216-related P2-like prophage in B. pseudomallei strains. Genomic DNA preparations of B. pseudomallei strains were used as PCR templates in multiplex PCR. Upper and lower fragments only (B. pseudomallei

2698a and 2704a) indicates presence of a P2-like (P2L) prophage. The presence of three fragments (B. pseudomallei 2692a and 2717a) indicates presence of a P2-like subgroup A prophage (P2L-A). The three marked DNA fragments correspond (top-to-bottom) to the fels-2 PCR product (418 bp), the int gene PCR product (316 bp), and the capsid gene N PCR product (248 bp). Lanes M, Hi-Lo molecular size ladder from Minnesota Molecular (Minneapolis, MN). There is a strong correlation between P2-like prophage-positive B. pseudomallei

strains and high efficiency 4SC-202 plaquing by φX216 on those strains (specificity 79.5%, positive predicative value 73.3%). In other words, it seems as though many B. pseudomallei 3-Methyladenine in vivo strains SB-715992 order that can be efficiently infected by φX216 have been previously infected by one of its P2-like relatives and, strictly speaking, have been converted into lysogens. Conclusions Phage φX216 has one of the highest strain infectivity rates reported among the B. pseudomallei phages characterized to date. Our results indicate that in contrast to previously isolated phages, φX216 infects and propagates only on strains belonging to the B. pseudomallei clade. This is a desirable diagnostic trait and we believe φX216 represents a good candidate platform for the development of phage-based B. pseudomallei diagnostic tools. Although φX216 infects both B. pseudomallei and B. mallei, these two species can be distinguished using φ1026b which is B. mallei-specific [10]. The independent isolation of nearly identical φX216 and φ52237 phages from Thai and Vietnamese isolates, respectively, combined with the apparent broad distribution of P2-like prophage elements in B. pseudomallei highlights the success of this closely-related clade of lysogenic phages at infection and spread among a diverse spectrum of B. pseudomallei strains [16]. Methods

Bacterial growth and preparation of phage lysates Burkholderia sp. used in this study are listed in Additional file 1. Burkholderia sp. and Escherichia coli strains were grown at 37°C with aeration in Lennox LB media as previously described [17]. For growth of B. mallei, LB was supplemented click here with 2-4% glycerol. Growth media for Bp82 and its derivatives were augmented with 80 μg/mL adenine [18]. All procedures involving B. pseudomallei and B. mallei were performed in Select Agent approved Biosafety Level 3 (BSL3) facilities in the Rocky Mountain Regional Biosafety Laboratory (CSU) and the United States Army Medical Research Institute of Infectious Diseases using Select Agent compliant procedures and protocols. Phage plaque plates were prepared by adding 200 μl of a Burkholderia sp. overnight culture to 4 mL of molten top agar (0.6% agar, 0.