φX216, a P2-like bacteriophage with broad Burkholderia pseudomallei and B. malleistrain infectivity

Abstract

BackgroundBurkholderia pseudomallei and B. mallei 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 B. pseudomallei genome content due in part to the presence or absence of integrated prophages. Several phages have previously been isolated from B. pseudomallei lysogens, for example φK96243, φ1026b and φ52237.ResultsWe have isolated a P2-like bacteriophage, φX216, which infects 78% of all B. pseudomallei strains tested. φX216 also infects B. mallei, but not other Burkholderia species, including the closely related B. thailandensis and B. oklahomensis. The nature of the φX216 host receptor remains unclear but evidence indicates that in B. mallei φX216 uses lipopolysaccharide O-antigen but a different receptor in B. pseudomallei. 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 B. pseudomallei strains but both φX216 and φ52237 readily infect prophage carrying strains.ConclusionsThe broad strain infectivity and high specificity for B. pseudomallei and B. mallei indicate that φX216 will provide a good platform for the development of phage-based diagnostics for these bacteria.

RapidBurkholderia pseudomalleiidentification and antibiotic resistance determination by bacteriophage amplification and MALDI-TOF MS

“…In comparison, the ϕX216 spectrum shown in Figure 1B contained only 3 peaks: a 37.6 kDa peak corresponding to the phage major capsid protein, its doubly charged ion at 18.8 kDa, and the phage tail protein at 22.1 kDa. These masses were in agreement with our previously published report describing the isolation, characterization, genome sequencing and annotation of ϕX216 27 . The MALDI-TOF MS ϕX216 limit of detection was determined to be 2.6 × 10 7 pfu/mL.…”

“…In addition to its utility for B. pseudomallei ID, ϕX216 lyses 100% (9/9) of B. mallei strains tested 27 . Although not explicitly assessed in this study, the described approach should therefore also be applicable for identification of B. mallei but not for differentiation of the two species.…”

“…Phage resistance in some strains is a notable caveat to the application of phage amplification for identification and antibiotic resistance determination. Although ϕX216 only infected 78% of B. pseudomallei strains tested, it has one of the broadest ranges of infectivity for a single Burkholderia phage 27 . To cover any remaining subset of resistant strains, one or more additional phages that infect these organisms must be isolated and characterized.…”

Contributions of P2- and P22-like prophages to understanding the enormous diversity and abundance of tailed bacteriophages

“…These include the following phages: Pseudomonas aeruginosa phage ∅CTX (Nakayama et al , 1999), Haemophilus influenzae phages HP1 and HP2 (Esposito et al , 1996) , Pasteurella multocida phage F108 (Campoy et al , 2006) , Mannheimia haemolytica phage ∅MHaA1 (Highlander et al , 2006), Stenotrophomonas maltophilia phage Smp131 (Lee et al , 2014), Aeromonas media phage ∅O18P (Beilstein and Dreiseikelmann, 2008), and Vibrio cholerae phage K139 (Kapfhammer et al , 2002) (hosts in the Gammaproteobacteria Pseudomonadaceae, Pasteurellaceae, Aeoromonadaceae, Xanthomonadaceae a nd Vibrionaceae families), as well as several Burkholderia cepacia and Ralstonia solanacearum Betaproteobacteria phages (Fujiwara et al , 2008; Lynch et al , 2010 and 2012; Kvitko et al , 2012; Niu et al , 2015). Although all well-characterized P2 supercluster phages are temperate, the “P2-like” Burkholderia phages ST79 and ∅E12-2 have no clearly recognizable integrase genes; ST79 has been reported to be lytic (Yordpratum et al , 2011; Kulsuwan et al , 2014), while it has been suggested that ∅E12-2 is temperate (Nakornpakdee et al , 2015).…”

The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage

“…BCC resistance to phage infection was considered as possible lysogeny by AP3 temperate phage during the bacterial culture infection. The presence of the AP3 genome within the bacterial cell was confirmed by PCR according to Kvitko et al (2012). First, the unique AP3 gene vB_BceM_AP3_0002 encoding cytotoxic translational repressor of toxin-antitoxin stability system was chosen for confirmation of AP3 genome presence inside the host cell (F: 5′-ATGAACTCGATCAAATGGACCCC-3′; R: 5′-TTAGTACGTGCGTTCATCGCGTTTC-3′; annealing temperature 57 °C; expected product size 254 bp).…”