Appl Microbiol Biotechnol DOI 10.1007/s00253-016-7924-7 APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage Bartosz Roszniowski1 & Agnieszka Latka 1 & Barbara Maciejewska1 & Dieter Vandenheuvel2 & Tomasz Olszak1 & Yves Briers2,3 & Giles S. Holt4 & Miguel A. Valvano5 & Rob Lavigne2 & Darren L. Smith4 & Zuzanna Drulis-Kawa1 Received: 8 July 2016 /Revised: 2 October 2016 /Accepted: 9 October 2016 # The Author(s) 2016. This article is published with open access at Springerlink.com Abstract Burkholderia phage AP3 (vB_BceM_AP3) is a remaining infective particles after 24 h of treatment. AP3 ly- temperate virus of the Myoviridae and the Peduovirinae sub- sogeny can occur by stable genomic integration and by pseu- family (P2likevirus genus). This phage specifically infects do-lysogeny. The lysogenic bacterial mutants did not exhibit multidrug-resistant clinical Burkholderia cenocepacia lineage any significant changes in virulence compared to wild-type IIIA strains commonly isolated from cystic fibrosis patients. host strain when tested in the Galleria mellonella moth wax AP3 exhibits high pairwise nucleotide identity (61.7 %) to model. Moreover, AP3 treatment of larvae infected with Burkholderia phage KS5, specific to the same B. cenocepacia revealed a significant increase (P <0.0001) B. cenocepacia host, and has 46.7–49.5 % identity to phages in larvae survival in comparison to AP3-untreated infected infecting other species of Burkholderia. The lysis cassette of larvae. AP3 showed robust lytic activity, as evidenced by its these related phages has a similar organization (putative broad host range, the absence of increased virulence in lyso- antiholin, putative holin, endolysin, and spanins) and shows genic isolates, the lack of bacterial gene disruption condi- 29–98 % homology between specific lysis genes, in contrast tioned by bacterial tRNA downstream integration site, and to Enterobacteria phage P2, the hallmark phage of this genus. the absence of detected toxin sequences. These data suggest The AP3 and KS5 lysis genes have conserved locations and that the AP3 phage is a promising potent agent against bacte- high amino acid sequence similarity. The AP3 bacteriophage ria belonging to the most common B. cenocepacia IIIA line- particles remain infective up to 5 h at pH 4–10 and are stable at age strains. 60 °C for 30 min, but are sensitive to chloroform, with no Keywords Temperate phage . Peduovirinae . Burkholderia Electronic supplementary material The online version of this article cepacia lineage IIIA (doi:10.1007/s00253-016-7924-7) contains supplementary material, which is available to authorized users. * Zuzanna Drulis-Kawa Introduction [email protected] Gram-negative, non-fermentative bacilli of the Burkholderia 1 Institute of Genetics and Microbiology, University of Wroclaw, cepacia complex (BCC) are inherently difficult to eradicate Przybyszewskiego 63/77, 51-148 Wroclaw, Poland clinically. They are dangerous opportunistic pathogens that 2 Laboratory of Gene Technology, KU Leuven, Kasteelpark Arenberg infect patients suffering from cystic fibrosis (CF). The BCC 21, box 2462, 3001 Leuven, Belgium includes 17 closely related bacterial species (B. cepacia, 3 Present address: Department of Applied Biosciences, Ghent Burkholderia multivorans, Burkholderia cenocepacia, University, Valentin Vaerwyckweg 1, 9000 Ghent, Belgium Burkholderia stabilis, Burkholderia vietnamiensis, 4 Applied Sciences, University of Northumbria, Ellison Building Burkholderia dolosa, Burkholderia ambifaria, Burkholderia EBD222, Newcastle upon Tyne NE1 8ST, UK anthina, Burkholderia pyrrocinia, Burkholderia ubonensis, 5 Center for Experimental Medicine, Queen’s University of Belfast, 97 Burkholderia latens, Burkholderia diffusa, Burkholderia Lisburn Rd., Belfast BT9 7BL, UK arboris, Burkholderia seminalis, Burkholderia metallica, Appl Microbiol Biotechnol Burkholderia contaminans,andBurkholderia lata), which biofilm formation, leading to significant decrease in the mor- can only be discriminated from each other using molecular tality of Pseudomonas-infected animals. The changes in bac- methods (Drevinek and Mahenthiralingam 2010; Medina- terial pathogenicity after phage integration into bacterial ge- Pascualetal.2012). The BCC bacteria survive for long pe- nome can be explained by host gene disruption, CRISPR/Cas riods in moist environments and can cause hospital outbreaks system interaction, or by mechanisms independent of the host especially in immunocompromised patients; they also con- background (Chung et al. 2012; Zegans et al. 2009;Cadyetal. taminate pharmaceutical products and water supplies 2012; Drulis-Kawa et al. 2015). (Gilligan et al. 2003). The most prevalent isolates in CF pa- To date, 37 Burkholderia specific phages have been depos- tients, B. multivorans and B. cenocepacia, are responsible for ited into GenBank (status on 28.06.2016), including members 85–97 % of infections (Drevinek and Mahenthiralingam of the Myoviridae (18), Siphoviridae (9), and Podoviridae 2010). B. cenocepacia also infects patients suffering from (10) families. The presence of a recombinase or integrase in chronic granulomatous disease (CGD; Bylund et al. 2005). the bacteriophage genome, indicating the temperate nature of Lung infection of CF patients by BCC leads to Bcepacia the phage (Gill and Young 2011), was confirmed for 22 of syndrome,^ an acute, necrotizing pneumonia leading to death these 37 sequenced genomes (phiE202, phi52237, KS14, (Isles et al. 1984). Based on recA gene polymorphisms, KL3, KS5, BEK, and φX216 from Myoviridae, B. cenocepacia isolates can be subdivided into four lineages. Peduovirinae,andP2likevirus;AH2,KL1,Bcep176, Lineages IIIA–D and IIIA/IIID are exclusively found in clin- Phi1026b, phiE125, and KS9 from Siphoviridae;and ical samples, IIIC occurs only in soil, and IIIB occurs in both BcepMigl, DC1, BcepC6B, BcepIL02, Bcep22 Bp-AMP1, environmental and clinical samples (Mahenthiralingam et al. Bp-AMP2, Bp-AMP3, and Bp-AMP4 from Podoviridae). 2000;Vandammeetal.2003; Manno et al. 2004). Also several other viruses including Enterobacteria phages: B. cenocepacia has multiple virulence factors, including P2, 186, PsP3, and Wφ; Yersinia phage L-413C; Salmonella cepacian exopolysaccharide (crucial for chronic infections), phages Fels-2 and SopEφ; Pseudomonas phage φCTX; adhesins, pili and flagella, biofilm, and type III and IV secre- Mannheimia phage φ-MhaA1_PHL101; and Ralstonia phage tion systems, recently reviewed in detail (Leitão et al. 2010; RSA1 belong to the P2likevirus genus (Lavigne et al. 2009). Casey and McClean 2015). The B. cenocepacia genome in- Even though these phages were classified as temperate, they cludes large, horizontally transferred genomic islands span- could be engineered as lytic mutants (Lynch et al. 2010r )o ning up to 9.3 % of the chromosome, which are considered provide a source for proteins (e.g., endolysins and to be important in adaptation to different environments depolymerases), which could potentially be used as enzyme- (Holden et al. 2009). based antibacterials. Here, we report a newly discovered Bacteriophages have been proposed as effective antibacte- B. cenocepacia IIIA-specific temperate phage, designated rial agents to eradicate bacterial pathogens (Hyman and AP3 (vB_BceM_AP3), and classified to the P2likevirus ge- Abedon 2010; Abedon 2011; Drulis-Kawa et al. 2012 and nus. This phage was characterized in terms of genome orga- 2015). Treatment with bacteriophages allows targeting patho- nization including lysis cassette, the integration site and lysog- gens that are resistant to conventional drugs without damaging eny event analysis, and the tail fiber protein amino acids com- the host’s natural flora. The efficacy of bacteriophages against position with the other P2-like phages and prophages found in CF pathogens has been shown in vivo using the Galleria host sequences. Furthermore, the efficacy of AP3 in the erad- mellonella insect model and various mouse pulmonary infec- ication of B. cenocepacia infection in moth larvae and the tion models (Seed and Dennis 2009; Carmody et al. 2010; emergence of resistant clones was studied. Lynch et al. 2010; Saussereau et al. 2014; Olszak et al. 2015;Danis-Włodarczyk et al. 2016). The synergistic effect of combining bacterial viruses with antibiotics may lead to higher production of phage progeny and increased phage ac- Materials and methods tivity, improving bacterial killing (Kamal and Dennis 2015). Phage-based treatment poses some risks and limitations; Phage isolation and propagation therefore, a principle requirement is phage precise characteri- zation not only in terms of biology, but also in genomic as- Phage AP3 was isolated from a natural wastewater treatment pects (Merril et al. 2003; Drulis-Kawa et al. 2012 and 2015). plant (irrigated fields) in Wroclaw (Poland) as previously de- There are reports that reinforce the idea of possible temperate scribed (Olszak et al. 2015). AP3 was isolated from and prop- phage application with approaches in using them for bacterial agated in the clinical isolate 7780 of B. cenocepacia IIIA virulence modification. The lysogenization process of temper- lineage (Table 1). The titer and plaque morphology were ate Pseudomonas phages DMS3, MP22, and D3112 caused assessed by the double-agar layer technique (Adams 1959). the inhibition in the expression of virulence factors, including