Inactivation of Burkholderia cepacia complex phage KS9 gp41 identifies the phage repressor and generates lytic virions.

The Burkholderia cepacia complex (BCC) is made up of at least 17 species of gram-negative opportunistic bacterial pathogens that cause fatal infections in patients with cystic fibrosis and chronic granulomatous disease. KS9 (vB_BcenS_KS9), one of a number of temperate phages isolated from BCC species, is a prophage of Burkholderia pyrrocinia LMG 21824. Transmission electron micrographs indicate that KS9 belongs to the family Siphoviridae and exhibits the B1 morphotype. The 39,896-bp KS9 genome, comprised of 50 predicted genes, integrates into the 3' end of the LMG 21824 GTP cyclohydrolase II open reading frame. The KS9 genome is most similar to uncharacterized prophage elements in the genome of B. cenocepacia PC184 (vB_BcenZ_ PC184), as well as Burkholderia thailandensis phage phiE125 and Burkholderia pseudomallei phage phi1026b. Using molecular techniques, we have disrupted KS9 gene 41, which exhibits similarity to genes encoding phage repressors, producing a lytic mutant named KS9c. This phage is incapable of stable lysogeny in either LMG 21824 or B. cenocepacia strain K56-2 and rescues a Galleria mellonella infection model from experimental B. cenocepacia K56-2 infections at relatively low multiplicities of infection. These results readily demonstrate that temperate phages can be genetically engineered to lytic form and that these modified phages can be used to treat bacterial infections in vivo.

New temperate DNA phage BcP15 acts as a drug resistance vector.

This study was designed to determine the role of a new temperate DNA phage BcP15 in relation to drug resistance. The multidrug resistant Shigella flexneri NK1925 was isolated from a patient of Infectious Diseases Hospital, Kolkata, India. This strain contained five plasmids ranging in size from 3 to 212 kb. After curing of five plasmids, this strain became sensitive to antibiotics. A plasmidless multidrug-resistant strain Burkholderia cepacia DR11 was isolated during the survey of microorganisms from coastal waters of deltaic Sunderbans. This strain always released a temperate phage BcP15 into culture supernatant. Turbid plaque formation was observed on the lawn of a plasmidless version (Pl(-)35) of Shigella flexneri NK1925. A few distinct clones (Pl(-)35R) appeared within the region of each plaque after 18 h incubation. S. flexneri NK1925, Pl(-)35, and Pl(-)35R clones showed the same PFGE band pattern of XbaI-digested chromosomal DNA. However, Pl(-)35R clones were resistant to co-trimoxazole, trimethoprim, and eryth- romycin, to which B. cepacia DR11 was also resistant. Southern hybridization results indicated that these three antibiotic resistances in Pl(-)35R clones were due to a BcP15 phage lysogen in the Pl(-)35 version of S. flexneri NK1925.

Isolation and characterization of bacteriophages of the Burkholderia cepacia complex.

The Burkholderia cepacia complex consists of nine phenotypically similar but genotypically distinct beta-proteobacteria that are metabolically diverse and highly antibiotic resistant. Because of this exceptional intrinsic antibiotic resistance, infections with B. cepacia complex members are difficult to treat clinically and new alternative therapies are required. One strategy that holds some promise is the use of naturally occurring antibacterial bacteriophages that could potentially bind to and lyse B. cepacia complex cells in vivo. Towards that end, we used enrichment techniques to isolate lytic and lysogenic bacteriophages specific to the B. cepacia complex. The newly isolated bacteriophages were characterized by host range analysis, electron microscopy, genome restriction analysis, and partial DNA sequencing. These isolates include a bacteriophage with one of the broadest host ranges yet identified for any bacteriophage specific to the B. cepacia complex, and the first description of bacteriophages capable of lysing B. ambifaria.

A new small temperate DNA phage BcP15 isolated from Burkholderia cepacia DR11.

A Burkholderia cepacia DR11 strain was isolated during the survey of microorganisms from coastal water of deltaic Sunderbans. This strain always released temperate phage BcP15 into culture supernatant. UV irradiation of the strain also induced phage induction. The phage titer was 2.3 x 10(8). New temperate phage BcP15 has unusual structure. It has a hexagonal head, 65 nm in diameter and a tail 200 nm long, attached with single thick wavy tail fiber (424-705 nm). Phage DNA is double stranded 11.9 kb long. Southern hybridization result indicated that the phage DNA was in lysogenic state into the B. cepacia DR11 genome. SDS-PAGE of phage protein showed two major bands of molecular weight 20 kDa and 40 kDa.

Burkholderia cenocepacia phage BcepMu and a family of Mu-like phages encoding potential pathogenesis factors.

We have isolated BcepMu, a Mu-like bacteriophage whose host range includes human pathogenic Burkholderia cenocepacia (formally B. cepacia genomovar III) isolates, and determined its complete 36748 bp genomic sequence. Like enteric bacteriophage Mu, the BcepMu genomic DNA is flanked by variable host sequences, a result of transposon-mediated replication. The BcepMu genome encodes 53 proteins, including capsid assembly components related to those of Mu, and tail sheath and tube proteins related to those of bacteriophage P2. Seventeen of the BcepMu genes were demonstrated to encode homotypic interacting domains by using a cI fusion system. Most BcepMu genes have close homologs to prophage elements present in the two published Salmonella typhi genomes, and in the database sequences of Photorhabdus luminescens, and Chromobacterium violaceum. These prophage elements, designated SalMu, PhotoMu and ChromoMu, respectively, are collinear with BcepMu through nearly their entire lengths and show only limited mosaicism, despite the divergent characters of their hosts. The BcepMu family of Mu-like phages has a number of notable differences from Mu. Most significantly, the critical left end region of BcepMu is inverted with respect to Mu, and the BcepMu family of transposases is clearly of a distinct lineage with different molecular requirements at the transposon ends. Interestingly, a survey of 33 B.cepacia complex strains indicated that the BcepMu prophage is widespread in human pathogenic B.cenocepacia ET12 lineage isolates, but not in isolates from the PHDC or Midwest lineages. Identified members of the BcepMu family all contain a gene possibly involved in bacterial pathogenicity, a homolog of the type-two-secretion component exeA, but only BcepMu also carries a lipopolysaccharide modification acyltransferase which may also contribute a pathogenicity factor.

Lysogeny and bacteriophage host range within the Burkholderia cepacia complex.

The Burkholderia cepacia complex comprises a group of nine closely related species that have emerged as life-threatening pulmonary pathogens in immunocompromised patients, particularly individuals with cystic fibrosis or chronic granulomatous disease. Attempts to explain the genomic plasticity, adaptability and virulence of the complex have paid little attention to bacteriophages, particularly the potential contribution of lysogenic conversion and transduction. In this study, lysogeny was observed in 10 of 20 representative strains of the B. cepacia complex. Three temperate phages and five lytic phages isolated from soils, river sediments or the plant rhizosphere were chosen for further study. Six phages exhibited T-even morphology and two were lambda-like. The host range of individual phages, when tested against 66 strains of the B. cepacia complex and a representative panel of other pseudomonads, was not species-specific within the B. cepacia complex and, in some phages, included Burkholderia gladioli and Pseudomonas aeruginosa. These new data indicate a potential role for phages of the B. cepacia complex in the evolution of these soil bacteria as pathogens of plants, humans and animals, and as novel therapeutic agents.

Sensitivity of the Burkholderia cepacia complex and Pseudomonas aeruginosa to transducing bacteriophages.

Burkholderia cepacia is now recognised as a life-threatening pathogen among several groups of immunocompromised patients. In this context, the proposed large-scale use of these bacteria in agriculture has increased the need for a better understanding of the genetics of the species forming the B. cepacia complex. Until now, little information has been available on the bacteriophages of the B. cepacia complex. Transducing phages, named NS1 and NS2, were derived from the lysogenic B. cepacia strains ATCC 29424 and ATCC 17616. The frequency of transduction per phage particle ranged from 1.0x10(-8) to 7.0x10(-6) depending on the phage and recipient strain used. The host range of NS1 and NS2 differed but in each case included environmental and clinical isolates, and strains belonging to several species and genomovars of the B. cepacia complex. The host range of both phages also included Pseudomonas aeruginosa. Some B. cepacia complex isolates were sensitive to the well-characterised P. aeruginosa transducing phages, B3, F116L and G101. The lytic activity of NS1 and NS2 was inhibited by B. cepacia lipopolysaccharide suggesting that this moiety is a binding site for both phages. The molecular size of the NS1 and NS2 genomes was approximately 48 kb.