The isolation and characterization of two Stenotrophomonas maltophilia bacteriophages capable of cross-taxonomic order infectivity.

BACKGROUND: A rapid worldwide increase in the number of human infections caused by the extremely antibiotic resistant bacterium Stenotrophomonas maltophilia is prompting alarm. One potential treatment solution to the current antibiotic resistance dilemma is "phage therapy", the clinical application of bacteriophages to selectively kill bacteria. RESULTS: Towards that end, phages DLP1 and DLP2 (vB_SmaS-DLP_1 and vB_SmaS-DLP_2, respectively) were isolated against S. maltophilia strain D1585. Host range analysis for each phage was conducted using 27 clinical S. maltophilia isolates and 11 Pseudomonas aeruginosa strains. Both phages exhibit unusually broad host ranges capable of infecting bacteria across taxonomic orders. Transmission electron microscopy of the phage DLP1 and DLP2 morphology reveals that they belong to the Siphoviridae family of bacteriophages. Restriction fragment length polymorphism analysis and complete genome sequencing and analysis indicates that phages DLP1 and DLP2 are closely related but different phages, sharing 96.7 % identity over 97.2 % of their genomes. These two phages are also related to P. aeruginosa phages vB_Pae-Kakheti_25 (PA25), PA73, and vB_PaeS_SCH_Ab26 (Ab26) and more distantly related to Burkholderia cepacia complex phage KL1, which together make up a taxonomic sub-family. Phages DLP1 and DLP2 exhibited significant differences in host ranges and growth kinetics. CONCLUSIONS: The isolation and characterization of phages able to infect two completely different species of bacteria is an exciting discovery, as phages typically can only infect related bacterial species, and rarely infect bacteria across taxonomic families, let alone across taxonomic orders.

Genomic characterization of JG068, a novel virulent podovirus active against Burkholderia cenocepacia.

BACKGROUND: As is true for many other antibiotic-resistant Gram-negative pathogens, members of the Burkholderia cepacia complex (BCC) are currently being assessed for their susceptibility to phage therapy as an antimicrobial treatment. The objective of this study was to perform genomic and limited functional characterization of the novel BCC phage JG068 (vB_BceP_JG068). RESULTS: JG068 is a podovirus that forms large, clear plaques on Burkholderia cenocepacia K56-2. Host range analysis indicates that this phage can infect environmental, clinical, and epidemic isolates of Burkholderia multivorans, B. cenocepacia, Burkholderia stabilis, and Burkholderia dolosa, likely through interaction with the host lipopolysaccharide as a receptor. The JG068 chromosome is 41,604 base pairs (bp) in length and is flanked by 216 bp short direct terminal repeats. Gene expression originates from both host and phage promoters and is in the forward direction for all 49 open reading frames. The genome sequence shows similarity to Ralstonia phage ϕRSB1, Caulobacter phage Cd1, and uncharacterized genetic loci of blood disease bacterium R229 and Burkholderia pseudomallei 1710b. CoreGenesUniqueGenes analysis indicates that JG068 belongs to the Autographivirinae subfamily and ϕKMV-like phages genus. Modules within the genome encode proteins involved in DNA-binding, morphogenesis, and lysis, but none associated with pathogenicity or lysogeny. Similar to the signal-arrest-release (SAR) endolysin of ϕKMV, inducible expression of the JG068 SAR endolysin causes lysis of Escherichia coli that is dependent on the presence of an N-terminal signal sequence. In an in vivo assay using the Galleria mellonella infection model, treatment of B. cenocepacia K56-2-infected larvae with JG068 results in a significant increase in larval survival. CONCLUSIONS: As JG068 has a broad host range, does not encode virulence factors, is obligately lytic, and has activity against an epidemic B. cenocepacia strain in vivo, this phage is a highly promising candidate for BCC phage therapy development.

PHAST: a fast phage search tool.

PHAge Search Tool (PHAST) is a web server designed to rapidly and accurately identify, annotate and graphically display prophage sequences within bacterial genomes or plasmids. It accepts either raw DNA sequence data or partially annotated GenBank formatted data and rapidly performs a number of database comparisons as well as phage 'cornerstone' feature identification steps to locate, annotate and display prophage sequences and prophage features. Relative to other prophage identification tools, PHAST is up to 40 times faster and up to 15% more sensitive. It is also able to process and annotate both raw DNA sequence data and Genbank files, provide richly annotated tables on prophage features and prophage 'quality' and distinguish between intact and incomplete prophage. PHAST also generates downloadable, high quality, interactive graphics that display all identified prophage components in both circular and linear genomic views. PHAST is available at (http://phast.wishartlab.com).

Characterization of Bacteriophage cd2, a Siphophage Infecting Carnobacterium divergens and a Representative Species of a New Genus of Phage.

Carnobacterium divergens is frequently isolated from natural environments and is a predominant species found in refrigerated foods, particularly meat, seafood, and dairy. While there is substantial interest in using C. divergens as biopreservatives and/or probiotics, some strains are known to be fish pathogens, and the uncontrolled growth of C. divergens has been associated with food spoilage. Bacteriophages offer a selective approach to identify and control the growth of bacteria; however, to date, few phages targeting C. divergens have been reported. In this study, we characterize bacteriophage cd2, which we recently isolated from minced beef. A detailed host range study reveals that phage cd2 infects certain phylogenetic groups of C. divergens. This phage has a latent period of 60 min and a burst size of ~28 PFU/infected cell. The phage was found to be acid and heat sensitive, with a complete loss of phage activity when stored at pH 2 or heated to 60°C. Electron microscopy shows that phage cd2 is a siphophage, and while it shares the B3 morphotype with a unique cluster of Listeria and Enterococcus phages, a comparison of genomes reveals that phage cd2 comprises a new genus of phage, which we have termed as Carnodivirus. IMPORTANCE Currently, very little is known about phages that infect carnobacteria, an important genus of lactic acid bacteria with both beneficial and detrimental effects in the food and aquaculture industries. This report provides a detailed characterization of phage cd2, a novel siphophage that targets Carnobacterium divergens, and sets the groundwork for understanding the biology of these phages and their potential use in the detection and biocontrol of C. divergens isolates.

Comparative analysis of two phenotypically-similar but genomically-distinct Burkholderia cenocepacia-specific bacteriophages.

BACKGROUND: Genomic analysis of bacteriophages infecting the Burkholderia cepacia complex (BCC) is an important preliminary step in the development of a phage therapy protocol for these opportunistic pathogens. The objective of this study was to characterize KL1 (vB_BceS_KL1) and AH2 (vB_BceS_AH2), two novel Burkholderia cenocepacia-specific siphoviruses isolated from environmental samples. RESULTS: KL1 and AH2 exhibit several unique phenotypic similarities: they infect the same B. cenocepacia strains, they require prolonged incubation at 30°C for the formation of plaques at low titres, and they do not form plaques at similar titres following incubation at 37°C. However, despite these similarities, we have determined using whole-genome pyrosequencing that these phages show minimal relatedness to one another. The KL1 genome is 42,832 base pairs (bp) in length and is most closely related to Pseudomonas phage 73 (PA73). In contrast, the AH2 genome is 58,065 bp in length and is most closely related to Burkholderia phage BcepNazgul. Using both BLASTP and HHpred analysis, we have identified and analyzed the putative virion morphogenesis, lysis, DNA binding, and MazG proteins of these two phages. Notably, MazG homologs identified in cyanophages have been predicted to facilitate infection of stationary phase cells and may contribute to the unique plaque phenotype of KL1 and AH2. CONCLUSIONS: The nearly indistinguishable phenotypes but distinct genomes of KL1 and AH2 provide further evidence of both vast diversity and convergent evolution in the BCC-specific phage population.

Characterization of DC1, a broad-host-range Bcep22-like podovirus.

Bcep22-like phages are a recently described group of podoviruses that infect strains of Burkholderia cenocepacia. We have isolated and characterized a novel member of this group named DC1. This podovirus shows many genomic similarities to BcepIL02 and Bcep22, but it infects strains belonging to multiple Burkholderia cepacia complex (BCC) species.

Cangene gold medal award lecture - Genomic analysis and modification of Burkholderia cepacia complex bacteriophages.

The Burkholderia cepacia complex (Bcc) is a group of 17 Gram-negative predominantly environmental bacterial species that cause potentially fatal opportunistic infections in cystic fibrosis (CF) patients. Although its prevalence in these individuals is lower than that of Staphylococcus aureus and Pseudomonas aeruginosa , the Bcc remains a serious problem in the CF community because of the pathogenicity, transmissibility, and inherent antibiotic resistance of these organisms. An alternative treatment for Bcc infections that is currently being developed is phage therapy, the clinical use of viruses that infect bacteria. To assess the suitability of individual phage isolates for therapeutic use, the complete genome sequences of a panel of Bcc-specific phages were determined and analyzed. These sequences encode a broad range of proteins with a gradient of relatedness to phage and bacterial gene products from Burkholderia and other genera. The majority of these phages were found not to encode virulence factors, and despite their predominantly temperate nature, a proof-of-principle experiment has shown that they may be modified to a lytic form. Both the genomic characterization and subsequent engineering of Bcc-specific phages are fundamental to the development of an effective phage therapy strategy for these bacteria.

An antirepressor, SrpR, is involved in transcriptional regulation of the SrpABC solvent tolerance efflux pump of Pseudomonas putida S12.

Organic compounds exhibit various levels of toxicity toward living organisms based upon their ability to insert into biological membranes and disrupt normal membrane function. The primary mechanism responsible for organic solvent tolerance in many bacteria is energy-dependent extrusion via efflux pumps. One such bacterial strain, Pseudomonas putida S12, is known for its high tolerance to organic solvents as provided through the SrpABC resistance-nodulation-cell division (RND) family efflux pump. To determine how two putative regulatory proteins (SrpR and SrpS, encoded directly upstream of the SrpABC structural genes) influence SrpABC efflux pump expression, we conducted transcriptional analysis, ß-galactosidase fusion experiments, electrophoretic mobility shift assays, and pulldown analysis. Together, the results of these experiments suggest that expression of the srpABC operon can be derepressed by two distinct but complementary mechanisms: direct inhibition of the SrpS repressor by organic solvents and binding of SrpS by its antirepressor SrpR.

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.

Genomic analysis and relatedness of P2-like phages of the Burkholderia cepacia complex.

BACKGROUND: The Burkholderia cepacia complex (BCC) is comprised of at least seventeen Gram-negative species that cause infections in cystic fibrosis patients. Because BCC bacteria are broadly antibiotic resistant, phage therapy is currently being investigated as a possible alternative treatment for these infections. The purpose of our study was to sequence and characterize three novel BCC-specific phages: KS5 (vB_BceM-KS5 or vB_BmuZ-ATCC 17616), KS14 (vB_BceM-KS14) and KL3 (vB_BamM-KL3 or vB_BceZ-CEP511). RESULTS: KS5, KS14 and KL3 are myoviruses with the A1 morphotype. The genomes of these phages are between 32317 and 40555 base pairs in length and are predicted to encode between 44 and 52 proteins. These phages have over 50% of their proteins in common with enterobacteria phage P2 and so can be classified as members of the Peduovirinae subfamily and the "P2-like viruses" genus. The BCC phage proteins similar to those encoded by P2 are predominantly structural components involved in virion morphogenesis. As prophages, KS5 and KL3 integrate into an AMP nucleosidase gene and a threonine tRNA gene, respectively. Unlike other P2-like viruses, the KS14 prophage is maintained as a plasmid. The P2 E+E' translational frameshift site is conserved among these three phages and so they are predicted to use frameshifting for expression of two of their tail proteins. The lysBC genes of KS14 and KL3 are similar to those of P2, but in KS5 the organization of these genes suggests that they may have been acquired via horizontal transfer from a phage similar to λ. KS5 contains two sequence elements that are unique among these three phages: an ISBmu2-like insertion sequence and a reverse transcriptase gene. KL3 encodes an EcoRII-C endonuclease/methylase pair and Vsr endonuclease that are predicted to function during the lytic cycle to cleave non-self DNA, protect the phage genome and repair methylation-induced mutations. CONCLUSIONS: KS5, KS14 and KL3 are the first BCC-specific phages to be identified as P2-like. As KS14 has previously been shown to be active against Burkholderia cenocepacia in vivo, genomic characterization of these phages is a crucial first step in the development of these and similar phages for clinical use against the BCC.

Genomic sequence and activity of KS10, a transposable phage of the Burkholderia cepacia complex.

BACKGROUND: The Burkholderia cepacia complex (BCC) is a versatile group of Gram negative organisms that can be found throughout the environment in sources such as soil, water, and plants. While BCC bacteria can be involved in beneficial interactions with plants, they are also considered opportunistic pathogens, specifically in patients with cystic fibrosis and chronic granulomatous disease. These organisms also exhibit resistance to many antibiotics, making conventional treatment often unsuccessful. KS10 was isolated as a prophage of B. cenocepacia K56-2, a clinically relevant strain of the BCC. Our objective was to sequence the genome of this phage and also determine if this prophage encoded any virulence determinants. RESULTS: KS10 is a 37,635 base pairs (bp) transposable phage of the opportunistic pathogen Burkholderia cenocepacia. Genome sequence analysis and annotation of this phage reveals that KS10 shows the closest sequence homology to Mu and BcepMu. KS10 was found to be a prophage in three different strains of B. cenocepacia, including strains K56-2, J2315, and C5424, and seven tested clinical isolates of B. cenocepacia, but no other BCC species. A survey of 23 strains and 20 clinical isolates of the BCC revealed that KS10 is able to form plaques on lawns of B. ambifaria LMG 19467, B. cenocepacia PC184, and B. stabilis LMG 18870. CONCLUSION: KS10 is a novel phage with a genomic organization that differs from most phages in that its capsid genes are not aligned into one module but rather separated by approximately 11 kb, giving evidence of one or more prior genetic rearrangements. There were no potential virulence factors identified in KS10, though many hypothetical proteins were identified with no known function.

Development of a species-specific fur gene-based method for identification of the Burkholderia cepacia complex.

Burkholderia is an important bacterial genus with a complex taxonomy that contains species of both ecological and pathogenic importance, including nine closely related species collectively termed the Burkholderia cepacia complex (BCC). Unfortunately, 16S rRNA gene analysis has proven to be not sensitive enough to discriminate between species of the BCC. Alternative species identification strategies such as recA-based PCR followed by restriction fragment length polymorphism analysis, although initially useful, have proven to be inaccurate with the increasing species diversity of the BCC. recA gene sequence analysis is more discriminatory and corroborates other biochemical and polyphasic means of taxonomic differentiation. However, it is limited by the fact that certain BCC species are subdivided into discrete recA sequence subgroups that may confuse clinical diagnoses. In this study, an effective approach is described for the rapid differentiation of BCC species from both environmental and clinical sources by means of a single-locus sequencing and PCR assay using fur as a target gene that provides sequence phylogenies that are species specific and, with few exceptions, not divided into subspecies clusters. This assay is specific and can be used to correctly determine the species status of BCC strains tested following sequencing and amplification of the fur gene by both general and species-specific primers. Based on our results, this typing strategy is simpler than and as sensitive as established tests currently in use clinically. This assay is useful for the rapid, definitive identification of all nine current BCC species and potentially novel species groups.

Identification and characterization of ϕH111-1: A novel myovirus with broad activity against clinical isolates of Burkholderia cenocepacia.

Characterization of prophages in sequenced bacterial genomes is important for virulence assessment, evolutionary analysis, and phage application development. The objective of this study was to identify complete, inducible prophages in the cystic fibrosis (CF) clinical isolate Burkholderia cenocepacia H111. Using the prophage-finding program PHAge Search Tool (PHAST), we identified three putative intact prophages in the H111 sequence. Virions were readily isolated from H111 culture supernatants following extended incubation. Using shotgun cloning and sequencing, one of these virions (designated ϕH111-1 [vB_BceM_ϕH111-1]) was identified as the infective particle of a PHAST-detected intact prophage. ϕH111-1 has an extremely broad host range with respect to B. cenocepacia strains and is predicted to use lipopolysaccharide (LPS) as a receptor. Bioinformatics analysis indicates that the prophage is 42,972 base pairs in length, encodes 54 proteins, and shows relatedness to the virion morphogenesis modules of AcaML1 and "Vhmllikevirus" myoviruses. As ϕH111-1 is active against a broad panel of clinical strains and encodes no putative virulence factors, it may be therapeutically effective for Burkholderia infections.

Respirable bacteriophages for the treatment of bacterial lung infections.

This review article discusses the development of respiratory therapeutics containing bacteriophages indicated for lung infections, specifically those that have become increasingly difficult to treat because of antibiotic resistance. Recent achievements and remaining problems are presented for each step necessary to develop a bacteriophage-containing dosage form for respiratory drug delivery, including selection of appropriate bacteriophages for therapy, processing and purification of phage preparations, formulation into a stable, solid dosage form, and delivery device selection. Safety and efficacy studies in animals and human subjects are also reviewed.

The promise of bacteriophage therapy for Burkholderia cepacia complex respiratory infections.

In recent times, increased attention has been given to evaluating the efficacy of phage therapy, especially in scenarios where the bacterial infectious agent of interest is highly antibiotic resistant. In this regard, phage therapy is especially applicable to infections caused by the Burkholderia cepacia complex (BCC) since members of the BCC are antibiotic pan-resistant. Current studies in BCC phage therapy are unique from many other avenues of phage therapy research in that the investigation is not only comprised of phage isolation, in vitro phage characterization and assessment of in vivo infection model efficacy, but also adapting aerosol drug delivery techniques to aerosol phage formulation delivery and storage.

Spray-dried respirable powders containing bacteriophages for the treatment of pulmonary infections.

Myoviridae bacteriophages were processed into a dry powder inhalable dosage form using a low-temperature spray-drying process. The phages were incorporated into microparticles consisting of trehalose, leucine, and optionally a third excipient (either a surfactant or casein sodium salt). The particles were designed to have high dispersibility and a respirable particle size, and to preserve the phages during processing. Bacteriophages KS4- M, KS14, and cocktails of phages ΦKZ/D3 and ΦKZ/D3/KS4-M were spray-dried with a processing loss ranging from 0.4 to 0.8 log pfu. The aerosol performance of the resulting dry powders as delivered from an Aerolizer® dry powder inhaler (DPI) exceeded the performance of commercially available DPIs; the emitted mass and the in vitro total lung mass of the lead formulation were 82.7% and 69.7% of filled capsule mass, respectively. The total lung mass had a mass median aerodynamic diameter of 2.5-2.8 µm. The total in vitro lung doses of the phages, delivered from a single actuation of the inhaler, ranged from 10(7) to 10(8) pfu, levels that are expected to be efficacious in vivo. Spray drying of bacteriophages into a respirable dry powder was found to be feasible.