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Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm.

Danis-Wlodarczyk K, Olszak T, Arabski M, Wasik S, Majkowska-Skrobek G, Augustyniak D, Gula G, Briers Y, Jang HB, Vandenheuvel D, Duda KA, Lavigne R, Drulis-Kawa Z - PLoS ONE (2015)

Bottom Line: Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages.Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms.It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathogen Biology and Immunology, Institute of Genetics and Microbiology, University of Wroclaw, Wroclaw, Poland; Division of Gene Technology, Catholic University of Leuven, Leuven, Belgium.

ABSTRACT
We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

No MeSH data available.


Related in: MedlinePlus

In silico analysis of KTN6 and KT28 phages and their comparison to PB1 phage. The predicted open reading frames of presented phages (rectangles) and their amino acid identity to the corresponding ORF in PB1 phage, indicated with different colors in the legend. Predicted terminators and promoters are shown as stem-loop structures and black arrows, respectively.
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pone.0127603.g006: In silico analysis of KTN6 and KT28 phages and their comparison to PB1 phage. The predicted open reading frames of presented phages (rectangles) and their amino acid identity to the corresponding ORF in PB1 phage, indicated with different colors in the legend. Predicted terminators and promoters are shown as stem-loop structures and black arrows, respectively.

Mentions: Using high throughput sequencing by the Illumina MiSeq platform, the complete genome sequences were determined (Fig 6, S2 Table). Phages show 96.43% nucleotide similarity to each other, but when compared to other Pb1-like phages (14–1, BcepF1, F8, LBL3, LMA2, PB1, SN) they display nucleotide similarity between 47.74–96.19% for KT28 and 46.94–96.55% for KTN6 (S3 Table), with most similarity to LMA2, 96.19% and 96.55%, respectively. BcepF1 phage showed a more distant relationship to Pb1-like phages, showing only 47.74% and 46.94% similarity to KT28 and KTN6, respectively and also below 48% to the other tested phages. When comparing in silico the protein identity of these new phages to PB1, 71% of the proteins showed more than 90% similarity, especially in the regions responsible for particle formation, host lysis, and DNA metabolism and replication (Fig 6). It confirms that the main core of Pb1-like phage genomes is very conserved. The largest variation of proteins was observed at the beginning and the end of the genomes. The tRNAscan-SE program did not reveal tRNA in genomes. We were also able to identify 15 putative factor-independent terminators in KT28 and 12 in KTN6 as well as 4 and 5 promoters, respectively (Fig 6). GC (%) was 55.6 for KT28 and 55.51 for KTN6.


Characterization of the Newly Isolated Lytic Bacteriophages KTN6 and KT28 and Their Efficacy against Pseudomonas aeruginosa Biofilm.

Danis-Wlodarczyk K, Olszak T, Arabski M, Wasik S, Majkowska-Skrobek G, Augustyniak D, Gula G, Briers Y, Jang HB, Vandenheuvel D, Duda KA, Lavigne R, Drulis-Kawa Z - PLoS ONE (2015)

In silico analysis of KTN6 and KT28 phages and their comparison to PB1 phage. The predicted open reading frames of presented phages (rectangles) and their amino acid identity to the corresponding ORF in PB1 phage, indicated with different colors in the legend. Predicted terminators and promoters are shown as stem-loop structures and black arrows, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4440721&req=5

pone.0127603.g006: In silico analysis of KTN6 and KT28 phages and their comparison to PB1 phage. The predicted open reading frames of presented phages (rectangles) and their amino acid identity to the corresponding ORF in PB1 phage, indicated with different colors in the legend. Predicted terminators and promoters are shown as stem-loop structures and black arrows, respectively.
Mentions: Using high throughput sequencing by the Illumina MiSeq platform, the complete genome sequences were determined (Fig 6, S2 Table). Phages show 96.43% nucleotide similarity to each other, but when compared to other Pb1-like phages (14–1, BcepF1, F8, LBL3, LMA2, PB1, SN) they display nucleotide similarity between 47.74–96.19% for KT28 and 46.94–96.55% for KTN6 (S3 Table), with most similarity to LMA2, 96.19% and 96.55%, respectively. BcepF1 phage showed a more distant relationship to Pb1-like phages, showing only 47.74% and 46.94% similarity to KT28 and KTN6, respectively and also below 48% to the other tested phages. When comparing in silico the protein identity of these new phages to PB1, 71% of the proteins showed more than 90% similarity, especially in the regions responsible for particle formation, host lysis, and DNA metabolism and replication (Fig 6). It confirms that the main core of Pb1-like phage genomes is very conserved. The largest variation of proteins was observed at the beginning and the end of the genomes. The tRNAscan-SE program did not reveal tRNA in genomes. We were also able to identify 15 putative factor-independent terminators in KT28 and 12 in KTN6 as well as 4 and 5 promoters, respectively (Fig 6). GC (%) was 55.6 for KT28 and 55.51 for KTN6.

Bottom Line: Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages.Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms.It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathogen Biology and Immunology, Institute of Genetics and Microbiology, University of Wroclaw, Wroclaw, Poland; Division of Gene Technology, Catholic University of Leuven, Leuven, Belgium.

ABSTRACT
We here describe two novel lytic phages, KT28 and KTN6, infecting Pseudomonas aeruginosa, isolated from a sewage sample from an irrigated field near Wroclaw, in Poland. Both viruses show characteristic features of Pbunalikevirus genus within the Myoviridae family with respect to shape and size of head/tail, as well as LPS host receptor recognition. Genome analysis confirmed the similarity to other PB1-related phages, ranging between 48 and 96%. Pseudomonas phage KT28 has a genome size of 66,381 bp and KTN6 of 65,994 bp. The latent period, burst size, stability and host range was determined for both viruses under standard laboratory conditions. Biofilm eradication efficacy was tested on peg-lid plate assay and PET membrane surface. Significant reduction of colony forming units was observed (70-90%) in 24 h to 72 h old Pseudomonas aeruginosa PAO1 biofilm cultures for both phages. Furthermore, a pyocyanin and pyoverdin reduction tests reveal that tested phages lowers the amount of both secreted dyes in 48-72 h old biofilms. Diffusion and goniometry experiments revealed the increase of diffusion rate through the biofilm matrix after phage application. These characteristics indicate these phages could be used to prevent Pseudomonas aeruginosa infections and biofilm formation. It was also shown, that PB1-related phage treatment of biofilm caused the emergence of stable phage-resistant mutants growing as small colony variants.

No MeSH data available.


Related in: MedlinePlus