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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

Histograms of imageJ software analysis of PAO1 biofilm (stained by CV) formed on PET membrane.(A) In left panel grey line presents cut-off point in which pixels from total analyzed (membrane with biofilm) have the grey level lower than control (native membrane). The % of membrane covering by biofilm formed for 72 h is presented. (B) Right panel presents images of analyzed membranes with/without biofilm. Analysis were done in two independent experiments.
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pone.0127603.g003: Histograms of imageJ software analysis of PAO1 biofilm (stained by CV) formed on PET membrane.(A) In left panel grey line presents cut-off point in which pixels from total analyzed (membrane with biofilm) have the grey level lower than control (native membrane). The % of membrane covering by biofilm formed for 72 h is presented. (B) Right panel presents images of analyzed membranes with/without biofilm. Analysis were done in two independent experiments.

Mentions: The increase of TSB diffusion through the biofilm might indicate its structure disruption. ImageJ software analysis of membrane images with biofilm (degree of membrane covering by biofilm; %) showed that the highest degree of PET membrane covering by PAO1 biofilm was obtained after 72 h (92.58% ± 6.35) (Fig 3). We decided to use PET membrane with a biofilm which was formed for 72 h for laser interferometry analysis. Fig 4 shows the laser interferometry analysis of growth medium diffusion through PAO1 biofilm treated with KT28 or KTN6 phage (active and inactivated) for 4 h. Native PET has hydrophobic properties and diffusion of water-soluble compounds (as TSB medium) through this membrane was limited. The amount of TSB medium transported through PET (2.56×10–4 mg/h) was statistically lower than through membrane with hydrophilic biofilm (2.51×10–3 mg/h) (p<0.001). After the biofilm incubation with four types of tested phages, and the amount of transported medium was lower compared to untreated PAO1 biofilm (p<0.001). This correlates to biofilm degradation. This indicates that degradation of biofilm structure by phages might be associated with: (i) the loss of biofilm matrix elements on membrane surface and the reversion to hydrophobic properties of membrane, or (ii) partial degradation of matrix/biofilm structure resulting on higher diffusion rate. This observation was partially confirmed by goniometry analysis (Fig 5). The value of contact angle for uncovered PET membrane (43.3) was similar to the angle value of membrane covered by biofilm and treated with both active phages (41.6 for KT28 and 40.3 for KTN6). The inactive form of phages gave more hydrophilic feature of membrane with the angle value equal to non-treated biofilm suggesting reduced eradication compared to active phages.


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)

Histograms of imageJ software analysis of PAO1 biofilm (stained by CV) formed on PET membrane.(A) In left panel grey line presents cut-off point in which pixels from total analyzed (membrane with biofilm) have the grey level lower than control (native membrane). The % of membrane covering by biofilm formed for 72 h is presented. (B) Right panel presents images of analyzed membranes with/without biofilm. Analysis were done in two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4440721&req=5

pone.0127603.g003: Histograms of imageJ software analysis of PAO1 biofilm (stained by CV) formed on PET membrane.(A) In left panel grey line presents cut-off point in which pixels from total analyzed (membrane with biofilm) have the grey level lower than control (native membrane). The % of membrane covering by biofilm formed for 72 h is presented. (B) Right panel presents images of analyzed membranes with/without biofilm. Analysis were done in two independent experiments.
Mentions: The increase of TSB diffusion through the biofilm might indicate its structure disruption. ImageJ software analysis of membrane images with biofilm (degree of membrane covering by biofilm; %) showed that the highest degree of PET membrane covering by PAO1 biofilm was obtained after 72 h (92.58% ± 6.35) (Fig 3). We decided to use PET membrane with a biofilm which was formed for 72 h for laser interferometry analysis. Fig 4 shows the laser interferometry analysis of growth medium diffusion through PAO1 biofilm treated with KT28 or KTN6 phage (active and inactivated) for 4 h. Native PET has hydrophobic properties and diffusion of water-soluble compounds (as TSB medium) through this membrane was limited. The amount of TSB medium transported through PET (2.56×10–4 mg/h) was statistically lower than through membrane with hydrophilic biofilm (2.51×10–3 mg/h) (p<0.001). After the biofilm incubation with four types of tested phages, and the amount of transported medium was lower compared to untreated PAO1 biofilm (p<0.001). This correlates to biofilm degradation. This indicates that degradation of biofilm structure by phages might be associated with: (i) the loss of biofilm matrix elements on membrane surface and the reversion to hydrophobic properties of membrane, or (ii) partial degradation of matrix/biofilm structure resulting on higher diffusion rate. This observation was partially confirmed by goniometry analysis (Fig 5). The value of contact angle for uncovered PET membrane (43.3) was similar to the angle value of membrane covered by biofilm and treated with both active phages (41.6 for KT28 and 40.3 for KTN6). The inactive form of phages gave more hydrophilic feature of membrane with the angle value equal to non-treated biofilm suggesting reduced eradication compared to active phages.

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