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Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains.

Urban-Chmiel R, Wernicki A, Stęgierska D, Dec M, Dudzic A, Puchalski A - PLoS ONE (2015)

Bottom Line: The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101.The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments.Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.

View Article: PubMed Central - PubMed

Affiliation: Sub-department of Veterinary Prevention and Avian Diseases, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, 20-033, Lublin, Poland.

ABSTRACT

Aim of study: The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves.

Material and methods: The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR).

Results: Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101.

Conclusions: The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments. Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.

No MeSH data available.


Related in: MedlinePlus

Restriction analysis profiles of phages from M. haemolytica isolates single-digested with TaqI (lines 1–7).Legend: φPHL-1 (from strain BAA-410); φA1; φA2; φA5; φA6; φA7; φ25; M—DNA markers (100–1000 bp, Fermentas).
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pone.0140140.g005: Restriction analysis profiles of phages from M. haemolytica isolates single-digested with TaqI (lines 1–7).Legend: φPHL-1 (from strain BAA-410); φA1; φA2; φA5; φA6; φA7; φ25; M—DNA markers (100–1000 bp, Fermentas).

Mentions: The electrophoretic profiles of the bacteriophages following digestion with the enzymes ClaI and HindIII contained from 4 to 10 restriction fragments ranging from 114 to 1,418 bp. The P2-like bacteriophage was characterized by a band of high intensity, 1,150 bp in size, and two smaller bands with specific weights of 525 and 336 bp. A restriction fragment of 1,150 bp was also present in the profiles of phages φ1, 5, 6 and 25, but was not observed in the profile of phage φ2. In the case of phage φ7, application of ClaI and HindIII did not result in a restriction cut (Fig 4). The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the reference P2-like phage (Fig 4). Analysis of the genetic material of the phages digested with TaqI showed no significant differences in the restriction products which would enable differentiation of the phages. All of the profiles contained a main restriction fragment 810 bp in size and smaller fragments ranging from 66 to 350 bp. It should be emphasized that the phages analysed were varied in terms of morphology (Fig 5).


Isolation and Characterization of Lytic Properties of Bacteriophages Specific for M. haemolytica Strains.

Urban-Chmiel R, Wernicki A, Stęgierska D, Dec M, Dudzic A, Puchalski A - PLoS ONE (2015)

Restriction analysis profiles of phages from M. haemolytica isolates single-digested with TaqI (lines 1–7).Legend: φPHL-1 (from strain BAA-410); φA1; φA2; φA5; φA6; φA7; φ25; M—DNA markers (100–1000 bp, Fermentas).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4599942&req=5

pone.0140140.g005: Restriction analysis profiles of phages from M. haemolytica isolates single-digested with TaqI (lines 1–7).Legend: φPHL-1 (from strain BAA-410); φA1; φA2; φA5; φA6; φA7; φ25; M—DNA markers (100–1000 bp, Fermentas).
Mentions: The electrophoretic profiles of the bacteriophages following digestion with the enzymes ClaI and HindIII contained from 4 to 10 restriction fragments ranging from 114 to 1,418 bp. The P2-like bacteriophage was characterized by a band of high intensity, 1,150 bp in size, and two smaller bands with specific weights of 525 and 336 bp. A restriction fragment of 1,150 bp was also present in the profiles of phages φ1, 5, 6 and 25, but was not observed in the profile of phage φ2. In the case of phage φ7, application of ClaI and HindIII did not result in a restriction cut (Fig 4). The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the reference P2-like phage (Fig 4). Analysis of the genetic material of the phages digested with TaqI showed no significant differences in the restriction products which would enable differentiation of the phages. All of the profiles contained a main restriction fragment 810 bp in size and smaller fragments ranging from 66 to 350 bp. It should be emphasized that the phages analysed were varied in terms of morphology (Fig 5).

Bottom Line: The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101.The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments.Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.

View Article: PubMed Central - PubMed

Affiliation: Sub-department of Veterinary Prevention and Avian Diseases, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, 20-033, Lublin, Poland.

ABSTRACT

Aim of study: The objective of this study was isolation and morphological characterization of temperate bacteriophages obtained from M. haemolytica strains and evaluation of their lytic properties in vitro against M. haemolytica isolated from the respiratory tract of calves.

Material and methods: The material for the study consisted of the reference strain M. haemolytica serotype 1 (ATCC®) BAA-410™, reference serotypes A1, A2, A5, A6, A7, A9 and A11, and wild-type isolates of M. haemolytica. Bacteriophages were induced from an overnight bacterial starter culture of all examined M. haemolytica strains treated with mitomycin C. The lytic properties and host ranges were determined by plaque assays. The morphology of the bacteriophages was examined in negative-stained smears with 5% uranyl acetate solution using a transmission electron microscope. The genetic analysis of the bacteriophages was followed by restriction analysis of bacteriophage DNA. This was followed by analysis of genetic material by polymerase chain reaction (PCR).

Results: Eight bacteriophages were obtained, like typical of the families Myoviridae, Siphoviridae and Podoviridae. Most of the bacteriophages exhibited lytic properties against the M. haemolytica strains. Restriction analysis revealed similarities to the P2-like phage obtained from the strain M. haemolytica BAA-410. The most similar profiles were observed in the case of bacteriophages φA1 and φA5. All of the bacteriophages obtained were characterized by the presence of additional fragments in the restriction profiles with respect to the P2-like reference phage. In the analysis of PCR products for the P2-like reference phage phi-MhaA1-PHL101 (DQ426904) and the phages of the M. haemolytica serotypes, a 734-bp phage PCR product was obtained. The primers were programmed in Primer-Blast software using the structure of the sequence DQ426904 of reference phage PHL101.

Conclusions: The results obtained indicate the need for further research aimed at isolating and characterizing bacteriophages, including sequence analysis of selected fragments. Moreover, standardization of methods for obtaining them in order to eliminate M. haemolytica bacteria involved in the etiopathogenesis of BRDC is essential.

No MeSH data available.


Related in: MedlinePlus