Vaid BC Bera Sanjay Barua Riyesh T and Praveen Malik Veterinary Type Culture Collection National Research Centre on Equines Sirsa Road Hisar Haryana India ID: 928131
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(Taruna Anand*, Rajesh.K. Vaid, B.C. Bera, Sanjay Barua, Riyesh T. and Praveen Malik)Veterinary Type Culture Collection, National Research Centre on Equines, Sirsa Road, Hisar, Haryana, Indiatarunandri@gmail.com
Isolation and characterization of
lytic
bacteriophages
against bacteria of veterinary importance
Slide2Phages have been preserved worldwideName of culture collectionAmerican Type Culture Collection (ATCC), USA
National Biological Resource
Center
(NBRC), JapanLaboratoire de Microbiologie et Génétique Moléculaires, France- largest collection of T4 superfamily of bacteriophages Felix d’Herelle Reference Center for bacterial viruses, CanadaDeutsche Sammlung von Mikroorganismen und Zellkulturen – DSMZ – (German Collection of Microorganisms and Cell Cultures)The Netherlands Culture Collection of Bacteria (NCCB)National Collections of Industrial, Food and Marine Bacteria, NCIMB, ScotlandKorea National Research Resource Center- Bacteriophage BankChina Center For Virus Culture Collection
9/16/2014
VTCC, National Research Centre on Equines
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Slide3ObjectivesTo isolate and characterize bacteriophages of different bacteria.To develop repository of bacteriophages.9/16/2014VTCC, National Research Centre on Equines
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Slide4Sample of raw sewage, soil, sludge etc.Bacteriophage enrichmentPFU assay by double layer agar method
Confirmation of bacteriophage activity on host cells
Phage titre determination
Investigation of phage susceptibility on a variety of host rangePlaque purificationCharacterization: Molecular characterization of nucleic acid: Restriction enzymes/PCR/PAGEBacteriophage morphology- TEM studyPhage propagation in bulk and preservation
Isolation of different bacteria
Bacterial identification
Preservation of bacteria
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Slide5E. coli Aeromonas
hydrophila
Bacillus
subtilis ssp. subtilisBacillus sp.Bacillus sp.Staphylococcus sciuri Bacillus spp. Bacillus spp.Bacterial host against which bacteriophages isolated9/16/2014VTCC, National Research Centre on Equines5
Slide6Plaque characteristics
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Slide7FO48E
Clear plaques of 1-2mm
dia
Clear plaques with rough margins, with a halo zone, 3-4mm diaClear plaques with a halo zone, 5-6mm diaBacillus pumilusPseudomonas mendocinaPaenibacillus sp.9/16/2014VTCC, National Research Centre on Equines7
Slide8Phages isolated-Plaque characteristicsBacteria- lab ID
Source of phage
Plaque characteristics
Size TypeEq37-2 Sewage1mm
Very clear with smooth margins
Eq16
Soil
<1mm
Very clear in middle with hazy margin, circular
Eq53
Soil
1-2mm
Clear with hazy margins
Eq73-2
Soil
<1mm
Very clear with smooth margins
C1/Fo38A
Soil
1-2mm
Irregular shape, clear
C2/ Fo38B
Soil
3mm
Clear plaques
Eq12B
Soil
2mm
Clear with smooth margins, circular
Fo37
Soil
<1mm,
Very small clear plaques
Fo23c
Sewage
1-2mm
Turbid plaques
Fo34c
Soil
2-3mm
Clear plaques
Fo32B (1)
Soil
2-3mm
Clear plaques with a zone of halo
Fo32B (2)
Soil
2-3mm
Clear plaques
Fo32c
Soil
2-3mm
Clear plaques with hazy margins
Fo35B
Soil
2-3mm
Clear plaques
Fo33A
Soil
6mm,
Turbid plaques with a clear central zone of 1-2mm
The phage stocks have been preserved at
-80°C
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Slide9Eq37-2Capsid dia:88nmTail length: 5nmBase plate width: 45nmTail width: 8nmPodoviridaeElectron Microscopy
Capsid
dia: 78 nmTail length: 308 nmTail width: 16 nmBase Plate width: 24nmBase Plate Length:36 nmSiphoviridaeFO34cCapsid dia: 86nmTail length: 100nmTail width: 20nmBase Plate width: 33nmBase Plate Length: 17nmMyoviridae
Eq16
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VTCC, National Research Centre on Equines
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Slide10Capsid dia: 56nmTail length: 5 nmPodoviridaeFO44ca
Length: 1.25-2.5um
Width: 17.7nm
InoviridaeFO44aCapsid dia: 69 nmTail length: 265 nmTail width: 10 nmSiphoviridae
FO35b
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Slide11FO23c-Aeromonas hydrophilaCapsid dia: 62nmTail length: 138nmTail width: 13nm (contracted)Tail width: 19nm (relaxed)Base plateMyoviridae
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Slide12Sample NameHostPhage Type/ characteristics observed by TEMEq37-2E. coliPodoviridaeFO34CStaphylococcus
sciuri
Siphoviridae
FO35BBacillus spp.SiphoviridaeFO44ABacillus spp.InoviridaeEq16E. coliMyoviridaeFO44CaAeromonas hydrophilaPodoviridae (Novel phage candidate)?FO23cAeromonas hydrophilaMyoviridaePhage Types9/16/2014VTCC, National Research Centre on Equines12
Slide13Protein profile of phage isolatesFo34c
38kDa
62kDa
32kDaFo35b58kDaFo44aFo44ca48kDa63kDa9/16/2014VTCC, National Research Centre on Equines13
Slide1442kDaEq16
Fo23c
54kDa
50kDaEq37-248kDa9/16/2014VTCC, National Research Centre on Equines14
Slide15Fo47D
Fo48E
Fo48B
35kDa38kDa40kDa50kDa9/16/2014VTCC, National Research Centre on Equines15
Slide16Phage DNA9/16/2014VTCC, National Research Centre on Equines16
Slide17Phage identification by PCR (gp23 gene)850bp9/16/2014
VTCC, National Research Centre on Equines
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Slide18Phylogenetic analysis9/16/2014VTCC, National Research Centre on Equines18
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Slide21(Group) ds DNA (Order)
Caudovirales
(Family)
Myoviridae (Sub Family) Tenevirinae (Genus) T4-like virus9/16/2014VTCC, National Research Centre on Equines21
Slide22Aeromonas hydrophila phage
^
*
PCR detection of (A) aerolysin gene product (252bp) and (B) lip gene product (760bp).AB9/16/2014VTCC, National Research Centre on Equines22
Slide23Biological activity against diarrhoeal isolates of E. coli analysed by Spot TestPhage isolateAccession no. of test host bacteriaActivityEq16(4/9)LLR001B1+++
LLR002A2
+++
LLR003B1+SDF007A1+Eq37-2(1/9)LLR002A2+++9/16/2014VTCC, National Research Centre on Equines23
Slide24ConclusionsDiverse kind of bacteriophages were isolated from sewage and soil samples of animal farms.Phages varied in plaque characteristics, morphology and protein profiles.Electron microscopic analysis classified phages mainly into caudovirales-Myoviridae, Podoviridae and Siphoviridae families.Few phages showed biological activity against E. coli isolated from calf diarrhoea………….potential application in phage therapy.
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Slide25Thankyou…………….9/16/2014VTCC, National Research Centre on Equines25