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5/20/2010

Isolation and Characterization of Lactic Acid Bacteria from Different Bee Species in the Philippines and Screening for Activity against the Causal Organism of American Foulbrood Disease (Paenibacillus larvae ssp. larvae)
March 2008 – March 2009

Prof. Noel G. Sabino Mr. Andrew D. Montecillo
Microbiology Division, Institute of Biological Sciences College of Arts and Sciences, UPLB

Collaborating Agencies

The UPLB BEE Program

Institute of Biological Sciences, UPLB

http://beeprogram.uplb.edu.ph

The Beekeepers Network Foundation Philippines, Inc. (BEENET)

Institute of Biological Sciences, UPLB

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Objectives
1. Isolation of lactic acid bacteria from three bee species collected from different sampling sites; 2. Characterization and identification of antagonistic LAB isolates and; 3. Screening for antagonistic activity against Paenibacillus larvae

Sampling Sites

Bee samples were provided by BEENET Philippines, Inc.

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Bee Species Used

Sample Processing

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9 of the 28 isolates were observed to have antagonistic activity against Paenibacillus larvae ssp. larvae

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Isolate

Source

Location Los Banos, Laguna Los Banos, Laguna Los Banos, Laguna Los Banos, Laguna Los Banos, Laguna Sta. Maria, Bulacan Guinobatan, Albay Los Banos, Laguna Sta. Maria, Bulacan

Average diameter of zone of inhibition (mm) 10 6 3 4 4 3 2.5 2 3

LB1-1 LB1-2 LB1-3 LB1-5 LB1-6 BUL1-6 AL1-1 PA BUL1-9

Apis mellifera A. mellifera A. mellifera A. mellifera A. mellifera A. mellifera Trigona sp. A. mellifera A. mellifera

Screening for Antagonistic Activity

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Screening for Antagonistic Activity

Isolates LB1-1 and LB1-2 showed the strongest antagonism of the 9 isolates. They were identified using API CHL 50: LB1-1 and LB1-2 were identified as Lactobacillus plantarum

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Screening for Bacteriocin-like Activity against P. larvae

No bacteriocin-like activity against P. larvae was observed using cell-free pH-adjusted and unadjusted broth culture supernatant. Antagonistic activity was only observed in the presence of actively-growing cells of LB1-1 and LB1-2. Some other factors (other than bacteriocins) might be exerting the antagonistic effect.

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Screening for Bacteriocin-like Activity against Enterococcus faecium

LB1-1 and LB1-2 exhibited bacteriocin-like activity against E. faecium (on agar-well diffusion assay)
Antimicrobial spectrum of LB1-1 and LB1-2 should be further characterized by using other bee and food-borne pathogens as test organisms

LB1-1 and LB1-2

Gram-stain preparation of isolate LB1-1 obtained from A. mellifera (1000x). Cells are Gram-positive, rod-shaped, occurring in singly or in pairs.

Gram-stain preparation of isolate LB1-2 obtained from A. mellifera (1000x). Cells are Gram-positive, rod-shaped, occurring in singly or in pairs.

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What's next?

Test for in vivo efficacy of the antagonistic isolates (as probiotic supplement)

Laboratory-scale trials using broods challenged with the bee pathogen Field trials using bee colonies

Identification of the antimicrobial substance(s) produced by the antagonistic isolates

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