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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Average diameter of
Isolate Source Location zone of inhibition
(mm)
Los Banos,
LB1-1 Apis mellifera 10
Laguna
Los Banos,
LB1-2 A. mellifera 6
Laguna
Los Banos,
LB1-3 A. mellifera 3
Laguna
Los Banos,
LB1-5 A. mellifera 4
Laguna
Los Banos,
LB1-6 A. mellifera 4
Laguna
Sta. Maria,
BUL1-6 A. mellifera 3
Bulacan

AL1-1 Trigona sp. Guinobatan, Albay 2.5

Los Banos,
PA A. mellifera 2
Laguna
Sta. Maria,
BUL1-9 A. mellifera 3
Bulacan

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

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 Gram-stain preparation of isolate

LB1-1 obtained from A. mellifera LB1-2 obtained from A. mellifera
(1000x). Cells are Gram-positive, (1000x). Cells are Gram-positive,
rod-shaped, occurring in singly or in rod-shaped, occurring in singly or in
pairs. 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