Transcript Characterization of Antibiotic-resistant Bacteria in Pear and Apple

Characterization of Antibioticresistant Bacteria in Pear and
Apple Orchards
Sarah R. Jean: Department of Biochemistry and Biophysics
Dr. Virginia Stockwell: Department of Botany and Plant Pathology
October 6, 2007
Antibiotic use in the U.S.
 Streptomycin and oxytetracycline are the only
antibiotics permitted for use in the U.S., including
organic crops
 90% of antibiotics used on plants in the U.S. are
for the treatment and prevention of fire blight
 No new antibiotics have been registered for use on
plants since the 1970s
 Streptomycin is ~90% effective and oxytetracycline
is ~60% effective for the treatment of fire blight
Antibiotic-resistance can
be costly and concerning
 The fire blight-causing bacterium
Erwinia amylovora can become
resistant to antibiotics
 Resistant to streptomycin in the
western states and Michigan
 As a result, fire blight has become a
real problem for orchard growers,
resulting in an estimated annual
loss and cost of control of over
$100 million in the U.S.
Fire Blight is caused by a
bacterium
Caused by a bacterium (Erwinia amylovora)
that attacks the flowers of pear and apple
trees and moves through the twigs and then
into the branches of the tree.
Gets its name from the burnt appearance of
affected flowers and twigs. Flowers turn
brown and wilt; twigs shrivel and blacken.
As disease advances, bacterial ooze is
exuded, and cankers form on branches.
Fire Blight moves up to the twigs
and then to the branches of the tree
Diseased branches
Healthy growth
Flowers turn brown and wilt; twigs
shrivel and blacken
branch
fruit and flowers
Cankers and discolored patches
form on branches
canker
Hypothesis
I hypothesize that trees in orchards,
which been exposed to the antibiotics
streptomycin and oxytetracycline, are
more likely to harbor antibiotic-resistant
bacteria.
I also hypothesize that bacteria from
antibiotic-treated trees will carry
transmissible genes for antibiotic
resistance
Plan of action
Isolate bacteria from treated and non-treated
flowers and/or fruit
Determine resistance profile of bacteria by
culturing isolates on media with and without
antibiotics
Use PCR to determine the genes associated
with the resistance
Determine if the antibiotic resistance genes
are present on plasmids that could be
transferred to the fire blight pathogen
Analyze any trends in antibiotic resistance
within the orchards studied
Determine resistance profile of
bacteria by culturing isolates on media
with and without antibiotics
no antibiotics
oxytetracycline
streptomycin
Use PCR to determine the genes
responsible for the resistance
Use primers to determine if streptomycin
resistance gene (strB) or tetracycline
resistance genes tetA, B, C, D, E, and/or
G are present in the antibiotic-resistant
bacteria
If evidence of gene is found, determine
DNA sequence for confirmation of
identity
Use PCR to determine genes
responsible for resistance
No evidence of streptomycin resistance
gene (strB) in the selected samples
It is likely that the bacteria were resistant
to streptomycin due to intrinsic resistance
not associated with strB or a
chromosomal mutation, which is unlikely
to be transferred to Erwinia amylovora
Use PCR to determine genes
associated with tetracycline
resistance
There was evidence of tetracycline
resistance genes tetA, B, and E in the
selected samples
PCR of Tc-resistance genes
+ +
1
2
3
4
5
6
7
8
possible tetB
650 bp
9 10 11
PCR of Tc-resistant genes
1
2
3
4
5
6
7
possible tetA
210 bp
8
9
PCR of Tc-resistant genes
+++
1
2
3
4
5
6
7
possible tetE
278 bp
8
9
10 11
Determine if the genes are present on
plasmids that could be transferred to
the fire blight pathogen
Amplicons of the anticipated size for
several tetracycline resistance genes
from orchard bacteria are awaiting
sequencing
Determination of presence on plasmid
will continue after confirmation of gene
sequence
Correlation between treatments and
isolation of antibiotic resistant
bacteria
Two, random-block, orchards: Bartlett pear
and Gala apple
4 treatments (water, tetracycline, streptomycin,
and sm/tet mix) 5-6 replicate trees per
treatment
Sprayed at 70% bloom, and at early petal fall
Sampled 5 times (early April, late April, May,
July, and September)
60 samples per treatment, per time
Trends in antibiotic resistance
Sm resistance in Bartlett Pears
OTc
Sm
Sm and oTC
Water
70
70
60
60
Samples per harvast
Samples per harvest
Water
Sm resistance in Gala apples
50
40
30
20
10
Sm
Sm and oTc
50
40
30
20
10
0
0
early April
late April
May
July
early April
September
Tc resistance in Bartlett Pears
Water
oTc
Sm
late April
May
July
September
Tc resistance in Gala apples
Sm and oTc
Water
70
70
60
60
Samples per harvast
Samples per harvest
oTc
50
40
30
20
10
oTc
Sm
Sm and oTc
50
40
30
20
10
0
0
early April
late April
May
July
September
early April
late April
May
July
September
Conclusions and Status
No evidence of streptomycin resistance
gene (strB) was present in any of the
investigated isolates
Evidence of tetracycline resistance
genes awaits DNA sequencing for
confirmation, then determination if
present on a plasmid
Exposure to antibiotics does not correlate
to selection for antibiotic-resistant
bacteria within a growing season
Acknowledgements
Howard Hughes Medical Institute
Center for Disease Control
United States Department of Agriculture
Loper Lab
Dr. Virginia O. Stockwell
Dr. Kevin Ahern
THANK YOU!!!