1. Sarah R. Jean: Department of Biochemistry and Biophysics Dr. Virginia Stockwell: Department of Botany and Plant Pathology October 6, 2007 Characterization of Antibiotic- resistant Bacteria in Pear and Apple Orchards

2. 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

3. 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.

4. 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.  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.  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.

5. Fire Blight moves up to the twigs and then to the branches of the tree Healthy growth Diseased branches

6. Flowers turn brown and wilt; twigs shrivel and blacken fruit and flowers branch

7. Cankers and discolored patches form on branches canker

8. 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

9. 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

10. Determine resistance profile of bacteria by culturing isolates on media with and without antibiotics no antibiotics streptomycinoxytetracycline

11. 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

12. 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

13. Use PCR to determine genes associated with tetracycline resistance  There was evidence of tetracycline resistance genes tetA, B, and E in the selected samples

14. PCR of Tc-resistance genes + 1 2 3 4 5 6 7 8 9 10 11 tetB 659 bp tetC 418 bp possible tetB 650 bp

15. PCR of Tc-resistant genes 1 2 3 4 5 6 7 8 9 possible tetA 210 bp

16. PCR of Tc-resistant genes + + + 1 2 3 4 5 6 7 8 9 10 11 tetG 468 bp tetA 210 bp possible tetE 278 bp

17. 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

18. 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

19. Trends in antibiotic resistance

20. 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

21. 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!!!