Bacterial Resistance Problem Space

1. Bacterial ResistanceProblem Space Ehren Bucholtz Saint Mary-of-the-Woods College Mark Gallo Niagara University

2. Background • Microbial drug resistance (DR) is a growing concern among health professionals and others around the world. • DR Issues that can be studied in a problem space environment include • Types of Drug classes • Types of Bacteria • Mechanism of Action • Mechanisms of DR

3. Microbial drug resistance Biological Principles Next slide Drug Resistance Data Sets CDC FDA Drug Digest RxList Tools Biology Workbench NCBI

4. Coevolution Genetic exchange Antibacterials Ecological Factors Phylogeny Drug Classes Bacteria types Antibiotics Host interaction Drug Resistance Resistance Mechanisms Mechanism of Action Mutations Bacteriocidal Cellular level Bacteriostatic

5. Resources • Centers for Disease Control • Case studies • Outbreak data • Genbank • Biology Workbench

6. Multidrug Resistance Region of Salmonella enterica Serovar Typhimurium DT104 • Outbreaks of MDR Typhimurium DT104 have also been reported in poultry, beef, cheese, and swine in numerous countries • Resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline • however, isolates have been identified which are also resistant to fluoroquinolones, trimethoprim, and kanamycin

7. antimicrobial resistance gene is clustered on a fragment of less than 46 kb

8. Task: Identify Resistance ORFs • Students are given pieces of nucleotide sequence of SGi1 to identify homology • beta-lactamase • Tetracycline resistance • Integrase • Transposase • beta_lactamase (partial sequence shown) NEGKLGDLRDTTTPKAIASTLNKFLFGSALSEMNQKKLESWMVNNQVTGNLLRSVLPAGW

9. Find results for • 4377727_4377728Vibrio cholerae non O1, non O139 plasmid class A beta-lactamase • 216842_216843Proteus mirabilis plasmid pCS229 blaP gene for bata-lactamase, • 30314015_30314018Pasteurella multocida plasmid pJR2, complete sequence. • 9719055_9719057Vibrio cholerae class 1 integron integrase (intI1) gene,partial • 12719011_12719033Salmonella enterica subsp. enterica serovar Typhimurium genomic • 151074_151076P.aeruginosa beta-lactamase (PSE-1) gene, complete cds • 47647_47648S.typhimurium beta-lactamase gene.

10. BlastP and Clustal for beta lactamase unknown • 30314015_3031401 NEGKLGDLRDTTTPKAIASTLNKFLFGSALSEMNQKKLESWMVNNQVTGNLLRSVLPAGW • 216842_216843 NEGKLGDLRDTTTPKAIASTLNKFLFGSALSEMNKKKLESWMVNNQVTGNLLRSVLPAGW • 47647_47648 NEGKLGDLRDTTTPKAIASTLNKFLFGSALSEMNQKKLESWMVNNQVTGNLLRSVLPAGW • 151074_151076 NEGKLGDLRDTTTPKAIASTLNKFLFGSALSEMNQKKLESWMVNNQVTGNLLRSVLPAGW • 12719011_12719033 NEGKLGDLRDTTTPKAIASTLNKFLFGSALSEMNQKKLESWMVNNQVTGNLLRSVLPAGW • 9719055_9719057 NEGKLGDLRDTTTPKAIASTLNKFLFGSALSEMNQKKLESWMVNNQVTGNLLRSVLPAGW • beta_lactamase NEGKLGDLRDTTTPKAIASTLNKFLFGSALSEMNQKKLESWMVNNQVTGNLLRSVLPAGW • 4377727_4377728 NEGKLGDLRDTTTPKAIASTLNQLLFGSTLSEASQKKLESWMVNNQVTGNLLRSVLPVKW • **********************::****:*** .:**********************. *

11. Rooted Tree from ClustalW

12. Other project ideas • Use wealth of CDC case study data to fill out project space

13. CDC Case Study • Variant Salmonella Genomic Island 1 Antibiotic Resistance Gene Cluster in Salmonella enterica Serovar Albany • Benoît Doublet et.al. Emerg Infect Dis 2003 May Available from: URL: http://www.cdc.gov/ncidod/EID/vol9no5/02-0609.htm

14. Something’s fishy • A variant SGI1, Salmonella genomic island 1, was identified as an antibiotic-resistance gene cluster in a multidrug-resistant strain of S. enterica serovar Albany isolated from food fish from Thailand and imported to France.  • In this strain, the streptomycin resistance aadA2 gene cassette in one of the SGI1 integrons was replaced by a dfrA1 gene cassette, conferring resistance to trimethoprim and an open reading frame of unknown function.

15. Gene Function Treasure Hunt

16. Clustering of Salmonella isolates