Drug Resistance

1. Drug Resistance Mechanism • The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are: • Drug inactivation or modification: e.g. enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of β-lactamases. • Alteration of target site : e.g. alteration of PBP—the binding target site of penicillins—in MRSA and other penicillin-resistant bacteria. • Alteration of metabolic pathway: e.g. some sulfonamide-resistant bacteria do not require para-aminobenzoic acid (PABA), an important precursor for the synthesis of folic acid and nucleic acids in bacteria inhibited by sulfonamides. Instead, like mammalian cells, they turn to utilizing preformed folic acid. • Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface.

2. Many of these mechanisms result from • Genetic mutation. • The acquisition of resistance genes from other microorganisms via gene transfer, and combinations of these two types of events • Genetic material can be transferred between bacteria by several means, most often by: • Conjugation – Transfer from one Bacterium to another • Transformation – As Naked DNA • And, Transduction – From a Bacteriophage Bottom Line: Genetic Make Up Changes

3. Detecting Genetic Changes Hybridization of DNA to a specific Probe PCR Quantitative PCR PCR – Single Strand Conformation polymorphism (PCR-SSCP)Migration in a denaturing gel where the mobility of the ssDNA depends on;i) Size and ii) Secondary Structure Branched DNA

4. Restriction Fragment Length Polymorphism - RFLP Results from Restriction Digest

5. Branched DNA (bDNA)

6. DNA Sequencing – Method of Choice in determining resistance in HIV DNA Arrays: - Hybridization – Mass screening of sequences using DNA Chip arrays http://www.bio.davidson.edu/Courses/genomics/chip/chip.html

9. Viruses as Vectors Obligate intra-cellular parasites – can illicite host immune response Retroviruses – lentiviruses Adenoviruses Adeno-associated viruses Herpes simples virus

10. Retrovirus Vectors Enveloped - single stranded RNA molecule Infection requires Reverse Transcription – dsDNA dsDNA integrates into host genome Gag – Core proteins Pol – Reverse Transcription Env – Envelope protein LTR – promoter/enhancer Psi –Packaging Carrying capacity – 7.5kb

11. Env can be modified to increase target cell range • Target cells should be dividing • Retroviruses inactivated by c1 complement protein – reduced byinflammatory interferons IFNα and IFNγ Lentiviruses – subclass of retroviruses Able to infect both proliferating and non-proliferating cells containing an additional six proteins, tat, rev, vpr, vpu, nef & vif

12. Adenovirus vecors • non-enveloped viruses containing a linear double stranded DNA genome • cause benign respiratory tract infections • The life cycle does not normally involve integration into the host genome • No risk of insertional mutagenesis • Efficient transducing vectors – Transcient • MHC class I restricted immune response occurs, using CD8+ CTLs to eliminate virus infected cells & CD4+ cells to secrete IFN-alpha which results in anti-adenoviral antibody • transient immunosupressive therapies have been successful in prolonging transgene expression

13. Adenovirus vecors four early transcriptional units (E1, E2, E3 & E4), which have regulatory functions, & a late transcript, which codes for structural proteins 35 kb Either E1 or E3 Gene inactivated

14. Drug Resistance occurs in: • bacteria -- antibiotic resistance • endoparasites • viruses -- resistance to antiviral drugs • fungi • cancer cells

15. Table 4.1. Restriction endonucleases