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Drug Resistance Mechanism The four main mechanisms by which microorganisms exhibit resistance to antimicrobials are: 1. Drug inactivation or modification: e.g. enzymatic deactivation of Penicillin G in some penicillin-resistant bacteria through the production of β-lactamases. 2. Alteration of target site : e.g. alteration of PBP—the binding target site of penicillins—in MRSA and other penicillin-resistant bacteria. 3. Alteration of metabolic pathway: e.g. some sulfonamideresistant 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. 4. Reduced drug accumulation: by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell surface. Many of these mechanisms result from I. Genetic mutation. II. 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: 1. Conjugation – Transfer from one Bacterium to another 2. Transformation – As Naked DNA 3. And, Transduction – From a Bacteriophage Bottom Line: Genetic Make Up Changes 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 Restriction Fragment Length Polymorphism - RFLP Results from Restriction Digest Branched DNA (bDNA) 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 Molecular methods for detecting antimicrobial resistancea Organism(s) Antimicrobial agent(s) Gene Detection method Staphylococci Methicillin Oxacillin mec Ab Standard DNA probe Branched chain DNA probe PCR Enterococci Vancomycin van A, B, C, Dc Standard DNA probe PCR Enterobacteriaceae Haemophilus influenzae Neisseria gonorrhoeae Beta-lactams blaTEM and blaSHVd Standard probe PCR and RFLP PCR and sequencing Enterobacteriaceae and gram-positive cocci Quinolones Point mutations in gyr A, gyr B, par C and par E PCR and sequencing Mycobacterium tuberuclosise Rifampin Isoniazid Ethambutol Streptomycin Point mutations in rpo B Point mutations in kat G, inh A, and ahp C Point mutations in emb B Point mutations in rps L and rrs PCR and SSCP PCR and sequencing PCR and SSCP PCR and sequencing PCR and RFLP Herpes virusesf Acyclovir and related drugs Foscarnet Mutations or deletions in the TK gene Point mutations in DNA polymerase gene PCR and sequencing PCR and sequencing HIVg Nucleoside reverse transcriptase inhibitors Protease inhibitors Point mutations in RT gene Point mutations in PROT gene PCR and sequencing PCR and LIPA PCR and sequencing Viruses as Vectors Obligate intra-cellular parasites – can illicite host immune response Retroviruses – lentiviruses Adenoviruses Adeno-associated viruses Herpes simples virus 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 Env can be modified to increase target cell range Target cells should be dividing Retroviruses inactivated by c1 complement protein – reduced by inflammatory 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 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 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 Drug Resistance occurs in: 1. 2. 3. 4. 5. bacteria -- antibiotic resistance endoparasites viruses -- resistance to antiviral drugs fungi cancer cells Table 4.1. Restriction endonucleases Source Sequence cut Enzyme Average expected fragment size (kb) in human DNAa AluI Arthrobacter luteus AGCT 0.3 HaeIII Hemophilus aegyptus GGCC 0.6 TaqI Thermus aquaticus TCGA 1.4 MnlI Moraxella nonliquefaciens CCTC/GAGG 0.4 HindIII Hemophilus influenzae Rd AAGCTT 3.1 EcoRI Escherichia coli R factor GAATTC 3.1 BamHI Bacillus amyloliquefaciens H GGATCC 7.0 PstI Providencia stuartii CTGCAG 7.0 MstI Microcoleus species CCTNAGGc 7.0 SmaI Serratia marcescens CCCGGG 78 BssHII Bacillus stearothermophilus GCGCGC 390b NotI Norcadia otitidis-caviarum GCGGCCGC 9766b Table 1. FDA-approved molecular diagnostic tests for infectious diseasea Test Method Companyb Chlamydia trachomatis detection PCRc LCR TMA Hybrid capture Roche Abbott Gen-Probe Digene Neisseria gonorrhoeae detection LCR Hybrid capture Abbott Digene C. trachomatis/N. gonorrhoeae screening/detection Hybridization SDR Gen-Probe Becton-Dickinson Mycobacterium tuberculosis detection PCR TMA Roche Gen-Probe HPV screening Hybrid capture Digene CMV Hybrid capture NASBA Digene Organon Teknika Grp A strep detection Hybridization Gen-Probe HIV quantitation PCR Roche Gardnerella, T. vaginalis, and Candida Hybridization Becton-Dickinson Culture confirmation for bacteria and fungi Hybridization Gen-Probe