Transcript Molecular Diagnostics - Institute Of Tropical & Infectious

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