Penicillin G: β-lactamases PBP Mrsa

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 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.
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 Gene Detection method
agent(s)
Staphylococci Methicillin mec Ab Standard DNA
Oxacillin probe
Branched chain
DNA probe
PCR
Enterococci Vancomycin van A, B, C, Dc Standard DNA
probe
PCR
Enterobacteriaceae Beta-lactams blaTEM Standard probe
Haemophilus and PCR and RFLP
influenzae blaSHVd PCR and
Neisseria sequencing
gonorrhoeae
Enterobacteriaceae Quinolones Point mutations in gyr A, PCR and
and gram-positive gyr B, par C and par E sequencing
cocci
Mycobacterium Rifampin Point mutations in rpo B PCR and SSCP
tuberuclosise Isoniazid Point mutations in kat G, PCR and
Ethambutol inh A, and ahp C sequencing
Streptomycin Point mutations in emb B PCR and SSCP
Point mutations in rps L PCR and
and rrs sequencing
PCR and RFLP
Herpes virusesf Acyclovir and related Mutations or deletions PCR and sequencing
drugs in the TK gene PCR and sequencing
Foscarnet Point mutations in
DNA polymerase gene
HIVg Nucleoside reverse Point mutations in RT PCR and sequencing

transcriptase inhibitors gene PCR and LIPA
Protease inhibitors Point mutations in PCR and sequencing
PROT gene
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. bacteria -- antibiotic resistance

2. endoparasites
3. viruses -- resistance to antiviral drugs
4. fungi
5. cancer cells
Table 4.1. Restriction endonucleases
Source Sequence cut Average expected fragment size

Enzyme (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 Roche
LCR Abbott
TMA Gen-Probe
Hybrid capture Digene
Neisseria gonorrhoeae detection LCR Abbott
Hybrid capture Digene
C. trachomatis/N. gonorrhoeae Hybridization Gen-Probe
screening/detection SDR Becton-Dickinson
Mycobacterium tuberculosis detection PCR Roche
TMA Gen-Probe
HPV screening Hybrid capture Digene
CMV Hybrid capture Digene
NASBA 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

Penicillin G: β-lactamases PBP Mrsa

Uploaded by

Copyright:

Available Formats

Penicillin G: β-lactamases PBP Mrsa

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Penicillin G: β-lactamases PBP Mrsa

Uploaded by

Copyright:

Available Formats

Drug Resistance

The four main mechanisms by which microorganisms exhibit

Genetic material can be transferred between bacteria

Bottom Line: Genetic Make Up Changes

Hybridization of DNA to a specific Probe

PCR – Single Strand Conformation polymorphism (PCR-SSCP)

Results from Restriction Digest

DNA Arrays: - Hybridization – Mass screening of sequences using

HIVg Nucleoside reverse Point mutations in RT PCR and sequencing

Obligate intra-cellular parasites – can illicite host immune response

Herpes simples virus

Enveloped - single stranded RNA molecule

Gag – Core proteins

Carrying capacity – 7.5kb

Lentiviruses – subclass of retroviruses

non-enveloped viruses containing a linear double stranded DNA

four early transcriptional units (E1, E2, E3 & E4),

1. bacteria -- antibiotic resistance

Source Sequence cut Average expected fragment size

AluI Arthrobacter luteus AGCT 0.3

Test Method Companyb

You might also like