Antibiotics

Uses & Resistance

Department of Microbiology
BVPMedical College
Pune
 “MICROBES”
WHAT DOES NOT KILL
THEM MAKES THEM
STRONGER
 ISSUES AT STAKE

n Bacteria Baffle Antibiotics !!

n How do microbes “LEARN” to defeat
Antibiotics !
n Is it All a question of “Team work”?
n How much has Molecular biology unravelled
about resistance?
n What is the role of Mutations in resistant bugs
 Definition
n Anti Microbial agent:-Is a chemical substance inhibiting
the growth or causing the death of a micro-organism.
n Antibiotic:-Natural metabolic products of fungi
actinomycetes and bacteria that kill or inhibit the
growth of micro-organisms.
n Antibiotic (Modified) :-Substances produced by a micro-
organism or a similar substance produced wholly or
partially by chemical synthesis that is capable of
inhibiting the growth or causing death of microorganisms
in low conc.
 PRE-ANTIBIOTIC ERA
n Origin & source of Resistance an Enigma ?
n Existed in soil ! Saprophytic Actinomycetes
n Naturally synthesised Antibacterial agents
n Mechanism self protection–
”My own defence”
n Not a single phenomenon -all antibiotics
n Spread due to Use/Misuse antibiotics
 Microorganisms that Produce
Antibiotics
n BACTERIA-
Streptomyces spp.-
griseus,venezualae,erythreus,orientalis Bacillus spp.-
polymxa,subtilis Micromonospora spp.-
purpurae
n FUNGI-
Penicillium spp.- notatum, chrysogenum,
griseofulvum Cephalosporium spp.-
acremonium
 Mechanisms of Action

n Inhibition of Bacterial cell wall synthesis

n Inhibition of Bacterial Cytoplasmic
membrane function
n Inhibition of Bacterial Protein synthesis
n Inhibition of Bacterial Nucleic acid
synthesis
n Inhibition of Metabolic Pathways.
 Drug Resistance
n Definition – An Organism that will not be inhibited
or killed by an antibacterial agent at concentrations
of the drug achievable in the body after normal
dosage.
n Types :- Innate
Acquired
n Innate- Born Resistant to family of antibiotics as
they lack a susceptible target or are impermeable to
to the antibacterial agent.
n Example-Gram negative bacteria with their outer
membrane layer exterior to the peptidoglycan layer
are less permeable to larger molecules as compared
to gram positive bacteria.
 Acquired Resistance

n Chromosomal Mutation –single/Multiple

n Plasmid mediated
n Transposons
n Integrons –gene cassettes
 Mechanisms of Resistance
n Enzymes that modify or destroy the antibiotic-
Drug Inactivation
n Access to the target site may be altered
Altered uptake / increased exit
n The target site may be altered-
Lowered affinity or additional targets
 Antibiotics & Resistance
n Beta Lactums -
Beta lactamase production
Alteration of target site-PBP’s-MRSA
Alteration in access to target site-
Porins
n Aminoglycosides-
n Modifying enzyme-acetyltransferase
n Alteration of specific ribosomal binding protein
n Permeability barrier (rare)
 Antibiotics & Resistance
n Tetracycline:-
Decreased uptake due to change in surface & rapid
efflux
n Chloramphenicol:-
Acetyltransferase
Permeability barrier determined by plasmid
n Macrolides:-
Induce enzymatic activitymethylating ribosomal
RNA
n Trimethoprim &Sulphonamides:-
Take up Folic acid from environment
Inhibition of bacterial dihydrofolate reductase enzyme
 Antibiotics & Resistance

n Quinolones:-
Alteration of subunit A ot B of DNA Gyrase
Decrease in Outer memebrane Porins
Mutant multiple resistant strains
n Rifampicin :-
Mutational modification of B subunit of RNA
polymerase
 Antimicrobial Resistance

n Biological Vs Clinical Resistance

n Environmentally MediatedResistance

n Micro-organism mediated Resistance

 BIOLOGICAL Vs CLINICAL
RESISTANCE
n Biological-changes org. less susceptible
n Clinical- drug not effective - clinical use
n Lab. methods focus on clinical resistance
n Substantial change with out notice
n Strep. pneumoniae-0.03ug/ml
n clinical detection-2ug/ml
n Develop biological resistance 10-50 times
higher without being detected.
ENVIRONMENTALLY MEDIATED
RESISTANCE
n Resistance that directly results from
physical or chemical changes environment
n alter drug/org. response
n pH,anaerobic staus,cation conc Mg,Ca
n Eryth./Aminoglycoside with pH
n Pseudomonas –aminoglycoside vs cations
n Testing conditions cannot recreate in-
vivo physiology
n Only resistance expression known.
 MICRO-ORGANISM MEDIATED
RESISTANCE
n Anti microbial resistance that is due to
genetically encoded traits of the micro-
organism and is the type of resistance
that in-vitro susceptibility testing
methods are targetted to detect.
n Two types:-
– INTRINSIC
– ACQUIRED
 INTRINSIC RESISTANCE
n Resistance due to normal genetic,
structural or physiologic state.
n usually expressed by
chromosomal genes within
bacterial group/species
n Predictable-determines battery of
drugs
 INTRINSIC RESISTANCE
EXAMPLES
n Anaerobic-Aminoglycocide - No oxidation
n Aerobic- Metronidazole- Inability reduce
n GPB - Aztreonam - Lack PBP’s
n Klebsiella –Ampicillin – lactamases—PBP’s
n Enterococci -All Cephalosporin –Lack PBP
n GNB -Vancomycin-Inability to penetrate
outer membrane
 Genetic determinants
n Intrinsic resistance:-
Resistance determined by chromosomal
genes eg- beta lactamase of gram -ve
bacteria inactivate beta lactum
antibiotics
n Acquired resistance:-
-Resistance determined by chromosomal
mutations eg-Nalidixic acid resistance
in E coli is due to point mutation at N
terminus of the GyrA protein reducing
nalidixic acid binding
 ACQUIRED RESISTANCE
n Resistance that results from altered
cellular physiology and structure due to
changes in its genetic make up
n Unpredictable
n Acquired by :-
– Successful genetic mutation
– Gene transfer
– Combination gene transfer & mutation
 ACQUIRED
BACTERIAL RESISTANCE
n SPONTANEOUS MUTATION
n MICROBIAL SEX-TRANSFORMATION
CONJUGATION
n ACQUISITION OF SMALL CIRCLE OF
DNA – PLASMIDS
1968,12,500 GUATEMALANS DIED OF
RESISTANT SHIGELLA DIARRHEA
n MUTATION IS CHANCY –SO SHARE
RESISTANT GENES
 DIABOLICAL BARTERING
n SHARE PLASMIDS
n GENE CASSETTES-INTEGRONS :-
INTEGRASES SPLICE INTO
FUNCTIONAL GENES
n RETRO TRANSFER
n JUMPING GENES
n PERMANENCE-DISTRIBUTED TO
PROGENY
 Associated Variables in
Resistance
n Location - chromosomal
- Extra chromosomal
- Transposon
n Transfer - Conjugation
- Transduction
-Transformation
n Expression - Constitutive
- Inducible
- Constitutive- Inducible
 FACTORS CONTRIBUTING
TO RESISTANCE
n Non-clinical use of Antibiotics:
agriculture,vetinary sciences
n Normal Intestinal flora healthy
animals
n Contaminated food for animals
n Aquatic environment
n Resistant bacteria in human flora
 Development of Resistance
n Hospital Environment:-
- Epidemic GNB-”Super bacteria”
- chromosomal mutations
- In-vivo transfer plasmids
- PBP-mediated resistance
n In the Community:-
increasing beta-lactamase presence
n Inappropriate antibiotic usage
n Antibiotic consumption
UNDERSTANDING MECHANICS
OF RESISTANCE

n Bacterial structure & physiology

n Molecular biology of Infective
agents
n Pharmacology of Antimicrobial
drugs
Cell Wall of GPB
Cell Wall of GNB
 MODES OF RESISTANCE

n Enzymatic degradation/ modification

n Decreased uptake/accumulation
n Altered antimicrobial target
n Circumvention of antimicrobial
action
n Uncoupling of antimicrobial agent-
target interaction
n Combination of any of the above
 Efflux of Antibiotic from cell

n Not allow antibiotic conc. to rise

n Energy dependant efflux mechanism-tetracycline

n Staph. -Quinolones efflux

n Transporter protein oprK

 Altered
Receptors/Targets
n altered protein -appearance of
Resistance
n Alteration of Penicillin binding proteins
n -PBP 2 -->PBP2a
n alteration one step ---single gene
control Mec A
n Imp.for GPB mostly,but do effect GNB
n Loss of variant protein restores
susceptibility
One point mutation changes
specificity

“In one stroke bacteria can

undo millions of dollars”
 Altered Antibiotic Transport
n Structural differences - cell wall GPB & GNB
n Tpt. across cell memb. NOT an issue Gram + bacteria
n GNB- Lipid layer difficult to clear, permeability important
n Outer memb. Porins: OmpF,OmpC, PhoE(mutants)
n Charge of Molecule & Hydrophobicity
n Negative Charge Slow movement-eg Methicillin in GNB
n Imipenem -zwitterion efficient
n Bulky side chain -crossing poor eg-pipericillin,cefaperazone
n Proton motive force-energy dependant
Action of Beta-Lactamase
SURVIVAL OF THE FITTEST

n Emergence of “new” genes -MRSA,VRE

n Spread of “old” genes to new host

n Mutations of old genes-more potent

resistance

n Emergence of intrinsically resistant

opportunistic bacteria