Mechanisms of antimicrobial resistance

Antimicrobial • are chemotherapeutic agents for treating infections

agents o eg antibiotics

Antibiotics • Historically term as products of the earth

• substances produced naturally by microorganisms that kill or inhibit other
microorganisms.
• are secondary metabolites of microorganisms
• microorganisms are resistant to the action of their own antibiotic
• tend to be rather large, low molecular weight, complicated structurally, organic molecules
• may require up to 30 enzymatic steps to synthesize
o this requires a substantial part of the bacterial genome as this is important for
survival of these organisms

Bacteria
producing
common
antibiotics

Fungi producing
common
antibiotics

Antibiotics • must be able to reach infection site

• should target microbial constituents not found on human cells
o to minimize toxicity
• Bacteriostatic: Capable of inhibiting bacterial growth
o eg tetracycline, chloramphenicol, macrolides
• Bacteriocidal: Capable of killing bacteria
o eg penicillin, isonazid

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• both are effective treatment for immunocompetent patient
• bacteriostatic antibiotics should not be used for immunocompromised individual
• broad-spectrum antibiotic
o kill resident flora, a drawback
• Best choice of antibiotic
o narrow spectrum and short duration course
• more importantly – accuracy of infection diagnosis
Mechanism of
action of
antibiotics

Antimicrobial • ability of a micoorganism to survive & multiply in the presence of antimicrobial agent that
resistance would normally inhibit or kill it
- Non-resistant bacteria multiply, and upon drug treatment, the bacteria die.
- Drug resistant bacteria multiply as well, but upon drug treatment, the bacteria
continue to spread.

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 Mechanisms of antimicrobial resistance

Causes of • Natural causes

antimicrobial • ‘Societal’ pressures
resistance

Superbacterium
Natural Selective Pressure
(Biological) • in the presence of an antimicrobial, microbes are either
Causes o killed
o remained alive only if they carry resistance genes
• the survivors will replicate and their progeny quickly become the dominant type

Mutation
• Microbes evolve rapidly and adapt quickly to ensure survival in new
environmental conditions
• during replication, mutations arise
o some of these mutations may help an individual microbe survive antimicrobial

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exposure

Gene Transfer
• microbes may also get genes from each other, including genes that make
the microbe drug resistant.
Societal Inappropriate use of antimicrobials in treatment of patient
pressures • who has a viral infection
• with as-yet an undiagnosed condition

Inadequate diagnostics
• incomplete or imperfect information diagnosis of an infection
• a broad-spectrum antimicrobial prescription when a specific antibiotic might be better

Hospital Use
• the extensive use of antimicrobials and close contact among patients creates a fertile
environment for the spread of resistant microbes

Agricultural Use
• the practice of adding antibiotics to agricultural feed
• more than half of the antibiotics produced in the US are used for agricultural purposes
The basis of • Inherent Resistance
microbial • Acquired Resistance
resistance to oVertical evolution
antibiotics oHorizontal evolution
Inherent • Natural resistance
Resistance • Bacteria may be inherently resistant to an antibiotic
• examples:
o some bacteria has some gene that is responsible for resistance to its own
antibiotic
• eg a streptomycete has a trans-membrane protein channel that pumps the
antibiotic out of the cells
o a Gram-negative bacterium has an outer membrane that establishes a
permeability barrier against the antibiotic
o an organism lacks a transport system for the antibiotic
o an organism lacks the target that is hit by the antibiotic
Acquired • bacteria can develop resistance to antibiotics
Resistance • due to genetic modifications that arise when a drug is introduced into the environment
o this acts as a selective pressure
• is driven by two genetic processes in bacteria:
o mutation and selection (vertical evolution)
o exchange of genes between strains and species (horizontal evolution)
Vertical • is strictly a matter of Darwinian evolution driven by principles of
evolution natural selection
o a spontaneous mutation in the bacterial chromosome imparts resistance to a member of
the bacterial population
• In the selective environment of the antibiotic, the wild type (non-mutants) are killed
o this allows the resistant mutant to grow and flourish
• the mutation rate for most bacterial genes is approximately 10-8
• if a bacterial population doubles from 108 cells to 2 x 108 cells, there is likely to be a
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mutant present for any given gene
• Since bacteria grow to reach population densities far in excess of 109 cells, such a mutant
could develop from a single generation during 15 minutes of growth
Horizontal • is the acquisition of genes for resistance from another organism
evolution o for eg, a streptomycete has a gene for resistance to streptomycin (its own antibiotic), but
somehow that gene escapes and gets into E. coli or Shigella
• some bacterium develops genetic resistance through the process of mutation and selection
(vertical evolution)
o it then donates these genes to some other bacterium through one of several processes for
genetic exchange that exist in bacteria
Resistance genes • are carried on:
o Chromosomes
o Plasmids
o Transposons
Resistance genes • stable (genes are not easily lost)
on chromosomes • allow vertical transmission of resistance only (descending)
• allow no horizontal transmission
o not transferable from one bacterium to another
• these genes cause intrinsic resistance
Resistance genes • unstable in absence of antibiotic
on plasmids • transferable between bacteria of different species
• these genes often cause acquired resistance
• plasmids can also carry genes for antibiotic production

Transposons • found on either chromosomes or plasmids

• genes capable of "jumping" from a chromosome/plasmid to a chromosome/plasmid

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Transfer of • Bacteria are able to exchange genes in nature by three processes:

resistance genes o conjugation
o transduction
o transformation
Mechanisms of A. Transfer of free DNA from a dead bacterium to recipient
gene B. Transfer of a plasmid or conjugative transposon from a donor bacterium to recipient
transfer in C. Transfer of gene from a bacterium infected with virus via a viral delivery
bacteria system/bacteriophage

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Conjugative • complex mobile bacterial elements which are capable of both transposition
transposons and conjugation
• can be transferred across large phylogenetic distances.
• mediate antibiotic resistance
• some evidence - contain genes encoding virulence factors.
• often found in pathogens
• in some cases, they are more important than plasmids in mediating antibiotic
resistance
• antibiotic resistance genes must be transferred by conjugation
Transfer of • bacteria evolve fast because of:
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resistance genes o fast growth rates
o high concentrations of cells
o genetic processes of mutation and selection
o the ability to exchange genes
• Hence, bacterial adaptation (resistance) to the antibiotic environment seems to take place
very rapidly in evolutionary time.
• genetic recombination can follow the transfer of DNA from one cell
to another
o leads to the emergence of a new genotype (recombinant)
• common for DNA to be transferred as plasmids between mating bacteria
• bacteria usually develop their genes for drug resistance on plasmids (resistance transfer
factors, or RTFs)
o hence, they are able to spread drug resistance to other strains and species
during genetic exchange processes
Mechanisms of • Bacteria developed several mechanisms to avoid the action of antibiotics
antimicrobial o Degradation/inactivation of antimicrobial agents
resistance o Modifying antimicrobial targets
o Altered transport
o Altered metabolic pathway

Degradation of • bacteria preserve themselves by destroying active components of

antimicrobial the antimicrobial agents
agents • classic example
o hydrolytic deactivation of b-lactam ring in penicillin by b-lactamase
oinactivated penicillin will not bind to penicillin binding proteins, cell wall
synthesis will be protected
• observed in:
o Enterobacteriaceae – chloramphenicol
oGram neg and pos – aminoglycosides
• aminoglycoside-modifying enzymes inactivate antibiotic by
adding groups eg phosphoryl, adenyl or acetyl to the antibiotic
Penicillin • a b-lactam antibiotic
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 Mechanisms of antimicrobial resistance
• discovered in 1928 by Scottish scientist Alexander Fleming
• Penicillin G
o the first penicillin
o prior to its discovery, post-operative infections were largely untreatable
resulting in many death
o Many soldiers died from wound infections than from any other cause in
1940 at the beginning of World War II
o major problem is that it is sensitive to stomach acid
o E. coli is intrinsically resistant to it because it cannot pass through the outer
membrane of Gram negative cell wall to reach the PBPs
• acts by inhibiting the formation of peptidoglycan cross-links in the bacterial cell wall
Bacterial cell
wall synthesis

Mechanism of
action of
Penicillin

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Penicillin
resistance

Modifying • evade antimicrobials by changing target sites to avoid recognition

antimicrobial 1. Resistance to drugs that target the ribosomal subunits may occur via
targets o ribosomal mutation (aminoglycosides, oxazolidinones)
o methylation (aminoglycosides, macrolides)
o ribosomal protection (tetracyclines)
o interfere with the ability of the drug to bind to the ribosome

2. Resistance to drugs that target nucleic acid synthesis (eg fluoroquinolones)

is via modifications in
o DNA gyrase (gram negative bacteria—e.g. gyrA)
o topoisomerase IV (gram positive bacteria— e.g. grlA)
o cause changes in the structure of gyrase and topoisomerase which decrease or
eliminate the ability of the drug to bind to these components
Altered • peptidoglycan – too porous to be an antibiotic barrier
transport: • outer membrane of Gram-negative bacteria
Reduce o limits antibiotic access to cytoplasmic membrane
penetration into o a minor advantage for Gram-negative bacteria
cell • porin are passage by which antibiotics can cross the outer membrane
• Gram neg bacteria reduce uptake of antibiotics by modifying porin channel by altering :
o Frequency
o Size
o Selectivity

• observed in:
o Pseudomonas aeruginosa - imipenem (b-lactam)
o Enterobacter aerogenes & Klebsiella spp. – imipenem
o many Gram neg – aminoglycosides, quinolones
Altered • to be effective, antibiotics must be of sufficient concentration
transport: • efflux pumps are membrane proteins that move lipophilic or amphipathic
Expulsion by molecules in and out of bacterial cell
efflux pumps • some bacteria possess efflux pumps that selectively extrude specific antibiotics
• observed in:
o E. coli & other Enterobacteriaceae – tetracyclines
o Enterobacteriaceae – cloramphenicol
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o Staphylococci – macrolides
o Staph. aureus & Strep. pneumoniae - fluoroquinolones

Altered metabolic • bacteria developed resistance from mutations favouring alternative pathway
pathway o hence, "bypass" the normal metabolic pathway
• antibiotic inhibiting metabolic pathway is trimethoprim-sulfamethoxazole (TMP-SMX /
co-trimoxazole)
o both work sequentially to inhibit enzyme systems in the bacterial synthesis of
tetrahydrofolic acid
• ∼20 different TMP-resistant transferable dhfr genes
o are most frequently found in gram-negative enteric bacteria
o also been found in staphylococci, Listeria monocytogenes
• transferable resistance to SMX is mediated by 2 drug-resistant DHPS enzymes, encoded
by sulI or sulII genes
o eg gram-negative enteric bacteria
Antimicrobial • ideally, every strain isolated from a clinical specimen should have its precise
susceptibility level of susceptibility determined
determination • done by testing the organism at diminishing concentrations of test antibiotic
• Minimum inhibitory concentration (MIC)
o lowest concentration the organisms do not produce visible growth
• Minimum bactericidal concentration (MBC)
o lowest concentration the organisms do not grow
• The wider the margin between MIC and MBC, the more tolerant is the
organism to the drug
• performing MIC to every specimen is laborious
Detection of • Antimicrobial susceptibility tests
antimicrobial oin vitro procedures used to detect antimicrobial resistance in individual bacterial isolates
resistance • Determine resistance or susceptibility of bacterial isolate against
possible therapeutic candidates
o useful in the selection of best antibiotic treatment option
• Can also monitor emergence & spread of resistant microorganism in population

Antimicrobial
susceptibility
tests

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• The size of the zones of inhibition of microbial growth surrounding the
antibiotic disks indicates microbial susceptibility to the antibiotic
• level of resistance indicated
o sensitive, intermediate, resistant
Standards for • international bodies that provide standards and guidelines for medical professionals
AST o Clinical and Laboratory Standards Institute (CLSI)
• Originally formed as the National Committee for Clinical Laboratory
Standards (NCCLS)
o European committee of antimicrobial susceptibility testing (EUCAST)
Control of Surveillance
antimicrobial • to monitor ‘how we are doing’
resistance • to provide data on resistant organisms, illness caused and antimicrobial therapy

Prudent antimicrobial use

• to reduce ‘pressure for resistance’ in clinical practice, veterinary, animal
husbandry, agriculture

Infection control
• to reduce the spread of infection in general and in antimicrobial resistant
microorganisms in particular
Control of • doctors should not prescribe and patients should not take antibiotics for which
antimicrobial there is no medical value
resistance in the • adhere to appropriate prescribing guidelines
medical o complete antibiotic course
environment • give combinations of antibiotics, when necessary, to minimize the development
of resistance to a single antibiotic
o as in the case of TB
• give another antibiotic or combination of antibiotics if the first is not working
Antimicrobial Important considerations:
therapy • Accurate diagnosis of infection
• Empirical vs definitive therapy
• Broad vs narrow-spectrum
• Duration of treatment

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