Chapter-9:

Antibiotic and emergence of

resistance -2

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Expected Learning Outcome

 Describe the antimicrobial medications that interfere with the following: protein synthesis,

nucleic acid synthesis, metabolic pathways, or cell membrane integrity.

 Describe the antibacterial medications used to treat Mycobacterium tuberculosis infections.

 Describe how the minimum inhibitory concentration (MIC) and the minimum bactericidal

concentration (MBC) are determined

 Compare and contrast the Kirby-Bauer disc diffusion test with commercial modifications of

antimicrobial susceptibility testing

 Describe four general mechanisms of antimicrobial resistance.

 Describe how antimicrobial resistance can be acquired.

 List five examples of emerging antimicrobial resistance.

 Describe how the emergence and spread of antimicrobial resistance can be slowed
Mechanisms of Action of Antibacterial Medications

 Antibacterial
medications target
specific bacterial
processes and
structures
1. Cell wall synthesis
2. Protein synthesis
3. Nucleic acid
synthesis
4. Metabolic pathways
5. Cell membranes
Mechanisms of Action of Antibacterial Medications

2. Antibacterial Medications That Inhibit Protein Synthesis

All cells synthesize proteins
Can exploit differences between prokaryotic and
eukaryotic ribosomes
Prokaryotes have 70S (the 70S ribosome is made up of a 50S
and 30S subunits), eukaryotes have 80S ribosomes
Mitochondria also have 70S ribosomes (may account for some
toxicity of these antibiotics

30S

Eucaryotes Procaryotes
Mechanisms of Action of Antibacterial Medications

2. Antibacterial Medications That Inhibit Protein Synthesis

Examples of
30S
aminoglycosides
include
streptomycin,
gentamycin, and
tobramycin.
Mechanisms of Action of Antibacterial Medications

 Antibacterial
medications target
specific bacterial
processes and
structures
1. Cell wall synthesis
2. Protein synthesis
3. Nucleic acid
synthesis
4. Metabolic pathways
5. Cell membranes
Mechanisms of Action of Antibacterial Medications

3. Antibacterial Medications That

Inhibit Nucleic Acid Synthesis
 For example Fluoroquinolones
inhibit topoisomerases
Enzymes that maintain
supercoiling of DNA
The topoisomerase DNA gyrase
breaks, rejoins strands to relieve
strain from localized unwinding of
DNA; function is essential
Bactericidal against wide variety
of bacteria
Resistance usually due to
alteration in DNA gyrase target
Mechanisms of Action of Antibacterial Medications

 Antibacterial
medications target
specific bacterial
processes and
structures
1. Cell wall synthesis
2. Protein synthesis
3. Nucleic acid
synthesis
4. Metabolic pathways
5. Cell membranes
Mechanisms of Action of Antibacterial Medications

4. Antibacterial Medications That Interfere with Metabolic

Pathways
 Few antibacterial antibiotics interfere with metabolism
Folate inhibitors are among most useful
Sulfonamides, trimethoprim inhibit different steps in synthesis of
folic acid and coenzyme required
for nucleotide synthesis
 Animals lack enzymes to
synthesize folic acid;
required in diet
Resistance often from
plasmid-encoded enzymes
with lower affinity to these
chemicals; genes for both often
carried on same plasmid
Mechanisms of Action of Antibacterial Medications

 Antibacterial Medications That Interfere with Metabolic

Pathways (continued…)
 Sulfonamides and related are called sulfa drugs
Inhibit many Gram-positives and Gram-negatives
Structurally similar to PABA, so enzyme binds chemical
Example of competitive inhibition
Human cells lack enzyme
 Trimethoprim inhibits
enzyme in later step
Has little effect on enzyme’s
counterpart in human cells
Combination of trimethoprim
and sulfonamide has
synergistic effect
Often used to treat UTIs
Mechanisms of Action of Antibacterial Medications

 Antibacterial
medications target
specific bacterial
processes and
structures
1. Cell wall synthesis
2. Protein synthesis
3. Nucleic acid
synthesis
4. Metabolic pathways
5. Cell membranes
Mechanisms of Action of Antibacterial Medications

5. Antibacterial Medications That Interfere with Cell

Membrane Integrity
 A few antimicrobials damage bacterial membranes
Cause cells to leak, leading to cell death
Limits usefulness to topical applications
Also bind to eukaryotic cells, though to a lesser extent
Daptomycin inserts into cytoplasmic membrane
 Used against Gram-positives resistant to other antibiotics
 Ineffective against Gram-negatives; cannot penetrate
outer membrane
 Mechanisms of Action of Antibacterial Medications

 Antibacterial Medications Effective Against

Mycobacterium species (e.g. Mycobacterium
tuberculosis)
 Few antimicrobials effective against
Mycobacterium
Waxy cell prevents entry of many chemicals; growth is
slow
Group of five medications preferred: first-line antibiotics
Most effective, least toxic; given as combination
therapy
 Typically two or more at a time
 Decrease chance of development of resistant
mutants
Some target unique cell wall of mycobacteria Mycobacterium
tuberculosis (acid-
(mycolic acid synthesis and other cell wall fast stain rods)
components).
Some interferes with protein synthesis
Second-line antibiotics used for resistant strains
 Less effective or have greater toxicity risk
Antimicrobial Susceptibility Testing
 Susceptibility of
pathogens often
unpredictable
MIC (Minimum Inhibitory
Concentration) is lowest
concentration that
prevents growth in vitro
Serial dilutions of chemical
in suitable growth medium
used; cultures added,
incubated, examined for
turbidity
Inhibition does not
necessarily mean successful
treatment; level may not be
achieved in person’s blood
Antimicrobial Susceptibility Testing

 Conventional Disc Diffusion Method

Kirby-Bauer disc diffusion test routinely used to determine
susceptibility of bacterial strain to antibiotics
Standard concentration of strain uniformly spread on agar
plate; discs containing different antibiotics placed on surface
Drugs diffuse outward, establish gradient
Resulting zone of inhibition compared with specially
prepared charts to determine
whether strain is susceptible,
intermediate, or resistant
Drug characteristics must
be taken into account (for example
molecular weight, stability,
amount)
Antimicrobial Susceptibility Testing

 Commercial Modifications of Susceptibility Testing

Less labor-intensive, often faster results
One automated system determines growth rate via turbidity
in cards, interprets results to determine MICs in 6–15 hours
Resistance to Antimicrobial Medications

 Increasing use, misuse selects

for resistant microorganisms
Only 3% of Staphylococcus
aureus originally resistant to
penicillin G; now more than 90%
are resistant
Hundreds of tons used each year
Antimicrobial resistance alarming
Impact on cost, complications,
and outcomes of treatment
Dealing with problem requires
understanding of mechanisms and
spread of resistance
Resistance to Antimicrobial Medications

 Mechanisms of Acquired
Resistance
1. Antibiotic-Inactivating Enzymes
e. g. Penicillinase
2. Alteration in Target Molecule
Minor structural changes
can prevent binding (e.g. PBP)
3. Decreased Uptake of the Medication
Changes in porin proteins of outer 1
4
membrane of Gram-negatives
4. Increased Elimination of Medication
Increased production of efflux pumps
allows faster removal
Structural changes can 2
influence range of antibiotics
3
 Resistance of this type
particularly worrisome;
might allow resistance
to multiple antibiotics
Resistance to Antimicrobial Medications
 Acquisition of Resistance
1. Spontaneous Mutations
Occur during replication
Happen at low rate but can have significant
effect
Just a single base-pair change in gene
encoding a ribosomal protein yields resistance
to streptomycin
2. Gene Transfer
Genes encoding resistance can spread to
different strains, species, even genera
 Most commonly through conjugative
transfer of R plasmids, which often carry
several resistance genes
 May also originate from the soil microbes
that naturally produce the antibiotic
 Gene coding for enzyme that modifies
aminoglycoside likely originated from the
Streptomyces species that produces the
antibiotic
 Resistance to Antimicrobial Medications

 Enterobacteriaceae
intrinsically resistant to Enterobacteriaceae are
a large family of Gram

many antibiotics negative bacteria

o Outer membrane prevents

entry (gram negative)
o Some enterics developed
ability to produce β-
lactamases
o Some further developed
ability to produce extended-
spectrum β-lactamases
(ESBLs); resist
cephalosporins and
aztreonam in addition to
penicillins
Examples of Emerging Resistance

 Mycobacterium Mycobacterium
tuberculosis (acid-
tuberculosis requires fast stain rods)

long treatment
o Can become resistant to first-
line antibiotics via mutation
o Large numbers of cells found in
active infection, so likely at
least one cell has developed
resistance
o Combination therapy is
therefore required
An anteroposterior X-ray of a patient
o 6 months or more of treatment diagnosed with advanced bilateral pulmonary
tuberculosis. This AP X-ray of the chest
necessary due to slow rate of reveals the presence of bilateral pulmonary
growth; many patients do not infiltrate (white triangles), and „caving
formation“ (black arrows) present in the right
comply apical region.The diagnosis is far-advanced
tuberculosis
Examples of Emerging Resistance

 Neisseria gonorrhoeae was

susceptible to penicillin
o Some strains developed
resistance to penicillin through
mutation
o others acquired a plasmid that Neisseria gonorrhoeae (Gram negative diplococci)

encoded production of
penicillinase
o Only certain cephalosporins
are reliably effective
o Combination therapy used now

Newborn with gonococcal ophthalmia

neonatorum caused by a maternally transmitted
gonococcal infection. Image courtesy of CDC
Examples of Emerging Resistance

 Staphylococcus aureus:
Gram-stained
increasingly resistant Staphylococcus aureus

o Common cause of healthcare-

associated infections
o Most strains resistant to
penicillin, encode penicillinase
o New strains recently emerged,
have PBPs with low affinity to
all β-lactam antibiotics,
methicillin and others
o Methicillin-resistant
Staphylococcus aureus
(MRSA)
CA-MRSA on face
Examples of Emerging Resistance

 Streptococcus
pneumoniae
o Historically susceptible
o some recently acquired
penicillin resistance
Streptococcus pneumoniae.
Gram-positive diplococci are
located outside neutrophils.
Penicillin-resistant S.
pneumoniae makes a clinical
problem

o produce PBPs with lower

affinity, likely via
transformation from other
Streptococcus species
Slowing Emergence and Spread of Resistance

 Will require cooperation from everyone globally

 Responsibilities of Physicians and Healthcare Workers

Increase efforts to identify cause of infection
Only prescribe suitable antimicrobials if appropriate
Educate patients about proper use of antibiotics

 Responsibilities of Patients
Carefully follow instructions even if inconvenient
Essential to maintain adequate blood levels of antibiotic over
time; skipping a dose may reduce levels, allowing less-
sensitive microbes a chance to grow and spread
Failure to complete treatment may not kill least-sensitive,
similarly allowing subsequent spread
Slowing Emergence and Spread of Resistance

 The Importance of an Educated Public

Antibiotics ineffective against viruses
 Cannot cure common cold!
Misuse selects for antibiotic-resistant bacteria in normal
microbiota; they can eventually transfer to pathogens
Rate of Antimicrobial Prescriptions (per 1000 Persons) in the United States, by State
Slowing Emergence and Spread of Resistance

Global Impacts of the Use of Antimicrobial

Medications
Overuse is a worldwide concern; resistant microbes
recognize no political boundaries
Antimicrobial antibiotics available without prescription in
many parts of the world, may allow improper use
Antimicrobial antibiotics used in animal feeds at low levels to
enhance growth; selects for antibiotic-resistant microbes
 Resistant Salmonella strains linked to animals