Download inhibition of bacterial growth with antibiotics professor tj foster

INHIBITION OF BACTERIAL
GROWTH WITH
ANTIBIOTICS
PROFESSOR T.J. FOSTER
MICROBIOLOGY LECTURE 3
PENICILLIN is secreted by fungal
moulds of the Genus Penicillium
Penicillium
Fleming’s observation
Modern antibiotic-impregnated
discs
Lawn of confluent Antibiotic diffuses
bacterial growth from disc into agar.
Clear zone due to
inhibition of growth.
Penicillin Production Scale-up
Many different antibiotics are produced by Streptomyces
species isolated from soil. Competition for resources
Different indicator bacteria
Lawn of indicator bacteria
Streptomyces
Mic142d3.jpg 204 KB
Streptomyces: scanning EM
Young
vegetative
hyphae
Transition
stage
Aerial
hyphae,
young spores
Mature
spores
Streptomyces in the soil
Molecular Basis of Selective Toxicity
Target is absent in mammal or is sufficiently
different to be inhibited specifically
PROKAYOTE-EUKARYOTE DIFFERENCES
“MAGIC BULLET”
Cell Wall (peptidoglycan)
Polypeptide
Folic acid
Biosynthesis
Ribosome.
translation
DNA
replication
mRNA
RNA polymerase
transcription
ANTIBIOTICS ARE EITHER
BACTERICIDAL OR BACTERISTATIC
Growth of bacteria in liquid medium
Exponential
Stationary
109
Cells / ml
108
Tetracycline
Bacteristatic
107
Log scale
Add antibiotic
Time
Penicillin
Bactericidal
BACTERIAL GROWTH CURVE
Arithmetic no plotted
on a logarithmic scale
9 109
STATIONARY
8 108
Log10
no of
cells/
7
7
10
ml
6
DECLINE
EXPONENTIAL
LAG
2
4
6
8
16
Time (hours)
(Reminder of topic covered in Lecture 1)
SPECTRUM OF ACTIVITY
•  NARROW SPECTRUM
–  Gram positive only (eg penicillin)
•  BROAD SPECTRUM
–  Both Gram positive & Gram negative
(eg tetracycline)
BROAD SPECTRUM
PENICILLINS
•  AMPICILLIN
•  CHEMICAL MODIFICATION OF PENICILLIN
–  NARROW SPECTRUM ----> BROAD SPECTRUM
–  EASY PENETRATION OF OUTER MEMBRANE OF
GRAM-NEGATIVE BACTERIA
CH3
R- CH2 – CO -NH
CH3
NH2
Ampicillin
O
N
COOH
GRAM NEGATIVE BACTERIAL CELL WALL
Lipopolysaccharide
(endotoxin)
Outer membrane
Fimbria (pilus)
Pore
Outer membrane protein
Peptidoglycan
Periplasm
Cytoplasmic membrane
Cytoplasm
Gram Negative Bacterial Wall Structure
Porin
Polysaccharide chain
Lipopolysaccharide
LPS, endotoxin
Outer Membrane
Thin peptidoglycan
Periplasmic space
Cytoplasmic membrane
Penicillin is a highly effective antibiotic because it inhibits the
biosynthesis of cell wall peptidoglycan.
Disaccharide repeat: N-acetyl glucosamine – N acetyl muramic acid
G
G
M
G
G
M
M
G
G
M
M
Cross bridge
Stem peptide
4 amino acids
Amino acid
G
M
M
G
M
MECHANISM OF ACTION OF
PENICILLIN.
INHIBITION OF FINAL STAGE OF
CROSS-LINKING
G
G
M
G
Nascent
peptide
bridge
G
M
M
G
G
M
M
G
M
M
G
M
D-Ala
D-Ala
D-Ala = D isomer of alanine
5 amino acid stem
G
G
M
G
Transpeptidation
(Enzyme inhibited
by penicillin)
G
M
M
G
G
M
M
G
M
M
G
M
G
G
M
G
Cross bridge
formed
Loss of 5th
Amino acid
G
M
M
G
G
M
M
G
M
M
G
M
Penicillin is a highly effective antibiotic because it inhibits the
biosynthesis of cell wall peptidoglycan
Disaccharide repeat: N-acetyl glucosamine – N acetyl muramic acid
G
G
M
G
G
M
M
G
G
M
M
Cross bridge
Stem peptide
4 amino acids
Amino acid
G
M
M
G
M
RESISTANCE TO ANTIBIOTICSPENICILLIN
Destruction of the antibiotic by a bacterial enzyme.
NH
O
CH3
CH3
N
β-LACTAM
RING
+H20
NH
CH3
CH3
HN
COOH
COOH
O OH
Hydrolysis of
β-lactam bond PENICILLOIC ACID
β-LACTAMASE
Inactive
ANTIBIOTICS THAT INHIBIT
PROTEIN BIOSYNTHESIS.
TRANSLATION
In prokaryotes the ribosomes are smaller than eukaryotes
and have sufficient differences in structure that antibiotics
can exhibit selective toxicity
TETRACYCLINE
•  INHIBITS TRANSLATION OF mRNA
INTO PROTEIN
•  BINDS SMALL (30S) RIBOSOMAL
SUBUNIT AT A SITE
•  PREVENTS AMINO-ACYL TRANSFER
RNA FROM BINDING
•  REVERSIBLE, BACTERISTATIC
Large subunit
RIBOSOME
Small subunit
Amino acyl tRNA
Translation
Elongation Cycle
Translocation
Peptide bond
formation
Tetracycline binds
Small subunit.
Blocks AA-tRNA
binding
Translation
Elongation Cycle
TETRACYCLINE
X
Prevents AA-tRNA
binding
Tetracycline
Binds 30S subunit
Blocks A site
Ribosome stalled.
Reversible, bacteristatic
Tetracycline binds
small subunit.
Blocks AA-tRNA
binding
Ribosome is a
Target for
Several Antibiotics
Erythromycin
blocks
translocation
Streptomycin
distorts
codon-anticodon
recognition
Chloramphenicol
blocks
peptide bond
formation
RESISTANCE TO TETRACYCLINE.
DRUG-SPECIFIC EFFLUX PUMP
Tetracycline
Slow diffusion
into cell
Cytoplasmic
membrane
Cell
wall
Rapid and specific
efflux
Membrane efflux
protein
GENETIC BASIS OF ANTIBIOTIC
RESISTANCE IN BACTERIA
Mutation
Reduce affinity of target for inhibitor
eg. Ribosome S12 protein – streptomycin binding site
Streptomycin resistant
mutant. Amino acid change
in S12 prevents streptomycin
binding
Streptomycin binds protein S12 in small subunit
Distorts codon-anticodon recognition.
Allows incorrect aminoacyl tRNA to bind.
THE BACTERIAL CHROMOSOME
•  Growth by binary fission - chromosome
replication.
•  Offspring genetically identical.
•  Mutation in any gene 1 x 10-7/cell division.
•  Great potential for diversity.
•  Adaptation by mutation.
5 colonies
ie 5 mutants in
108 cells
Agar containing
streptomycin
0.1ml culture
109/ml
incubate
DRUG RESISTANCE (R) PLASMIDS
Acquisition of extrachromosomal DNA
• 
Plasmid
• 
Transfer by conjugation (cell-cell
transfer)
• 
Specify resistance to one or more
antibiotics
plasmid
Tetracycline efflux
chromosome
β-lactamase
PLASMIDS
•  Autonomous replication. Not part
of chromosome
•  Some large plasmids promote
conjugation
•  Carry genes encoding pilus and
DNA transfer
•  Some plasmids (R) carry several
antibiotic resistance genes.
•  Multiple resistance transferred in a
single event
Conjugation.
Transfer of F or R (resistance)
Plasmid from Cell to Cell
A single strand of plasmid DNA is peeled off the double-stranded parent plasmid.
The parent is restored to double stranded.
The SS molecule is converted to DS in the recipient.
Both cells have a copy of the plasmid.
Cells separate.
Is the End of the Antibiotic Era
Approaching?
•  Multiple resistance in some pathogens (M. tuberculosis,
S. pneumoniae) and some hospital pathogens (MRSA).
•  Over-use /abuse of antibiotics leads to resistance
– 
– 
– 
– 
Treatment of viral infections - consumer “demand”
Over-the-counter antibiotics in developing countries
Animal husbandry – growth promotion with low antibiotic concs
Lack of control of antibiotic usage in hospitals
•  Pharmaceutical companies
–  Few new drugs in pipeline. Few novel drugs introduced in past
20 years
–  Low profits, increased legislation and regulation, limitations on
usage
–  New technologies (genomics, targeted drug design) has failed to
produce a drug in the clinic
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Penicillin was the first antibiotic to be used in treatment of bacterial infectious diseases. The classic
experiment of Fleming in which a fungal mould Penicillium was shown to produce an inhibitor of bacterial
growth is performed in the laboratory practicals that accompany this series of lectures.
The major targets for antibiotic action are (i) cell wall peptidoglycan (ii) nucleic acid synthesis (iii)
biosynthesis of folic acid and (iv) the ribosome (protein synthesis). Inhibitors each exhibit SELECTIVE
TOXICITY which can be understood in terms of differences between prokaryotic cells and eukaryotic
cells
Penicillin is a narrow spectrum antibiotic which is only active against Gram positive bacteria. This is
because it cannot penetrate the outer membrane of Gram negative bacteria. The mode of action of
penicillin is to inhibit the biosynthesis of cell wall peptidoglycan resulting in a bactericidal effect. It inhibits
the penultimate step in cell wall peptidoglycan biosynthesis: transpeptidation. Semi-synthetic derivatives of
penicillin (eg ampicillin) have a broader spectrum of activity because they can penetrate the outer
membrane of Gram negative bacteria (more hydrophilic, pass through outer membrane pores).
In contrast, tetracycline is a bacteristatic, broad spectrum antibiotic. Its mode of action is to bind to the
small subunit of the bacterial ribosome to inhibit translation of mRNA into protein. It is important that you
revise the process of translation and understand fully the inhibitory effect of this drug. Note that different
antibiotics target other steps in translation. Eg streptomycin binds to a protein S12 in the small subunit
and distorts codon-anticodon recognition causing faulty proteins to be made (messenger misreading).
Resistance has developed very rapidly to all antibiotics used in medical practice. Resistance to penicillin
is mediated by an enzyme that destroys the drug. Tetracycline resistance is caused by a membranelocated pump that keeps the concentration of the drug in the cytoplasm below the inhibitory level. Both
resistance determinants are specified by genes on extrachromosomal DNA elements called plasmids that
can be transferred between bacterial cells by conjugation. Resistance can also be acquired by
mutations that prevent the antibiotic from binding to its target (eg streptomycin resistance and ribosomal
protein S12)
• 
Unfortunately Campbell has nothing on penicillin action. It deals with protein synthesis (translation) in some detail. It also
covers R plasmids and conjugation
See: Totora 6th edn chapter 7 (Control) and chapter 20 (Antimicrobial drugs)
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http://www.sumanasinc.com/scienceinfocus/sif_antibiotics.html