ANTIBIOTIC RESISTANCE

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ANTIBIOTIC RESISTANCE
1940 1947 1956 1987
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INCREASES IN ANTIBIOTIC RESISTANCE

• Bacterium Resistance Year
• M.tuberculosis multidrug1 1981 0.6
• S.aureus (MRSA) methicillin 1990 2.2
• Enterococci vancomycin 1989 0.4
• S.pneumoniae penicillin 1989 0.3
• erythromycin 1989 3.3
• Klebsiella ceftazidime 1989 2.7
• ciprofloxacin 1989 2.9
• S.typhimurium multidrug2 1985 6.0
• ciprofloxacin 1994 1.0

Year 1995 1.2 1996 13.7 1995
10.8 1995 2.9 1995 10.9 1994 5.7 1994
6.5 1996 gt70 1996 12.0
1 INH, rifampicin, ethambutol 2
ampicillin, chloramphenicol, sulphonamides,
tetracycline
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ANTIBIOTIC RESISTANCE MECHANISMS

• Antibiotic inactivation
• Altered antibiotic target/Overproduction of
  target
• Reduced antibiotic accumulation
• Impaired uptake
• Enhanced efflux
• Bypass antibiotic-sensitive step

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ANTIBIOTIC RESISTANCE MECHANISMS
Inactivation ?-lactams Chloramphenicol Aminoglyc
osides
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ANTIBIOTIC RESISTANCE

• Intrinsic resistance
• Inherent features of the bacterial species which
  prevent antibiotic action
• Usually expressed by chromosomal genes
• i.e. Beta-lactamases of gram -ve bacteria
  inactivate beta-lactam antibiotics

• Acquired resistance
• Resistant strains emerge from previously
  sensitive bacterial populations
• Caused by mutations in chromosomal genes(the
  spontaneous mutation frequency is 10 -7) i.e.
  Nalidixic acid resistance in E.coli
• Or by acquisition of plasmids or transposons

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ACQUIRED RESISTANCE

• Resistance determined by plasmids
• Extrachromosomal genetic elements that replicate
  independently of the chromosome
• Resistance determined by transposons
• Mobile genetic elements capable of transferring
  (transposing) themselves from one DNA molecule to
  another
• Not capable of independent replication
• Repeats at end of transposon act as recognition
  sequences for transposition enzymes (transposases)

Acquired resistance is much more significant
clinically - why ?
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PLASMIDS

• Extrachromosomal genetic elements that replicate
  independently
• Plasmids are mobile by conjugation
• Frequently carry antibiotic resistance genes
• Up to 7 different resistance genes on one plasmid
• In the absence of antibiotic the plasmid is often
  lost from the majority of cells
• Cost of carrying a resistance gene
• Exposure to the antibiotic results in all cells
  having the plasmid
• Sensitive cells are killed and plasmid is
  mobilized

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PLASMID CONJUGATION
Chromosome AmpicillinR plasmid
StreptomycinR plasmid
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PLASMID MOBILIZATION

• Small non-conjugative plasmids also carry
  antibiotic-resistance genes and can be mobilized
  by a large conjugative plasmid

X
X
AmpicillinR congugative plasmid
StreptomycinR non-conjugative plasmid
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TRANSPOSONS

• Mobile DNA sequences which can transpose onto
  another DNA molecule
• Classified as Tn551, Tn4291 etc.
• Central region of transposon often carries an
  antibiotic-resistance gene(s)
• Results in the spread of antibiotic resistance

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RANGE OF RESISTANCE EXCHANGE
Amabile-Cuevas Chicurel . Bacterial plasmids
and gene flux. Cell 70 189-199 (1992)
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RESISTANCE TO SULPHONAMIDES

• Chromosomal-encoded
• Hyperproduction of PABA
• Mutation in dihydropteroate synthetase (DHPS)
  lowers affinity for sulphonamides

• Plasmid-encoded
• Duplication of DHPS enzyme
• Bind sulphonamides 10,000 fold less efficiently
• At least two types (I and II) of DHPS enzymes
  have been found which are only 50 homologous in
  sequence

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RESISTANCE TO QUINOLONES

• Only chromosomal mutations found

• gyrA mutations confer nalidixic acid resistance
  only
• N-terminal point mutations in DNA gyrase which
  reduce affinity of binding of quinolones

• gyrB mutations confer resistance to nalidixic
  acid and to ciprofloxacin
• Amino-acid substitutions which reduce affinity of
  binding

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RESISTANCE TO RIFAMPICIN

• Only chromosomal mutations found

• Altered DNA-dependent RNA polymerase
• Beta sub-unit does not bind rifampicin

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RESISTANCE TO AMINOGLYCOSIDES

• Enzymatic modification of the antibiotic
• Effect antibiotic uptake
• Plasmid/transposon-encoded enzymes
• Three classes of enzyme
• Acetyltransferases (AAC)
• Adenyltransferases (AAD)
• Phosphotransferases (APH)
• Enzymes divided into sub-types on the basis of
  the sites they modify in the antibiotics (gt30 in
  total)

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RESISTANCE TO CHLORAMPHENICOL

• Enzymatic inactivation
• Plasmid/transposon-encoded chloramphenicol acetyl
  transferases (cat)

• Impaired uptake
• Plasmid-encoded cml gene encodes a protein which
  reduces uptake of the antibiotic

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RESISTANCE TO TETRACYCLINE

• Plasmid/transposon-encoded
• Membrane proteins are encoded by the resistance
  genes
• Mechanism involves energy-dependent efflux
• Decreased accumulation of the antibiotic

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RESISTANCE TO BETA-LACTAMS

• Reductions in permeability
• Altered Porins in outer membrane effect
  permeability

• Alteration of the target
• Penicillin-binding-proteins involved in murein
  assembly

• Enzymatic inactivation
• Beta-lactamase enzymes (penicillinase,
  cephalosporinase)

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VANCOMYCIN RESISTANCE

• Vancomycin binds to Dal-D-Ala of murein
  precursors
• Inducible resistance mechanism - transposon
  encoded

• Vancomycin-resistant enterococci (VRE) sense
  presence of vancomycin and induce the synthesis
  of D-Ala-D-Lac, which are insensitive to
  vancomycin inhibition

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VANCOMYCIN RESISTANCE

• NAM NAG
• L-Ala
• D-Glu
• DAP
• D-Ala
• D-Lac

• VanA type transposons encode
• Dehydrogenase (VanH)
• Reduces pyruvate to lactate

• D,D-dipeptidase (VanX)
• Hydrolyses any D-Ala-D-Ala present

• D,D-carboxypeptidase (VanY)
• Removes the terminal D-Ala- D-Ala

Reviewed in Arthur et al., Trends in Microbiology
4, 401-407 (1996)
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BETA-LACTAM ANTIBIOTICS
Penicillins Cephalosporins penicillin G,
ampicillin cephalexin,
cofotaxime, cefuroxime
Cephamycins Carbapenems Monobactams cefox
itin imipenem aztreonam
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BETA-LACTAMASES

• Principal mechanism of resistance in clinical
  isolates
• Attack the beta-lactam ring to inactivate the
  antibiotic

?-lactamase
Inactive Penicillin derivative
b-lactamase Penicillin
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PENICILLINS

• Derivatives made by addition of precursors to the
  fermentation
• phenylacetic acid penicillin G
• phenoxyacetic acid penicillin V
• Active against Gram ves. Less active against
  Gram -ves
• Enzymic modification of penicillins yielded
  6-aminopenicillanic acid (6-APA)
• Derivatives are made from this molecule
• Some show resistance to b-lactamases probably
  due to the bulky R group protecting the b-lactam
  ring from cleavage

Penicillin G Penicillin V 6 APA Methicillin Cl
oxacillin Ampicillin
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CEPHALOSPORINS

• Cephalosporin C was first member of this group
• Enzymic modification resulted in
  7-aminocephalosporanic acid
• 7-ACA can be chemically acylated to give
  derivatives
• Many cephalosporins were resistant to the
  beta-lactamases which made penicillins
  inneffective
• Most cephalosporins are not acid-resistant, so
  have to be injected

Cephalosporin C 7 ACA Cephaloridine Cephalot
hin
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CLASSIFICATION OF BETA-LACTAMASES

• Matthew (1975) showed that ?-lactamase enzymes
  could be resolved on isoelectric-focussing gels
• Run crude bacterial cell extracts against
  standards

• Overlay with nitrocefin - a chromogenic
  cephalosporin. Observe pink bands

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CLASSIFICATION OF BETA-LACTAMASES II

• Matthew described 11 types of plasmid-encoded
  ?-lactamases
• Sub-divided into three classes
• BROAD-SPECTRUM PENICILLINASES Tem-1. Tem-2,
  Shv-1, Hms-1
• OXACILLINASES Oxa-1, Oxa-2, Oxa-3
• CARBENICILLINASES Pse-1, Pse-2, Pse-3, Pse-4
• Each have characteristic isoelectric point
• Antibody vs Tem-1 cross-reacts with Tem-2
• Antibody vs Pse-1 cross-reacts with Pse-4

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BETA-LACTAMASES AND RESISTANCE
?-lactamase MIC (?g/ml) Amp Cef Ctx Aztr
Imi Tem-1 4,000 16 0.125 0.125
0.5 Tem-2 16,000 16 0.125 0.25
0.5 Shv-1 8,000 16 0.125 0.25 1 Oxa-1
500 16 0.25 0.125 1 Oxa-6 32
16 0.125 0.25 1 Pse-1 2,000 16 0.125
0.125 1 Cep-1 1,000 500 8 32 1
Amp Ampicillin Cef Cefoxitin Ctx
Cefotaxime Aztr Aztreonam Imi Imipenem
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CLASSIFICATION OF ?-LACTAMASES IV

• Four classes of ?-lactamase by protein sequence
  homology
• Class A, C and D are serine-proteases - contain
  the Ser-X-X-Lys motif at the active site
• Sequence homology with
• a transpeptidase enzyme
• penicillin-binding proteins
• Class B require a catalytic zinc atom for
  activity
• The 3D structures of several ?-lactamases are now
  known i.e. Tem-1

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Tem-1 BETA-LACTAMASE

• Class A ?-lactamase enzyme
• Ser70 attacks the ?-lactam ring
• Lys73, Ser130 and Glu166 are also important
  residues
• Present on transposons Tn2 and Tn3
• Most common plasmid-encoded enzyme

Ser70
3-D structure of Tem-1 ? -lactamase
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EVOLUTION OF BETA-LACTAMASE ENZYMES
SOUGAKOFF et al., Rev. Infect. Dis.. 10, 879-884
(1988)

• Resistance of Gram -ve bacteria to 3rd
  generationcephalosporins observed in France and
  Germany
• Extended spectrum ?-lactamases were identified
  Ctx-1
• DNA sequencing of cloned gene revealed homology
  with Tem-1 and Tem-2

Gln39 Glu104 Gly238
Tem-1 Tem-2 Ctx-1
Lys39 Glu104 Gly238
Gln39 Lys104 Ser238
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WHY ARE THERE SO MANY ?-LACTAMASE ENZYMES

• The Glu104 and Gly238 residues in the
  Tem-1?-lactamase are locatednear the active
  site Ser70.
• Presumably the mutationsin the Tem-3 enzyme
  alter the substrate specificity of the enzyme
• The selection pressure of antibiotic use has led
  to the appearance of suchmutants
• Many more have now been identified

Glu104
Gly238
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NEW ?-LACTAMASES ARE STILL BEING FOUND

• Kinetic analysis of an inhibitor-resistant
  variant of the Ohio-1 ?-lactamase, an
  Shv-1-family class A enzymeLin, et al. Biochem
  J. 333, 395-400 (1998)
• Ctx-M-5, a novel cefotaxime-hydrolysing
  ?-lactamase from an outbreak of Salmonella
  typhimurium in LatviaBradford, et al. Antimicrob
  Agents Chemother. 2, 1980-1984 (1998)
• Cloning and sequencing of the gene encoding
  Toho-2, a class A ?-lactamase preferentially
  inhibited by tazobactamMa, et al. Antimicrob
  Agents Chemother. 2, 1181-1186 (1998)

75 Tem type ?-lactamases have now been
identified. See the WWW site http//www.lahey.org
/studies/webt.htm for further information.
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IMPACT OF ANTIBIOTIC RESISTANCE

• Acquired rather than intrinsic resistance poses
  the greatest threat to antibiotic therapy
• Many valuable antibiotic treatments are no longer
  available or under threat
• Sulphonamides for meningitis
• Ampicillin for H.influenzae infections
• Low dose penicillin for gonorrhoea
• Ampicillin for hospital-acquired coliform
  infections
• Many bacteria are resistant to almost all
  clinically useful antibiotics
• MRSA - Methicillin-resistant Staphylococcus
  aureus
• Vancomycin is often the only useful antibiotic
  left
• Vancomycin resistance has now been reported in
  enterococci

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STRATEGIES FOR COUNTERACTING RESISTANCE

• Develop new antibiotics
• Assisted by genome sequencing projects?

• Limit antibiotic use
• Save potent antibiotics for when they are needed
  ( who pays the pharmaceutical companies ?)
• Antibiotic rotation encourages loss of resistance

• Prevent cross-infection between hospital patients
• Requires epidemiological investigation of
  hospital-acquired infections