Title: ANTIBIOTIC RESISTANCE
1ANTIBIOTIC RESISTANCE
1940 1947 1956 1987
2INCREASES 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
3ANTIBIOTIC RESISTANCE MECHANISMS
- Antibiotic inactivation
- Altered antibiotic target/Overproduction of
target - Reduced antibiotic accumulation
- Impaired uptake
- Enhanced efflux
- Bypass antibiotic-sensitive step
4ANTIBIOTIC RESISTANCE MECHANISMS
Inactivation ?-lactams Chloramphenicol Aminoglyc
osides
5ANTIBIOTIC 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
6ACQUIRED 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 ?
7PLASMIDS
- 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
8PLASMID CONJUGATION
Chromosome AmpicillinR plasmid
StreptomycinR plasmid
9PLASMID 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
10TRANSPOSONS
- 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
11RANGE OF RESISTANCE EXCHANGE
Amabile-Cuevas Chicurel . Bacterial plasmids
and gene flux. Cell 70 189-199 (1992)
12RESISTANCE 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
13RESISTANCE 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
14RESISTANCE TO RIFAMPICIN
- Only chromosomal mutations found
- Altered DNA-dependent RNA polymerase
- Beta sub-unit does not bind rifampicin
15RESISTANCE 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)
16RESISTANCE 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
17RESISTANCE TO TETRACYCLINE
- Plasmid/transposon-encoded
- Membrane proteins are encoded by the resistance
genes - Mechanism involves energy-dependent efflux
- Decreased accumulation of the antibiotic
18RESISTANCE 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)
19VANCOMYCIN 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
20VANCOMYCIN 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)
21BETA-LACTAM ANTIBIOTICS
Penicillins Cephalosporins penicillin G,
ampicillin cephalexin,
cofotaxime, cefuroxime
Cephamycins Carbapenems Monobactams cefox
itin imipenem aztreonam
22BETA-LACTAMASES
- Principal mechanism of resistance in clinical
isolates - Attack the beta-lactam ring to inactivate the
antibiotic
?-lactamase
Inactive Penicillin derivative
b-lactamase Penicillin
23PENICILLINS
- 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
24CEPHALOSPORINS
- 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
25CLASSIFICATION 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
26CLASSIFICATION 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
27BETA-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
28CLASSIFICATION 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
29Tem-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
30EVOLUTION 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
31WHY 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
32NEW ?-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.
33IMPACT 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
34STRATEGIES 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