Antibiotic resistance a mechanistic overview Neil Woodford · 2015-12-25 · Streptogramins Tetracyclines + tigecycline Courtesy of Ian Chopra Resistance is as old as antibiotics
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Antibiotic resistance a mechanistic overview
Neil Woodford
1The screen versions of these slides have full details of copyright and acknowledgements
Antibiotic Resistance a Mechanistic Overview
1
Neil Woodford BSc PhD FRCPathConsultant Clinical Scientist
Mechanisms of antibiotic action
Cytoplasmicmembrane PolypeptideTopo-
isomerase
DNA
PolymyxinColistinDaptomycin
Folic acid
MupirocinNitrofuransNitroimidazoles
Quinolones β-lactamsBacitracinCycloserineFosfomycinGlycopeptidesRamoplanin
Wall synthesis
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RibosomeRNAPolymerase
mRNA
Rifamycins
Folic acid synthesis
SulphonamidesTrimethoprim
AminoglycosidesChloramphenicolFusidic AcidKetolidesLincosamidesMacrolidesOxazolidinonesStreptograminsTetracyclines + tigecycline
Courtesy of Ian Chopra 2
Resistance is as old as antibiotics (not just human use of them)
Penicillin isolated in 1928
Resistant E. coli ‘discovered’ in 1940
…but antibiotics and bacteriahave co-existed for millions of years
3
Fleming
Antibiotic resistance a mechanistic overview
Neil Woodford
2The screen versions of these slides have full details of copyright and acknowledgements
Antibiotic resistance mechanisms
reduced uptake
antibiotics
antibiotics
4www.scq.ubc.ca
antibiotics
Types of resistance
Intrinsic (or inherent) resistanceResistance to an agent is normal for a genus, species or bacterial group (lack the target, or drug can’t get to target)• Glycopeptide resistance in Gram-negatives
• Aztreonam resistance in Gram positives
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• Aztreonam resistance in Gram-positives
Acquired resistanceMost isolates of a genus, species or bacterial group are susceptible, but resistance may arise via:• Mutation (usually of a chromosomal gene) e.g., Rif R; FQ R
• Acquisition of new DNA conferring resistance (horizontal spread)
Defining resistance
Cou
rtesy
: bio
Mer
ieux
6
Biological - “the inhibition zone is smaller (or MIC is higher) than normal for the species, so it’s resistant”
Pharmacological - “the MIC is 32 mg/L, but the drug has a serum peak of 150 mg/L, so it’s sensitive”
Clinical - “I know that strains like this don’t respond in the patient”
Antibiotic resistance a mechanistic overview
Neil Woodford
3The screen versions of these slides have full details of copyright and acknowledgements
Specific Pore (D2)
Non-Specific
Porin
Efflux Pump
VEntry + VEfflux
External [drug]
Periplasmic [drug]
Susceptibility / resistance of every bacterialisolate reflects interplay of multiple factors
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VHydrolysis
VBinding
Periplasmic [drug]
Antibiotics select resistant bacteria:mutational resistance
A mutant emerges
randomly
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Sensitive bacteria
killed by antibiotic
Mutant’s progeny
survive and growI have called this principle, by which each slight variation, if useful, is preserved, “Natural Selection”
Bacteria carry resistance in their DNA
Mutations in chromosomal DNA can cause resistance
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Many bacteria have extra DNA in small rings, known as plasmids
plasmids can also carry resistance
Antibiotic resistance a mechanistic overview
Neil Woodford
4The screen versions of these slides have full details of copyright and acknowledgements
…and they don’t keep resistance to themselves
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Three routes that bacteria useto transfer resistance
Most efficient between closely related bacteria
11http://bioinfo.bact.wisc.edu/themicrobialworld/homepage
related bacteria
…, but unrelated bacteria often exchange DNA too
The Red Queen Principle
Evolution is often an “arms race”
Antibiotic development vs. antibiotic resistance
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Bacteria evolve in “real-time”
“Now, here, you see, it takes all the running
you can do, to keep in the same place"
Van Valen L. A New Evolutionary Law. Evolutionary Theory 1973;1:1-30
Antibiotic resistance a mechanistic overview
Neil Woodford
5The screen versions of these slides have full details of copyright and acknowledgements
Resistance is inevitable… even to new antibiotics
Considered by all companies developing new agents
Search for cross-resistanceAssess activity vs. clinical strains resistant to other agents
? resistance reservoir if product is natural (or semi-synthetic)
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In-vitro development of resistance by mutation
Not reliable predictors; Ideal scenarios:Penicillin vs. Strep. pyogenes: no resistance
Vancomycin: resistance emerged after c. 30 years use
Will resistance to compound ‘X’ emerge:
Quickly, in target species,… and will it be transferable?
How quickly does resistance emerge?
Linezolid: a synthetic drugBacteria have never ‘seen’ anything like it
Excellent activity against almost all Gram-positive species
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Clinical use sets them a new challenge
O N
F
NO
O
NH
CH3
O
Oxazolidinone timeline
Class discovered
UK licensing of linezolid
Influenced by many factors, including:Use of agent (how much, by whom?)Cross-resistance to other antibioticsType of resistance
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Investigation restarted
1st LRE in UK
1985 1990 2000 20022001 2015
Will we have a major
resistance problem?
Antibiotic resistance a mechanistic overview
Neil Woodford
6The screen versions of these slides have full details of copyright and acknowledgements
Mechanism 1: alteration of the target Linezolid resistance and 23S rRNA
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Only G2576U in clinical isolates(MICs, 8-128 mg/L)
Prystowsky et al., AAC 2001;45:2145-56
The forensics of antibiotic resistance
Resistance involvesEmergence of mutations
Spread of resistance genes (plasmids, transposons, integrons)
Spread of resistant strains and clones of bacteria
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Spread of resistant strains and clones of bacteria
Tracking and characterizingThe resistant strains: in hospitals and in the community
Their resistance genes
Surveillance and good microbiology
Genes
Gene carriersIS, In, Tn, plasmids
Epidemiological investigation can be applied to every level
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Host speciesStrains, clones, phylogenetic
groups, virulence traits, co-resistance
PatientsHospital / community setting; risk factors
Courtesy of Rafael Canton
Antibiotic resistance a mechanistic overview
Neil Woodford
7The screen versions of these slides have full details of copyright and acknowledgements
Surveillance of resistance
Informs on prevalence and changes in antibiotic resistance
Guides empirical prescribing & control strategies
Assess if control is working
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Surveillance shortfallsLack of clinical denominators
Need more community based surveillance
Need to link antibiotic consumption to resistance
Must be supported by good microbiology (not just number crunching)
Similar trend, but is there a causal association?
A 3035404550
% MRSA bacteraemias
150
125E-15 (ST22)E-16 (ST36)
pita
ls
Rise of clones
20Data: HPA
% M
RSA
05
1015202530
91 '92 '93 '94 '95 '96 '97 '98 '99 '00 '01 '02 '03 '04Year
100
75
50
25
0
’93 ’94 ’95 ’96 ‘97’93 ’94 ’95 ’96 ‘97
E-3 (ST05)Num
ber o
f Hos
p
Year
Mechanism 2: metabolic by-passβ-lactam resistance in MRSA
New peptidoglycan Cross-linked wall
Methicillin inhibits PBPs 1,2,3
MRSA produce PBP2’, decreased binding, clinical resistancet t il bl
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to most availableβ-lactams
Ceftobiprole
Antibiotic resistance a mechanistic overview
Neil Woodford
8The screen versions of these slides have full details of copyright and acknowledgements
Cephalosporin-resistant E. colifrom bacteraemias
An explosive increase recorded since start of 21st century
In the UK, c. 20,000 cases E. coli bacteraemia p.a. (voluntary)
c. 12% CTX and/or CAZ resistance = c. 2400 cases p.a.
22http://www.earss.nivm.nl
20082001
…, but hang on a minute; Why might resistance rates rise?
Technical (artefacts)Change in surveillance methods (e.g., mandatory vs. voluntary)
Lowering of breakpoints (isolates previously S, now R)
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Education / awareness (more people look, and so find)
Better screening methods
Biological (real)Expansion of resistant clones / strains
Emergence of resistance in new clones / strains• De novo emergence (mutation)
• Horizontal spread of plasmids between strains
Understanding rising prevalence: SE England, 2004
400
500
600CTX-M Other ESBL AmpC Other
24
0
100
200
300
E. coli K. pneumoniae Enterobacter spp.
Antibiotic resistance a mechanistic overview
Neil Woodford
9The screen versions of these slides have full details of copyright and acknowledgements
Mechanism 3: drug destruction β-lactamases:
Oxyimino-aminothiazolyl or methoxy groups:
Evade classical penicillinases
Hinders access to active site
O
O
N
SNH2
O
CH3
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Hinders access to active site
No hydrolysis
ESBLs are able to hydrolyse β-lactam bond
Allow hydrolysis
Confer resistance
S
R
OOH
N
O
NHN
O
R
O
Global explosion of CTX-M ESBLsin Enterobacteriaceae
2001-2002
Endemic Spordic reports
2620052007
Courtesy of Rafael Canton
Multi-resistance plasmids: how bad is bad?
Antibiotic classes Genes Mechanism
Aminoglycosidesaac6’-Ib-cr
aadA5Modify drug
β-lactams
blaCTX-M-15
blaOXA-1 Destroy drug
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blaTEM-1
Chloramphenicol catB4 Modify drug
Macrolides mph(A) Efflux
Fluoroquinolones aac6’-Ib-cr Modify drug
Sulfonamides sulI By-pass
Trimethoprim dhfrXVII By-pass
Tetracycline tet(A) Efflux
pEK499 (118 kb) encodes CTX-M-15 ESBL in a prevalent UK strain of E. coli
Woodford, Carattoli et al., AAC
Antibiotic resistance a mechanistic overview
Neil Woodford
10The screen versions of these slides have full details of copyright and acknowledgements
E. coli & Klebsiella with ESBLs or AmpC: can it get worse?
Carbapenems
Standard i.v. therapy for ESBL / AmpC producers
400
500
600 ErtapenemImipenemMeropenem
28…which leads to an all-too-familiar situation
↑↑ use = ↑↑ selective pressure = ↑↑ resistant isolates
0
100
200
300
<=0.12 0.25 0.5 1 2 4 8
>=16
Mechanism 4: reduced uptakeporin-mediated carbapenem resistance
E. coli ‘strain A’ “usually” encodes 3 β-lactamases:
CTX-M-15; OXA-1; TEM-1
A1 A2 B C D E
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In 1 centre it has acquired an additional AmpC β-lactamase:
CMY-23
…and carbapenem resistanceisolate A2: ETP, 4 mg/L, IPM / MEM, 0.5-1 mg/L
OmpC loss
Restore porins and reverse carbapenem resistance
Strain PlasmidMIC (mg/L)
ETP IPM MEM
- 16 0.5 2
30
Klebsiella pTR 16 0.5 2
pTRompK36 0.5 0.25 0.125
Doumith et al., JAC 2009; 63: 659-67
Antibiotic resistance a mechanistic overview
Neil Woodford
11The screen versions of these slides have full details of copyright and acknowledgements
Carbapenemase-mediated resistance in the UK
40
50
60
Carbapenemase producers: ARMRL referrals
31HPA ARMRL, Unpublished data
0
10
20
30
40
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
Enterobacteriaceae Non-Fermenter
Another multi-resistant epidemic bacterial clone: A. baumannii, OXA-23 clone 1
Prevalent UK strain
First appeared in 2002
>50 UK t ff t d
OXA-23 clone 1 in UK (and Ireland)
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>50 UK centres affected
Multi-resistant lineage
Typically susceptible only to COL and TIG
Mechanism 5: up-regulated efflux Tigecycline resistance in A. baumannii:
adeB expression
0.946
0.70.80.9
1
ssio
n
33
0.003
0.131
00.10.20.30.40.50.6
Pre Rx Post Rx Lab mutant
Rel
ativ
e ex
pres
Adapted, Lomovskyaya et al., 2007
16 0.5 64TIG MIC:
Antibiotic resistance a mechanistic overview
Neil Woodford
12The screen versions of these slides have full details of copyright and acknowledgements
Recap: mechanisms of resistance
Target site modification / protection (mutation or enzymic)e.g., changes in a PBP, the ribosome, DNA gyrase
By-pass (acquired target unaffected by antibiotic)e.g., PBP2’; mupirocin HL resistance; trimethoprim resistance
Enzymic inactivation / modification of antibiotic
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Enzymic inactivation / modification of antibiotice.g., β-lactamases, aminoglycoside-modifying enzymes
Impermeability (porin loss)e.g., ETP R Enterobacteriaceae; OprD (D2) in Ps. aeruginosa
Active efflux e.g., tigecycline resistance in A. baumannii; non-specific (affects multiple drug classes); diverse pump types
Undefined mechanism of action (daptomycin) …
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= undefined resistance mechanisms
36Sabol et al., Antmicrob Agents Chemother 2005;49:1664-5
Antibiotic resistance a mechanistic overview
Neil Woodford
13The screen versions of these slides have full details of copyright and acknowledgements
Summary
Resistance is complexNot a new phenomenon; ‘discovered’ by us in last 70 years
New drug = new selective pressure = bacterial response; mechanisms are diverse
ESBLs (CTX-M types) in E. coli are a major new resistance problem for the 21st century
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Resistance associated with plasmids encoding multi-resistance
Potential to develop further resistance; mutation and other plasmids
Surveillance & microbiology to understand dominant & emerging resistances
Rational antibiotic usage needed to limit increasing resistance
Acknowledgements
Colleagues at the Health Protection Agency
Students and collaborators 1988-2009
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Students and collaborators, 1988 2009
Thank you for listening
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