Antibiotic resistance a mechanistic overview Neil Woodford · 2015-12-25 · Streptogramins Tetracyclines + tigecycline Courtesy of Ian Chopra Resistance is as old as antibiotics

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

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Fleming


Neil Woodford


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”


Neil Woodford


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


Neil Woodford


…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


Neil Woodford


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?


Neil Woodford


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


Neil Woodford


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


Neil Woodford


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

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0

100

200

300

E. coli K. pneumoniae Enterobacter spp.


Neil Woodford


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


Neil Woodford


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

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Klebsiella pTR 16 0.5 2

pTRompK36 0.5 0.25 0.125

Doumith et al., JAC 2009; 63: 659-67


Neil Woodford


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:


Neil Woodford


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


Neil Woodford


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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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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