Drug Resistance inDrug Resistance inMycobacterium tuberculosis Mycobacterium tuberculosis ––

Fitness, Laboratory TestingFitness, Laboratory Testingand Beyondand Beyond

Erik C. Böttger

Universität ZürichInstitut für Medizinische MikrobiologieNationales Zentrum für MykobakterienSwitzerland

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• Drug resistance and clinical implications

• Drug resistance and fitness

Drug Resistance inMycobacterium tuberculosis

- genetic basis of resistance

- principles of mycobacterial drug susceptibility testing

- laboratory versus clinical resistance

- biological cost of drug resistance

- epidemiological aspects

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1. Why is standard short-course therapy reportedly an effectivetreatment for a significant part of multidrug resistant (MDR)tuberculosis?Böttger, E.C. Lancet 2001, 357: 1288-1289

2. Does DOTS (directly observed therapy short-course) work as ameans to control TB in populations with drug-resistanttuberculosis?”Even in settings with moderate rates of MDR tuberculosis, directly observedtherapy (DOTS) can rapidly reduce the transmission and incidence of both drug-susceptible and drug-resistant tuberculosis.“De Riemer, K. et al. Lancet 2005, 365: 1235-1245

Laboratory versus ClinicalResistance

“We found that many control programs that have been the most successful inreducing wild-type sensitive TB (as a result of high case detection and treatmentrates) have paradoxically been the most likely to evolve into hot zones of MDR.”Blower, S.M. and Chou, T. Nature Med. 2004, 10: 1111-1116

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1. Critical proportion(proportion method)

2. Critical concentration(drug concentration))

Pecularities of MycobacterialDrug Susceptibility Testing (DST)

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Principle of the method

"All strains of tuberculosis contain some bacilli that areresistant to antibacillary drugs - in resistant strains, theproportion of such bacilli is considerably higher than insensitive strains."

Canetti et al. Bull. WHO 1969, 41: 21-43

Drug Critical Proportionfor Resistance %

IsoniazidRifampicinPyrazinamidEthambutolStreptomycin

Concentration(µg/ml)

0.2410024

11101010

The Proportion Method

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Antimicrobialagent

Conc(µg/ml)

in serum

Conc (µg/ml)

INHRMPPZAEMBSM

0.05 - 0.20.520

1 - 51

71045

2 - 525 - 50

0.12

1002.52

0.4

7.56

mostly a single drug concentration (termed critical concentration) is used to define susceptibility vs. resistance

The Critical Concentration

MIC(µg/ml) of

susceptibleM. tuberculosis

BACTEC 12Blow

BACTEC 12Bhigh

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Wild Type Population

Proportion and CriticalConcentration

% P

opul

atio

n

Drug Concentration

Drug Critical ProportionConcentration for Resistance(mg/L) (% of population )

INH 0.2 1.0INH 1.0 0.1SM 4.0 10.0SM 8.0 0.1

Drug Critical ProportionConcentration for Resistance(mg/L) (% of population )

INH 0.2 1.0INH 1.0 0.1SM 4.0 10.0SM 8.0 0.1

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Resistance is defined as a decrease in sensitivity

of sufficient degree to be reasonably certain that

the strain concerned is different from a sample of

wild strains of human type that have never come

into contact with the drug.

Mitchison, 1962

Drug Resistance inM. tuberculosis

…, strains that are resistant in this sense do notnecessarily fail to respond.

1 5 10 50

wt population

Drug Concen-tration mg/L

% P

opul

atio

nhigh level resistance,various rpoB mutationse.g. rpoB S531L, H526Y, H526D

1 5 10 20

wt population

low-level resistance,various mutationse.g. Δ gldB

moderate resistancee.g. rrs, rpsL K87 mut

high level resistance,i.e. rpsL K42L

Drug Concen-tration mg/L

% P

opul

atio

n

0.1 1 3 10

wt population

low-level resistance,various mutationse.g. inhA

moderate and high level resistance,various katG mutationse.g. katG S315T

"critical concentrations"

Drug Concen-tration mg/Ldrug serum level

% P

opul

atio

n

C

RIFAMPICIN

STREPTOMYCIN

ISONIAZID

Schematized changes in drug susceptibility uponmutational alterations – exemplary Gaussiandistributions of a population’s drug susceptibility

Rifampicin: predominantly high-level drug resistanceassociated with mutations in rpoB.

Streptomycin: various levels of phenotypic resistance –low-, moderate-, and high-level drug resistance;different phenotypic resistance levels are associatedwith distinct chromosomal mutations.

Isoniazid: various levels of phenotypic resistance –low-, moderate-, and high-level drug resistance;different chromosomal mutations are associated withdistinct phenotypic resistance levels; in addition, agiven resistance mutation may be associated withvariable phenotypic expression of drug resistance.

= “critical concentration”= drug serum level

from Böttger, E.C. and Springer, B. 2009, Mycobacterium tuberculosis: drugresistance and genetic mechanisms – facts, artifacts and fallacies. In: HIVand tuberculosis: a deadly liaison, eds. S.H.E. Kaufmann and B. Walker,Wiley VCH, p. 103-121

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>30%tlyACapreomycin

embBeis

gldB

inhA

Geneticlocus

>50%20-30%

20-30%20-30%

Frequency inclinical isolates

EthambutolKanamycin

StreptomycinIsoniazid

Drug

Low-Level Drug Resistance inM. tuberculosis

With the possible exception ofRifampicin and Pyrazinamidlow-level resistance is asignificant part of drug resistantM. tuberculosis

Regardless of low-, moderate, or high-level drug resistance any correspondingclinical isolate is categorized as resistant in the diagnostic laboratory. However, thebiological impact of low- versus high-level drug resistance is to be different.• drug concentrations present in-vivo• tuberculosis chemotherapy is a combination therapy of compounds which

act in part in an additive / synergistic fashion and which includes agentstargeting cell wall synthesis

Banerjee et al. Science 1994, 263: 227 Johansen et al. Mol. Cell 2006, 23: 173Meier et al. Antimicrob. Agents Chemother. 1996, 40:2452 Zaunbrecher et al. Proc. Natl. Acad. Sci. USA 2009, 106: 20004Okamoto et al. Mol. Microbiol. 2007, 63: 1096 Telenti et al. Nat. Med. 1997, 3: 567

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1. Drug resistance is exclusively due to chromo-somal mutations and these occur in a stochasticfashion.

2. Drug resistance mutations found in clinicalstrains are a result of randomly occurringmutations and selection.

The Nature of Drug Resistance inMycobacterium tuberculosis

Are different phylogenetic lineages of M.tuberculosis associated with different resistancemutations and if – what are the explanations?

Are Different Phylogenetic Lineages Associated withDifferent Resistance Mutations?

RU2

USA1

Geogr.Area

29(10%)

3(1%)

287(89%)

319(100%)

21(22%)

20(21%)

51(55%)

92(100%)

nomut

mutinhA

mutkatG

Isoniazid resistantisolates (n)

absolute concentration method 1.0 mg/L

proportion method 0.1 mg/L, 0.4 mg/L

Laboratory Drug Susceptibility Testing

Isolates from United States of America: New York State1Somoski et al. J. Clin. Microbiol. 2006, 44: 4459-4463

Isolates from Russian Federation: Central region, Ural region, West Siberian region2Afamas‘ev et al. J. Antimicrob. Chemother. 2007, 59: 1057-1064

15(42%)

6(10%)

18(52%)

2(4%)

2(6%)

49(86%)

35(100%)

57(100%)

proportion testing < 0.4 mg/L

proportion testing > 0.4 mg/L

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“We consider that the best type of sensitivity test is a fully quantitativedetermination in which the organisms‘ capability of growth on mediumcontaining a wide range of drug concentrations is known. This type of testwould provide full information on the degree of resistance. However, sincesuch a test requires large amounts of medium and is time-consuming, itcannot be recommended as a routine procedure.“Canetti et al. Bull. WHO 1963, 29: 565-579

≥ 16.08.04.0≤ 2.0Ciprofloxacin

≥ 16.08.04.0≤ 2.0Ofloxacin

≥ 16.08.04.0≤ 2.0Streptomycin

≥ 10.05.02.5≤ 1.25Ethionamide

≥ 16.08.01.0-4.0≤ 0.5Rifampicin

≥ 4.02.00.2-1.0≤ 0.1Isoniazid

≥ 16.08.04.0≤ 2.0Ethambutol

ResistantModeratelyresistant

ModeratelysusceptibleSusceptibleDrug

Heifets, L. Am. Rev. Resp. Dis. 1988, 137: 1217-1222Tunis 2010 - 13

What are the implications of this phenotypic heterogeneity forclinical outcome?Predictive value of laboratory drug susceptibility testing

Mycobacterial Drug Resistance and Susceptibility Testing –Drug Resistance in M. tuberculosis is a Mixed Bag:

significant heterogeneity is present, i.e. low-,moderate- and high-level drug resistance

• Successful treatment outcome despite a resistant phenotype – as defined byroutine drug susceptibility testing – reflects limitations of the procedures usedto determine drug susceptibility

• Low-level drug resistance does not correspond to clinical resistance

• Treatment of high-level drug resistance with standardized therapies will contributeto the build-up of further resistance and facilitate the development of XDRtuberculosis

screen with critical concentration testing

implement semi-quantitative measures of drug susceptibility testingTunis 2010 - 14

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Antibiotic Resistance, FitnessCost and Compensatory Mutations

Schrag et al., Nature 1996; 381: 120Björkman et al., Proc. Natl. Acad. Sci. USA 1998; 95: 3949Nagaev et al., Mol. Microbiol. 2001; 40: 433

Drug resistance comes at a biological cost, e.g. reduced growth inthe absence of antibiotics. In-vitro, a resistance mutation associatedwith a fitness burden may be counteracted by secondary site,compensatory mutations ameliorating the fitness cost (frequency10-5 – 10-8).

Compensatory mutations have been suggested to maintain thespread of resistance in the absence of antibiotics.Björkman et al., Science 2000; 287: 1479Björkholm et al., Proc. Natl. Acad. Sci. USA 2001; 98: 14607Besier et al., Antimicrob. Agents Chemother. 2005; 49: 1426

What is the evidence for this scenario to take placein-vivo, e.g. in the infected patient or duringtransmission?

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What is the evidence …

• that transmission of resistant strains is dependent onthe type of genetic mutation leading to resistance?

• that compensatory evolution mitigates the fitnessdeficits associated with drug resistance?

• that the impact of a resistance mutation on bacterialfitness is greatly influenced by the strains’ geneticbackground?

Antibiotic Resistance, FitnessCost and Compensatory Mutations

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Use in-vitro experimental data on the genetics andcosts associated with a resistance determinant in theinterpretation of resistant clinical isolates in-vivo

• assess frequency of resistance mutations in clinicaldrug resistant isolates

• introduce mutations by genetic engineering into asuitable model system to generate isogenic mutants

• study mutants for drug susceptibility, generationtime and in fitness assays (in-vitro competitiongrowth experiments)

single rRNA allelic M. smegmatis

Experimental Procedure

-0.663216S rRNA 524G C

2%0.901616S rRNA 526C T

3%0.913216S rRNA 522C T

6%0.941616S rRNA 523A C

< 1%0.85> 128RpsL 42Lys Asp

< 1%0.86> 128RpsL 42Lys Thr

88%0.99> 128RpsL 42Lys Arg

-1.01.0wild-type

Frequency in clinicalSmR M. tuberculosis

isolatesFitnessMIC mg / LAlteration

Böttger et al., Nature Med. 1998; 12: 1343, Sander et al., Antimicrob. Agents Chemother. 2002; 46: 1204

Resistance mutations in rpsL aa position 88 and in rrs positions 501, 912 and 913 are not included;these mutations account for approximately 10% of clinical streptomycin resistant isolates in M.tb

Streptomycin Resistance Mutations inMycobacteria and Fitness

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Relationship between Frequency of StreptomycinResistance Mutations in Clinical M. tuberculosis Isolates

and Relative Fitness

The vast majority of drug resistance in M. tuberculosis is primary resistance, indicating activetransmission. Thus, resistance mutations in clinical strains reflect the outcome of natural infectionexperiments, which capture the complexity of the life cycle of M. tuberculosis and its epidemiology,including development of drug resistance, competition between different resistance mutations,transmission and infection.

Böttger et al. Nature Med. 1998, 12: 1343, Sander et al. Antimicrob. Agents Chemother. 2002, 46: 1204 Tunis 2010 - 19

Chemical structure of streptomycin, showinginteractions of the various groups with

specific residues of the ribosome

A view of the 30S ribosomal subunitshowing streptomycin in a space-fillingmodel, and the surrounding RNA and

protein elements

Carter et al., Nature 2000, 407: 340-348

Crystal structures provide an explanation why the RpsL 42 Argalteration is the only streptomycin resistance mutation which does not

carry a fitness cost RpsL aa 42 is involved in intraribosomal contacts which affect translation accuracy

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Primary Resistance versusAcquired Resistance

Primary resistance: infection with a resistant strain

Acquired resistance: infection with a drug susceptible strain withresistance developing under therapy

… Primary resistance is not a mere replication of acquired resistance. Other factors, such as altered virulence of the resistant strains, or instability inherent to certain types of resistance, may also be at work in producing the difference.

... If some of the strains with acquired resistance are incapable(through insufficient virulence) of producing new cases of

tuberculosis ... the relative frequency of resistance must necessarily be lower in primary than in acquired resistance.G. Canetti, Am. Rev. Respirat. Dis. 1965, 92: 687

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Primary Resistance and AcquiredResistance to Streptomycin

G. Canetti, Am. Rev. Respirat. Dis. 1965, 92: 687

Concentration of Streptomycin(µg/ml)

≥ 4 10 ≥ 100Total of Cases

% % %% # # ##

Primary resistanceAcquired resistance

163426

100100

94267

5863

34102

2124

3557

2113

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Primary Resistance and AcquiredResistance to Isoniazid

G. Canetti, Am. Rev. Respirat. Dis. 1965, 92: 687

Concentration of Isoniazid (µg/ml)

≥ 0.2 1 ≥ 10Total of Cases

% % %% # # ##

Primary resistanceAcquired resistance

98443

100100

49114

5026

3895

3921

11234

1153

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Multiple Chromosomal Alterations inkatG May Result in Isoniazid Resistance

> 32.0> 32.0

2.0 – 16.0

0.1 – 0.2

MIC mg/L

yes ayes a

> 1%> 1%

deletionframeshift

little b> 50%S 315 T

-100%wild-type

Fitness CostCatalase/Peroxidase ActivityAlteration (examples)

the low-cost katG S315T alteration is the most frequent isoniazid resistancemutation found in clinical strains (40 to 60%); this mutation confers an intermediatelevel of resistance

isoniazid resistant mutants of M. tuberculosis selected in-vitro do not reflect the in-vivo mechanism of isoniazid resistance; in the majority of in-vitro selected isoniazidresistant mutants katG was partially or completely deleted and the katG S315Talteration was present at <1% (0/120 mutants analyzed)c

a Li et al. J. Infect. Dis. 1998, 177: 1030b Pym et al. Infect. Immun. 2002, 70: 4955c Bergval et al. J. Antimicrob. Chemother. 2009, 64: 515

The rare presence of high cost resistance mutations in clinical isolates can best be explained by

It is under these circumstances that the scenario of costly resistance mutations ameliorated bycompensatory mutations may take place in-vivo.

i. stochastic probability of a resistance mutation under circumstances of a size limitedbacterial population

ii. bottleneck phenomena

n = 1691-716S rRNA alteration

21491G→U / 1409C→A

11491G→U

61409C→U

1601408A→G

View of the three-dimensional structure of the A-sitecomplexed with 4,5- and 4,6 disubstituted 2-deoxy-streptamines: the common neamine core is denoted inyellow; ring III of the 4,6 compounds is denoted in red;rings II and IV of the 4,5-compounds are denoted in blue.Key nucleotides of the binding site, which have beenaltered by site-directed mutagenesis to probe structuraldrug-target interactions, are given in bold.

1Sander et al. Mol. Microbiol. 1996, 22: 841-8482Alangaden et al. AAC 1998, 42: 1295-12973Suzuki et al. JCM 1998, 36: 1220-12254Krüüner et al. AAC 2003, 47: 2971-29735Maus et al. AAC 2005, 49: 3192-31976Feuerrigel et al. AAC 2009, 53: 3353-33567Jugheli et al. AAC 2009, 53: 5064-5068

Frequency of Resistance Mutations in Aminoglycoside Resistant Clinical M. tuberculosis

16S rRNA A-site

1408

1409 1491

A

GU UC

C G

AA

G C

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4,5 Aminoglycosides 4,6 Aminoglycosides

Paramomycin (R = OH)Neomycin (R = NH 3

+)Geneticin (R = OH)Gentamicin (R = NH 3

+)Neamine (R = NH 3

+)

4,5 Aminoglycosides 4,6 Aminoglycosides

Paramomycin (R = OH)Neomycin (R = NH 3

+)Geneticin (R = OH)Gentamicin (R = NH 3

+)Neamine (R = NH 3

+)

4,5-Aminoglycosides

4,5 Aminoglycosides 4,6 Aminoglycosides

Paramomycin (R = OH)Neomycin (R = NH 3

+)Geneticin (R = OH)Gentamicin (R = NH 3

+)Neamine (R = NH 3

+)

4,5 Aminoglycosides 4,6 Aminoglycosides

Paramomycin (R = OH)Neomycin (R = NH 3

+)Geneticin (R = OH)Gentamicin (R = NH 3

+)Neamine (R = NH 3

+)

4,5 Aminoglycosides 4,6 Aminoglycosides

Paramomycin (R = OH)Neomycin (R = NH 3

+)Geneticin (R = OH)Gentamicin (R = NH 3

+)Neamine (R = NH 3

+)

4,5 Aminoglycosides 4,6 Aminoglycosides

Paramomycin (R = OH)Neomycin (R = NH 3

+)Geneticin (R = OH)Gentamicin (R = NH 3

+)Neamine (R = NH 3

+)

4,5 Aminoglycosides 4,6 Aminoglycosides

Paramomycin (R = OH)Neomycin (R = NH 3

+)Geneticin (R = OH)Gentamicin (R = NH 3

+)Neamine (R = NH 3

+)

4,5 Aminoglycosides 4,6 Aminoglycosides

Paramomycin (R = OH)Neomycin (R = NH 3

+)Geneticin (R = OH)Gentamicin (R = NH 3

+)Neamine (R = NH 3

+)

4,6-Aminoglycosides

• bind to the decoding A-site of the small ribosomal subunit(helix 44 in bacterial 16S rRNA)

• affect ribosomal accuracy by interfering with decoding to resultin misreading

• inhibit translocation

2-deoxystreptamines

Aminoglycoside Antibiotics

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1.08.0-16.08.0-16.01.5 ± 2.93.1 ± 0.10C1409A/G1491U

0.51.01.04.5 ± 3.13.2 ± 0.06C1409U/G1491A

0.52.04.031.9 ± 1.93.8 ± 0.06C1409G/G1491C

1.0-2.08.0-16.04.0-8.09.3 ± 2.63.3 ± 0.05C1409U

8.016.0-32.02.050.4 ± 1.54.3 ± 0.06C1409G

16.0-32.032.0-64.032.0-64.011.7 ± 5.93.7 ± 0.06G1491U

2.0-4.016.0-32.016.0-32.067.1 ± 5.75.8 ± 0.15G1491C

0.51.0-2.02.0-4.045.5 ± 3.64.9 ± 0.12G1491A

>1024.0>1024.0>1024.05.0 ± 2.83.1 ± 0.06A1408G

0.50.5-1.00.5-1.0-3.0 ± 0.05ΔrrnB (parental)

AmikacinKanamycin AGentamycinCost per generation% ± SD

Generation timeh ± SDStrain

MIC (mg/L)

M. smegmatis strains constructed: generation time measurements, cost pergeneration and drug susceptibility

Results

Shcherbakov et al. Mol. Microbiol. 2010, 77: 830-840

Mutation Development and Compensatory Evolutionwt

A1408, C1409, G1491

1491C

RR: 32cpg: 67.1 ±5.7

1491A

RR: 2cpg: 45.5 ±3.6

1491U

RR: 64cpg: 11.7 ±5.9

1409U

RR: 16cpg: 9.3 ±2.6

1409G

RR: 32cpg: 50.4 ±1.3

1408G

RR: >1024cpg: 5.0 ±2.8

1491C/1409G

RR: 2 -4cpg: 31.9 ±1.9

1491A/1409U

RR: 1 -2cpg: 4.5 ±3.1

1491U/1409A

RR: 16cpg: 0.1 ±2.9

1409G/1491C

RR: 2 -4cpg: 31.9 ±1.9

1409U/1491A

RR: 1 -2cpg: 4.5 ±3.1

1

2

3

1408Ulethal

1408Clethal

1409Alethal

! rare clinicalmutation

! clinicallythe mostprevalentmutation

! clinicallyobserved rarecompensatorymutation

! rare clinicalmutation

1in green: genotypes observed in clinical drug resistant M. tuberculosis strains; in red:genotypes not found in clinical strains; in blue: lethal genotype2RR (relative resistance): MIC kanamycin mutant divided by MIC kanamycin wild type;green – observed in clinical strains, black – not observed in clinical strains, red –resistance too low (RR <4) to provide a selective advantage and thus not found inclinical strains3cpg (cost per generation): green – observed in clinical strains, black – not observed inclinical strains, red – too costly (>15% cpg) to be observed in clinical strains

View of the stacking of ring I on G1491(pairing to C1409 in thin lines) with the twoH-bonding contacts between ring I andA1408: O5′ of ring I and N6 of A1408, N6′ ofring I and position N1 of A1408. TheH-bonding contacts between ring I andA1408 are indicated by dark grey dots, thosebetween 1409 and 1491 by light grey lines.

Guanine

Adenine

[6]

[1]

[2]

[6]

[1]

OHOH

NH3+

H3N1‘

2‘ 3‘ 4‘

5‘ 6‘Ring I

C1409

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Drug Resistance, Fitnessand Transmission

Reconstructing the in-vivo evolution of drug resistance inM. tuberculosis – some general conclusions:

The presence of a dominant resistance mutation in clinical isolates(e.g. rpsL K42R, rrs A1408G) versus multiple different resistancemutations (e.g. pncA) presumably reflects different constraints onresistance alterations to maintain gene function.

1. The biological cost of resistance is a characteristic of both the drugtarget and the resistance mechanism involved.

2. The frequency of a resistance mutation in clinical isolates is a result ofits resistance level conferred and its fitness cost associated.

3. A high selection exists in clinical isolates for those resistancemutations which carry the least fitness cost.

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Drug Resistance, Fitnessand Transmission

4. The finding of a significant correlation between the frequency of aresistance mutation in genetically heterogeneous clinical isolates andthe fitness cost – as assessed by study of genetically constructedisogenic mutants – argues against the belief that the strain geneticbackground plays a major role in resistance-related fitness cost inM. tuberculosis. Rather, the fitness cost is a dominant characteristicof the specific resistance mutation itself.

Mutations become highly selected during resistance evolution andthus occur in unrelated M. tuberculosis strains from around the world.The observation that common resistance mutations can be found indifferent and unrelated populations reflects convergent evolution.

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Drug Resistance, Fitnessand Transmission

Considerations of fitness cost also have to address the periodavailable for transmission; i.e. any fitness deficit associated withresistance might easily be compensated for by longer infectivitydue to improper treatment and consequently extended timeperiods for transmission – explaining the emergence of MDRepidemics in competition with drug susceptible strains.

5. When a costly resistance mutation becomes dominant in a bacterialpopulation, compensatory secondary-site mutations amelioratingresistance-associated fitness cost may evolve and contribute tomutation fixation.

Acknowledgements

Grant Support:European Commission, Bundesamt für Gesundheit,

Swiss National Science Foundation, University of Zurich,Deutsche Forschungsgemeinschaft

D. AnderssonM. Pletschette

D. ScherbakovR. AkbergenovT. MattT. Janusic

B. SpringerP. Sander