:ילאיבורקימ יטנא לופיטל תוליבסו תודימע לע ?םישדח םילדומב ךרוצ שי םאה

Prof. Judith Berman, Shmunis School, Life Science, TAUIsrael Ministry of Health lecture/discussion series

On resistance and tolerance to antimicrobials: Do we need new models?

vFungal threats to human health

§ 1,000,000,000 skin infections

§ > 100,000,000 mucosal infections

§ > 10,000,000 allergies & asthmas

§ > 1,6M deaths per year

Aspergillus

Candida

Cryptococcus

The big 4

Pneumocystis

MRC Centre for Medical Mycology

Fungal threats to human health

Serious fungal infections are becoming more prevalent

Brown, G.D.et al. Science Translational Medicine.

Impact of fungal infections

Data from Brown et al. 2012 Science Transl. Med and WHO 2015.

MalariaTB

HIV/AIDSCryptococcosis

Inv. Cand.Inv. Asperg.Elizabeth Ballou, 2017. Nature Research Microbiology Community

Patie

nts

(# d

eath

s)

Perc

ent M

orta

lity

Pneumocystis

Clinically available antifungal drug classes

Mambro et al., 2019 Front. Pharmacol.

Azoles• Orally available• Few side effects• Fungistatic• Targets membrane

biosynthesis

Pyrimidine analogues• High frequency of

resistance• Used only in

combination with other antifungals

Polyenes• Intravenous

administration• High nephro-

toxicity• Binds ergostrol

Echinocandins• Intravenous

administration• Low toxicity• Inhibits cell wall

biosynthesis

expertise

Is antifungal drug resistance increasing?

Amir Arastehfar, et al. 2020. Antibiotics 2020, 9, 877; doi:10.3390/antibiotics9120877

• Increase in prevalence of non-albicans species

Is antifungal drug resistance increasing?

https://www.cdc.gov/fungal/candida-auris/tracking-c-auris.html

• Increase in non-albicans species• Emergence of new, intrinsically resistant & adaptable species

Resistance mechanisms directly affect drug-target interactions

• Horizontal/lateral transfer of resistance genes• Within and between

species

• NO Horizontal/lateral transfer• Resistance spreads vertically• Aerial spores or yeast cell

dispersal

Routes to transmit/spread resistance

How are fungal diseases transmitted?

• NO plasmid transmission

Image of Aspergillus fumigatus. From: Alunos Online

Environmental fungi: acquisition due to dispersal

• 30% of agricultural fungicides are azoles

• ~300X more azoles used in agriculture than in medicine

• <1% of infections with resistant isolates

• Therapies fail for ~10% of susceptible isolates

• Therapies fail for ~40% of resistant isolates

Is antifungal drug resistance increasing?

Has resistance been increasing?

• <1% of infections with resistant isolates

• Therapies fail for ~40% of resistant isolates

• Therapies fail for ~10% of susceptible isolates

• Far more infection failure with susceptible, than resistant isolates

CDC USA www.cdc.gov/fungal/index.html

Case

s of r

esist

ant C

andi

da in

fect

ions

in

hos

pita

lized

pat

ient

s

year

Some Candida cells can persist in the host, despite drug susceptibility

Fungal propertiesResistanceToleranceCell typeFungal burden

Adapted from Masiá Canuto & Gutiérrez 2002. The Lancet

Host factorsImmune statusNon-complianceforeign devicesabscesses

Drug propertiesAntifungal drug dosepharmacokinetics/dynamicsdrug/drug interactionsfungistatic vs fungicidal

Treatment failure

How are resistance and tolerance defined?

Clinical definition: The minimum inhibitory concentration (MIC) is above a clinical breakpoint (drug/species), such that therapeutic failure is more likely.

Research definition: The MIC of a resistant strain is higher than that of susceptible isolates (usually a control or reference strain)

Susceptible Resistant Disk diffusion assays

Resistance is concentration-dependent

Newly resistant isolates carry a heritable mutation

drug

No drug

Resistance is usually heritable and due to specific mutations

Pick and plate

drug

Susceptible cells do not grow at concentrations above the MIC

Susceptible

All resistant cells grow at concentrations above the MIC

Resistant

Susceptible

All cells exhibit similar drug-concentration-dependent response

• Perturb the drug – target interaction• Altered binding

• Drug inactivation or failure to activate a pro-drug

Susceptible Resistant

=• Perturb the drug - target interaction

• Reduce intracellular drug concentration • Reduced import• Increased export/efflux

Susceptible Resistant

Resistance mechanisms directly affect drug-target interactions

• High MIC after 24 hours of growth• Concentration-dependent• Genetic feature - similar in all cells

Defining Resistance: Minimal Inhibitory Concentration

Isochromosome (5L)

azole drug target ERG11 TAC1

Positive regulator of efflux pumps

Susceptible

Susceptible

ResistantSelmecki et al, 2016 & 2008

Anna Selmecki

Aneuploidy can give rise to antifungal drug resistance

Resistance is:--concentration dependent (MIC)

--due to mutations in target-drug interaction

--heritable for ~all progeny cells

Why do susceptible strains cause treatment failures?

Take home point #1

0 0.5 1 2 4 8 16 32 64 128

Resistant

Susceptible

MIC

CFU

/ml

00 0.5 1 2 4 8 16 32 64 128 256 Fluconazole µg/ml

106

104

102

100

Alex Rosenberg

0 0.5 1 2 4 8 16 32 64 128

Resistant

Tolerant

Susceptible

CFU

/ml

00 0.5 1 2 4 8 16 32 64 128 256 Fluconazole µg/ml

106

104

102

100

A subpopulation of cells (>1%) are tolerant to the drug

MIC

C. albicans FL: ‘Ignore lawn of microcolonies, read at 80% inhibition’

AB Biodisk, 2006

Susceptible Resistant Susceptible

Tolerance(trailing growth)

Measuring responses to antifungal azoles

MIC 0.125 µg/ml

Should we ignore tolerance? Can we quantify tolerance?

Resistance vs Tolerance at the single cell level

Resistant isolate Tolerant isolate

Is tolerance due to genetic adaptation?

RAD 18FoG 0.69

RAD 18.5FoG 0.65

Inside Outside

Parental

RAD 18.5FoG 0.63

Rosenberg et al, Nature Comm. 2018Genetic differences Phenotypic differences

Low High

Different isolates have different tolerance levels

Isogenic cells have different growth in drug

Genetic differences

Why are some strains more tolerant than others?

CFU

with

FLC

CFU

with

out d

rug

%

Tolerance (FoG)

%CFU on drug/total

Higher tolerance: larger subpopulation growing on drug

Take home point #2

Tolerance (trailing) is a function of:

--the proportion of cells that grow in drug

-- time for colonies to appear NOT correlated with MIC

Is tolerance clinically relevant?

Jules EneRichard Bennett

Does tolerance level have clinical relevance?

resistant

suscep

tible

nonpersistent

persistent

0.0

0.5

1.0

FoG20

***

resistant

susceptible

Non-persistent

persistent

FoG

Tolerance

Persistent candidemia is associated with higher initial levels of tolerance

Non-persistent isolates

Infection clearance

Persistent isolates

Persistent infection

Non-persistent isolates Tolerance levels

With Dr. A. Columbo, Sao Paulo Brazil

Rosenberg, Ene et al, Nature Comm. 2018, Berman and Kryzan, 2020. Nat. Reviews Micro.

FLC

MIC

(mg/

l) P=0.19

FLC

tole

ranc

e P=0.038

Does tolerance level have clinical relevance?• Retrospective study, 44 patients. • 41 were susceptible, mortality was 29.5% (30 d)4, 3.1% (12 wks)• High FoG was associated with mortality for patients treated with FLC

within <24 h of onset of candidemia

Death from candidemia is associated with tolerance (not resistance)Levinson, Dahan et al., 2020 Mycoses

Take home point #3

Higher tolerance is associated with --more infection persistence --higher mortality

…But more clinical studies are needed…

Rosenberg, Ene et al. 2018. Nat. Comm.

Fluoxetine

Rapamycin

Aureobasidin A

Fluphenazine

Geldanamycin

replica plateno drug

Disk25 µg FLC

RadicicolCyclosporine A

FK506

Adjuvant drugs + fluconazole

Several adjuvant drugs increase FLC cidality, not MIC

No adjuvant

Rosenberg, Ene et al. Nature Comm. 2018; Berman & Krysan, 2020 Nature Reviews Microbio.

Stress response pathways contribute to tolerance

Why do stress responses require function of all these pathways???

Testing an adjuvant drug + FLC in an animal modelStrain with low tolerance level

Adjuvant provides little benefit

Adjuvant provides large benefit

Strain with high tolerance level

Rosenberg, Ene et al. Nature Comm. 2018; Berman & Krysan, 2020 Nature Reviews Microbio.

Fluconazole + adjuvant may improve treatment outcomes BUT only for infections with high tolerance strains

Mechanisms of azole drug responses

Alter mediators of stress responses

(Hsp90, PKC, TOR)

Resistance

Robbins et al, 2017

Tolerance

Robbins et al, 2017

Susceptible ResistantSusceptible Resistant

Altered target Increased drug efflux

Mechanisms of azole drug responses

Altered targetAlter mediators of stress responses

(Hsp90, PKC, TOR)

Resistance

Robbins et al, 2017

Tolerance

Robbins et al, 2017

Susceptible ResistantSusceptible Resistant

Increased drug efflux

Tolerance

Altered stress response pathways (cAMP, Hsp90, PKC,

TOR…)

Stress response pathways mediate tolerance

Take home point #4

--Drugs that inhibit tolerance increase FLC cidality

--High tolerance strains à poorer patient outcomes

--Tolerance relies on stress response pathways

--Tolerance inhibitors reduce animal death

Measuring tolerance may be clinically relevant

Tel Aviv University*Alex Rosenberg*Maayan Bibi*Feng Yang*Aleeza GersteinSwati BijlaniNaomi Lyons*Esther WeindlingCristina AvilaAviva KatzVladimir GritsenkoAnton LevitanNaomi LyonsDTally MergenerChen BibiNir Even

TAU Chemistry*Micha Fridman*Raphael Benhamou

Brown University*Iuliana IneRichard Bennett

Federal University Sao Paulo*Arnaldo Columbo

A*STAR *Norman Pavelka*Flora Teoh*Alrina Tan

Acknowledgements