1022018

1

MSGERC Biennial Meeting Big Sky Montana

25-28 September 2018

David S Perlin PhDExecutive Director and Professor

Public Health Research Institute

Rutgers Biomedical and Health Sciences Newark New Jersey USA

Antifungal resistance among Candida spp

Disclosures

DSP receives funding from US National Institutes of Health and contracts from the CDC Astellas Scynexis Cidara and Amplyx He serves on advisory boards for Astellas Cidara Amplyx Scynexis and Matinas In addition DSP has an issued US patent concerning echinocandin resistance and patents for the detection antifungal drug resistance

1022018

2

Non-microbiological Unfavorable PK Poor drug penetration Co-morbidities Host reservoirs

bull GI tract intra-abdominal abscesses

Microbiological Drug resistancebull Inherentbull Acquired

Clinical response is a blend of drug host and microbial factors

Why Patients Fail Antimicrobial Therapy

Resistance occurs with all antifungal agents but

resistance to triazole and echinocandin drugs is the

most clinically significant and strains resistant to

multiple drug classes have emerged

What are the current trends and underlying genetic mechanisms that confer acquired drug resistance

ANTIFUNGAL DRUG RESISTANCE

1022018

3

Castanheira et al 2017 AAC

Global view of echinocandin and azole susceptibility (ECVs) among

Candida

2809 Candida spp isolates collected in 29 countries worldwide in 2014 and 2015

Susceptibility remains high among most Candida species

Antifungal activity using CLSI reference method against invasive

yeasts collected in 29 countries worldwide in 2014 and 2015

Castanheira et al 2017 AACResistance among Candida species is low

1022018

4

Int J Antimicrob Agents 2011 Jul38(1)65-9 Candida bloodstream infections comparison of species distribution and resistance to echinocandin and azole antifungal agents in Intensive Care Unit (ICU) and non-ICU settings in the SENTRY Antimicrobial Surveillance Program (2008-2009)Pfaller MA1 Messer SA Moet GJ Jones RN Castanheira M

AbstractMinimum inhibitory concentration (MIC) data from the SENTRY Antimicrobial Surveillance Program generated by reference methodswere analysed to compare the antifungal resistance profiles and species distribution of Candida bloodstream infection (BSI) isolates obtained from patients in the Intensive Care Unit (ICU) and those from non-ICU locations Results from

79 medical centres between 2008 and 2009 were tabulated MIC values were obtained for anidulafungin

caspofungin micafungin fluconazole posaconazole and voriconazole Recently revised Clinical and Laboratory Standards Institute

breakpoints for resistance were employed A total of 1752 isolates of Candida spp were obtained from ICU (779 445) and non-ICU (973 555) settings The frequency of ICU-associated Candida BSI was

higher in Latin America (565) compared with Europe (444) and North America (396) The frequency of candidaemia in the ICU decreased both in Latin America and North America over the 2-year study period Approximately 96 of isolates both in ICU and non-ICU settings were caused by only five species (Candida albicans Candida glabrata Candida parapsilosis Candida tropicalis and Candida

krusei) Resistance both to azoles and echinocandins was uncommon in ICU and non-ICU settings Overall fluconazole resistance was detected in 50 of ICU isolates and 44 of non-ICU isolates Candida glabrata was the only species in which resistance to azoles and echinocandins was noted and this multidrug-resistant phenotype was found in both settings In conclusion the

findings from this global survey indicate that invasive candidiasis can no longer be considered to be just an ICU-related infection and efforts to design preventive and diagnostic strategies must be expanded to include other at-risk populations and hospital environments Concern regarding C glabrata must now include resistance to echinocandins as well as azole antifungal agents

Prevalence comparable to that reported by SENTRY Antimicrobial Surveillance Program for 2008-09

Antimicrob Agents Chemother 2018 May 2562(6)

Absence of Azole or Echinocandin Resistance in Candida glabrata Isolates in India despite Background Prevalence of Strains with Defects in the DNA Mismatch Repair PathwaySingh A1 Healey KR2 Yadav P1 Upadhyaya G1 Sachdeva N3 Sarma S4 Kumar A5 Tarai B6 Perlin DS2 Chowdhary A7

AbstractCandida glabrata infections are increasing worldwide and exhibit greater rates of antifungal resistance than those with other species DNA mismatch repair (MMR) gene deletions such as msh2Δ in C glabrata resulting in a mutator phenotype have recently been

reported to facilitate rapid acquisition of antifungal resistance This study determined the antifungal susceptibility profiles of 210 C glabrata isolates in 10 hospitals in India and

investigated the impact of novel MSH2 polymorphisms on mutation potential No echinocandin- or azole-resistant strains and no mutations in FKS hot spot regions were detected among the C glabrata isolates supporting our in vitro susceptibility testing results CLSI antifungal susceptibility data showed that the MICs of anidulafungin (geometric mean [GM] 012 μgml) and

micafungin (GM 001 μgml) were lower and below the susceptibility breakpoint compared to that of caspofungin (CAS) (GM 131 μgml) Interestingly 69 of the C glabrata strains sequenced contained six nonsynonymous mutations in MSH2 ie V239L and the novel mutations E459K R847C Q386K T772S and V239D946E Functional analysis of MSH2 mutations revealed that 49 of the tested strains (4081) contained a partial loss-of-function MSH2 mutation The novel MSH2 substitution Q386K produced higher frequencies of CAS-resistant colonies upon expression in the msh2Δ mutant However expression of two other novel MSH2 alleles ie E459K or R847C did not confer selection of resistant colonies confirming that not all mutations in the MSH2 MMR pathway affect its function or generate a phenotype of resistance to antifungal drugs The lack of drug resistance prevented any correlations from being drawn with respect to MSH2 genotype

All C glabrata isolates were fully susceptiblemdashno drug resistance detected

INDIA

1022018

2

Non-microbiological Unfavorable PK Poor drug penetration Co-morbidities Host reservoirs

bull GI tract intra-abdominal abscesses

Microbiological Drug resistancebull Inherentbull Acquired

Clinical response is a blend of drug host and microbial factors

Why Patients Fail Antimicrobial Therapy

Resistance occurs with all antifungal agents but

resistance to triazole and echinocandin drugs is the

most clinically significant and strains resistant to

multiple drug classes have emerged

What are the current trends and underlying genetic mechanisms that confer acquired drug resistance

ANTIFUNGAL DRUG RESISTANCE

1022018

3

Castanheira et al 2017 AAC

Global view of echinocandin and azole susceptibility (ECVs) among

Candida

2809 Candida spp isolates collected in 29 countries worldwide in 2014 and 2015

Susceptibility remains high among most Candida species

Antifungal activity using CLSI reference method against invasive

yeasts collected in 29 countries worldwide in 2014 and 2015

Castanheira et al 2017 AACResistance among Candida species is low

1022018

4

Int J Antimicrob Agents 2011 Jul38(1)65-9 Candida bloodstream infections comparison of species distribution and resistance to echinocandin and azole antifungal agents in Intensive Care Unit (ICU) and non-ICU settings in the SENTRY Antimicrobial Surveillance Program (2008-2009)Pfaller MA1 Messer SA Moet GJ Jones RN Castanheira M

AbstractMinimum inhibitory concentration (MIC) data from the SENTRY Antimicrobial Surveillance Program generated by reference methodswere analysed to compare the antifungal resistance profiles and species distribution of Candida bloodstream infection (BSI) isolates obtained from patients in the Intensive Care Unit (ICU) and those from non-ICU locations Results from

79 medical centres between 2008 and 2009 were tabulated MIC values were obtained for anidulafungin

caspofungin micafungin fluconazole posaconazole and voriconazole Recently revised Clinical and Laboratory Standards Institute

breakpoints for resistance were employed A total of 1752 isolates of Candida spp were obtained from ICU (779 445) and non-ICU (973 555) settings The frequency of ICU-associated Candida BSI was

higher in Latin America (565) compared with Europe (444) and North America (396) The frequency of candidaemia in the ICU decreased both in Latin America and North America over the 2-year study period Approximately 96 of isolates both in ICU and non-ICU settings were caused by only five species (Candida albicans Candida glabrata Candida parapsilosis Candida tropicalis and Candida

krusei) Resistance both to azoles and echinocandins was uncommon in ICU and non-ICU settings Overall fluconazole resistance was detected in 50 of ICU isolates and 44 of non-ICU isolates Candida glabrata was the only species in which resistance to azoles and echinocandins was noted and this multidrug-resistant phenotype was found in both settings In conclusion the

findings from this global survey indicate that invasive candidiasis can no longer be considered to be just an ICU-related infection and efforts to design preventive and diagnostic strategies must be expanded to include other at-risk populations and hospital environments Concern regarding C glabrata must now include resistance to echinocandins as well as azole antifungal agents

Prevalence comparable to that reported by SENTRY Antimicrobial Surveillance Program for 2008-09

Antimicrob Agents Chemother 2018 May 2562(6)

Absence of Azole or Echinocandin Resistance in Candida glabrata Isolates in India despite Background Prevalence of Strains with Defects in the DNA Mismatch Repair PathwaySingh A1 Healey KR2 Yadav P1 Upadhyaya G1 Sachdeva N3 Sarma S4 Kumar A5 Tarai B6 Perlin DS2 Chowdhary A7

AbstractCandida glabrata infections are increasing worldwide and exhibit greater rates of antifungal resistance than those with other species DNA mismatch repair (MMR) gene deletions such as msh2Δ in C glabrata resulting in a mutator phenotype have recently been

reported to facilitate rapid acquisition of antifungal resistance This study determined the antifungal susceptibility profiles of 210 C glabrata isolates in 10 hospitals in India and

investigated the impact of novel MSH2 polymorphisms on mutation potential No echinocandin- or azole-resistant strains and no mutations in FKS hot spot regions were detected among the C glabrata isolates supporting our in vitro susceptibility testing results CLSI antifungal susceptibility data showed that the MICs of anidulafungin (geometric mean [GM] 012 μgml) and

micafungin (GM 001 μgml) were lower and below the susceptibility breakpoint compared to that of caspofungin (CAS) (GM 131 μgml) Interestingly 69 of the C glabrata strains sequenced contained six nonsynonymous mutations in MSH2 ie V239L and the novel mutations E459K R847C Q386K T772S and V239D946E Functional analysis of MSH2 mutations revealed that 49 of the tested strains (4081) contained a partial loss-of-function MSH2 mutation The novel MSH2 substitution Q386K produced higher frequencies of CAS-resistant colonies upon expression in the msh2Δ mutant However expression of two other novel MSH2 alleles ie E459K or R847C did not confer selection of resistant colonies confirming that not all mutations in the MSH2 MMR pathway affect its function or generate a phenotype of resistance to antifungal drugs The lack of drug resistance prevented any correlations from being drawn with respect to MSH2 genotype

All C glabrata isolates were fully susceptiblemdashno drug resistance detected

INDIA

1022018

3

Castanheira et al 2017 AAC

Global view of echinocandin and azole susceptibility (ECVs) among

Candida

2809 Candida spp isolates collected in 29 countries worldwide in 2014 and 2015

Susceptibility remains high among most Candida species

Antifungal activity using CLSI reference method against invasive

yeasts collected in 29 countries worldwide in 2014 and 2015

Castanheira et al 2017 AACResistance among Candida species is low

1022018

4

Int J Antimicrob Agents 2011 Jul38(1)65-9 Candida bloodstream infections comparison of species distribution and resistance to echinocandin and azole antifungal agents in Intensive Care Unit (ICU) and non-ICU settings in the SENTRY Antimicrobial Surveillance Program (2008-2009)Pfaller MA1 Messer SA Moet GJ Jones RN Castanheira M

AbstractMinimum inhibitory concentration (MIC) data from the SENTRY Antimicrobial Surveillance Program generated by reference methodswere analysed to compare the antifungal resistance profiles and species distribution of Candida bloodstream infection (BSI) isolates obtained from patients in the Intensive Care Unit (ICU) and those from non-ICU locations Results from

79 medical centres between 2008 and 2009 were tabulated MIC values were obtained for anidulafungin

caspofungin micafungin fluconazole posaconazole and voriconazole Recently revised Clinical and Laboratory Standards Institute

breakpoints for resistance were employed A total of 1752 isolates of Candida spp were obtained from ICU (779 445) and non-ICU (973 555) settings The frequency of ICU-associated Candida BSI was

higher in Latin America (565) compared with Europe (444) and North America (396) The frequency of candidaemia in the ICU decreased both in Latin America and North America over the 2-year study period Approximately 96 of isolates both in ICU and non-ICU settings were caused by only five species (Candida albicans Candida glabrata Candida parapsilosis Candida tropicalis and Candida

krusei) Resistance both to azoles and echinocandins was uncommon in ICU and non-ICU settings Overall fluconazole resistance was detected in 50 of ICU isolates and 44 of non-ICU isolates Candida glabrata was the only species in which resistance to azoles and echinocandins was noted and this multidrug-resistant phenotype was found in both settings In conclusion the

findings from this global survey indicate that invasive candidiasis can no longer be considered to be just an ICU-related infection and efforts to design preventive and diagnostic strategies must be expanded to include other at-risk populations and hospital environments Concern regarding C glabrata must now include resistance to echinocandins as well as azole antifungal agents

Prevalence comparable to that reported by SENTRY Antimicrobial Surveillance Program for 2008-09

Antimicrob Agents Chemother 2018 May 2562(6)

Absence of Azole or Echinocandin Resistance in Candida glabrata Isolates in India despite Background Prevalence of Strains with Defects in the DNA Mismatch Repair PathwaySingh A1 Healey KR2 Yadav P1 Upadhyaya G1 Sachdeva N3 Sarma S4 Kumar A5 Tarai B6 Perlin DS2 Chowdhary A7

AbstractCandida glabrata infections are increasing worldwide and exhibit greater rates of antifungal resistance than those with other species DNA mismatch repair (MMR) gene deletions such as msh2Δ in C glabrata resulting in a mutator phenotype have recently been

reported to facilitate rapid acquisition of antifungal resistance This study determined the antifungal susceptibility profiles of 210 C glabrata isolates in 10 hospitals in India and

investigated the impact of novel MSH2 polymorphisms on mutation potential No echinocandin- or azole-resistant strains and no mutations in FKS hot spot regions were detected among the C glabrata isolates supporting our in vitro susceptibility testing results CLSI antifungal susceptibility data showed that the MICs of anidulafungin (geometric mean [GM] 012 μgml) and

micafungin (GM 001 μgml) were lower and below the susceptibility breakpoint compared to that of caspofungin (CAS) (GM 131 μgml) Interestingly 69 of the C glabrata strains sequenced contained six nonsynonymous mutations in MSH2 ie V239L and the novel mutations E459K R847C Q386K T772S and V239D946E Functional analysis of MSH2 mutations revealed that 49 of the tested strains (4081) contained a partial loss-of-function MSH2 mutation The novel MSH2 substitution Q386K produced higher frequencies of CAS-resistant colonies upon expression in the msh2Δ mutant However expression of two other novel MSH2 alleles ie E459K or R847C did not confer selection of resistant colonies confirming that not all mutations in the MSH2 MMR pathway affect its function or generate a phenotype of resistance to antifungal drugs The lack of drug resistance prevented any correlations from being drawn with respect to MSH2 genotype

All C glabrata isolates were fully susceptiblemdashno drug resistance detected

INDIA

1022018

4

Int J Antimicrob Agents 2011 Jul38(1)65-9 Candida bloodstream infections comparison of species distribution and resistance to echinocandin and azole antifungal agents in Intensive Care Unit (ICU) and non-ICU settings in the SENTRY Antimicrobial Surveillance Program (2008-2009)Pfaller MA1 Messer SA Moet GJ Jones RN Castanheira M

AbstractMinimum inhibitory concentration (MIC) data from the SENTRY Antimicrobial Surveillance Program generated by reference methodswere analysed to compare the antifungal resistance profiles and species distribution of Candida bloodstream infection (BSI) isolates obtained from patients in the Intensive Care Unit (ICU) and those from non-ICU locations Results from

79 medical centres between 2008 and 2009 were tabulated MIC values were obtained for anidulafungin

caspofungin micafungin fluconazole posaconazole and voriconazole Recently revised Clinical and Laboratory Standards Institute

breakpoints for resistance were employed A total of 1752 isolates of Candida spp were obtained from ICU (779 445) and non-ICU (973 555) settings The frequency of ICU-associated Candida BSI was

higher in Latin America (565) compared with Europe (444) and North America (396) The frequency of candidaemia in the ICU decreased both in Latin America and North America over the 2-year study period Approximately 96 of isolates both in ICU and non-ICU settings were caused by only five species (Candida albicans Candida glabrata Candida parapsilosis Candida tropicalis and Candida

krusei) Resistance both to azoles and echinocandins was uncommon in ICU and non-ICU settings Overall fluconazole resistance was detected in 50 of ICU isolates and 44 of non-ICU isolates Candida glabrata was the only species in which resistance to azoles and echinocandins was noted and this multidrug-resistant phenotype was found in both settings In conclusion the

findings from this global survey indicate that invasive candidiasis can no longer be considered to be just an ICU-related infection and efforts to design preventive and diagnostic strategies must be expanded to include other at-risk populations and hospital environments Concern regarding C glabrata must now include resistance to echinocandins as well as azole antifungal agents

Prevalence comparable to that reported by SENTRY Antimicrobial Surveillance Program for 2008-09

Antimicrob Agents Chemother 2018 May 2562(6)

Absence of Azole or Echinocandin Resistance in Candida glabrata Isolates in India despite Background Prevalence of Strains with Defects in the DNA Mismatch Repair PathwaySingh A1 Healey KR2 Yadav P1 Upadhyaya G1 Sachdeva N3 Sarma S4 Kumar A5 Tarai B6 Perlin DS2 Chowdhary A7

AbstractCandida glabrata infections are increasing worldwide and exhibit greater rates of antifungal resistance than those with other species DNA mismatch repair (MMR) gene deletions such as msh2Δ in C glabrata resulting in a mutator phenotype have recently been

reported to facilitate rapid acquisition of antifungal resistance This study determined the antifungal susceptibility profiles of 210 C glabrata isolates in 10 hospitals in India and

investigated the impact of novel MSH2 polymorphisms on mutation potential No echinocandin- or azole-resistant strains and no mutations in FKS hot spot regions were detected among the C glabrata isolates supporting our in vitro susceptibility testing results CLSI antifungal susceptibility data showed that the MICs of anidulafungin (geometric mean [GM] 012 μgml) and

micafungin (GM 001 μgml) were lower and below the susceptibility breakpoint compared to that of caspofungin (CAS) (GM 131 μgml) Interestingly 69 of the C glabrata strains sequenced contained six nonsynonymous mutations in MSH2 ie V239L and the novel mutations E459K R847C Q386K T772S and V239D946E Functional analysis of MSH2 mutations revealed that 49 of the tested strains (4081) contained a partial loss-of-function MSH2 mutation The novel MSH2 substitution Q386K produced higher frequencies of CAS-resistant colonies upon expression in the msh2Δ mutant However expression of two other novel MSH2 alleles ie E459K or R847C did not confer selection of resistant colonies confirming that not all mutations in the MSH2 MMR pathway affect its function or generate a phenotype of resistance to antifungal drugs The lack of drug resistance prevented any correlations from being drawn with respect to MSH2 genotype

All C glabrata isolates were fully susceptiblemdashno drug resistance detected

INDIA

1022018

5

Trends of fluconazole (A) and voriconazole (B) susceptibility and resistance rates of

Candida species determined through the CHIF-NET study (2010 to 2014)

Meng Xiao et al J Clin Microbiol 201856e00577-18

Sixty-five tertiary hospitals in China collected 8829 Candida isolates from August 2009 to July 2014

But not in China where significant azole resistance detected among certain Candida spp

CHINA

J Clin Microbiol 2018 Mar 2656(4)

Update from a 12-Year Nationwide Fungemia Surveillance Increasing Intrinsic and Acquired Resistance Causes ConcernAstvad KMT Johansen HK Roslashder BL Rosenvinge FS Knudsen JD Lemming L Schoslashnheyder HC Hare RK Kristensen L Nielsen L Gertsen JB Dzajic E Pedersen M Oslashstergaringrd C Olesen B Soslashndergaard TS Arendrup MC

Abstract

New data from the years 2012 to 2015 from the Danish National FungemiaSurveillance are reported and epidemiological trends are investigated in a 12-year perspective (2004 to 2015) During 2012 to 2015 1900 of 1939 (98) fungal bloodstream isolates were included The average incidence was 84100000 inhabitants and this appears to represent a

stabilizing trend after the increase to 101100000 in 2011 The incidence was higher in males than females (100 versus 68) and in patients above 50 years and those changes were mainly driven by an increasing incidence among 80-to-89-year-old males (653100000 in 2014 to 2015) The proportion of Candida albicans isolates decreased from 2004 to 2015 (644 to 424) in parallel with a doubling of the proportion of Candida glabrata isolates (165 to 346 P lt 00001) C glabrata was more common among females (340 versus 304 in males) Following an increase in 2004 to 2011 the annual drug use stabilized during the last 2 to 3 years of that time period but remained higher than in other Nordic countries This was particularly true for the fluconazole and itraconazole use in the primary health care sector which exceeded the combined national levels of use of these compounds in each of the other Nordic countries

Fluconazole susceptibility decreased (685 652 and 606 in 2004 to 2007 2008 to 2011 and 2012 to 2015 respectively P lt 00001) and echinocandin resistance emerged in Candida (0 06 and 17 respectively P lt 0001) Amphotericin B susceptibility remained high (987) Among 16 (27) echinocandin-resistant C glabrata isolates (2012 to 2015) 13 harbored FKS mutations and 5 (31) were multidrug resistant The epidemiological changes and the increased incidence of intrinsic and acquired resistance emphasize the

importance of continued surveillance and of strengthened focus on antifungal stewardship

In Denmark resistance is a growing problem especially among C glabrata

DENMARK

1022018

6

0

2

4

6

8

10

12

14

Resistance rate varies among different studies The rate reported from institutional studies are higher than that from population-based surveys

Echinocandin resistance in C glabrata

Perlin Shor and Zhao 2015 CCMR

Geographical variations in the distribution of

non-C albicans Candida species

Pappas P G et al (2018) Invasive candidiasis

Nat Rev Dis PrimersHigher prevalence correlates with greater resistance

Data presented are from Australia Brazil Canada Denmark France Japan and the United States6200ndash205

Data on C auris are from the Centers for Disease Control and Prevention (last accessed 27 March 2018)

1022018

7

Proportion of the five major Candida species responsible for fungemia in patients with (159) or without (2289) prior exposure to FLZ or with (61) or without (2387) prior exposure to CSF

Ca

Ca

Ca

Cg

Cg

Cg

Prior azole and echinocandin exposure is shifting the distribution of Candida spp recovered from severely ill patients with C glabrata as a major cause of disease

Lortholary et al 2011 AAC

Hachem et al 2008 Cancer

C glabrata has emerged as the dominant pathogen in certain immunocompromised patient populations

1022018

8

0

5

10

15

20

25

30

2001-2002 2003-2004 2005-2006 2007-2008 2009-2010

Res

ista

nce

()

Time Period

Anidulafungin

Caspofungin

Micafungin

Fluconazole

Azoles

Echinocandins

MDR

Concomitant rise in azole and echinocandin resistance among Candida glabrata isolates with emergence of multidrug (MDR) resistance

C glabratandashpositive blood cultures at MD Anderson Cancer Center Houston TX during March 2005ndashSeptember 2013

Farmakiotis et al EID 2014

146 Total Isolatesbull 205 FLUr

bull 103 CSFr

bull 68 MDR (CSF FLU AMB)

MDR increasingly more common

MDR

Resistance to echinocandins azoles and polyenes

1022018

9

Global and regional maps depicting rapid emergence of multidrug-resistant clinical Candida auris strains

httpswwwcdcgovfungalcandida-auristracking-c-aurishtml

Most of the isolates were resistant tobull Fluconazole (n ge 318 4429) bull Voriconazole (VRZ) (n ge 91 1267) bull Itraconazole (ITZ) (n ge 13 181)bull Isavuconazole (ISA) (n ge 11 153)bull Posaconazole (PSZ) (n ge 10 139)

bull AMB (n ge 111 1546)bull Flucytosine (FCN) (n ge 14 195)

bull Caspofungin (n ge 25 348) bull Anidulafungin (n ge 9 125)bull Micafungin (n ge 9 125)bull SCY‐078 (0 0)

Resistance to at least two of these drugs were frequently reported

Antifungal resistance profiles of the estimated 742 C auris isolates

Microbiologyopen 2018 Aug 7(4) e00578Candida auris A systematic review and meta‐analysis of current updates on an emerging multidrug‐resistant pathogenJohn Osei Sekyere

1022018

10

What are the mechanisms of azole and echinocandin resistance among Candida species

I Alteration of Drug TargetErg11 Candida sppErg5 Candida sppErg11 Cr neoformansCyp51A Aspergillus spp

Mdr1 C albicansFlu1 C albicansMcm1 C albicansMdr3 A fumigatus

II Over-expression of Efflux Transporters

Cdr1 C albicansCdr1 C glabrataPdh1 C glabrataMdr124 A fumigatusAfr1 C neoformans

H+

drug

N

N

A B A B

out

in

ATP

drug

ATP

ABC

MFS

III Over-expression of Target Site

Erg11 Candida sppCyp51A A fumigatus

Azole resistance mechanisms

IV Chromosomal abnormalitiesAneuploidy disomyIsochromosomes

C albicans Cr Neoformans

eg ERG11 TAC1

V Transcription factorsTAC1 MRR1 PDR1UPC2

V Heteroresistance(Genetic and epigenetic)C glabrata Cr neoformans C tropicalis

1022018

11

Azole resistance in Candida glabrata

a perfect storm for multidrug resistance

GOF Mutations in TF Pdr1

Upregulation of CDR1 Decreased adherence and

uptake by macrophages

Vermitsky et al 2006 MM Vale-Silva et al 2013 Infect Immun

Azole

resistance

Increased

burden

pdr1 GOF mutations identified within

fluconazole-resistant clinical isolates

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

12

India (n=40)

bull

Kordalewska 2018

Erg11 Prevalence Elevated MICs

WT 25 (n=1) - -

Y132F 55 (n=22) FLC VRC IVC

K143R 425 (n=17) FLC

WT 625 (n=35) WT

Y132F 35 (n=2) FLC VRC IVC

K143R 18 (n=1) FLC

I466M 304 (n=17) FLC

Y501H 18 (n=1) FLC VRC IVC

Colombia (n=56)

Target Site amino acid substitutions in Erg11 account for AZOLE Resistance in C auris

FLC VRC

48h 72h 48h 72h

BY4741 + empty 8 16 012 012

BY4741 + pCauErg11-WT 16 16 012 025

BY4741 + pCauErg11-Y132F 128 gt128 2 4

BY4741 + pCauErg11-K143R 64 gt128 05 1

BY4741 + pCauErg11-K177RN335SE343D

8 16 012 025

bull Expression of C auris erg11 mutations in S cerevisiae

‐ Saccharomyces cerevisiae strain -BY4741 (haploid strain)‐ Plasmid - pRS416 (S cerevisiae CENARS URA3 AMPr)‐ Amplified DNA fragment (C auris ERG11) and linearized plasmid co-transformed into BY4741

C auris Y132 = C albicans Y132 = S cerevisiae Y140C auris K143 = C albicans K143 = S cerevisiae K151

Sagatova et al 2015 AAC

HEALEY et al AAC 2018

Mutations in Erg11 confer resistance in S cerevisiae tester strain

1022018

13

bull Heteroresistant strains are not detected by standard susceptibility testing

bull Heteroresistance to FLZ is a graded phenotype associated with ABC

transporter upregulation and drug efflux- may reflect cellular andor

epigenetic adaptation

R Ben-Ami et al 2016 mBio

Candida and Cryptococcus

Heteroresistance refers to variability in the response to a drug

within a clonal cell population

Tolerance to antifungal drug concentrations above the minimal

inhibitory concentration (MIC) is rarely quantified and current clinical

recommendations suggest it should be ignored Here we quantify antifungal tolerance in Candida albicans isolates as the fraction of growth above the MIC and find that it is distinct from susceptibilityresistance Instead tolerance is due to the slow growth of subpopulations of cells that overcome drug stress more efficiently than the rest of the population and correlates inversely with intracellular drug accumulation Many adjuvant drugs used in combination with fluconazole a widely used fungistatic drug reduce tolerance without affecting resistance Accordingly in an invertebrate infection model adjuvant combination therapy is more effective than fluconazole in treating infections with highly tolerant isolates and does not affect infections with

low tolerance isolates Furthermore isolates recovered from immunocompetent patients with persistent candidemia display higher tolerance than isolates readily cleared by fluconazole Thus tolerance correlates with and may help predict patient responses to fluconazole therapy

Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemiaAlexander Rosenberg Iuliana V Ene Maayan Bibi Shiri Zakin Ella Shtifman Segal Naomi Ziv AlonM Dahan Arnaldo Lopes Colombo Richard J Bennett amp Judith Berman

Nat Commun 2018 Jun 259(1)2470

Fraction of growth (FoG) above fluconazole MIC may influence clinical response

1022018

14

Drug responses in disk diffusion assays (DDAs) and liquid broth microdilution assays (BMDAs) The fraction of growth inside the zone of inhibition (FoG) is the area under the curve (red) at the RAD threshold divided by the maximum area

Rosenberg et al 2018 Nat Comm

Echinocandin resistance resulting in clinical failures

involves mutations in two hot spot region of the Fks

catalytic subunit of glucan synthase

bull Two regions (HS1 and HS2) of Fks are associated with resistance

in Candida

bull Prominent mutations confer cross-resistance to all echinocandin

class drugs

bull Azole resistance mechanisms are not active on echinocandin

drugs

155Park et al 2005 AAC

0 1900AA

Fks1

1345-1365F641-P649HS1 HS2

1022018

7

Proportion of the five major Candida species responsible for fungemia in patients with (159) or without (2289) prior exposure to FLZ or with (61) or without (2387) prior exposure to CSF

Ca

Ca

Ca

Cg

Cg

Cg

Prior azole and echinocandin exposure is shifting the distribution of Candida spp recovered from severely ill patients with C glabrata as a major cause of disease

Lortholary et al 2011 AAC

Hachem et al 2008 Cancer

C glabrata has emerged as the dominant pathogen in certain immunocompromised patient populations

1022018

8

0

5

10

15

20

25

30

2001-2002 2003-2004 2005-2006 2007-2008 2009-2010

Res

ista

nce

()

Time Period

Anidulafungin

Caspofungin

Micafungin

Fluconazole

Azoles

Echinocandins

MDR

Concomitant rise in azole and echinocandin resistance among Candida glabrata isolates with emergence of multidrug (MDR) resistance

C glabratandashpositive blood cultures at MD Anderson Cancer Center Houston TX during March 2005ndashSeptember 2013

Farmakiotis et al EID 2014

146 Total Isolatesbull 205 FLUr

bull 103 CSFr

bull 68 MDR (CSF FLU AMB)

MDR increasingly more common

MDR

Resistance to echinocandins azoles and polyenes

1022018

9

Global and regional maps depicting rapid emergence of multidrug-resistant clinical Candida auris strains

httpswwwcdcgovfungalcandida-auristracking-c-aurishtml

Most of the isolates were resistant tobull Fluconazole (n ge 318 4429) bull Voriconazole (VRZ) (n ge 91 1267) bull Itraconazole (ITZ) (n ge 13 181)bull Isavuconazole (ISA) (n ge 11 153)bull Posaconazole (PSZ) (n ge 10 139)

bull AMB (n ge 111 1546)bull Flucytosine (FCN) (n ge 14 195)

bull Caspofungin (n ge 25 348) bull Anidulafungin (n ge 9 125)bull Micafungin (n ge 9 125)bull SCY‐078 (0 0)

Resistance to at least two of these drugs were frequently reported

Antifungal resistance profiles of the estimated 742 C auris isolates

Microbiologyopen 2018 Aug 7(4) e00578Candida auris A systematic review and meta‐analysis of current updates on an emerging multidrug‐resistant pathogenJohn Osei Sekyere

1022018

10

What are the mechanisms of azole and echinocandin resistance among Candida species

I Alteration of Drug TargetErg11 Candida sppErg5 Candida sppErg11 Cr neoformansCyp51A Aspergillus spp

Mdr1 C albicansFlu1 C albicansMcm1 C albicansMdr3 A fumigatus

II Over-expression of Efflux Transporters

Cdr1 C albicansCdr1 C glabrataPdh1 C glabrataMdr124 A fumigatusAfr1 C neoformans

H+

drug

N

N

A B A B

out

in

ATP

drug

ATP

ABC

MFS

III Over-expression of Target Site

Erg11 Candida sppCyp51A A fumigatus

Azole resistance mechanisms

IV Chromosomal abnormalitiesAneuploidy disomyIsochromosomes

C albicans Cr Neoformans

eg ERG11 TAC1

V Transcription factorsTAC1 MRR1 PDR1UPC2

V Heteroresistance(Genetic and epigenetic)C glabrata Cr neoformans C tropicalis

1022018

11

Azole resistance in Candida glabrata

a perfect storm for multidrug resistance

GOF Mutations in TF Pdr1

Upregulation of CDR1 Decreased adherence and

uptake by macrophages

Vermitsky et al 2006 MM Vale-Silva et al 2013 Infect Immun

Azole

resistance

Increased

burden

pdr1 GOF mutations identified within

fluconazole-resistant clinical isolates

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

12

India (n=40)

bull

Kordalewska 2018

Erg11 Prevalence Elevated MICs

WT 25 (n=1) - -

Y132F 55 (n=22) FLC VRC IVC

K143R 425 (n=17) FLC

WT 625 (n=35) WT

Y132F 35 (n=2) FLC VRC IVC

K143R 18 (n=1) FLC

I466M 304 (n=17) FLC

Y501H 18 (n=1) FLC VRC IVC

Colombia (n=56)

Target Site amino acid substitutions in Erg11 account for AZOLE Resistance in C auris

FLC VRC

48h 72h 48h 72h

BY4741 + empty 8 16 012 012

BY4741 + pCauErg11-WT 16 16 012 025

BY4741 + pCauErg11-Y132F 128 gt128 2 4

BY4741 + pCauErg11-K143R 64 gt128 05 1

BY4741 + pCauErg11-K177RN335SE343D

8 16 012 025

bull Expression of C auris erg11 mutations in S cerevisiae

‐ Saccharomyces cerevisiae strain -BY4741 (haploid strain)‐ Plasmid - pRS416 (S cerevisiae CENARS URA3 AMPr)‐ Amplified DNA fragment (C auris ERG11) and linearized plasmid co-transformed into BY4741

C auris Y132 = C albicans Y132 = S cerevisiae Y140C auris K143 = C albicans K143 = S cerevisiae K151

Sagatova et al 2015 AAC

HEALEY et al AAC 2018

Mutations in Erg11 confer resistance in S cerevisiae tester strain

1022018

13

bull Heteroresistant strains are not detected by standard susceptibility testing

bull Heteroresistance to FLZ is a graded phenotype associated with ABC

transporter upregulation and drug efflux- may reflect cellular andor

epigenetic adaptation

R Ben-Ami et al 2016 mBio

Candida and Cryptococcus

Heteroresistance refers to variability in the response to a drug

within a clonal cell population

Tolerance to antifungal drug concentrations above the minimal

inhibitory concentration (MIC) is rarely quantified and current clinical

recommendations suggest it should be ignored Here we quantify antifungal tolerance in Candida albicans isolates as the fraction of growth above the MIC and find that it is distinct from susceptibilityresistance Instead tolerance is due to the slow growth of subpopulations of cells that overcome drug stress more efficiently than the rest of the population and correlates inversely with intracellular drug accumulation Many adjuvant drugs used in combination with fluconazole a widely used fungistatic drug reduce tolerance without affecting resistance Accordingly in an invertebrate infection model adjuvant combination therapy is more effective than fluconazole in treating infections with highly tolerant isolates and does not affect infections with

low tolerance isolates Furthermore isolates recovered from immunocompetent patients with persistent candidemia display higher tolerance than isolates readily cleared by fluconazole Thus tolerance correlates with and may help predict patient responses to fluconazole therapy

Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemiaAlexander Rosenberg Iuliana V Ene Maayan Bibi Shiri Zakin Ella Shtifman Segal Naomi Ziv AlonM Dahan Arnaldo Lopes Colombo Richard J Bennett amp Judith Berman

Nat Commun 2018 Jun 259(1)2470

Fraction of growth (FoG) above fluconazole MIC may influence clinical response

1022018

14

Drug responses in disk diffusion assays (DDAs) and liquid broth microdilution assays (BMDAs) The fraction of growth inside the zone of inhibition (FoG) is the area under the curve (red) at the RAD threshold divided by the maximum area

Rosenberg et al 2018 Nat Comm

Echinocandin resistance resulting in clinical failures

involves mutations in two hot spot region of the Fks

catalytic subunit of glucan synthase

bull Two regions (HS1 and HS2) of Fks are associated with resistance

in Candida

bull Prominent mutations confer cross-resistance to all echinocandin

class drugs

bull Azole resistance mechanisms are not active on echinocandin

drugs

155Park et al 2005 AAC

0 1900AA

Fks1

1345-1365F641-P649HS1 HS2

1022018

8

0

5

10

15

20

25

30

2001-2002 2003-2004 2005-2006 2007-2008 2009-2010

Res

ista

nce

()

Time Period

Anidulafungin

Caspofungin

Micafungin

Fluconazole

Azoles

Echinocandins

MDR

Concomitant rise in azole and echinocandin resistance among Candida glabrata isolates with emergence of multidrug (MDR) resistance

C glabratandashpositive blood cultures at MD Anderson Cancer Center Houston TX during March 2005ndashSeptember 2013

Farmakiotis et al EID 2014

146 Total Isolatesbull 205 FLUr

bull 103 CSFr

bull 68 MDR (CSF FLU AMB)

MDR increasingly more common

MDR

Resistance to echinocandins azoles and polyenes

1022018

9

Global and regional maps depicting rapid emergence of multidrug-resistant clinical Candida auris strains

httpswwwcdcgovfungalcandida-auristracking-c-aurishtml

Most of the isolates were resistant tobull Fluconazole (n ge 318 4429) bull Voriconazole (VRZ) (n ge 91 1267) bull Itraconazole (ITZ) (n ge 13 181)bull Isavuconazole (ISA) (n ge 11 153)bull Posaconazole (PSZ) (n ge 10 139)

bull AMB (n ge 111 1546)bull Flucytosine (FCN) (n ge 14 195)

bull Caspofungin (n ge 25 348) bull Anidulafungin (n ge 9 125)bull Micafungin (n ge 9 125)bull SCY‐078 (0 0)

Resistance to at least two of these drugs were frequently reported

Antifungal resistance profiles of the estimated 742 C auris isolates

Microbiologyopen 2018 Aug 7(4) e00578Candida auris A systematic review and meta‐analysis of current updates on an emerging multidrug‐resistant pathogenJohn Osei Sekyere

1022018

10

What are the mechanisms of azole and echinocandin resistance among Candida species

I Alteration of Drug TargetErg11 Candida sppErg5 Candida sppErg11 Cr neoformansCyp51A Aspergillus spp

Mdr1 C albicansFlu1 C albicansMcm1 C albicansMdr3 A fumigatus

II Over-expression of Efflux Transporters

Cdr1 C albicansCdr1 C glabrataPdh1 C glabrataMdr124 A fumigatusAfr1 C neoformans

H+

drug

N

N

A B A B

out

in

ATP

drug

ATP

ABC

MFS

III Over-expression of Target Site

Erg11 Candida sppCyp51A A fumigatus

Azole resistance mechanisms

IV Chromosomal abnormalitiesAneuploidy disomyIsochromosomes

C albicans Cr Neoformans

eg ERG11 TAC1

V Transcription factorsTAC1 MRR1 PDR1UPC2

V Heteroresistance(Genetic and epigenetic)C glabrata Cr neoformans C tropicalis

1022018

11

Azole resistance in Candida glabrata

a perfect storm for multidrug resistance

GOF Mutations in TF Pdr1

Upregulation of CDR1 Decreased adherence and

uptake by macrophages

Vermitsky et al 2006 MM Vale-Silva et al 2013 Infect Immun

Azole

resistance

Increased

burden

pdr1 GOF mutations identified within

fluconazole-resistant clinical isolates

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

12

India (n=40)

bull

Kordalewska 2018

Erg11 Prevalence Elevated MICs

WT 25 (n=1) - -

Y132F 55 (n=22) FLC VRC IVC

K143R 425 (n=17) FLC

WT 625 (n=35) WT

Y132F 35 (n=2) FLC VRC IVC

K143R 18 (n=1) FLC

I466M 304 (n=17) FLC

Y501H 18 (n=1) FLC VRC IVC

Colombia (n=56)

Target Site amino acid substitutions in Erg11 account for AZOLE Resistance in C auris

FLC VRC

48h 72h 48h 72h

BY4741 + empty 8 16 012 012

BY4741 + pCauErg11-WT 16 16 012 025

BY4741 + pCauErg11-Y132F 128 gt128 2 4

BY4741 + pCauErg11-K143R 64 gt128 05 1

BY4741 + pCauErg11-K177RN335SE343D

8 16 012 025

bull Expression of C auris erg11 mutations in S cerevisiae

‐ Saccharomyces cerevisiae strain -BY4741 (haploid strain)‐ Plasmid - pRS416 (S cerevisiae CENARS URA3 AMPr)‐ Amplified DNA fragment (C auris ERG11) and linearized plasmid co-transformed into BY4741

C auris Y132 = C albicans Y132 = S cerevisiae Y140C auris K143 = C albicans K143 = S cerevisiae K151

Sagatova et al 2015 AAC

HEALEY et al AAC 2018

Mutations in Erg11 confer resistance in S cerevisiae tester strain

1022018

13

bull Heteroresistant strains are not detected by standard susceptibility testing

bull Heteroresistance to FLZ is a graded phenotype associated with ABC

transporter upregulation and drug efflux- may reflect cellular andor

epigenetic adaptation

R Ben-Ami et al 2016 mBio

Candida and Cryptococcus

Heteroresistance refers to variability in the response to a drug

within a clonal cell population

Tolerance to antifungal drug concentrations above the minimal

inhibitory concentration (MIC) is rarely quantified and current clinical

recommendations suggest it should be ignored Here we quantify antifungal tolerance in Candida albicans isolates as the fraction of growth above the MIC and find that it is distinct from susceptibilityresistance Instead tolerance is due to the slow growth of subpopulations of cells that overcome drug stress more efficiently than the rest of the population and correlates inversely with intracellular drug accumulation Many adjuvant drugs used in combination with fluconazole a widely used fungistatic drug reduce tolerance without affecting resistance Accordingly in an invertebrate infection model adjuvant combination therapy is more effective than fluconazole in treating infections with highly tolerant isolates and does not affect infections with

low tolerance isolates Furthermore isolates recovered from immunocompetent patients with persistent candidemia display higher tolerance than isolates readily cleared by fluconazole Thus tolerance correlates with and may help predict patient responses to fluconazole therapy

Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemiaAlexander Rosenberg Iuliana V Ene Maayan Bibi Shiri Zakin Ella Shtifman Segal Naomi Ziv AlonM Dahan Arnaldo Lopes Colombo Richard J Bennett amp Judith Berman

Nat Commun 2018 Jun 259(1)2470

Fraction of growth (FoG) above fluconazole MIC may influence clinical response

1022018

14

Drug responses in disk diffusion assays (DDAs) and liquid broth microdilution assays (BMDAs) The fraction of growth inside the zone of inhibition (FoG) is the area under the curve (red) at the RAD threshold divided by the maximum area

Rosenberg et al 2018 Nat Comm

Echinocandin resistance resulting in clinical failures

involves mutations in two hot spot region of the Fks

catalytic subunit of glucan synthase

bull Two regions (HS1 and HS2) of Fks are associated with resistance

in Candida

bull Prominent mutations confer cross-resistance to all echinocandin

class drugs

bull Azole resistance mechanisms are not active on echinocandin

drugs

155Park et al 2005 AAC

0 1900AA

Fks1

1345-1365F641-P649HS1 HS2

1022018

9

Global and regional maps depicting rapid emergence of multidrug-resistant clinical Candida auris strains

httpswwwcdcgovfungalcandida-auristracking-c-aurishtml

Most of the isolates were resistant tobull Fluconazole (n ge 318 4429) bull Voriconazole (VRZ) (n ge 91 1267) bull Itraconazole (ITZ) (n ge 13 181)bull Isavuconazole (ISA) (n ge 11 153)bull Posaconazole (PSZ) (n ge 10 139)

bull AMB (n ge 111 1546)bull Flucytosine (FCN) (n ge 14 195)

bull Caspofungin (n ge 25 348) bull Anidulafungin (n ge 9 125)bull Micafungin (n ge 9 125)bull SCY‐078 (0 0)

Resistance to at least two of these drugs were frequently reported

Antifungal resistance profiles of the estimated 742 C auris isolates

Microbiologyopen 2018 Aug 7(4) e00578Candida auris A systematic review and meta‐analysis of current updates on an emerging multidrug‐resistant pathogenJohn Osei Sekyere

1022018

10

What are the mechanisms of azole and echinocandin resistance among Candida species

I Alteration of Drug TargetErg11 Candida sppErg5 Candida sppErg11 Cr neoformansCyp51A Aspergillus spp

Mdr1 C albicansFlu1 C albicansMcm1 C albicansMdr3 A fumigatus

II Over-expression of Efflux Transporters

Cdr1 C albicansCdr1 C glabrataPdh1 C glabrataMdr124 A fumigatusAfr1 C neoformans

H+

drug

N

N

A B A B

out

in

ATP

drug

ATP

ABC

MFS

III Over-expression of Target Site

Erg11 Candida sppCyp51A A fumigatus

Azole resistance mechanisms

IV Chromosomal abnormalitiesAneuploidy disomyIsochromosomes

C albicans Cr Neoformans

eg ERG11 TAC1

V Transcription factorsTAC1 MRR1 PDR1UPC2

V Heteroresistance(Genetic and epigenetic)C glabrata Cr neoformans C tropicalis

1022018

11

Azole resistance in Candida glabrata

a perfect storm for multidrug resistance

GOF Mutations in TF Pdr1

Upregulation of CDR1 Decreased adherence and

uptake by macrophages

Vermitsky et al 2006 MM Vale-Silva et al 2013 Infect Immun

Azole

resistance

Increased

burden

pdr1 GOF mutations identified within

fluconazole-resistant clinical isolates

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

12

India (n=40)

bull

Kordalewska 2018

Erg11 Prevalence Elevated MICs

WT 25 (n=1) - -

Y132F 55 (n=22) FLC VRC IVC

K143R 425 (n=17) FLC

WT 625 (n=35) WT

Y132F 35 (n=2) FLC VRC IVC

K143R 18 (n=1) FLC

I466M 304 (n=17) FLC

Y501H 18 (n=1) FLC VRC IVC

Colombia (n=56)

Target Site amino acid substitutions in Erg11 account for AZOLE Resistance in C auris

FLC VRC

48h 72h 48h 72h

BY4741 + empty 8 16 012 012

BY4741 + pCauErg11-WT 16 16 012 025

BY4741 + pCauErg11-Y132F 128 gt128 2 4

BY4741 + pCauErg11-K143R 64 gt128 05 1

BY4741 + pCauErg11-K177RN335SE343D

8 16 012 025

bull Expression of C auris erg11 mutations in S cerevisiae

‐ Saccharomyces cerevisiae strain -BY4741 (haploid strain)‐ Plasmid - pRS416 (S cerevisiae CENARS URA3 AMPr)‐ Amplified DNA fragment (C auris ERG11) and linearized plasmid co-transformed into BY4741

C auris Y132 = C albicans Y132 = S cerevisiae Y140C auris K143 = C albicans K143 = S cerevisiae K151

Sagatova et al 2015 AAC

HEALEY et al AAC 2018

Mutations in Erg11 confer resistance in S cerevisiae tester strain

1022018

13

bull Heteroresistant strains are not detected by standard susceptibility testing

bull Heteroresistance to FLZ is a graded phenotype associated with ABC

transporter upregulation and drug efflux- may reflect cellular andor

epigenetic adaptation

R Ben-Ami et al 2016 mBio

Candida and Cryptococcus

Heteroresistance refers to variability in the response to a drug

within a clonal cell population

Tolerance to antifungal drug concentrations above the minimal

inhibitory concentration (MIC) is rarely quantified and current clinical

recommendations suggest it should be ignored Here we quantify antifungal tolerance in Candida albicans isolates as the fraction of growth above the MIC and find that it is distinct from susceptibilityresistance Instead tolerance is due to the slow growth of subpopulations of cells that overcome drug stress more efficiently than the rest of the population and correlates inversely with intracellular drug accumulation Many adjuvant drugs used in combination with fluconazole a widely used fungistatic drug reduce tolerance without affecting resistance Accordingly in an invertebrate infection model adjuvant combination therapy is more effective than fluconazole in treating infections with highly tolerant isolates and does not affect infections with

low tolerance isolates Furthermore isolates recovered from immunocompetent patients with persistent candidemia display higher tolerance than isolates readily cleared by fluconazole Thus tolerance correlates with and may help predict patient responses to fluconazole therapy

Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemiaAlexander Rosenberg Iuliana V Ene Maayan Bibi Shiri Zakin Ella Shtifman Segal Naomi Ziv AlonM Dahan Arnaldo Lopes Colombo Richard J Bennett amp Judith Berman

Nat Commun 2018 Jun 259(1)2470

Fraction of growth (FoG) above fluconazole MIC may influence clinical response

1022018

14

Drug responses in disk diffusion assays (DDAs) and liquid broth microdilution assays (BMDAs) The fraction of growth inside the zone of inhibition (FoG) is the area under the curve (red) at the RAD threshold divided by the maximum area

Rosenberg et al 2018 Nat Comm

Echinocandin resistance resulting in clinical failures

involves mutations in two hot spot region of the Fks

catalytic subunit of glucan synthase

bull Two regions (HS1 and HS2) of Fks are associated with resistance

in Candida

bull Prominent mutations confer cross-resistance to all echinocandin

class drugs

bull Azole resistance mechanisms are not active on echinocandin

drugs

155Park et al 2005 AAC

0 1900AA

Fks1

1345-1365F641-P649HS1 HS2

1022018

10

What are the mechanisms of azole and echinocandin resistance among Candida species

I Alteration of Drug TargetErg11 Candida sppErg5 Candida sppErg11 Cr neoformansCyp51A Aspergillus spp

Mdr1 C albicansFlu1 C albicansMcm1 C albicansMdr3 A fumigatus

II Over-expression of Efflux Transporters

Cdr1 C albicansCdr1 C glabrataPdh1 C glabrataMdr124 A fumigatusAfr1 C neoformans

H+

drug

N

N

A B A B

out

in

ATP

drug

ATP

ABC

MFS

III Over-expression of Target Site

Erg11 Candida sppCyp51A A fumigatus

Azole resistance mechanisms

IV Chromosomal abnormalitiesAneuploidy disomyIsochromosomes

C albicans Cr Neoformans

eg ERG11 TAC1

V Transcription factorsTAC1 MRR1 PDR1UPC2

V Heteroresistance(Genetic and epigenetic)C glabrata Cr neoformans C tropicalis

1022018

11

Azole resistance in Candida glabrata

a perfect storm for multidrug resistance

GOF Mutations in TF Pdr1

Upregulation of CDR1 Decreased adherence and

uptake by macrophages

Vermitsky et al 2006 MM Vale-Silva et al 2013 Infect Immun

Azole

resistance

Increased

burden

pdr1 GOF mutations identified within

fluconazole-resistant clinical isolates

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

12

India (n=40)

bull

Kordalewska 2018

Erg11 Prevalence Elevated MICs

WT 25 (n=1) - -

Y132F 55 (n=22) FLC VRC IVC

K143R 425 (n=17) FLC

WT 625 (n=35) WT

Y132F 35 (n=2) FLC VRC IVC

K143R 18 (n=1) FLC

I466M 304 (n=17) FLC

Y501H 18 (n=1) FLC VRC IVC

Colombia (n=56)

Target Site amino acid substitutions in Erg11 account for AZOLE Resistance in C auris

FLC VRC

48h 72h 48h 72h

BY4741 + empty 8 16 012 012

BY4741 + pCauErg11-WT 16 16 012 025

BY4741 + pCauErg11-Y132F 128 gt128 2 4

BY4741 + pCauErg11-K143R 64 gt128 05 1

BY4741 + pCauErg11-K177RN335SE343D

8 16 012 025

bull Expression of C auris erg11 mutations in S cerevisiae

‐ Saccharomyces cerevisiae strain -BY4741 (haploid strain)‐ Plasmid - pRS416 (S cerevisiae CENARS URA3 AMPr)‐ Amplified DNA fragment (C auris ERG11) and linearized plasmid co-transformed into BY4741

C auris Y132 = C albicans Y132 = S cerevisiae Y140C auris K143 = C albicans K143 = S cerevisiae K151

Sagatova et al 2015 AAC

HEALEY et al AAC 2018

Mutations in Erg11 confer resistance in S cerevisiae tester strain

1022018

13

bull Heteroresistant strains are not detected by standard susceptibility testing

bull Heteroresistance to FLZ is a graded phenotype associated with ABC

transporter upregulation and drug efflux- may reflect cellular andor

epigenetic adaptation

R Ben-Ami et al 2016 mBio

Candida and Cryptococcus

Heteroresistance refers to variability in the response to a drug

within a clonal cell population

Tolerance to antifungal drug concentrations above the minimal

inhibitory concentration (MIC) is rarely quantified and current clinical

recommendations suggest it should be ignored Here we quantify antifungal tolerance in Candida albicans isolates as the fraction of growth above the MIC and find that it is distinct from susceptibilityresistance Instead tolerance is due to the slow growth of subpopulations of cells that overcome drug stress more efficiently than the rest of the population and correlates inversely with intracellular drug accumulation Many adjuvant drugs used in combination with fluconazole a widely used fungistatic drug reduce tolerance without affecting resistance Accordingly in an invertebrate infection model adjuvant combination therapy is more effective than fluconazole in treating infections with highly tolerant isolates and does not affect infections with

low tolerance isolates Furthermore isolates recovered from immunocompetent patients with persistent candidemia display higher tolerance than isolates readily cleared by fluconazole Thus tolerance correlates with and may help predict patient responses to fluconazole therapy

Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemiaAlexander Rosenberg Iuliana V Ene Maayan Bibi Shiri Zakin Ella Shtifman Segal Naomi Ziv AlonM Dahan Arnaldo Lopes Colombo Richard J Bennett amp Judith Berman

Nat Commun 2018 Jun 259(1)2470

Fraction of growth (FoG) above fluconazole MIC may influence clinical response

1022018

14

Drug responses in disk diffusion assays (DDAs) and liquid broth microdilution assays (BMDAs) The fraction of growth inside the zone of inhibition (FoG) is the area under the curve (red) at the RAD threshold divided by the maximum area

Rosenberg et al 2018 Nat Comm

Echinocandin resistance resulting in clinical failures

involves mutations in two hot spot region of the Fks

catalytic subunit of glucan synthase

bull Two regions (HS1 and HS2) of Fks are associated with resistance

in Candida

bull Prominent mutations confer cross-resistance to all echinocandin

class drugs

bull Azole resistance mechanisms are not active on echinocandin

drugs

155Park et al 2005 AAC

0 1900AA

Fks1

1345-1365F641-P649HS1 HS2

1022018

11

Azole resistance in Candida glabrata

a perfect storm for multidrug resistance

GOF Mutations in TF Pdr1

Upregulation of CDR1 Decreased adherence and

uptake by macrophages

Vermitsky et al 2006 MM Vale-Silva et al 2013 Infect Immun

Azole

resistance

Increased

burden

pdr1 GOF mutations identified within

fluconazole-resistant clinical isolates

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

12

India (n=40)

bull

Kordalewska 2018

Erg11 Prevalence Elevated MICs

WT 25 (n=1) - -

Y132F 55 (n=22) FLC VRC IVC

K143R 425 (n=17) FLC

WT 625 (n=35) WT

Y132F 35 (n=2) FLC VRC IVC

K143R 18 (n=1) FLC

I466M 304 (n=17) FLC

Y501H 18 (n=1) FLC VRC IVC

Colombia (n=56)

Target Site amino acid substitutions in Erg11 account for AZOLE Resistance in C auris

FLC VRC

48h 72h 48h 72h

BY4741 + empty 8 16 012 012

BY4741 + pCauErg11-WT 16 16 012 025

BY4741 + pCauErg11-Y132F 128 gt128 2 4

BY4741 + pCauErg11-K143R 64 gt128 05 1

BY4741 + pCauErg11-K177RN335SE343D

8 16 012 025

bull Expression of C auris erg11 mutations in S cerevisiae

‐ Saccharomyces cerevisiae strain -BY4741 (haploid strain)‐ Plasmid - pRS416 (S cerevisiae CENARS URA3 AMPr)‐ Amplified DNA fragment (C auris ERG11) and linearized plasmid co-transformed into BY4741

C auris Y132 = C albicans Y132 = S cerevisiae Y140C auris K143 = C albicans K143 = S cerevisiae K151

Sagatova et al 2015 AAC

HEALEY et al AAC 2018

Mutations in Erg11 confer resistance in S cerevisiae tester strain

1022018

13

bull Heteroresistant strains are not detected by standard susceptibility testing

bull Heteroresistance to FLZ is a graded phenotype associated with ABC

transporter upregulation and drug efflux- may reflect cellular andor

epigenetic adaptation

R Ben-Ami et al 2016 mBio

Candida and Cryptococcus

Heteroresistance refers to variability in the response to a drug

within a clonal cell population

Tolerance to antifungal drug concentrations above the minimal

inhibitory concentration (MIC) is rarely quantified and current clinical

recommendations suggest it should be ignored Here we quantify antifungal tolerance in Candida albicans isolates as the fraction of growth above the MIC and find that it is distinct from susceptibilityresistance Instead tolerance is due to the slow growth of subpopulations of cells that overcome drug stress more efficiently than the rest of the population and correlates inversely with intracellular drug accumulation Many adjuvant drugs used in combination with fluconazole a widely used fungistatic drug reduce tolerance without affecting resistance Accordingly in an invertebrate infection model adjuvant combination therapy is more effective than fluconazole in treating infections with highly tolerant isolates and does not affect infections with

low tolerance isolates Furthermore isolates recovered from immunocompetent patients with persistent candidemia display higher tolerance than isolates readily cleared by fluconazole Thus tolerance correlates with and may help predict patient responses to fluconazole therapy

Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemiaAlexander Rosenberg Iuliana V Ene Maayan Bibi Shiri Zakin Ella Shtifman Segal Naomi Ziv AlonM Dahan Arnaldo Lopes Colombo Richard J Bennett amp Judith Berman

Nat Commun 2018 Jun 259(1)2470

Fraction of growth (FoG) above fluconazole MIC may influence clinical response

1022018

14

Drug responses in disk diffusion assays (DDAs) and liquid broth microdilution assays (BMDAs) The fraction of growth inside the zone of inhibition (FoG) is the area under the curve (red) at the RAD threshold divided by the maximum area

Rosenberg et al 2018 Nat Comm

Echinocandin resistance resulting in clinical failures

involves mutations in two hot spot region of the Fks

catalytic subunit of glucan synthase

bull Two regions (HS1 and HS2) of Fks are associated with resistance

in Candida

bull Prominent mutations confer cross-resistance to all echinocandin

class drugs

bull Azole resistance mechanisms are not active on echinocandin

drugs

155Park et al 2005 AAC

0 1900AA

Fks1

1345-1365F641-P649HS1 HS2

1022018

12

India (n=40)

bull

Kordalewska 2018

Erg11 Prevalence Elevated MICs

WT 25 (n=1) - -

Y132F 55 (n=22) FLC VRC IVC

K143R 425 (n=17) FLC

WT 625 (n=35) WT

Y132F 35 (n=2) FLC VRC IVC

K143R 18 (n=1) FLC

I466M 304 (n=17) FLC

Y501H 18 (n=1) FLC VRC IVC

Colombia (n=56)

Target Site amino acid substitutions in Erg11 account for AZOLE Resistance in C auris

FLC VRC

48h 72h 48h 72h

BY4741 + empty 8 16 012 012

BY4741 + pCauErg11-WT 16 16 012 025

BY4741 + pCauErg11-Y132F 128 gt128 2 4

BY4741 + pCauErg11-K143R 64 gt128 05 1

BY4741 + pCauErg11-K177RN335SE343D

8 16 012 025

bull Expression of C auris erg11 mutations in S cerevisiae

‐ Saccharomyces cerevisiae strain -BY4741 (haploid strain)‐ Plasmid - pRS416 (S cerevisiae CENARS URA3 AMPr)‐ Amplified DNA fragment (C auris ERG11) and linearized plasmid co-transformed into BY4741

C auris Y132 = C albicans Y132 = S cerevisiae Y140C auris K143 = C albicans K143 = S cerevisiae K151

Sagatova et al 2015 AAC

HEALEY et al AAC 2018

Mutations in Erg11 confer resistance in S cerevisiae tester strain

1022018

13

bull Heteroresistant strains are not detected by standard susceptibility testing

bull Heteroresistance to FLZ is a graded phenotype associated with ABC

transporter upregulation and drug efflux- may reflect cellular andor

epigenetic adaptation

R Ben-Ami et al 2016 mBio

Candida and Cryptococcus

Heteroresistance refers to variability in the response to a drug

within a clonal cell population

Tolerance to antifungal drug concentrations above the minimal

inhibitory concentration (MIC) is rarely quantified and current clinical

recommendations suggest it should be ignored Here we quantify antifungal tolerance in Candida albicans isolates as the fraction of growth above the MIC and find that it is distinct from susceptibilityresistance Instead tolerance is due to the slow growth of subpopulations of cells that overcome drug stress more efficiently than the rest of the population and correlates inversely with intracellular drug accumulation Many adjuvant drugs used in combination with fluconazole a widely used fungistatic drug reduce tolerance without affecting resistance Accordingly in an invertebrate infection model adjuvant combination therapy is more effective than fluconazole in treating infections with highly tolerant isolates and does not affect infections with

low tolerance isolates Furthermore isolates recovered from immunocompetent patients with persistent candidemia display higher tolerance than isolates readily cleared by fluconazole Thus tolerance correlates with and may help predict patient responses to fluconazole therapy

Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemiaAlexander Rosenberg Iuliana V Ene Maayan Bibi Shiri Zakin Ella Shtifman Segal Naomi Ziv AlonM Dahan Arnaldo Lopes Colombo Richard J Bennett amp Judith Berman

Nat Commun 2018 Jun 259(1)2470

Fraction of growth (FoG) above fluconazole MIC may influence clinical response

1022018

14

Drug responses in disk diffusion assays (DDAs) and liquid broth microdilution assays (BMDAs) The fraction of growth inside the zone of inhibition (FoG) is the area under the curve (red) at the RAD threshold divided by the maximum area

Rosenberg et al 2018 Nat Comm

Echinocandin resistance resulting in clinical failures

involves mutations in two hot spot region of the Fks

catalytic subunit of glucan synthase

bull Two regions (HS1 and HS2) of Fks are associated with resistance

in Candida

bull Prominent mutations confer cross-resistance to all echinocandin

class drugs

bull Azole resistance mechanisms are not active on echinocandin

drugs

155Park et al 2005 AAC

0 1900AA

Fks1

1345-1365F641-P649HS1 HS2

1022018

13

bull Heteroresistant strains are not detected by standard susceptibility testing

bull Heteroresistance to FLZ is a graded phenotype associated with ABC

transporter upregulation and drug efflux- may reflect cellular andor

epigenetic adaptation

R Ben-Ami et al 2016 mBio

Candida and Cryptococcus

Heteroresistance refers to variability in the response to a drug

within a clonal cell population

Tolerance to antifungal drug concentrations above the minimal

inhibitory concentration (MIC) is rarely quantified and current clinical

recommendations suggest it should be ignored Here we quantify antifungal tolerance in Candida albicans isolates as the fraction of growth above the MIC and find that it is distinct from susceptibilityresistance Instead tolerance is due to the slow growth of subpopulations of cells that overcome drug stress more efficiently than the rest of the population and correlates inversely with intracellular drug accumulation Many adjuvant drugs used in combination with fluconazole a widely used fungistatic drug reduce tolerance without affecting resistance Accordingly in an invertebrate infection model adjuvant combination therapy is more effective than fluconazole in treating infections with highly tolerant isolates and does not affect infections with

low tolerance isolates Furthermore isolates recovered from immunocompetent patients with persistent candidemia display higher tolerance than isolates readily cleared by fluconazole Thus tolerance correlates with and may help predict patient responses to fluconazole therapy

Antifungal tolerance is a subpopulation effect distinct from resistance and is associated with persistent candidemiaAlexander Rosenberg Iuliana V Ene Maayan Bibi Shiri Zakin Ella Shtifman Segal Naomi Ziv AlonM Dahan Arnaldo Lopes Colombo Richard J Bennett amp Judith Berman

Nat Commun 2018 Jun 259(1)2470

Fraction of growth (FoG) above fluconazole MIC may influence clinical response

1022018

14

Drug responses in disk diffusion assays (DDAs) and liquid broth microdilution assays (BMDAs) The fraction of growth inside the zone of inhibition (FoG) is the area under the curve (red) at the RAD threshold divided by the maximum area

Rosenberg et al 2018 Nat Comm

Echinocandin resistance resulting in clinical failures

involves mutations in two hot spot region of the Fks

catalytic subunit of glucan synthase

bull Two regions (HS1 and HS2) of Fks are associated with resistance

in Candida

bull Prominent mutations confer cross-resistance to all echinocandin

class drugs

bull Azole resistance mechanisms are not active on echinocandin

drugs

155Park et al 2005 AAC

0 1900AA

Fks1

1345-1365F641-P649HS1 HS2

1022018

14

Drug responses in disk diffusion assays (DDAs) and liquid broth microdilution assays (BMDAs) The fraction of growth inside the zone of inhibition (FoG) is the area under the curve (red) at the RAD threshold divided by the maximum area

Rosenberg et al 2018 Nat Comm

Echinocandin resistance resulting in clinical failures

involves mutations in two hot spot region of the Fks

catalytic subunit of glucan synthase

bull Two regions (HS1 and HS2) of Fks are associated with resistance

in Candida

bull Prominent mutations confer cross-resistance to all echinocandin

class drugs

bull Azole resistance mechanisms are not active on echinocandin

drugs

155Park et al 2005 AAC

0 1900AA

Fks1

1345-1365F641-P649HS1 HS2

1022018

15

Kinetic inhibition of Glucan Synthase Activity-UDP-D-[1-3] glucose Incorporation Assay

S645FWT

IC50

Prominent HS1 mutation in FKS1 decreases sensitivity of glucan synthase to echinocandin drugs by 100-10000 fold

Park et al 2005 AAC

Altered drug-target interaction

Shields et al AAC 2012

Presence of an FKS mutation rather than MIC is the most

significant risk factor for clinical failure

1022018

16

Fks1 Fks2 Fks3 Organism Hot Spot1 Hot Spot 2 Hot Spot 1 Hot Spot 2 Hot Spot 1

C albicans F641LTLSLRDP DWIRRYTL NO NO NO

C krusei F655LILSIRDP DWIRRYTL NO NO NO

C glabrata F625LILSIRDP DWIRRYTL F659LILSLRDP DWIRRYTL NO

C guilliermondii F632MALSIRDP DWIRRYTL NO NO NO

C lypolytica F662LILSIRDP DWIRRCVL NO NO NO

C tropicalis F---LTLSIRDP DWIRRYTL NO NO NO

C dubliniensis F641LTLSIRDP DWIRRYTL NO NO NO

C auris F635LTLSLRDP DWIRRYTL NO NO NO

C parapsilosis F652LTLSIRDA DWIRRYTL NO NO NO

C orthopsilosis F---LTLSIRDA DWVRRYTL NO NO NO

C metapsilosis F---LTLSIRDA DWIRRYTL NO NO NO

NO Not Observed C parapsilosis sensu stricto --- Incomplete sequence designation

Acquired Mutation

Strong Resistance

Acquired Mutation

Weak Resistance

Naturally- occurring

polymorphism

Perlin 2015 MCMArendrup a and Perlin OID 2014Johnson and Edlind 2012 Euk Cell

Spectrum of Fks amino acid changes conferring clinical resistance- 17 year view

Resistance to glucan synthase inhibitor SCY-078 an enfumafungin is mediated by amino acid changes within and near echinocandin hot spots

Overlapping but not identical

SCY-078

1022018

17

Dose-response relationships for caspofungin against multiple C glabrataisolates including wild type and fks-containing mutants

Lepak et al 2012 AAC

fks mutants are insensitive to high doses of CSF in a pharmacodynamic model of infection-FKS status matters

Effect of FKS mutations on IC50 and MIC

0 0 1 0 1 1 1 0 1 0 0

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

lo g 1 0 M IC [ g m l]

GS

lo

g1

0 IC

50

[n

gm

l]

W T W T

W T

F 6 2 5 S

S 6 2 9 P

D 6 3 2 GD 6 3 2 E

D 6 3 2 Y + R 1 3 7 7 S T O P

F 6 5 9 d e l

F 6 5 9 V

F 6 5 9 S

S 6 6 3 P

D 6 6 6 G

D 6 6 6 E

P 6 6 7 T

W 1 3 7 5 L

0 0 1 0 1 1 1 0

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

lo g 1 0 M IC [ g m l]

GS

lo

g1

0 IC

50

[n

gm

l]

W T

W TW T

F 6 2 5 S

S 6 2 9 P

D 6 3 2 GD 6 3 2 E

D 6 3 2 Y + R 1 3 7 7 S T O P

F 6 5 9 d e l

F 6 5 9 V

F 6 5 9 S

S 6 6 3 P

D 6 6 6 G

D 6 6 6 E

P 6 6 7 TW 1 3 7 5 L

Caspofungin Anidulafungin

Astellas ECN Resistance Ref Center

1022018

18

90

10

HS1

Candida albicans (n=47 strains with mutations in FKS1 gene)

28

65

7

FKS1 HS1 mutations

F641

S645

HS2

In C albicans FKS1 hot spot 1 mutations occur 90 of the time two loci S645 and S641 account for 93 of resistance

Perlin ECNR Ref Lab

20

40

40

FKS1 HS2 mutations

R1361H

R1361RH

R1360RK

FKS126

FKS274

Candida glabrata (n=93 strains with mutations in FKS genes)

4

13

33

8

17

17

44

Mutations found in FKS1

D632Y

F625S

S629P

D632G

D632E

I634V

R631G

L660F

54

6

4

4

25

7

Mutations found in FKS2

S663P

F659V

F659S

delF659

others HS1

HS2

FKS2 mutations occur at nearly 3 times the rate of FKS1 mutations S663 (FKS2) and equivalent residue S629 (FKS1) are most prominent

Perlin ECNR Ref Lab

1022018

19

Caspofungin modal MIC variability among independent laboratories using CLSI broth

microdilution method for Candida spp (A Espinel-Ingroff et al AAC 2013)

Micafungin and anidulafungin behave more reliably in AFST (Arendrup et al Mycoses 2014 Pfaller et al JCM 2014)

15X

Caspofungin AFST for Candida spp may vary by

laboratory and is unreliable

CAS testing lsquoEagle effectrsquo (paradoxical growth effect) Difficult reading AFST with CSF should be viewed cautiously or avoided

Kordalewska et al 2018 AAC

Caspofungin Paradoxical Growth in C auris

1022018

20

bull 102 echinocandin sensitive isolates wild-type genotype

bull Echinocandin-resistant isolates S639F

ECHINOCANDINS - FKS1 gene analysis

Species Fks1 HS1

C auris F635LTLSLRDP

C albicans F641LTLSLRDP

C glabrata F625LILSIRDP

C parapsilosis F652LTLSLRDA

C tropicalis F650LTLSLRDP

Milena KORDALEWSKA

Most common site for high-level echinocandin resistance

Echinocandin resistance associated with FKS genotype

Kordalewska et al 2018 AAC

1022018

21

Azoles and echinocandins have non-overlapping

mechanisms of resistance

Mechanism of

resistance

Azoles Echinocandins

Target site modification

ERG11FKS

Target site overexpression

Drug pumps

If azoles and echinocandins have distinct non-

overlapping mechanisms of resistance then what

is driving multidrug resistance in C glabrata

Perlin 2015 Antimicrob Ther Rev

Evolution of Echinocandin Resistance

MIC

1022018

22

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 0016 003 006 012 025 05 1 2 4 8 16 32

Cell killing lt Adaptation

MIC

Cell killing gt Adaptation

Adaptation Persisters

Cell killing

ATCC 2001 HTL cells

Average of 4 independent experiments

24 hour growthsurvival of 1x107 cells

RPMI supplemented with His Trp Leu

Caspofungin microgml

Ce

lls R

em

ain

ing

(CFU

ml

Phases of in vitro cell killing and persistence with

echinocandins and Candida glabrata

Micafungin

Log C

FU K

idn

ey

Echinocandins are fungicidal yet they act as fungistatic agents in the human therapeutic range in a murine kidney infection model

Howard et al 2011 AAC

Static behavior reflects cell adaptation due to drug stress

1022018

23

Candida glabrata mechanisms of echinocandin tolerance

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slg1Δ (WSC1)

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slt2Δ (MAPK)

Fks1

Fks2 Chs

Rho1

Pkc1

Bck1

MAP2K

MAP1K

Slt2

Crz1Rlm1

Slg1Wsc1

GEFs

Fks1

Chs

Ssd1

Fks2

Bem2

Cnb

1

Cna

1

Fks2

Chs3B

Calcineurin

Inhibitors

Ca2+

HACS

Fks1

Secretory

Pathway

Tethering

Cell Wall

Integrity

PathwayCalcineurin

signaling

Healey and Perlin JoF 2018

HSP90

Disruption of tolerance-associated genes or

pathways lead to increased caspofungin killing

How do we reconcile the emergence of two

independent resistance mechanisms

1022018

24

Msh2

Pms1

MutS recognition of base-base mismatches or small insertionsdeletions

MutL endonuclease activity

Gupta et al 2012 Nat Struct Mol Biol

bull DNA polymerase misincorporation

rate ~10-7 per duplicated nucleotide

bull MMR removes 999 of these

mismatches

Msh6

An underlying defect that increases spontaneous mutation rates

would allow C glabrata to escape drug action by inducing critical

independent resistance mechanisms in real time

DNA mismatch repair

0E+00

1E-07

2E-07

3E-07

4E-07

5E-07

6E-07

7E-07

Casp

ofu

ng

in c

olo

ny f

req

uen

cy

1x 76x 21x 4x 37x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

40E-04

Flu

co

nazo

le c

olo

ny f

req

uen

cy

1x 8x 6x 2x 6x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

Am

pB

Co

lon

y F

req

uen

cy

1x 23x 16x 3x 10x

Fks1Fks2 hotspot

mutationsPdr1 GOF

mutations

Erg6

mutations

C glabrata msh2 Δ msh6Δ msh3Δ and pms1Δ lead to increased frequencies of antifungal-resistant colonies

Mutations in all four components enhance selection for MDR phenotypes

Healey et al 2016 Nat CommUnpubl

Echinocandins Azoles Polyene

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

16

Fks1 Fks2 Fks3 Organism Hot Spot1 Hot Spot 2 Hot Spot 1 Hot Spot 2 Hot Spot 1

C albicans F641LTLSLRDP DWIRRYTL NO NO NO

C krusei F655LILSIRDP DWIRRYTL NO NO NO

C glabrata F625LILSIRDP DWIRRYTL F659LILSLRDP DWIRRYTL NO

C guilliermondii F632MALSIRDP DWIRRYTL NO NO NO

C lypolytica F662LILSIRDP DWIRRCVL NO NO NO

C tropicalis F---LTLSIRDP DWIRRYTL NO NO NO

C dubliniensis F641LTLSIRDP DWIRRYTL NO NO NO

C auris F635LTLSLRDP DWIRRYTL NO NO NO

C parapsilosis F652LTLSIRDA DWIRRYTL NO NO NO

C orthopsilosis F---LTLSIRDA DWVRRYTL NO NO NO

C metapsilosis F---LTLSIRDA DWIRRYTL NO NO NO

NO Not Observed C parapsilosis sensu stricto --- Incomplete sequence designation

Acquired Mutation

Strong Resistance

Acquired Mutation

Weak Resistance

Naturally- occurring

polymorphism

Perlin 2015 MCMArendrup a and Perlin OID 2014Johnson and Edlind 2012 Euk Cell

Spectrum of Fks amino acid changes conferring clinical resistance- 17 year view

Resistance to glucan synthase inhibitor SCY-078 an enfumafungin is mediated by amino acid changes within and near echinocandin hot spots

Overlapping but not identical

SCY-078

1022018

17

Dose-response relationships for caspofungin against multiple C glabrataisolates including wild type and fks-containing mutants

Lepak et al 2012 AAC

fks mutants are insensitive to high doses of CSF in a pharmacodynamic model of infection-FKS status matters

Effect of FKS mutations on IC50 and MIC

0 0 1 0 1 1 1 0 1 0 0

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

lo g 1 0 M IC [ g m l]

GS

lo

g1

0 IC

50

[n

gm

l]

W T W T

W T

F 6 2 5 S

S 6 2 9 P

D 6 3 2 GD 6 3 2 E

D 6 3 2 Y + R 1 3 7 7 S T O P

F 6 5 9 d e l

F 6 5 9 V

F 6 5 9 S

S 6 6 3 P

D 6 6 6 G

D 6 6 6 E

P 6 6 7 T

W 1 3 7 5 L

0 0 1 0 1 1 1 0

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

lo g 1 0 M IC [ g m l]

GS

lo

g1

0 IC

50

[n

gm

l]

W T

W TW T

F 6 2 5 S

S 6 2 9 P

D 6 3 2 GD 6 3 2 E

D 6 3 2 Y + R 1 3 7 7 S T O P

F 6 5 9 d e l

F 6 5 9 V

F 6 5 9 S

S 6 6 3 P

D 6 6 6 G

D 6 6 6 E

P 6 6 7 TW 1 3 7 5 L

Caspofungin Anidulafungin

Astellas ECN Resistance Ref Center

1022018

18

90

10

HS1

Candida albicans (n=47 strains with mutations in FKS1 gene)

28

65

7

FKS1 HS1 mutations

F641

S645

HS2

In C albicans FKS1 hot spot 1 mutations occur 90 of the time two loci S645 and S641 account for 93 of resistance

Perlin ECNR Ref Lab

20

40

40

FKS1 HS2 mutations

R1361H

R1361RH

R1360RK

FKS126

FKS274

Candida glabrata (n=93 strains with mutations in FKS genes)

4

13

33

8

17

17

44

Mutations found in FKS1

D632Y

F625S

S629P

D632G

D632E

I634V

R631G

L660F

54

6

4

4

25

7

Mutations found in FKS2

S663P

F659V

F659S

delF659

others HS1

HS2

FKS2 mutations occur at nearly 3 times the rate of FKS1 mutations S663 (FKS2) and equivalent residue S629 (FKS1) are most prominent

Perlin ECNR Ref Lab

1022018

19

Caspofungin modal MIC variability among independent laboratories using CLSI broth

microdilution method for Candida spp (A Espinel-Ingroff et al AAC 2013)

Micafungin and anidulafungin behave more reliably in AFST (Arendrup et al Mycoses 2014 Pfaller et al JCM 2014)

15X

Caspofungin AFST for Candida spp may vary by

laboratory and is unreliable

CAS testing lsquoEagle effectrsquo (paradoxical growth effect) Difficult reading AFST with CSF should be viewed cautiously or avoided

Kordalewska et al 2018 AAC

Caspofungin Paradoxical Growth in C auris

1022018

20

bull 102 echinocandin sensitive isolates wild-type genotype

bull Echinocandin-resistant isolates S639F

ECHINOCANDINS - FKS1 gene analysis

Species Fks1 HS1

C auris F635LTLSLRDP

C albicans F641LTLSLRDP

C glabrata F625LILSIRDP

C parapsilosis F652LTLSLRDA

C tropicalis F650LTLSLRDP

Milena KORDALEWSKA

Most common site for high-level echinocandin resistance

Echinocandin resistance associated with FKS genotype

Kordalewska et al 2018 AAC

1022018

21

Azoles and echinocandins have non-overlapping

mechanisms of resistance

Mechanism of

resistance

Azoles Echinocandins

Target site modification

ERG11FKS

Target site overexpression

Drug pumps

If azoles and echinocandins have distinct non-

overlapping mechanisms of resistance then what

is driving multidrug resistance in C glabrata

Perlin 2015 Antimicrob Ther Rev

Evolution of Echinocandin Resistance

MIC

1022018

22

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 0016 003 006 012 025 05 1 2 4 8 16 32

Cell killing lt Adaptation

MIC

Cell killing gt Adaptation

Adaptation Persisters

Cell killing

ATCC 2001 HTL cells

Average of 4 independent experiments

24 hour growthsurvival of 1x107 cells

RPMI supplemented with His Trp Leu

Caspofungin microgml

Ce

lls R

em

ain

ing

(CFU

ml

Phases of in vitro cell killing and persistence with

echinocandins and Candida glabrata

Micafungin

Log C

FU K

idn

ey

Echinocandins are fungicidal yet they act as fungistatic agents in the human therapeutic range in a murine kidney infection model

Howard et al 2011 AAC

Static behavior reflects cell adaptation due to drug stress

1022018

23

Candida glabrata mechanisms of echinocandin tolerance

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slg1Δ (WSC1)

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slt2Δ (MAPK)

Fks1

Fks2 Chs

Rho1

Pkc1

Bck1

MAP2K

MAP1K

Slt2

Crz1Rlm1

Slg1Wsc1

GEFs

Fks1

Chs

Ssd1

Fks2

Bem2

Cnb

1

Cna

1

Fks2

Chs3B

Calcineurin

Inhibitors

Ca2+

HACS

Fks1

Secretory

Pathway

Tethering

Cell Wall

Integrity

PathwayCalcineurin

signaling

Healey and Perlin JoF 2018

HSP90

Disruption of tolerance-associated genes or

pathways lead to increased caspofungin killing

How do we reconcile the emergence of two

independent resistance mechanisms

1022018

24

Msh2

Pms1

MutS recognition of base-base mismatches or small insertionsdeletions

MutL endonuclease activity

Gupta et al 2012 Nat Struct Mol Biol

bull DNA polymerase misincorporation

rate ~10-7 per duplicated nucleotide

bull MMR removes 999 of these

mismatches

Msh6

An underlying defect that increases spontaneous mutation rates

would allow C glabrata to escape drug action by inducing critical

independent resistance mechanisms in real time

DNA mismatch repair

0E+00

1E-07

2E-07

3E-07

4E-07

5E-07

6E-07

7E-07

Casp

ofu

ng

in c

olo

ny f

req

uen

cy

1x 76x 21x 4x 37x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

40E-04

Flu

co

nazo

le c

olo

ny f

req

uen

cy

1x 8x 6x 2x 6x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

Am

pB

Co

lon

y F

req

uen

cy

1x 23x 16x 3x 10x

Fks1Fks2 hotspot

mutationsPdr1 GOF

mutations

Erg6

mutations

C glabrata msh2 Δ msh6Δ msh3Δ and pms1Δ lead to increased frequencies of antifungal-resistant colonies

Mutations in all four components enhance selection for MDR phenotypes

Healey et al 2016 Nat CommUnpubl

Echinocandins Azoles Polyene

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

17

Dose-response relationships for caspofungin against multiple C glabrataisolates including wild type and fks-containing mutants

Lepak et al 2012 AAC

fks mutants are insensitive to high doses of CSF in a pharmacodynamic model of infection-FKS status matters

Effect of FKS mutations on IC50 and MIC

0 0 1 0 1 1 1 0 1 0 0

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

lo g 1 0 M IC [ g m l]

GS

lo

g1

0 IC

50

[n

gm

l]

W T W T

W T

F 6 2 5 S

S 6 2 9 P

D 6 3 2 GD 6 3 2 E

D 6 3 2 Y + R 1 3 7 7 S T O P

F 6 5 9 d e l

F 6 5 9 V

F 6 5 9 S

S 6 6 3 P

D 6 6 6 G

D 6 6 6 E

P 6 6 7 T

W 1 3 7 5 L

0 0 1 0 1 1 1 0

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

lo g 1 0 M IC [ g m l]

GS

lo

g1

0 IC

50

[n

gm

l]

W T

W TW T

F 6 2 5 S

S 6 2 9 P

D 6 3 2 GD 6 3 2 E

D 6 3 2 Y + R 1 3 7 7 S T O P

F 6 5 9 d e l

F 6 5 9 V

F 6 5 9 S

S 6 6 3 P

D 6 6 6 G

D 6 6 6 E

P 6 6 7 TW 1 3 7 5 L

Caspofungin Anidulafungin

Astellas ECN Resistance Ref Center

1022018

18

90

10

HS1

Candida albicans (n=47 strains with mutations in FKS1 gene)

28

65

7

FKS1 HS1 mutations

F641

S645

HS2

In C albicans FKS1 hot spot 1 mutations occur 90 of the time two loci S645 and S641 account for 93 of resistance

Perlin ECNR Ref Lab

20

40

40

FKS1 HS2 mutations

R1361H

R1361RH

R1360RK

FKS126

FKS274

Candida glabrata (n=93 strains with mutations in FKS genes)

4

13

33

8

17

17

44

Mutations found in FKS1

D632Y

F625S

S629P

D632G

D632E

I634V

R631G

L660F

54

6

4

4

25

7

Mutations found in FKS2

S663P

F659V

F659S

delF659

others HS1

HS2

FKS2 mutations occur at nearly 3 times the rate of FKS1 mutations S663 (FKS2) and equivalent residue S629 (FKS1) are most prominent

Perlin ECNR Ref Lab

1022018

19

Caspofungin modal MIC variability among independent laboratories using CLSI broth

microdilution method for Candida spp (A Espinel-Ingroff et al AAC 2013)

Micafungin and anidulafungin behave more reliably in AFST (Arendrup et al Mycoses 2014 Pfaller et al JCM 2014)

15X

Caspofungin AFST for Candida spp may vary by

laboratory and is unreliable

CAS testing lsquoEagle effectrsquo (paradoxical growth effect) Difficult reading AFST with CSF should be viewed cautiously or avoided

Kordalewska et al 2018 AAC

Caspofungin Paradoxical Growth in C auris

1022018

20

bull 102 echinocandin sensitive isolates wild-type genotype

bull Echinocandin-resistant isolates S639F

ECHINOCANDINS - FKS1 gene analysis

Species Fks1 HS1

C auris F635LTLSLRDP

C albicans F641LTLSLRDP

C glabrata F625LILSIRDP

C parapsilosis F652LTLSLRDA

C tropicalis F650LTLSLRDP

Milena KORDALEWSKA

Most common site for high-level echinocandin resistance

Echinocandin resistance associated with FKS genotype

Kordalewska et al 2018 AAC

1022018

21

Azoles and echinocandins have non-overlapping

mechanisms of resistance

Mechanism of

resistance

Azoles Echinocandins

Target site modification

ERG11FKS

Target site overexpression

Drug pumps

If azoles and echinocandins have distinct non-

overlapping mechanisms of resistance then what

is driving multidrug resistance in C glabrata

Perlin 2015 Antimicrob Ther Rev

Evolution of Echinocandin Resistance

MIC

1022018

22

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 0016 003 006 012 025 05 1 2 4 8 16 32

Cell killing lt Adaptation

MIC

Cell killing gt Adaptation

Adaptation Persisters

Cell killing

ATCC 2001 HTL cells

Average of 4 independent experiments

24 hour growthsurvival of 1x107 cells

RPMI supplemented with His Trp Leu

Caspofungin microgml

Ce

lls R

em

ain

ing

(CFU

ml

Phases of in vitro cell killing and persistence with

echinocandins and Candida glabrata

Micafungin

Log C

FU K

idn

ey

Echinocandins are fungicidal yet they act as fungistatic agents in the human therapeutic range in a murine kidney infection model

Howard et al 2011 AAC

Static behavior reflects cell adaptation due to drug stress

1022018

23

Candida glabrata mechanisms of echinocandin tolerance

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slg1Δ (WSC1)

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slt2Δ (MAPK)

Fks1

Fks2 Chs

Rho1

Pkc1

Bck1

MAP2K

MAP1K

Slt2

Crz1Rlm1

Slg1Wsc1

GEFs

Fks1

Chs

Ssd1

Fks2

Bem2

Cnb

1

Cna

1

Fks2

Chs3B

Calcineurin

Inhibitors

Ca2+

HACS

Fks1

Secretory

Pathway

Tethering

Cell Wall

Integrity

PathwayCalcineurin

signaling

Healey and Perlin JoF 2018

HSP90

Disruption of tolerance-associated genes or

pathways lead to increased caspofungin killing

How do we reconcile the emergence of two

independent resistance mechanisms

1022018

24

Msh2

Pms1

MutS recognition of base-base mismatches or small insertionsdeletions

MutL endonuclease activity

Gupta et al 2012 Nat Struct Mol Biol

bull DNA polymerase misincorporation

rate ~10-7 per duplicated nucleotide

bull MMR removes 999 of these

mismatches

Msh6

An underlying defect that increases spontaneous mutation rates

would allow C glabrata to escape drug action by inducing critical

independent resistance mechanisms in real time

DNA mismatch repair

0E+00

1E-07

2E-07

3E-07

4E-07

5E-07

6E-07

7E-07

Casp

ofu

ng

in c

olo

ny f

req

uen

cy

1x 76x 21x 4x 37x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

40E-04

Flu

co

nazo

le c

olo

ny f

req

uen

cy

1x 8x 6x 2x 6x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

Am

pB

Co

lon

y F

req

uen

cy

1x 23x 16x 3x 10x

Fks1Fks2 hotspot

mutationsPdr1 GOF

mutations

Erg6

mutations

C glabrata msh2 Δ msh6Δ msh3Δ and pms1Δ lead to increased frequencies of antifungal-resistant colonies

Mutations in all four components enhance selection for MDR phenotypes

Healey et al 2016 Nat CommUnpubl

Echinocandins Azoles Polyene

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

18

90

10

HS1

Candida albicans (n=47 strains with mutations in FKS1 gene)

28

65

7

FKS1 HS1 mutations

F641

S645

HS2

In C albicans FKS1 hot spot 1 mutations occur 90 of the time two loci S645 and S641 account for 93 of resistance

Perlin ECNR Ref Lab

20

40

40

FKS1 HS2 mutations

R1361H

R1361RH

R1360RK

FKS126

FKS274

Candida glabrata (n=93 strains with mutations in FKS genes)

4

13

33

8

17

17

44

Mutations found in FKS1

D632Y

F625S

S629P

D632G

D632E

I634V

R631G

L660F

54

6

4

4

25

7

Mutations found in FKS2

S663P

F659V

F659S

delF659

others HS1

HS2

FKS2 mutations occur at nearly 3 times the rate of FKS1 mutations S663 (FKS2) and equivalent residue S629 (FKS1) are most prominent

Perlin ECNR Ref Lab

1022018

19

Caspofungin modal MIC variability among independent laboratories using CLSI broth

microdilution method for Candida spp (A Espinel-Ingroff et al AAC 2013)

Micafungin and anidulafungin behave more reliably in AFST (Arendrup et al Mycoses 2014 Pfaller et al JCM 2014)

15X

Caspofungin AFST for Candida spp may vary by

laboratory and is unreliable

CAS testing lsquoEagle effectrsquo (paradoxical growth effect) Difficult reading AFST with CSF should be viewed cautiously or avoided

Kordalewska et al 2018 AAC

Caspofungin Paradoxical Growth in C auris

1022018

20

bull 102 echinocandin sensitive isolates wild-type genotype

bull Echinocandin-resistant isolates S639F

ECHINOCANDINS - FKS1 gene analysis

Species Fks1 HS1

C auris F635LTLSLRDP

C albicans F641LTLSLRDP

C glabrata F625LILSIRDP

C parapsilosis F652LTLSLRDA

C tropicalis F650LTLSLRDP

Milena KORDALEWSKA

Most common site for high-level echinocandin resistance

Echinocandin resistance associated with FKS genotype

Kordalewska et al 2018 AAC

1022018

21

Azoles and echinocandins have non-overlapping

mechanisms of resistance

Mechanism of

resistance

Azoles Echinocandins

Target site modification

ERG11FKS

Target site overexpression

Drug pumps

If azoles and echinocandins have distinct non-

overlapping mechanisms of resistance then what

is driving multidrug resistance in C glabrata

Perlin 2015 Antimicrob Ther Rev

Evolution of Echinocandin Resistance

MIC

1022018

22

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 0016 003 006 012 025 05 1 2 4 8 16 32

Cell killing lt Adaptation

MIC

Cell killing gt Adaptation

Adaptation Persisters

Cell killing

ATCC 2001 HTL cells

Average of 4 independent experiments

24 hour growthsurvival of 1x107 cells

RPMI supplemented with His Trp Leu

Caspofungin microgml

Ce

lls R

em

ain

ing

(CFU

ml

Phases of in vitro cell killing and persistence with

echinocandins and Candida glabrata

Micafungin

Log C

FU K

idn

ey

Echinocandins are fungicidal yet they act as fungistatic agents in the human therapeutic range in a murine kidney infection model

Howard et al 2011 AAC

Static behavior reflects cell adaptation due to drug stress

1022018

23

Candida glabrata mechanisms of echinocandin tolerance

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slg1Δ (WSC1)

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slt2Δ (MAPK)

Fks1

Fks2 Chs

Rho1

Pkc1

Bck1

MAP2K

MAP1K

Slt2

Crz1Rlm1

Slg1Wsc1

GEFs

Fks1

Chs

Ssd1

Fks2

Bem2

Cnb

1

Cna

1

Fks2

Chs3B

Calcineurin

Inhibitors

Ca2+

HACS

Fks1

Secretory

Pathway

Tethering

Cell Wall

Integrity

PathwayCalcineurin

signaling

Healey and Perlin JoF 2018

HSP90

Disruption of tolerance-associated genes or

pathways lead to increased caspofungin killing

How do we reconcile the emergence of two

independent resistance mechanisms

1022018

24

Msh2

Pms1

MutS recognition of base-base mismatches or small insertionsdeletions

MutL endonuclease activity

Gupta et al 2012 Nat Struct Mol Biol

bull DNA polymerase misincorporation

rate ~10-7 per duplicated nucleotide

bull MMR removes 999 of these

mismatches

Msh6

An underlying defect that increases spontaneous mutation rates

would allow C glabrata to escape drug action by inducing critical

independent resistance mechanisms in real time

DNA mismatch repair

0E+00

1E-07

2E-07

3E-07

4E-07

5E-07

6E-07

7E-07

Casp

ofu

ng

in c

olo

ny f

req

uen

cy

1x 76x 21x 4x 37x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

40E-04

Flu

co

nazo

le c

olo

ny f

req

uen

cy

1x 8x 6x 2x 6x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

Am

pB

Co

lon

y F

req

uen

cy

1x 23x 16x 3x 10x

Fks1Fks2 hotspot

mutationsPdr1 GOF

mutations

Erg6

mutations

C glabrata msh2 Δ msh6Δ msh3Δ and pms1Δ lead to increased frequencies of antifungal-resistant colonies

Mutations in all four components enhance selection for MDR phenotypes

Healey et al 2016 Nat CommUnpubl

Echinocandins Azoles Polyene

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

19

Caspofungin modal MIC variability among independent laboratories using CLSI broth

microdilution method for Candida spp (A Espinel-Ingroff et al AAC 2013)

Micafungin and anidulafungin behave more reliably in AFST (Arendrup et al Mycoses 2014 Pfaller et al JCM 2014)

15X

Caspofungin AFST for Candida spp may vary by

laboratory and is unreliable

CAS testing lsquoEagle effectrsquo (paradoxical growth effect) Difficult reading AFST with CSF should be viewed cautiously or avoided

Kordalewska et al 2018 AAC

Caspofungin Paradoxical Growth in C auris

1022018

20

bull 102 echinocandin sensitive isolates wild-type genotype

bull Echinocandin-resistant isolates S639F

ECHINOCANDINS - FKS1 gene analysis

Species Fks1 HS1

C auris F635LTLSLRDP

C albicans F641LTLSLRDP

C glabrata F625LILSIRDP

C parapsilosis F652LTLSLRDA

C tropicalis F650LTLSLRDP

Milena KORDALEWSKA

Most common site for high-level echinocandin resistance

Echinocandin resistance associated with FKS genotype

Kordalewska et al 2018 AAC

1022018

21

Azoles and echinocandins have non-overlapping

mechanisms of resistance

Mechanism of

resistance

Azoles Echinocandins

Target site modification

ERG11FKS

Target site overexpression

Drug pumps

If azoles and echinocandins have distinct non-

overlapping mechanisms of resistance then what

is driving multidrug resistance in C glabrata

Perlin 2015 Antimicrob Ther Rev

Evolution of Echinocandin Resistance

MIC

1022018

22

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 0016 003 006 012 025 05 1 2 4 8 16 32

Cell killing lt Adaptation

MIC

Cell killing gt Adaptation

Adaptation Persisters

Cell killing

ATCC 2001 HTL cells

Average of 4 independent experiments

24 hour growthsurvival of 1x107 cells

RPMI supplemented with His Trp Leu

Caspofungin microgml

Ce

lls R

em

ain

ing

(CFU

ml

Phases of in vitro cell killing and persistence with

echinocandins and Candida glabrata

Micafungin

Log C

FU K

idn

ey

Echinocandins are fungicidal yet they act as fungistatic agents in the human therapeutic range in a murine kidney infection model

Howard et al 2011 AAC

Static behavior reflects cell adaptation due to drug stress

1022018

23

Candida glabrata mechanisms of echinocandin tolerance

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slg1Δ (WSC1)

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slt2Δ (MAPK)

Fks1

Fks2 Chs

Rho1

Pkc1

Bck1

MAP2K

MAP1K

Slt2

Crz1Rlm1

Slg1Wsc1

GEFs

Fks1

Chs

Ssd1

Fks2

Bem2

Cnb

1

Cna

1

Fks2

Chs3B

Calcineurin

Inhibitors

Ca2+

HACS

Fks1

Secretory

Pathway

Tethering

Cell Wall

Integrity

PathwayCalcineurin

signaling

Healey and Perlin JoF 2018

HSP90

Disruption of tolerance-associated genes or

pathways lead to increased caspofungin killing

How do we reconcile the emergence of two

independent resistance mechanisms

1022018

24

Msh2

Pms1

MutS recognition of base-base mismatches or small insertionsdeletions

MutL endonuclease activity

Gupta et al 2012 Nat Struct Mol Biol

bull DNA polymerase misincorporation

rate ~10-7 per duplicated nucleotide

bull MMR removes 999 of these

mismatches

Msh6

An underlying defect that increases spontaneous mutation rates

would allow C glabrata to escape drug action by inducing critical

independent resistance mechanisms in real time

DNA mismatch repair

0E+00

1E-07

2E-07

3E-07

4E-07

5E-07

6E-07

7E-07

Casp

ofu

ng

in c

olo

ny f

req

uen

cy

1x 76x 21x 4x 37x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

40E-04

Flu

co

nazo

le c

olo

ny f

req

uen

cy

1x 8x 6x 2x 6x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

Am

pB

Co

lon

y F

req

uen

cy

1x 23x 16x 3x 10x

Fks1Fks2 hotspot

mutationsPdr1 GOF

mutations

Erg6

mutations

C glabrata msh2 Δ msh6Δ msh3Δ and pms1Δ lead to increased frequencies of antifungal-resistant colonies

Mutations in all four components enhance selection for MDR phenotypes

Healey et al 2016 Nat CommUnpubl

Echinocandins Azoles Polyene

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

20

bull 102 echinocandin sensitive isolates wild-type genotype

bull Echinocandin-resistant isolates S639F

ECHINOCANDINS - FKS1 gene analysis

Species Fks1 HS1

C auris F635LTLSLRDP

C albicans F641LTLSLRDP

C glabrata F625LILSIRDP

C parapsilosis F652LTLSLRDA

C tropicalis F650LTLSLRDP

Milena KORDALEWSKA

Most common site for high-level echinocandin resistance

Echinocandin resistance associated with FKS genotype

Kordalewska et al 2018 AAC

1022018

21

Azoles and echinocandins have non-overlapping

mechanisms of resistance

Mechanism of

resistance

Azoles Echinocandins

Target site modification

ERG11FKS

Target site overexpression

Drug pumps

If azoles and echinocandins have distinct non-

overlapping mechanisms of resistance then what

is driving multidrug resistance in C glabrata

Perlin 2015 Antimicrob Ther Rev

Evolution of Echinocandin Resistance

MIC

1022018

22

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 0016 003 006 012 025 05 1 2 4 8 16 32

Cell killing lt Adaptation

MIC

Cell killing gt Adaptation

Adaptation Persisters

Cell killing

ATCC 2001 HTL cells

Average of 4 independent experiments

24 hour growthsurvival of 1x107 cells

RPMI supplemented with His Trp Leu

Caspofungin microgml

Ce

lls R

em

ain

ing

(CFU

ml

Phases of in vitro cell killing and persistence with

echinocandins and Candida glabrata

Micafungin

Log C

FU K

idn

ey

Echinocandins are fungicidal yet they act as fungistatic agents in the human therapeutic range in a murine kidney infection model

Howard et al 2011 AAC

Static behavior reflects cell adaptation due to drug stress

1022018

23

Candida glabrata mechanisms of echinocandin tolerance

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slg1Δ (WSC1)

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slt2Δ (MAPK)

Fks1

Fks2 Chs

Rho1

Pkc1

Bck1

MAP2K

MAP1K

Slt2

Crz1Rlm1

Slg1Wsc1

GEFs

Fks1

Chs

Ssd1

Fks2

Bem2

Cnb

1

Cna

1

Fks2

Chs3B

Calcineurin

Inhibitors

Ca2+

HACS

Fks1

Secretory

Pathway

Tethering

Cell Wall

Integrity

PathwayCalcineurin

signaling

Healey and Perlin JoF 2018

HSP90

Disruption of tolerance-associated genes or

pathways lead to increased caspofungin killing

How do we reconcile the emergence of two

independent resistance mechanisms

1022018

24

Msh2

Pms1

MutS recognition of base-base mismatches or small insertionsdeletions

MutL endonuclease activity

Gupta et al 2012 Nat Struct Mol Biol

bull DNA polymerase misincorporation

rate ~10-7 per duplicated nucleotide

bull MMR removes 999 of these

mismatches

Msh6

An underlying defect that increases spontaneous mutation rates

would allow C glabrata to escape drug action by inducing critical

independent resistance mechanisms in real time

DNA mismatch repair

0E+00

1E-07

2E-07

3E-07

4E-07

5E-07

6E-07

7E-07

Casp

ofu

ng

in c

olo

ny f

req

uen

cy

1x 76x 21x 4x 37x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

40E-04

Flu

co

nazo

le c

olo

ny f

req

uen

cy

1x 8x 6x 2x 6x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

Am

pB

Co

lon

y F

req

uen

cy

1x 23x 16x 3x 10x

Fks1Fks2 hotspot

mutationsPdr1 GOF

mutations

Erg6

mutations

C glabrata msh2 Δ msh6Δ msh3Δ and pms1Δ lead to increased frequencies of antifungal-resistant colonies

Mutations in all four components enhance selection for MDR phenotypes

Healey et al 2016 Nat CommUnpubl

Echinocandins Azoles Polyene

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

21

Azoles and echinocandins have non-overlapping

mechanisms of resistance

Mechanism of

resistance

Azoles Echinocandins

Target site modification

ERG11FKS

Target site overexpression

Drug pumps

If azoles and echinocandins have distinct non-

overlapping mechanisms of resistance then what

is driving multidrug resistance in C glabrata

Perlin 2015 Antimicrob Ther Rev

Evolution of Echinocandin Resistance

MIC

1022018

22

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 0016 003 006 012 025 05 1 2 4 8 16 32

Cell killing lt Adaptation

MIC

Cell killing gt Adaptation

Adaptation Persisters

Cell killing

ATCC 2001 HTL cells

Average of 4 independent experiments

24 hour growthsurvival of 1x107 cells

RPMI supplemented with His Trp Leu

Caspofungin microgml

Ce

lls R

em

ain

ing

(CFU

ml

Phases of in vitro cell killing and persistence with

echinocandins and Candida glabrata

Micafungin

Log C

FU K

idn

ey

Echinocandins are fungicidal yet they act as fungistatic agents in the human therapeutic range in a murine kidney infection model

Howard et al 2011 AAC

Static behavior reflects cell adaptation due to drug stress

1022018

23

Candida glabrata mechanisms of echinocandin tolerance

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slg1Δ (WSC1)

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slt2Δ (MAPK)

Fks1

Fks2 Chs

Rho1

Pkc1

Bck1

MAP2K

MAP1K

Slt2

Crz1Rlm1

Slg1Wsc1

GEFs

Fks1

Chs

Ssd1

Fks2

Bem2

Cnb

1

Cna

1

Fks2

Chs3B

Calcineurin

Inhibitors

Ca2+

HACS

Fks1

Secretory

Pathway

Tethering

Cell Wall

Integrity

PathwayCalcineurin

signaling

Healey and Perlin JoF 2018

HSP90

Disruption of tolerance-associated genes or

pathways lead to increased caspofungin killing

How do we reconcile the emergence of two

independent resistance mechanisms

1022018

24

Msh2

Pms1

MutS recognition of base-base mismatches or small insertionsdeletions

MutL endonuclease activity

Gupta et al 2012 Nat Struct Mol Biol

bull DNA polymerase misincorporation

rate ~10-7 per duplicated nucleotide

bull MMR removes 999 of these

mismatches

Msh6

An underlying defect that increases spontaneous mutation rates

would allow C glabrata to escape drug action by inducing critical

independent resistance mechanisms in real time

DNA mismatch repair

0E+00

1E-07

2E-07

3E-07

4E-07

5E-07

6E-07

7E-07

Casp

ofu

ng

in c

olo

ny f

req

uen

cy

1x 76x 21x 4x 37x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

40E-04

Flu

co

nazo

le c

olo

ny f

req

uen

cy

1x 8x 6x 2x 6x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

Am

pB

Co

lon

y F

req

uen

cy

1x 23x 16x 3x 10x

Fks1Fks2 hotspot

mutationsPdr1 GOF

mutations

Erg6

mutations

C glabrata msh2 Δ msh6Δ msh3Δ and pms1Δ lead to increased frequencies of antifungal-resistant colonies

Mutations in all four components enhance selection for MDR phenotypes

Healey et al 2016 Nat CommUnpubl

Echinocandins Azoles Polyene

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

22

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 0016 003 006 012 025 05 1 2 4 8 16 32

Cell killing lt Adaptation

MIC

Cell killing gt Adaptation

Adaptation Persisters

Cell killing

ATCC 2001 HTL cells

Average of 4 independent experiments

24 hour growthsurvival of 1x107 cells

RPMI supplemented with His Trp Leu

Caspofungin microgml

Ce

lls R

em

ain

ing

(CFU

ml

Phases of in vitro cell killing and persistence with

echinocandins and Candida glabrata

Micafungin

Log C

FU K

idn

ey

Echinocandins are fungicidal yet they act as fungistatic agents in the human therapeutic range in a murine kidney infection model

Howard et al 2011 AAC

Static behavior reflects cell adaptation due to drug stress

1022018

23

Candida glabrata mechanisms of echinocandin tolerance

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slg1Δ (WSC1)

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slt2Δ (MAPK)

Fks1

Fks2 Chs

Rho1

Pkc1

Bck1

MAP2K

MAP1K

Slt2

Crz1Rlm1

Slg1Wsc1

GEFs

Fks1

Chs

Ssd1

Fks2

Bem2

Cnb

1

Cna

1

Fks2

Chs3B

Calcineurin

Inhibitors

Ca2+

HACS

Fks1

Secretory

Pathway

Tethering

Cell Wall

Integrity

PathwayCalcineurin

signaling

Healey and Perlin JoF 2018

HSP90

Disruption of tolerance-associated genes or

pathways lead to increased caspofungin killing

How do we reconcile the emergence of two

independent resistance mechanisms

1022018

24

Msh2

Pms1

MutS recognition of base-base mismatches or small insertionsdeletions

MutL endonuclease activity

Gupta et al 2012 Nat Struct Mol Biol

bull DNA polymerase misincorporation

rate ~10-7 per duplicated nucleotide

bull MMR removes 999 of these

mismatches

Msh6

An underlying defect that increases spontaneous mutation rates

would allow C glabrata to escape drug action by inducing critical

independent resistance mechanisms in real time

DNA mismatch repair

0E+00

1E-07

2E-07

3E-07

4E-07

5E-07

6E-07

7E-07

Casp

ofu

ng

in c

olo

ny f

req

uen

cy

1x 76x 21x 4x 37x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

40E-04

Flu

co

nazo

le c

olo

ny f

req

uen

cy

1x 8x 6x 2x 6x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

Am

pB

Co

lon

y F

req

uen

cy

1x 23x 16x 3x 10x

Fks1Fks2 hotspot

mutationsPdr1 GOF

mutations

Erg6

mutations

C glabrata msh2 Δ msh6Δ msh3Δ and pms1Δ lead to increased frequencies of antifungal-resistant colonies

Mutations in all four components enhance selection for MDR phenotypes

Healey et al 2016 Nat CommUnpubl

Echinocandins Azoles Polyene

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

23

Candida glabrata mechanisms of echinocandin tolerance

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slg1Δ (WSC1)

1E+00

1E+02

1E+04

1E+06

1E+08

0

00

16

00

3

00

6

01

2

02

5

05 1 2 4 8

16

32

CF

Um

l

[caspofungin] microgml

wt

slt2Δ (MAPK)

Fks1

Fks2 Chs

Rho1

Pkc1

Bck1

MAP2K

MAP1K

Slt2

Crz1Rlm1

Slg1Wsc1

GEFs

Fks1

Chs

Ssd1

Fks2

Bem2

Cnb

1

Cna

1

Fks2

Chs3B

Calcineurin

Inhibitors

Ca2+

HACS

Fks1

Secretory

Pathway

Tethering

Cell Wall

Integrity

PathwayCalcineurin

signaling

Healey and Perlin JoF 2018

HSP90

Disruption of tolerance-associated genes or

pathways lead to increased caspofungin killing

How do we reconcile the emergence of two

independent resistance mechanisms

1022018

24

Msh2

Pms1

MutS recognition of base-base mismatches or small insertionsdeletions

MutL endonuclease activity

Gupta et al 2012 Nat Struct Mol Biol

bull DNA polymerase misincorporation

rate ~10-7 per duplicated nucleotide

bull MMR removes 999 of these

mismatches

Msh6

An underlying defect that increases spontaneous mutation rates

would allow C glabrata to escape drug action by inducing critical

independent resistance mechanisms in real time

DNA mismatch repair

0E+00

1E-07

2E-07

3E-07

4E-07

5E-07

6E-07

7E-07

Casp

ofu

ng

in c

olo

ny f

req

uen

cy

1x 76x 21x 4x 37x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

40E-04

Flu

co

nazo

le c

olo

ny f

req

uen

cy

1x 8x 6x 2x 6x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

Am

pB

Co

lon

y F

req

uen

cy

1x 23x 16x 3x 10x

Fks1Fks2 hotspot

mutationsPdr1 GOF

mutations

Erg6

mutations

C glabrata msh2 Δ msh6Δ msh3Δ and pms1Δ lead to increased frequencies of antifungal-resistant colonies

Mutations in all four components enhance selection for MDR phenotypes

Healey et al 2016 Nat CommUnpubl

Echinocandins Azoles Polyene

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

24

Msh2

Pms1

MutS recognition of base-base mismatches or small insertionsdeletions

MutL endonuclease activity

Gupta et al 2012 Nat Struct Mol Biol

bull DNA polymerase misincorporation

rate ~10-7 per duplicated nucleotide

bull MMR removes 999 of these

mismatches

Msh6

An underlying defect that increases spontaneous mutation rates

would allow C glabrata to escape drug action by inducing critical

independent resistance mechanisms in real time

DNA mismatch repair

0E+00

1E-07

2E-07

3E-07

4E-07

5E-07

6E-07

7E-07

Casp

ofu

ng

in c

olo

ny f

req

uen

cy

1x 76x 21x 4x 37x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

40E-04

Flu

co

nazo

le c

olo

ny f

req

uen

cy

1x 8x 6x 2x 6x

00E+00

50E-05

10E-04

15E-04

20E-04

25E-04

30E-04

35E-04

Am

pB

Co

lon

y F

req

uen

cy

1x 23x 16x 3x 10x

Fks1Fks2 hotspot

mutationsPdr1 GOF

mutations

Erg6

mutations

C glabrata msh2 Δ msh6Δ msh3Δ and pms1Δ lead to increased frequencies of antifungal-resistant colonies

Mutations in all four components enhance selection for MDR phenotypes

Healey et al 2016 Nat CommUnpubl

Echinocandins Azoles Polyene

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

25

n strains carrying msh2 mutationtotal n

()

Center Susceptible FLC-R ECH-R MDR

MD Anderson Cancer

Center414 (29) 99 (100) 01 (0) 01 (0)

Univ Hospital

Lausanne

(Switzerland)

1325 (52) 1115 (73) - -

Hamad Med Corp

(Qatar)2138 (55) 2034 (59) - 22 (100)

Duke Hospital 1936 (53) 613 (46) 28 (25) 48 (50)

Centers for Disease

Control5299 (53) 02 (0) - 917 (53)

Wayne State Univ 79 (78) 79 (78) - -

Miscellaneous 14 (25) 11 (100) 16 (17) 66 (100)

Univ Delhi (India) 4182 (51) 11 (100) - -

TOTAL 158307

(52)

5584

(65)

315

(20)

2134

(62)

0

10

20

30

40

50

60

70

80

90

100

s

tra

ins

wit

h m

uta

ted

ms

h2

Msh2 nonsynonymous mutations identified in 55

of 440 geographically diverse clinical isolates

P lt 005

P lt 001

Healey et al 2016 Nat Comm

DNA

Cg P208S

Cg E231G

Cg V239L

Cg L269FS

HTH dimer

contacts

Core

Clamp

Mismatch

ATPase

Connector

Bacterial Msh2 crystal structure

Meindert et al 2000 Nature Obmolova et al 2000 Nature

C glabrata clinical isolatesrsquo msh2 mutations map

to the connector domain

Msh2 connector

domain essential for

interaction with the

MutL complex and

MMR activity (Mendillo et al 2009 PNAS)

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

26

C glabrata gastrointestinal

colonizationprophylaxis model

PTZ (daily)

Antifungal treatment (ip or oral)

Day -2 0 1 3 5 7 9 11 13 15

15 x 108 CFU

C glabrata

fecal collection GI burden and antifungal resistance plating

blood kidney

liver and spleen

collection

DMX

(2x daily)

GI tract model for breakthrough resistance

Healey et al 2017 AAC Healey et al 2016 Nat Comm

00E+00

10E-01

20E-01

30E-01

40E-01

50E-01

60E-01

70E-01

0 1 3 5 7 9 11 13 15 17 19 21

Fre

qu

en

cy o

f re

sis

tan

ce

Days post inoculation

CSF 05 CSF 5

CSF 20 C-466 05

C-466 15 C-466 30 C-466 30

C-466 15

CSF 20

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

1E+10

0 1 3 5 7 9 11 13 15 17 19 21

CF

Ug

ram

sto

ol

Day post inoculation

CSF 05 CSF 5CSF 20 C-466 05C-466 15 C-466 30vehicle

Caspofungin - daily Dexamethasone

Resistance acquisition (Fks2-S663P)

High GI tract drug exposure leads to resistance among

colonized organisms

Immunosuppression breaches GIT leading to systemic infection with

resistant isolates

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

27

1E+00

1E+01

1E+02

1E+03

1E+04

1E+05

1E+06

1E+07

1E+08

1E+09

0 1 3 5 7 9 11 13 15

CF

Ug

ram

sto

ol

Days post inoculation

ATCC 2001 (Msh2-WT)Isolate 1 (Msh2-P208SN890IIsolate 2 (Msh2-E231GL269F)Isolate 3 (Msh2-V239L)Isolate 4 (Msh2-E456D)Isolate 5 (Msh2-E459K)

Daily caspofungin treatment (20 mgkg ip)

Resistance acquisition (fks mutation) in ge 1 mouse

Average GI burdens of mice colonized with the indicated strain and treated with high dose (20 mgkg) of caspofungin

Note 5 mgkg of CSF is considered the equivalent humanized dose

Clinical isolates harboring specific Msh2 alleles show enhanced capacity to develop drug resistance

pdr1 GOF mutations identified within fluconazole-

resistant clinical isolates- MSH2 prevalence

Msh2 wild type genotype

Msh2 mut genotype

Healey unpubl

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

28

Mismatch repair is not an absolute driver in all patients

populations for mono-resistance but appears to be more strongly

linked to MDR phenotypes and resistance sequence types

Fluconazole and Echinocandin Resistance of Candida glabrata Correlates Better with Antifungal Drug Exposure Rather than with MSH2 Mutator Genotype in a French Cohort of Patients Harboring Low Rates of ResistanceDelliegravere S Healey K Gits-Muselli M Carrara B Barbaro A Guigue N LecefelC Touratier S Desnos-Ollivier M Perlin DS Bretagne S Alanio AFront Microbiol 2016 Dec 2372038

Front Microbiol 2018 Jul 1391523 doi 103389fmicb201801523 eCollection 2018Multilocus Sequence Typing (MLST) Genotypes of Candida glabrataBloodstream Isolates in Korea Association With Antifungal Resistance Mutations in Mismatch Repair Gene (Msh2) and Clinical OutcomesByun SA1 Won EJ1 Kim MN2 Lee WG3 Lee K4 Lee HS5 Uh Y6 Healey KR7 Perlin DS7 Choi MJ1 Kim SH1 Shin JH1

Different C glabrata sequence types (ST) carry various msh2 alleles and may have different capacity for evolving drug resistance- ST correlates with unique karyotypes

Lott et al Eukaryotic Cell 2010Healey et al Frontiers in Microbiology 2016

Erika ShorCristina Jiminez-Ortogosa

Healey et al 2016 Nat Comm

1022018

29

Sequence type (ST) percentages of susceptible fluconazole-resistant

echinocandin-resistant and multidrug (MDR) resistant strains isolated from

various US centers Yellow highlight encompasses STs with partial LOF Msh2

alleles (see Table 1) ST10 and ST16 are nearly exclusive to US isolates

Sequence type distribution of US-based isolates

Genomic plasticity in C glabrata aids genetic diversity

1022018

30

To define the genomes of prevalent C glabrata STrsquos we performed

bull PacBio WG sequencing of clinical strains of 7 predominant STs from different hospitals in the US and from Qatar (CDC)

bull Optical restriction mapping of the same strains (CDC)

bull WGS of 25 strains 6 predominant STs (Duke collection) ST46 (Qatar) and Indian isolates of new STs

C glabrata reference strain (CBS138) Chr D

C glabrata isolate 1 Chr D

C glabrata isolate 2 Chr D

C glabrata isolate 3 Chr D

BamHI sites

C glabrata isolate 1 Chr L

C glabrata isolate 3 hybrid chromosome L-I with an inversion in the Chr L-derived region

C glabrata reference strain Chr L

C glabrata reference strain Chr I

Erika Shor PHRI-Rutgers)Vladimir Loparev (CDCOIDNCEZID)Shawn Lockhart (CDCOIDNCEZID)

ATCC2001 BamHI optical map generated in silico

Clinical isolate BamHI optical map generated experimentally

Reciprocal translocation between Chr I and M

ATCC2001 chromosomes

Clinical isolate chromosomes

Deep dive chromosomal analyses using linear optical mapping and and WGS of C glabrata ST

Erika Shor PHRI-Rutgers)Vladimir Loparev (CDCOIDNCEZID)Shawn Lockhart (CDCOIDNCEZID)

1022018

31

Optical maps and long-read PacBio sequencing reveals numerous crossover and recombinational events that define sequence types

Erika Shor PHRI-Rutegrs)Loparev Vladimir (CDCOIDNCEZID)Shawn Lockhart (CDCOIDNCEZID)

1022018

32

bull Optical mapping detect large scale genome changes

bull Significant rearrangements detected between STs and within STs

bull Sub-telomeric regions are highly subject to changes that result in scrambling of restriction patterns and appearance of novel patterns

bull Maximizing genetic diversity through chromosomal plasticity is a hallmark of C glabrata and other fungal species

Preliminary conclusions from optical mapping

Summary

bull Overall rates of antifungal resistance among Candida species are low

bull Drug resistance is a growing problem with C glabrata

bull DNA mismatch repair (MMR) defects lead to greater frequencies of

antifungal-resistant mutants- may be a factor for genetic diversity

bull Certain clades of C glabrata carry a greater potential for resistance

development

bull The ability of C glabrata to maximize genetic diversity allows for rapid

adaptable to drugs and environment

1022018

33

Acknowledgments

Perlin lab K Healey Y Zhao W Perez C

Jimenez Ortigosa E Shor P Paderu M

Kordalewska G Rasic M Mina E Dolgov I

Kolesnikova G Weiderman M He Lee S

Satish E Feketeova A Lee

Collaborators

bull SR Lockhart US Centers for Disease

Control and Prevention

bull DP Kontoyiannis- MD Anderson CC

bull B Alexander Duke University

bull D Sanglard University Hospital of

Lausanne

bull SJ Taj-Aldeen Hamad Medical

Corporation

bull Anuradha Chowdhary New Delhi

bull Alexandre Alaino Pasteur Institute

Funding

DP ndash NIH R01AI109025 AstellasCidara CDC

KH ndashArnold and Mabel Beckman Foundation

1022018

30

To define the genomes of prevalent C glabrata STrsquos we performed

bull PacBio WG sequencing of clinical strains of 7 predominant STs from different hospitals in the US and from Qatar (CDC)

bull Optical restriction mapping of the same strains (CDC)

bull WGS of 25 strains 6 predominant STs (Duke collection) ST46 (Qatar) and Indian isolates of new STs

C glabrata reference strain (CBS138) Chr D

C glabrata isolate 1 Chr D

C glabrata isolate 2 Chr D

C glabrata isolate 3 Chr D

BamHI sites

C glabrata isolate 1 Chr L

C glabrata isolate 3 hybrid chromosome L-I with an inversion in the Chr L-derived region

C glabrata reference strain Chr L

C glabrata reference strain Chr I

Erika Shor PHRI-Rutgers)Vladimir Loparev (CDCOIDNCEZID)Shawn Lockhart (CDCOIDNCEZID)

ATCC2001 BamHI optical map generated in silico

Clinical isolate BamHI optical map generated experimentally

Reciprocal translocation between Chr I and M

ATCC2001 chromosomes

Clinical isolate chromosomes

Deep dive chromosomal analyses using linear optical mapping and and WGS of C glabrata ST

Erika Shor PHRI-Rutgers)Vladimir Loparev (CDCOIDNCEZID)Shawn Lockhart (CDCOIDNCEZID)

1022018

31

Optical maps and long-read PacBio sequencing reveals numerous crossover and recombinational events that define sequence types

Erika Shor PHRI-Rutegrs)Loparev Vladimir (CDCOIDNCEZID)Shawn Lockhart (CDCOIDNCEZID)

1022018

32

bull Optical mapping detect large scale genome changes

bull Significant rearrangements detected between STs and within STs

bull Sub-telomeric regions are highly subject to changes that result in scrambling of restriction patterns and appearance of novel patterns

bull Maximizing genetic diversity through chromosomal plasticity is a hallmark of C glabrata and other fungal species

Preliminary conclusions from optical mapping

Summary

bull Overall rates of antifungal resistance among Candida species are low

bull Drug resistance is a growing problem with C glabrata

bull DNA mismatch repair (MMR) defects lead to greater frequencies of

antifungal-resistant mutants- may be a factor for genetic diversity

bull Certain clades of C glabrata carry a greater potential for resistance

development

bull The ability of C glabrata to maximize genetic diversity allows for rapid

adaptable to drugs and environment

1022018

33

Acknowledgments

Perlin lab K Healey Y Zhao W Perez C

Jimenez Ortigosa E Shor P Paderu M

Kordalewska G Rasic M Mina E Dolgov I

Kolesnikova G Weiderman M He Lee S

Satish E Feketeova A Lee

Collaborators

bull SR Lockhart US Centers for Disease

Control and Prevention

bull DP Kontoyiannis- MD Anderson CC

bull B Alexander Duke University

bull D Sanglard University Hospital of

Lausanne

bull SJ Taj-Aldeen Hamad Medical

Corporation

bull Anuradha Chowdhary New Delhi

bull Alexandre Alaino Pasteur Institute

Funding

DP ndash NIH R01AI109025 AstellasCidara CDC

KH ndashArnold and Mabel Beckman Foundation

1022018

31

Optical maps and long-read PacBio sequencing reveals numerous crossover and recombinational events that define sequence types

Erika Shor PHRI-Rutegrs)Loparev Vladimir (CDCOIDNCEZID)Shawn Lockhart (CDCOIDNCEZID)

1022018

32

bull Optical mapping detect large scale genome changes

bull Significant rearrangements detected between STs and within STs

bull Sub-telomeric regions are highly subject to changes that result in scrambling of restriction patterns and appearance of novel patterns

bull Maximizing genetic diversity through chromosomal plasticity is a hallmark of C glabrata and other fungal species

Preliminary conclusions from optical mapping

Summary

bull Overall rates of antifungal resistance among Candida species are low

bull Drug resistance is a growing problem with C glabrata

bull DNA mismatch repair (MMR) defects lead to greater frequencies of

antifungal-resistant mutants- may be a factor for genetic diversity

bull Certain clades of C glabrata carry a greater potential for resistance

development

bull The ability of C glabrata to maximize genetic diversity allows for rapid

adaptable to drugs and environment

1022018

33

Acknowledgments

Perlin lab K Healey Y Zhao W Perez C

Jimenez Ortigosa E Shor P Paderu M

Kordalewska G Rasic M Mina E Dolgov I

Kolesnikova G Weiderman M He Lee S

Satish E Feketeova A Lee

Collaborators

bull SR Lockhart US Centers for Disease

Control and Prevention

bull DP Kontoyiannis- MD Anderson CC

bull B Alexander Duke University

bull D Sanglard University Hospital of

Lausanne

bull SJ Taj-Aldeen Hamad Medical

Corporation

bull Anuradha Chowdhary New Delhi

bull Alexandre Alaino Pasteur Institute

Funding

DP ndash NIH R01AI109025 AstellasCidara CDC

KH ndashArnold and Mabel Beckman Foundation

1022018

32

bull Optical mapping detect large scale genome changes

bull Significant rearrangements detected between STs and within STs

bull Sub-telomeric regions are highly subject to changes that result in scrambling of restriction patterns and appearance of novel patterns

bull Maximizing genetic diversity through chromosomal plasticity is a hallmark of C glabrata and other fungal species

Preliminary conclusions from optical mapping

Summary

bull Overall rates of antifungal resistance among Candida species are low

bull Drug resistance is a growing problem with C glabrata

bull DNA mismatch repair (MMR) defects lead to greater frequencies of

antifungal-resistant mutants- may be a factor for genetic diversity

bull Certain clades of C glabrata carry a greater potential for resistance

development

bull The ability of C glabrata to maximize genetic diversity allows for rapid

adaptable to drugs and environment

1022018

33

Acknowledgments

Perlin lab K Healey Y Zhao W Perez C

Jimenez Ortigosa E Shor P Paderu M

Kordalewska G Rasic M Mina E Dolgov I

Kolesnikova G Weiderman M He Lee S

Satish E Feketeova A Lee

Collaborators

bull SR Lockhart US Centers for Disease

Control and Prevention

bull DP Kontoyiannis- MD Anderson CC

bull B Alexander Duke University

bull D Sanglard University Hospital of

Lausanne

bull SJ Taj-Aldeen Hamad Medical

Corporation

bull Anuradha Chowdhary New Delhi

bull Alexandre Alaino Pasteur Institute

Funding

DP ndash NIH R01AI109025 AstellasCidara CDC

KH ndashArnold and Mabel Beckman Foundation

1022018

33

Acknowledgments

Perlin lab K Healey Y Zhao W Perez C

Jimenez Ortigosa E Shor P Paderu M

Kordalewska G Rasic M Mina E Dolgov I

Kolesnikova G Weiderman M He Lee S

Satish E Feketeova A Lee

Collaborators

bull SR Lockhart US Centers for Disease

Control and Prevention

bull DP Kontoyiannis- MD Anderson CC

bull B Alexander Duke University

bull D Sanglard University Hospital of

Lausanne

bull SJ Taj-Aldeen Hamad Medical

Corporation

bull Anuradha Chowdhary New Delhi

bull Alexandre Alaino Pasteur Institute

Funding

DP ndash NIH R01AI109025 AstellasCidara CDC

KH ndashArnold and Mabel Beckman Foundation