Colistin Heteroresistance in Carbapenem-Resistant Klebsiella Pneumoniae

Colistin heteroresistance in carbapenem-resistant Klebsiella pneumoniae

Jean-Marc Rolain Aix Marseille University, IRD, APHM, MEPHI, IHU-Méditerranée Infection – Marseille – France Email: [email protected]

ESCMIDSaturday, 13 April 019, Amsterdam,eLibrary Netherlands © by author No conflict of interest

ESCMID eLibrary © by author Outline of the lecture

– History of emergence of resistance to colistin – Mechanism of action and resistance • General mechanisms • Definition of heteroresistance – What is known in K. pneumoniae and related bacteria : a review of the literature – Heteroresistance: does it matter? – Conclusion

ESCMID eLibrary3 © by author Consequences of 3GC resistance: increase use of carbapenems and increase of carbapenem resistance in Gram-negative bacteria [ESBL or carbapenemase] and K. pneumoniae keywords 150 [PubMed, 1990-2011]

100 Klebsiella pneumoniae 50

0 NB ofNBpublications

Year

Nb of publications (ESBL) Nb of publications (carbapenemase)

Kempf et al, Int J Antimicrob Agents 2012

Increasing use of colistin

ESCMIDIncrease UseeLibrary of colistin And …… © by author Colistin therapy for 3 – 6 months for MDR P. aeruginosa orthopedic infections

ESCMID eLibrary © by author Colistin : mechanism of action and resistance

 Polymyxins:  Polymyxin B and colistin (polymyxin E)  discovered in 1947 in Paenibacillus polymyxa (formerly Bacillus polymyxa var. colistinus)  available since 1959 for treatment of Gram negative bacterial infections  cyclic polypeptide antibiotics  secondary metabolite from nonribosomal peptide synthase (NRPS)  mode of action : on bacterial cell membrane, binds to LPS and phospholipids  in the outer cell membrane and displaces divalent cations (Ca and Mg)  Cationic antimicrobial polypeptides  Bactericidal activity against most Gram-negative bacteria

 Spectrum of activity: Two major categories of Gram-negative bacteria i. Naturally susceptible bacteria (Escherichia coli, Klebsiella pneumoniae, Salmonella, Acinetobacter baumannii, Pseudomonas aeruginosa) Active

ii. Intrinsically resistant bacteria (Proteus, Providencia, Morganella, ESCMIDSerratia, Burkholderia) Not active eLibrary © by author General Mechanism of Colistin Resistance through Lipid A Modifications Review In Gram-negative Bacteria

+ 4-amino-4-deoxy-L-arabinose - arnT-pmrE operon -Phosphoethanolamine + + pmrA-pmrB mutation pmrC

pmrD Lipid A + phoP-phoQ mutation pmrA-pmrB mutation mgrB mutation

(negative regulator of phoPQ) ESCMIDOlaitan AO et al, FronteLibrary Microbiol 2014; Baron S et al, Int J Antimicrob Agents 2016 © by author Background Overall colistin resistance mechanism

1st description of a transferable gene confering colistin resistance - mcr-1 Liu YY, et al TheLancet Infectious Disease 2015

Molecular mechanisms of polymyxin resistance: knowns and unknowns. Baron S, Hadjadj L, Rolain JM, Olaitan AO. Int J AntimicrobESCMID Agents. 2016 Dec;48(6):583-591. eLibrary © by author Heteroresistance

• Heteroresistance : phenomenon wherein a subpopulation of bacteria in an otherwise homogenous population display antibiotic resistance

Difficulty to detect colistin heteroresistance with routine laboratory tests (broth microdilution or agar)

A. Brauner, etESCMID al. Nat. Rev. Microbiol., 2016 eLibrary © by author Constant Colistin Heteroresistance

• Disk diffusion method or E-test : first inhibition zone followed by regrowth in high colistin concentrations • Broth microdilution method : resistant or ‘skipped’ wells • Described in Gram negative bacteria for: – Serratia marcescens -> Intrinsic resistance Role of efflux? – Enterobacter spp.-> Acquired resistance

Telke A, J. of Antimicrob Chemother., 2017 ESCMIDBaron SA, J. of Antimicrob Chemother., 2018 eLibrary © by author PAPs : Population Analysis Profiles Colistin mg/L Control 0,125 0,25 0,5 1 2

MIC True Colistin susceptibility?

0,5 McF suspension

Colistin susceptible

Colistin Hetero- resistant Control 0,125 0,25 0,5 1 2 4 8 16 32 Colistin mg/L ESCMID eLibraryPersistance of colonies = Heteroresistance © by author Time Kill Assays of colistin against colistin-heteroresistant K. pneumoniae isolates

HR isolate HR isolate

• Regrowth observed after 4h in all the heteroresistant (HR) isolates • Concentrations needed to kill HR isolates unachievable in vivo

HR isolate Susceptible isolate ESCMID eLibrary © by author Development of Diagnostic tools : the problematic of colistin resistance

• Colistin Resistance defined as MIC > 2µg/ml –isolates with borderline MICs • Disk diffusion method is not reproducible for detection  broth microdilution

Carba R (MIC > 2mg/L) n = 209 n = 256 72,6 % 89 %

ESCMID eLibrary © by author Colistin heteroresistance in carbapenem-resistant K. pneumoniae – Review of the literature

20 carbapenem resistant K. pneumoniae

16 colistin-susceptible isolates by BMD

12 isolates colistin-heteroresistant ESCMID(PAPs) eLibrary © by author Heteroresistance in K. pneumoniae

2015

Emergence of a colistin-resistant subclone dueESCMID to mutation Asp191Tyr in protein PhoP eLibrary © by author ESCMID eLibrary © by author Mechanisms of Heteroresistance in K. pneumoniae

Mutations found in mgrB, phoQ, yciM, lpxM yciM contributes to cell wall integrity by regulating LPS biosynthesis LpxM is responsible for the addition of one of the secondary acyl chains to lipid A in Enterobacteriaceae HalabyESCMIDT, Antimicrob, Agents and Chemother, 2016 eLibrary © by author 2017 Emergence of a colistin-resistant subclone due to mutations in mgrB

KP_LB1 CMI > 48 µg/mL

KP_LB2 CMI = 0.125 µg/mL ESCMID eLibrary © by author Typing subpopulation

1) LB1 LB2 3) 4) AMX-6 AMC-14 FEP-34 TPZ-25 AMX-6 AMC-14 FEP-34 TPZ-25

ATM-32 CRO-31 ETP-30 IPM-30 ATM-32 CRO-31 ETP-30 IPM-30

RIF-10 NF-15 FF-20 CN-22 RIF-10 NF-15 FF-20 CN-22

CIP-30 SXT-6 Colistin-10 NA-22 CIP-30 SXT-6 Colistin-15 NA-22

1) Same AST 2) MLST : ST86 3) Same PFGE (XbaI) 4) Same MALDI-TOF Gel View BardetESCMIDet al, Antimicrob Agents Chemother., 2017 eLibrary © by author Other example in Enterobacter

Efflux pump overexpression due to a complex mechanism!!

Efflux pump inhibitor

E. cloacae NH52 E. asburiae E. cloacae NH52 E. asburiae

Temoin PAβN ESCMIDTelke A et al , J Antimicrob Chemother, 2017; Dandachi I et al submitted eLibrary © by author Carbonyl Cyanide m- 2,4-dinitrophenol (DNP) X X Chlorophenylhydrazine (CCCP)

ME: membrane externe Efflux pump inhibitor

-> Objectif: Evaluate the effect of CCCP on a collection of colistin-resistant strains ESCMIDof Gram negative eLibrary bacteria. © by author Role of efflux in Gram negative bacteria

[CCCP]= 10 mg/L

Strains tested: -90 Enterobacteriaceae including: -32 K. pneumoniae -24 E. coli -7 Enterobacter sp. -2 Salmonella sp. -13 S. marcescens -3 Morganella morganii -4 Providencia spp. -3 Pseudomonas aeruginosa

CCCP rescue also colistin heteroresistance

ESCMIDBaron S and Rolain JM , J Antimicrob Chemother, 2018 eLibrary © by author Heteroresistance and antibiotic failure Does it matter?

E-test

Nat. microbiol, 2016

• Suggested in Enterobacter cloacae • Not detected with E-test method • Responsible for treatment failure in mice

Survival % in infection model on mice caused by a susceptible strain (left) and the resistant subpopulation (right) PAP ESCMID eLibrary © by author Heteroresistance: does it matter?

• Not new but probably under-diagnosed • Need to improve the detection of heteroresistance in routine laboratory • Understanding the real impact of this heteroresistance : – Risk of therapeutic failure ? – Alternatives?

• Management of the use of colistin to treat carbapenem-resistant K. pneumoniae • Limit its use ? • Only in combination ? ESCMID eLibrary © by author ESCMID eLibraryStaff and students from my team © by author Thank you for your attention