Does this Meropenem E-test Point Carbapenem Heteroresistance in

OXA-48 producing Klebsiella pneumoniae?

E López-Camacho & J Mingorance

IDIPAZ, Madrid

March 1st, 2104

Meropenem E-test of a K.pneumoniae isolate

Meropenem E-test of colonies from the inhibition zone of

the same K.pneumoniae isolate

Heteroresistance phenomenon

Re-growth in blood-agar of

colonies from the inhibition zone of

the E-test

Heteroresistance phenomenon

Drug- sensitive population

Spread in high drug concentration plates (Ej: 64xMIC)

Resistant colonies

Drug- sensitive population

48h – 72h

• Resistance to certain antibiotics expressed by a ISOGENIC SUB-POPULATION of a microbial population that is generally considered to be susceptible to these antibiotics according to traditional in-vitro susceptibility testing

• Phenotypic manifestation!

– Not stable

– Not heritable

Heteroresistance

Heteroresistant Population• Mixed populations of drug-resistant and drug-sensitive cells in a

single clinical specimen or isolate where the proportion of resistant organims cannot be explained by the natural mutation rate

Drug- sensitive population

Mutant frequency: ~10-8 – 10-9

Heteroresistant population

Resistant (but NOT_MUTANT) frequency: ~10-5 – 10-6

Heteroresistance has been observed in a range of microbes and antibiotics:

• GLYCOPEPTIDES– S.aureus MRSA (vancomycin)– Coagulase-negative staphylococci

• PENICILLIN– S.pneumoniae

• CARBAPENEMS– K.pneumoniae - E.faecalis– A.baumanii - P.aeruginosa

• COLISTIN– K.pneumoniae– A.baumanii

• OTHERS– METRONIDAZOLE (Gadnerella vaginalis)– FLUCONAZOLE (Criptococcus neoformans)

- S.epidermidis - S.haemolyticus- S.auricularis - S.simulans- S.capitis - S.warneri

• Experimental design to quantify the resistant sub-population as a function of antibiotic concentration

Population analysis profiles (PAP-CURVES)

0 0,1250,25

0,51

24

816

32

Set of plates with increasing antibiotic concentrations (µg/ml)

1/10 Serial dilutions of a stationary-phase culture

0 -1 -2 -3 -4 -5 -60

• Spread culture

• Incubation (24-48h)

• Colony counting

• Graphical representation

109 108 107 106 105 104 103

0 5 10 15 20 25 30 35

Resistant-mutant

Heteroresistant

wild-type

10-9

10-8

10-7

10-6

10-5

10-4

10-3

10-2

10-1

1

Antibiotic concentration (µg/ml)

FR

EQ

UE

NC

Y

(res

ista

nts

cfu

/ml r

elat

ive

to v

iab

les

cfu

/ml)

Model PAP-curve

Mutation

frequency

Analysis of Carbapenem

Heteroresistance in

K.pneumoniae OXA-48

Carbapenem hetroresistance in K.pneumoniae OXA-48?

Clone 1 (ST405) Clone 11 (ST11)

PAP-curves of KPOXA-48 Clone 1 (ST405)

0 5 10 15 20 25 30 35

CLONE 1 (1670) CMI=1

CLONE 1 (3210) CMI=2

CLONE 1 Belgium CMI=0,5

CLONE 10 (porin mutant)

Meropenem Concentration (µg/ml)

FR

EQ

UE

NC

Y

10-9

10-8

10-7

10-6

10-5

10-4

10-3

10-2

10-1

1

PAP-curves of KPOXA-48 Clone 11

(ST11)

0 5 10 15 20 25 30 35

CLONE 11 (0973) CMI=4

CLONE 11 (5588) CMI=2

CLONE 11 (7533) CMI=2

CLONE 10 (porin mutant)

10-9

10-8

10-7

10-6

10-5

10-4

10-3

10-2

10-1

1

Meropenem Concentration (µg/ml)

FR

EQ

UE

NC

Y

PAP-curve of a KPOXA-48 sporadic Clon: Clon 13 (ST45)

0 5 10 15 20 25 30 35

CLONE 10 (porin mutant)

CLONE 13 CMI=4

10-9

10-8

10-7

10-6

10-5

10-4

10-3

10-2

10-1

1

Meropenem Concentration (µg/ml)

F

RE

QU

EN

CY

Conclusions

• No carbapenem heteroresistance in K.pneumoniae OXA-48

• Cells grown at high antibiotic concentrations were resistant mutants

• The presence of colonies in the inhibition zone of the E-test is not indicative of heteroresistance