can meropenem heteroresistance in oxa-48-producing k. pneumoniae be inferred from an e-test?
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TRANSCRIPT
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