This document discusses the phenomenon of heteroresistance in bacteria and analyzes whether it occurs in OXA-48 producing Klebsiella pneumoniae. It finds that population analysis profile curves did not show evidence of heteroresistance in various clones of OXA-48 K. pneumoniae, as cells growing at high carbapenem concentrations were resistant mutants rather than a heteroresistant subpopulation. The presence of colonies growing in the inhibition zone of meropenem E-tests was also not indicative of heteroresistance in this case. In conclusion, heteroresistance was not observed in OXA-48 producing K. pneumoniae.

1. Does this Meropenem E-test Point Carbapenem
Heteroresistance in
OXA-48 producing Klebsiella pneumoniae?
E López-Camacho & J Mingorance
IDIPAZ, Madrid
March 1st, 2104

2. Heteroresistance phenomenon
Re-growth in
blood-agar of
colonies from the
inhibition zone of
the E-test
Meropenem E-test of a
K.pneumoniae isolate
Meropenem E-test of colonies
from the inhibition zone of
the same K.pneumoniae
isolate

3. Heteroresistance phenomenon
Spread in high drug
concentration plates (Ej:
64xMIC)
Drug- sensitive population
Resistant colonies
48h – 72h
Drug- sensitive population

4. Heteroresistance
• 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

5. 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
Heteroresistant population
Mutant frequency: ~10-8 – 10-9
Resistant (but NOT_MUTANT)
frequency: ~10-5 – 10-6

6. Heteroresistance has been observed in a
range of microbes and antibiotics:
•
GLYCOPEPTIDES
– S.aureus MRSA (vancomycin)
– Coagulase-negative staphylococci
•
PENICILLIN
- S.epidermidis
- S.auricularis
- S.capitis
– S.pneumoniae
•
CARBAPENEMS
– K.pneumoniae
– A.baumanii
•
- E.faecalis
- P.aeruginosa
COLISTIN
– K.pneumoniae
– A.baumanii
•
OTHERS
– METRONIDAZOLE (Gadnerella vaginalis)
- S.haemolyticus
- S.simulans
- S.warneri

7. Population analysis profiles (PAP-CURVES)
• Experimental design to quantify the resistant subpopulation as a function of antibiotic concentration
1/10 Serial dilutions of a
stationary-phase culture
Set of plates with increasing
antibiotic concentrations
(µg/ml)
0 0,125
0,25
0,5
1
2
4
8
• Spread culture
• Incubation (24-48h)
• Colony counting
• Graphical representation
16
32
0
109
-1
-2
-3
-4
-5
-6
108 107 106 105 104 103

8. (resistants cfu/ml relative to viables cfu/ml)
FREQUENCY
Model PAP-curve
1
10-1
Resistant-mutant
Heteroresistant
wild-type
10-2
10-3
10-4
10-5
10-6
Mutation
frequency
10-7
10-8
10-9
0
5
10
15
20
25
Antibiotic concentration (µg/ml)
30
35

9. Analysis of Carbapenem
Heteroresistance in
K.pneumoniae OXA-48

10. Carbapenem hetroresistance in K.pneumoniae
OXA-48?
Clone 1 (ST405)
Clone 11 (ST11)

11. PAP-curves of KPOXA-48
Clone 1 (ST405)
1
10
CLONE 1 (1670) CMI=1
-1
CLONE 1 (3210) CMI=2
CLONE 1 Belgium CMI=0,5
FREQUENCY
10-2
10
CLONE 10 (porin mutant)
-3
10-4
10-5
10-6
10-7
10-8
10-9
0
5
10
15
20
25
Meropenem Concentration (µg/ml)
30
35

12. PAP-curves of KPOXA-48 Clone 11
(ST11)
1
CLONE 11 (0973) CMI=4
CLONE 11 (5588) CMI=2
10-1
CLONE 11 (7533) CMI=2
FREQUENCY
10
-2
CLONE 10 (porin mutant)
10-3
10-4
10-5
10-6
10-7
10-8
10-9
0
5
10
15
20
25
Meropenem Concentration (µg/ml)
30
35

13. PAP-curve of a KPOXA-48 sporadic
Clon: Clon 13 (ST45)
1
CLONE 10 (porin mutant)
10-1
CLONE 13 CMI=4
FREQUENCY
10-2
10-3
10-4
10-5
10-6
10-7
10-8
10-9
0
5
10
15
20
25
Meropenem Concentration (µg/ml)
30
35

14. 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 Etest is not indicative of heteroresistance