Antimicrobial

Antimicrobial susceptibility
susceptibility

Michael R. Jacobs, MD, PhD

Case Western Reserve University
University Hospitals of Cleveland
Cleveland, OH
 Determinants of
clinical success
Bacteria
Y
Y Host Antibiotics
defenses
Y

PK/PD
profile
Bacterial
eradication

Clinical
success
 Determining “potency” of
antimicrobial agents
Minimal inhibitory concentration (MIC)
determination
Minimal bactericidal concentration (MBC)
determination
Subinhibitory and post-antibiotic effects (SME
and PAE)
Interpreting MICs
• MIC50 and MIC90 values
• MIC distributions
• Interpretative breakpoints
Minimal inhibitory concentration
(MIC): measure of “potency”
MICs are the most common measures used for the
evaluation of antimicrobial activity

MICs are in vitro measurements:

• the antibiotic concentration required to
completely inhibit visible growth in a test tube
• Bacterial inoculum, medium and incubation
time are standardized
• Antibiotic concentration is constant
 Minimal inhibitory
concentration
Known quantity of bacteria
placed into each tube

0 0.25 0.5 1.0 2.0 4.0 8.0 16

µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
Increasing antibiotic
concentration
 Minimal inhibitory
concentration
Lowest concentration of an
Known quantity of bacteria
antimicrobial that results in the
placed into each tube
inhibition of visible growth of a
microorganism

0 0.25 0.5 1.0 2.0 4.0 8.0 16

µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
Increasing antibiotic
concentration
 Minimal inhibitory
concentration
The more “potent” the antibiotic, the less is
needed to kill the bacteria, and the MIC is
LOWER

0 0.25 0.5 1.0 2.0 4.0 8.0 16

µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL
Increasing antibiotic
concentration
MIC90: the lowest concentration that
includes at least 90% of the strains
tested
93%
25

20

15
% of
strains
10
5%

5
2%

0
0.06

0.25
0.12

4
0.5

2
0.01

0.02

0.03

16
8
MIC (µg/ml) MIC90=4 µg/ml
MIC50=0.25 µg/ml
MIC50 and MIC90 unimodal population

50% 90%

0.03 0.06 012 0.25 0.5 1 2 4 0.5

MIC (ug/ml) MIC50

50 MIC90
90
 MIC50 and MIC90 bimodal population

50% 90%

0.015 0.03 0.06 0.12 0.25 0.5 1 2 4 8 16 32

MIC (ug/ml) MIC50

50 MIC90
90
 Streptococcus pneumoniae
Penicillin MIC variation by method

E-test
50
Agar dilution
No.

25
Microdilution
0
0 .0 0 8
0 .0 1 5
0 .0 3
0 .0 6
0 .1 2
0 .2 5
0 .5
1
2
4
>4

From Jacobs et al., J. Clin. Micro. 1998, 36:179

 Streptococcus pneumoniae
Erythromycin MIC variation by method

Agar dil.-CO2
75 Agar dil.-air
50 Microdil.-CO2
25 Microdil.-air
0
< 0 .0 3
0 .0 6
0 .1 2
0 .2 5
0 .5
1
2
4
8
16
32
>32

From Fasola et al., Antimicr. Agents Chemother. 1997, 41:129-134

 S. pneumoniae Pen-R:
The Alexander Project 2000
100
Penicillin-resistant isolates (%)

90
80
70
60
50
40
30 22%
20
10
0
Ru Ge Cz Ne Br Ita Be UK Po Gr Sw So Po M Sl Sp US Sa Ja Si Fr Ho
ss rm ec th az ly lgi la ee it ut rtu exi ov ain A ud pa ng an ng
ia a h er il
ny Re la
um nd ce ze h g co ak
rla Af al i A n apo ce K
R ra re on
pu nd nd ric ep b g
bl s a ub ia
ic lic

Penicillin-resistant defined as penicillin MIC 2 µg/mL

Alexander Project data 2000 – GlaxoSmithKline, data on file.

Subinhibitory effects of
antibiotics
Odenhalt 2001
 One log
growth
increase

Odenhalt 2001
Odenhalt 2001
Prolonged in vivo PAE present

Odenhalt 2001
 PAE of 7 h present
PA SME increases PAE by a
further 4-7 h at different
subinhibitory drug
concentrations
Odenhalt 2001
 Subinhibitory effects of
antibiotics

Subinhibitory effects such as PAE

and PAE-SME can often be
demonstrated and may account in
part for concentration-dependent
PK/PD interactions in vivo
Bactericidal activity
 Bactericidal activity: Time-kill
determination
MICs do not provide data on whether the
isolate has only been inhibited or has been
“killed”

MBCs provide data on bacterial killing

(defined as a 3 log10 kill in 24 h)

Time-kill curves provide more detailed data

on extent and time course of killing
 Gemifloxacin time kill of a penicillin
resistant strain of S.pneumoniae
(gemifloxacin MIC 0.016 ug/ml)
8
MIC 0.008 ug/ml

7 MBC 0.015 ug/ml

8 X MIC
6
Log 10 CFU/ml

4 X MIC
5 2 X MIC
1 X MIC
4
0.5 X MIC
3 0.25 X MIC
0.125 X MIC
2
Growth control
1
0 6 12 18 24

Time (hours)
 Time Kill of S. pneumoniae for
telithromycin (MIC 0.125 mg/L)

MIC 0.125 ug/ml

9 8 x MIC
MBC 0.125 ug/ml
8 4 x MIC
7
L o g 1 0 c fu /m L

2 x MIC
6 MIC
5 0.5 x MIC
4 0.25 x MIC
3 Growth control
Threshold
2
0 6 12 18 24
Time (hours)
 Time Kill of S. pneumoniae for
pristinomycin (MIC 0.5 mg/L)
MIC 0.5 ug/ml
MBC 1 ug/ml
9 8 x MIC
8 4 x MIC
2 x MIC
L o g 1 0 c fu /m L

7
6 MIC
0.5 x MIC
5
0.25 x MIC
4
Growth control
3
Threshold
2
0 6 12 18 24
Time (hours)
 Time kill of S.pneumoniae
Bactericidal activity against most
strains at MICs for quinolones
3 log10 kill in 24 hours compared to at 2X MICs for beta-
lactams
12
Levofloxacin
11
10
Gatifloxacin
No. of strains

9 Sparfloxacin
8 Trovafloxacin
7 Ciprofloxacin
6
Amoxicillin
5
Cefuroxime
4
3 Ceftriaxone
2 Clarithromycin
1
0
1 X MIC 2 X MIC
 Time kill of S.pneumoniae
Little bactericidal activity at MIC at 3 h
Bactericidal activity against up to half of the strains at 6 h
Bactericidal activity against most strains at 12 h, with faster
killing by quinolones that by beta-lactams
12
Little change between 12 and 24 h
11
10
Bay 12-8039
9 3 log10 kill at MIC at
No. of strains

Ciprofloxacin
8 various time points
7 Ofloxacin
6 Sparfloxacin
5
Amoxicillin
4

3
Ampicillin
2 Cefuroxime
1 Cefpodoxime
0
3 6 12 24
Time (hours)
MIC distributions of RTI
pathogens
 MIC distributions of RTI
pathogens
Can MIC distributions provide a basis for
comparing susceptibilities of different
bacterial species causing infections at the
same sites?

Can MIC distributions show if discrimination

between isolates with different MICs is likely
to be possible in clinical studies?

Can MIC distributions be applied to clinically

determined breakpoints to determine
susceptibility of isolates?
MIC distributions
 Streptococcus pneumoniae
MIC distributions of penicillin
and selected cephalosporins

Cefaclor (0.03-4 mg/L)

75
Cefixime (0.03-64 mg/L)
50 Cefuroxime (0.015-32 mg/L
25 Ceftriaxone (0.004-4 mg/L)
Penicillin (0.004-8 mg/L)
0
< 0 .0 0 4
0 .0 0 8
0 .0 1 6
0 .0 3
0 .0 6
0 .1 2
0 .2 5

1
2
4
8
16
32
> 64
0 .5

Source: The Alexander Project 1996

 Haemophilus influenzae
MIC distributions of cephalosporins

Cefaclor (0.03-64 mg/L)

75 Cefuroxime (0.015-32 mg/L)
50 Cefixime (0.03-64 mg/L)
25 Ceftriaxone (0.004-4mg/L)
0
0 .0 0 4
0 .0 0 8
0 .0 1 6
0 .0 3
0 .0 6
0 .1 2
0 .2 5
0 .5
1
2
4
8
16
32
> 64

Source: The Alexander Project 1996

 Haemophilus Influenzae
MIC distributions of macrolides

Clarithromycin (0.015-32 mg/L)

75
50 Erythromycin (0.015-32 mg/L)

25 Azithromycin (0.015-32 mg/L)

0
0 .0 3
0 .0 6
0 .1 2
0 .2 5
0 .5
1
2
4
8
16
> 32

Source: The Alexander Project 1996

 Amoxicillin

60
% of strains

50 M. catarrhalis
40
30 S. pneumoniae
20
10 H. influenzae
0
0.02

0.03

0.06

0.12

0.25

0.5

16
MIC in ug/mL >16

Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),

M. catarrhalis 2000 (n=206)
 Amoxicillin-clavulanate

60
% of strains

50 M. catarrhalis
40
30 S. pneumoniae
20
10 H. influenzae
0
0.02

0.03

0.06

0.12

0.25

0.5

16
MIC in ug/mL >16

Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),

AugSR M. catarrhalis (n=972)
 Cefaclor

40 M. catarrhalis
35
% of strains

30
25
S. pneumoniae
20
15
10
5
H. influenzae
0
0.5 1 2 4 8 16 32 64 >64
MIC in ug/mL
Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),
M. catarrhalis 2000 (n=206)
 Ceftriaxone

80
70 M. catarrhalis
% of strains

60
50
40 S. pneumoniae
30
20
10 H. influenzae
0
0.004
0.008
0.015
0.030
0.060
0.120
0.250
0.500
1.000
2.000
4.000
8.000
MIC in ug/mL

Adapted from Alexander Project 2000; GlaxoSmithKline, data on file.

 Cefuroxime

45
40
% of strains

35 M. catarrhalis
30
25
20 S. pneumoniae
15
10
5 H. influenzae
0
0.02

0.03

0.06

0.12

0.25

0.5

16
MIC in ug/mL >16

Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),

M. catarrhalis 2000 (n=206)
 Cefdinir

60
M. catarrhalis
% of strains

50
40
30 H. influenzae
20
10 S. pneumoniae
0
0.02

0.03

0.06

0.12

0.25

0.5

16

MIC in ug/mL >16

Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),

M. catarrhalis 2000 (n=206)
 Cefprozil

50 M. catarrhalis
% of strains

40

30 S. pneumoniae
20

10 H. influenzae
0
0.12 0.25 0.5 1 2 4 8 16 >16
MIC in ug/mL
Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),
M. catarrhalis 2000 (n=206)
 Cefixime

70
% of strains

60 M. catarrhalis
50
40
30
S. pneumoniae
20
10 H. influenzae
0
0.02

0.03

0.06

0.12

0.25

0.5

16
MIC in ug/mL >16

Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),

M. catarrhalis 2000 (n=206)
 Cefditoren

50
% of strains

40 M. catarrhalis
30
S. pneumoniae
20

10 H. influenzae
0
0

0.01

0.02

0.03

0.06

0.12

0.25

0.5

4
MIC in ug/mL 8

Adapted from Jacobs ICAAC 1997 abstr E103, Kelly ICAAC 1999 abstr 2323,
and Spectracef Prescribing Information 2002
 Ceftibuten

80
70
% of strains

M. catarrhalis
60
50
40 S. pneumoniae
30
20
10 H. influenzae
0
0.02

0.03

0.06

0.12

0.25

0.5

16

>16
MIC in ug/mL
Adapted from Jacobs ICAAC 1997 abstr E103,
and Cedax Prescribing Information 2002
 Azithromycin

100
% of strains

80 M. catarrhalis
60
S. pneumoniae
40

20 H. influenzae
0
0.03

0.06

0.12

0.25

0.5

16

32
MIC in ug/mL >32

Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),

AugSR M. catarrhalis (n=969)
 Clarithromycin

70
% of strains

60 M. catarrhalis
50
40
30
S. pneumoniae
20
10 H. influenzae
0
0.03

0.06

0.12

0.25

0.5

16

32
MIC in ug/mL >32

Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),

AugSR M. catarrhalis (n=969)
 Clindamycin

M. catarrhalis
100
% of strains

80
S. pneumoniae
60

40

20 H. influenzae
0
0.03 0.06 0.12 0.25 0.5 1 >1
MIC in ug/mL
Alexander Project USA 2000: S. pneumoniae (n=1362)
AugSR H. influenzae (n=3793) M. catarrhalis 2000 (n=970)
 Telithromycin

80
70
% of strains

M. catarrhalis
60
50
40 S. pneumoniae
30
20
10 H. influenzae
0
0

0.01

0.02

0.03

0.06

0.12

0.25

0.5

4
MIC in ug/mL >4

Nagai AAC 2002, 46:371-7; Pankuch AAC 1998, 42:3032-34

 Doxycycline

70 M. catarrhalis
% of strains

60
50
40 S. pneumoniae
30
20
10 H. influenzae
0
0.03 0.06 0.12 0.25 0.5 1 2 4 8 >8
MIC in ug/mL
Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),
M. catarrhalis 2000 (n=206)
 Trimethoprim-
sulfamethoxazole

45 M. catarrhalis
40
% of strains

35
30
25 S. pneumoniae
20
15
10 H. influenzae
5
0
0.02 0.03 0.06 0.12 0.25 0.5 1 2 4 >4
MIC in ug/mL
Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),
M. catarrhalis AugSR (n=972)
 Ciprofloxacin

80
70
% of strains

60
M. catarrhalis
50
40 S. pneumoniae
30
20
10 H. influenzae
0
0.01

0.02

0.03

0.06

0.12

0.25

0.5

MIC in ug/mL >8

Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),

M. catarrhalis AugSR (n=972)
 Levofloxacin

90 M. catarrhalis
80
% of strains

70
60
50 S. pneumoniae
40
30
20 H. influenzae
10
0
0.03

0.06

0.12

0.25

0.5

>2
0.008

0.015

MIC in ug/mL
Alexander Project USA 2000: S. pneumoniae (n=1362), H. influenzae (n=634),
M. catarrhalis AugSR (n=972)
 MIC distributions of RTI
pathogens
MIC distributions can provide a basis for
comparing susceptibilities of different
bacterial species causing infections at the
same sites

MIC distributions can show if discrimination

between isolates with different MICs is likely
to be possible in clinical studies

MIC distributions can be applied to clinically

determined breakpoints to determine
susceptibility of isolates