BSAC Standardised Disc Susceptibility
Test User Group Day.
Royal College of Physicians, London.
8 June 2007
Susceptibility testing of mucoid Pseudomonas and
Burkholderia strains from patients with cystic
fibrosis including evaluation of the BSAC
standardised method.
J.D. Perry
Freeman Hospital
Newcastle upon Tyne
Contents:
• Direct susceptibility testing of whole sputum from
CF patients to detect resistant strains of P.
aeruginosa.
• Preliminary work to validate disc susceptibility
testing with P. aeruginosa and B. cepacia from CF
patients.
• MCBT testing of resistant strains of P. aeruginosa
and B. cepacia from CF patients.
Direct susceptibility testing of
Pseudomonas aeruginosa from
sputa of patients with cystic fibrosis
Phenotypic variability of Pseudomonas aeruginosa in
sputa from patients with acute infective exacerbation
of cystic fibrosis and its impact on the validity of
antimicrobial susceptibility testing
J. E. Foweraker*, C. R. Laughton, D. F. J. Brown and D. Bilton
Department of Microbiology, Papworth Hospital, Papworth Everard, Cambridge
CB3 8RE, UK;
Health Protection Agency, Clinical Microbiology and Public Health Laboratory,
Addenbrooke’s Hospital,
Cambridge CB2 2QW, UK; Department of Chest Medicine, Papworth Hospital,
Papworth Everard,
Cambridge CB3 8RE, UK
Journal of Antimicrobial Chemotherapy (2005) 55, 921–927
Foweraker et al. 2005.
Methods:
• One hundred and one sputa were
cultured. Four colonies of each
P.aeruginosa morphotype were
suspended.
• Susceptibility to 12 agents by disc
diffusion was tested individually or by
pooling the four suspensions.
Foweraker et al. 2005.
Results / Conclusions
• In some cases, all four colonies of a single
morphotype had different antibiograms.
• The susceptibility profiles of single isolates of P.
aeruginosa correlated poorly with pooled
cultures, with the pooled tests missing
resistance.
• A range of susceptibility patterns is seen, even
within a morphotype. Routine test results are
not reproducible and underestimate resistance.
Aim of the current investigation:
Can antimicrobial resistance be detected
more reliably by culture of whole sputum
onto media containing antimicrobials ?
Routine culture:
• Sputum samples were homogenized 1:1
with Sputasol.
• Routine culture:
• 10 µl aliquots of liquid sputum were plated
onto:
• Chocolate agar (+Bacitracin), Blood agar,
CLED agar, Isosensitest agar,
Pseudomonas Selective agar &
Burkholderia cepacia Selective agar.
Routine culture (continued):
• A 10 µl aliquot of liquid sputum was diluted by
addition to 9.99 ml sterile water (1/1000).
• 10 µl of this diluted sample was also plated onto:
• Chocolate agar (+Bacitracin), Blood agar, CLED
agar, Isosensitest agar, Pseudomonas Selective
agar & Burkholderia cepacia Selective agar.
Selective culture:
A 10 µl aliquot of liquid sputum was inoculated
onto 10 distinct Isosensitest agar plates
incorporating the following antimicrobials:
•
•
•
•
•
Amikacin (16 mg/L)
Gentamicin (4 mg/L)
Tobramycin (4 mg/L)
Aztreonam (8 mg/L)
Ceftazidime (8 mg/L)
•
•
•
•
•
Meropenem (4 mg/L)
Temocillin (16 mg/l)
Ciprofloxacin (1 mg/L).
Piperacillin-tazobactam (16
mg/L)
Ticarcillin-clavulanic acid (32
mg/L)
Summary of media used
Routine culture:
‘Selective’ culture
• 10 µl of Neat and diluted
• Culture of neat homogenized
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
homogenized sputa were
inoculated onto:
Cholcolate-Bacitracin
Blood agar.
CLED agar.
Isosensitest agar.
Pseudomonas selective agar.
B. cepacia selective agar.
(Total = 10 culture plates).
sputa on Isosensitest agar (x
10) containing:
Amikacin
Gentamicin
Tobramycin
Aztreonam
Ceftazidime
Meropenem
Temocillin
Ciprofloxacin
Piperacillin-tazobactam
Ticarcillin-clavulanic acid
Interpretation of cultures:
• All plates were incubated for 72 hours and
examined after 24, 48 and 72 hours.
• All colonial variants or ‘morphotypes’ of
Gram-negative bacteria were sub-cultured
onto a blood agar plate to obtain pure
cultures. These subcultures were used for
MIC testing, identification and storage in
glycerol for potential further studies.
Identification:
• P. aeruginosa was identified by inoculation of all
morphotypes onto PC agar, cetrimide agar and blood
agar to test for growth at 42°C.
• PC agar contains:
• 30 mg/L 9-chloro-9-[4-(diethylamino)phenyl]-9,10•
dihydro-10-phenylacridine hydrochloride (C-390) and
30 mg/L 1,10-phenantholine.
• Growth on this agar is diagnostic for P. aeruginosa with
100 % specificity1.
1
J Clin Microbiol. 1988 Sep;26(9):1910-2.
Identification:
• For this study:
• Growth on cetrimide + growth on PC agar +
growth at 42°C = P. aeruginosa.
• All other strains were identified by API 20 NE.
Susceptibility testing:
• All morphotypes (from any medium) were
referred for MIC testing against 10
antibiotics.
• MIC testing was performed using agar
dilution in Isosensitest agar with a final
inoculum of 10 000 cfu/spot.
• MIC’s were recorded after both 24 and 48
hours of incubation at 37°C.
Susceptibility testing:
Ranges of antibiotics
for MIC testing:
•
•
•
•
•
AMIK
GENT
TOBRA
ATM
CAZ
(64
(16
(16
(32
(32
-
2 mg/L)
0.5 mg/L)
0.5 mg/L)
1 mg/L)
1 mg/L)
•
•
•
•
•
CIPRO
TEM
TIM
PIPTAZO
MERO
(4 - 0.125 mg/L)
(64 - 2 mg/l)
(128 - 4 mg/L)
(64 - 2 mg/L)
(16 - 0.5 mg/L)
Results:
• From 45 sputum
samples, 705 bacterial
morphotypes were
referred for
identification and
susceptibility testing:
Identification of 705 bacterial
isolates:
Pseudomonas aeruginosa
645
Alcaligenes xylosoxidans
27
Pseudomonas fluorescens
22
Stenotrophomonas
maltophilia
8
Bulkholderia cenocepacia
1
Moraxella species
1
Sphingobacterium
spiritovorum
1
Results:
• 43 / 45 samples yielded P. aeruginosa
(one sample: B. cenocepacia only.
one sample: A. xylosoxidans only).
• An average of three morphotypes was
tested from routine media (range 1 – 5).
Table 1: Number of specimens containing strains of
P. aeruginosa resistant to antimicrobials
AMIK
ATM
CAZ
CIPRO
GENT
MERO
TAZO
31
26
29
30
35
30
33
35
29
13
No. detected:
13
23
18
24
20
21
20
23
26
7
% detected:
42
88
62
80
57
70
61
66
90
54
No. detected:
31
20
26
27
35
29
33
35
25
13
% detected:
100
77
90
90
100
97
100
100
86
100
Total
TIM
TEM
TOBRA
Routine method
Selective method
Example: 14 Yr old CF pateint: LTA
AMIK
ATM
CAZ
CIPRO
GENT
MERO
TAZO
TIM
TEM
TOBRA
Met ®
P.aeruginosa
<2 (4)
<2
<2
0.25 (0.5)
1
0.5 (2)
<2
<4 (64)
2 (4)
<0.5
L®
P.aeruginosa
<2
<2
<2
0.5 (1)
1
0.5
<2
<4
4 (8)
<0.5
muc ®
P.aeruginosa
<2
<2
<2
0.25 (0.5)
0.5 (1)
0.5
<2
<4
4
<0.5
L (Amik)
P.aeruginosa
64
L (Caz)
P.aeruginosa
L (Cip)
P.aeruginosa
gr (gent)
P.aeruginosa
L (Mero)
P.aeruginosa
M (Tazo)
P.aeruginosa
L (Tim)
P.aeruginosa
S (Tob)
P.aeruginosa
L (Atm)
P.fluorescens
S (Caz)
P.fluorescens
L (Gent)
P.fluorescens
L (Tazo)
S. spiritovorum
M (Tim)
P.fluorescens
L (Tem)
P.fluorescens
tiny (Tob)
P.fluorescens
32
2
16
8 (16)
>64
>128
16
32
32
4
32
>128
>64
8 (16)
Table 2: Positive predictive value of growth on
selective agars to predict antimicrobial resistance
by MIC testing.
PPV (%)
AMIK
ATM
CAZ
CIPRO
GENT
MERO
TAZO
TIM
TEM
TOBRA
92
100
93
94
88
93
96
95
96
89
Conclusions:
• Growth on selective media containing breakpoint
concentrations of antimicrobials can be used to
detect antimicrobial resistance in P. aeruginosa
(PPV: 88 – 100 %).
• For most antimicrobials, the use of selective
media facilitates detection of more antimicrobial
resistance when compared with routine methods
that involve selection of morphotypes for
susceptibility testing.
Disc susceptibility testing of
Pseudomonas aeruginosa from
sputa of patients with cystic fibrosis
Disc susceptibility testing was
performed by BSAC method:
• 38 strains of P. aeruginosa were tested. Each strain was isolated
from a distinct patient with CF.
• 0.5 McFarland suspension of strains cultured overnight on blood
agar.
• 1/100 dilution of suspension in sterile water.
• Dilution spread with a swab onto pre-poured Oxoid Isosensitest
agar (PO O779A).
• Incubation for 24 h at 37°C (48 h only if required) to obtain semiconfluent growth.
• Agar dilution MIC’s were performed in Isosensitest agar (Oxoid)
using the BSAC method using the same 0.5 McFarland suspensions.
Disc susceptibility testing was
performed by BSAC method:
• Controls:
• With each batch of disc susceptibility tests and
agar dilution tests we included:
• Pseudomonas aeruginosa NCTC 10662
• Escherichia coli NCTC 10418
This was performed to ensure that zones of
inhibition fell within published acceptable limits
and MIC values were within one dilution of
published acceptable values.
Results:
• 36 / 38 strains of P. aeruginosa grew well
within 24 h and produced an ideal semiconfluent inoculum.
• 1 strain generated a light growth within
the ‘acceptable range’.
• One strain produced no growth on
repeated disc susceptibility testing.
• (Control strains produced acceptable
results)
R or I by disc (6) = Resistant by MIC
Sensitive
Intermediate
Sensitive by disc (31):
≤ 8 mg/L
≥ 19 mm
16 mg/L
16-18 mm
Sensitive : 17
Intermediate: 6
Resistant :
8
Resistant
≥ 32 mg/L
≤ 15 mm
Amikacin 10 µg disc with P.aeruginosa
45
40
Zone diameter (mm)
35
30
25
20
15
10
5
0
0
10
20
30
40
MIC (mg/L)
50
60
70
Resistant by disc (8) = Resistant by MIC
Sensitive
Resistant
Sensitive by disc (28):
≤ 4 mg/L
≥ 18 mm
≥ 8 mg/L
≤ 17 mm
Sensitive :
Resistant :
18
10
Gentamicin 10 µg disc with P.aeruginosa
45
40
Zone diameter (mm)
35
30
25
20
15
10
5
0
0
10
20
30
40
MIC (mg/L)
50
60
70
Resistant by disc (1) = Resistant by MIC
Sensitive
Resistant
Sensitive by disc (35):
≤ 4 mg/L
≥ 20 mm
≥ 8 mg/L
≤ 19 mm
Sensitive :
Resistant :
34
1
Tobramycin 10 µg disc with P.aeruginosa
50
45
Zone diameter (mm)
40
35
30
25
20
15
10
5
0
0
2
4
6
8
10
MIC (mg/L)
12
14
16
18
Resistant by disc (8) = Resistant by
MIC
Sensitive by disc (17):
Sensitive
Intermediate
≤ 0.5 mg/L
≥ 30 mm
1 mg/L
20-29 mm
Resistant
≥ 2 mg/L
≤ 19 mm
Sensitive :
7
Intermediate: 9
Resistant :
1
Ciprofloxacin 5 µg disc with P.aeruginosa
50
45
Zone diameter (mm)
40
35
30
25
20
15
10
5
0
0
2
4
6
8
10
MIC (mg/L)
12
14
16
18
Resistant by disc (8) = Resistant by MIC
Sensitive
Resistant
Sensitive by disc (29):
≤ 8 mg/L
≥ 23 mm
≥ 16 mg/L
≤ 22 mm
Sensitive :
Resistant :
23
6
Aztreonam 30 µg disc with P.aeruginosa
70
Zone diameter (mm)
60
50
40
30
20
10
0
0
10
20
30
40
MIC (mg/L)
50
60
70
Resistant by disc (10) = 9 Resistant by MIC (1 S)
Sensitive
Resistant
Sensitive by disc (27):
≤ 8 mg/L
≥ 24 mm
≥ 16 mg/L
≤ 23 mm
Sensitive :
Resistant :
21
6
Ceftazidime 30 µg disc with P.aeruginosa
60
Zone diameter (mm)
50
40
30
20
10
0
0
10
20
30
40
MIC (mg/L)
50
60
70
Resistant by disc (11) = R or I by MIC
Sensitive by disc (22):
Sensitive : 19
Intermediate: 3 (MIC = 4)
Resistant :
0
Sensitive
Intermediate
Resistant
≤ 2 mg/L
≥ 27 mm
4-8 mg/L
22-26 mm
≥ 16 mg/L
≤ 21 mm
Meropenem 10 µg disc with P.aeruginosa
60
Zone diameter (mm)
50
40
30
20
10
0
0
5
10
15
20
MIC (mg/L)
25
30
35
Resistant by disc (7) = Resistant by MIC
Sensitive
Sensitive by disc (29):
≤ 16 mg/L
≥ 22 mm
Sensitive :
Resistant :
21
8 (MIC 16-32)
Resistant
≥ 32 mg/L
≤ 21 mm
Piperacillin / tazobactam 75 / 10 µg disc with P.aeruginosa
60
Zone diameter (mm)
50
40
30
20
10
0
0
50
100
150
MIC (mg/L)
200
250
300
Resistant by disc (17) Resistant by MIC: 16 R
1S
S ≤ 8 mg/L (Unconfirmed) R > 8 mg/L (Unconfirmed)
S ≥ 20 mm (Unconfirmed) R ≤ 19 mm (Unconfirmed)
Sensitive by disc : 19
P. aeruginosa
E. coli
Sensitive : 18
Resistant : 1
NCTC 10662 MIC = > 64 mg/L
NCTC 10418 MIC = 4 mg/L
Temocillin 30 µg disc with P.aeruginosa
60
Zone diameter (mm)
50
40
30
20
10
0
0
10
20
30
40
MIC (mg/L)
50
60
70
Disc susceptibility testing of
Burkholderia cepacia complex using
BSAC criteria for interpretation of
Pseudomonas spp.
Disc susceptibility testing was
performed by BSAC method:
• 0.5 McFarland suspension of strains cultured overnight
on blood agar.
• 1/100 dilution of suspension in sterile water.
• Dilution spread with a swab onto pre-poured Oxoid
Isosensitest agar (PO O779A).
• Incubation for 24 h at 37°C (48 h only if required) to
obtain semi-confluent growth.
• Agar dilution MIC’s were performed in Isosensitest agar
(Oxoid) using the BSAC method using the same 0.5
McFarland suspensions.
40 strains of Burkholderia cepacia complex used for disc
susceptibility testing :
LMG 16654
Burkholderia cenocepacia
LMG 13010
Burkholderia multivorans
LMG 16656
Burkholderia cenocepacia
LMG 16665
Burkholderia multivorans
LMG 16659
Burkholderia cenocepacia
LMG 17588
Burkholderia multivorans
LMG 18826
Burkholderia cenocepacia
LMG 18822
Burkholderia multivorans
LMG 18827
Burkholderia cenocepacia
LMG 18823
Burkholderia multivorans
LMG 18829
Burkholderia cenocepacia
LMG 18824
Burkholderia multivorans
LMG 18830
Burkholderia cenocepacia
LMG 18825
Burkholderia multivorans
LMG 18832
Burkholderia cenocepacia
LMG 18863
Burkholderia cenocepacia
40 strains of Burkholderia cepacia complex (cont…)
LMG 14086
Burkholderia stabilis
CEP 0092
Burkholderia cepacia complex
LMG 14294
Burkholderia stabilis
CEP 0408
Burkholderia cepacia complex
LMG 16660
Burkholderia stabilis
CEP 0591
Burkholderia cepacia complex
LMG 18888
Burkholderia stabilis
CEP 0615
Burkholderia cepacia complex
LMG 10929
Burkholderia vietnamiensis
CEP 0686
Burkholderia cepacia complex
LMG 16232
Burkholderia vietnamiensis
CEP 0769
Burkholderia cepacia complex
LMG 18835
Burkholderia vietnamiensis
CEP 0786
Burkholderia cepacia complex
LMG 18836
Burkholderia vietnamiensis
CEP 0891
Burkholderia cepacia complex
LMG 1222
Burkholderia cepacia
CEP 0945
Burkholderia cepacia complex
LMG 17997
Burkholderia cepacia
CEP 1012
Burkholderia cepacia complex
LMG 18821
Burkholderia cepacia
CEP 1190
Burkholderia cepacia complex
LMG 2162
Burkholderia cepacia
CEP 1235
Burkholderia cepacia complex
Results:
• 35 / 40 strains of B. cepacia complex grew
well within 24 h and produced an ideal
semi-confluent inoculum.
• 5 strains generated a light growth within
the ‘acceptable range’.
• One strain produced an unacceptable lawn
(too light) which required incubation for
48 h.
Resistant by disc = Resistant by MIC
Sensitive
Intermediate
Resistant
Sensitive by disc (31):
≤ 0.5 mg/L
≥ 30 mm
1 mg/L
20-29 mm
≥ 2 mg/L
≤ 19 mm
Sensitive : 17
Intermediate: 6
Resistant :
8
Ciprofloxacin 5 µg disc with B. cepacia complex
50
45
Zone diameter (mm)
40
35
30
25
20
15
10
5
0
0
2
4
6
8
10
MIC (mg/L)
12
14
16
18
Resistant by disc (7) = Resistant by MIC
Sensitive
Sensitive by disc (33):
≤ 8 mg/L
≥ 23 mm
Sensitive :
Resistant :
31
2
Resistant
≥ 16 mg/L
≤ 22 mm
Aztreonam 30 µg disc with B. cepacia complex
50
45
Zone diameter (mm)
40
35
30
25
20
15
10
5
0
0
10
20
30
40
MIC (mg/L)
50
60
70
Resistant by disc (4) = Resistant by MIC
Sensitive
Sensitive by disc (36):
≤ 8 mg/L
≥ 24 mm
Sensitive :
Resistant :
34
2
Resistant
≥ 16 mg/L
≤ 23 mm
Ceftazidime 30 µg disc with B. cepacia complex
50
45
Zone diameter (mm)
40
35
30
25
20
15
10
5
0
0
10
20
30
40
MIC (mg/L)
50
60
70
R or I by disc (6) = R or I by MIC
Sensitive
Intermediate
Sensitive by disc (34):
≤ 2 mg/L
≥ 27 mm
4-8 mg/L
22-26 mm
Sensitive :
Resistant :
32
2
Resistant
≥ 16 mg/L
≤ 21 mm
Meropenem 10 µg disc with B. cepacia complex
60
Zone diameter (mm)
50
40
30
20
10
0
0
2
4
6
8
10
MIC (mg/L)
12
14
16
18
No resistance by disc
Sensitive
Sensitive by disc (40):
≤ 16 mg/L
≥ 22 mm
Sensitive :
Resistant :
36
4
Resistant
≥ 32 mg/L
≤ 21 mm
Piperacillin / tazobactam 75 / 10 µg disc with B. cepacia
complex
60
Zone diameter (mm)
50
40
30
20
10
0
0
50
100
150
MIC (mg/L)
200
250
300
Resistant by disc (8) = Resistant by MIC
Sensitive by disc (32):
Sensitive :
Resistant :
S ≤ 8 mg/L (Unconfirmed) R > 8 mg/L (Unconfirmed)
S ≥ 20 mm (Unconfirmed) R ≤ 19 mm (Unconfirmed)
P. aeruginosa
E. coli
30
2
NCTC 10662 MIC = > 64 mg/L
NCTC 10418 MIC = 4 mg/L
Temocillin 30 µg disc with B. cepacia complex
70
Zone diameter (mm)
60
50
40
30
20
10
0
0
10
20
30
40
MIC (mg/L)
50
60
70
MULTIPLE ANTIBIOTIC
SYNERGY TESTING AGAINST
P. AERUGINOSA AND
B. CEPACIA COMPLEX
STRAINS FROM CYSTIC
FIBROSIS PATIENTS.
Principles:
• Inoculation of 106 cfu/ml test
bacteria into Isosensitest broth
with 78 distinct antimicrobial
combinations.
• Incubation for 48 h at 37°C.
Antimicrobial combinations
tested:
A
B
C
D
E
F
G
H
1
TIM
TIM/CAZ
TIM/CIP
TIM/MIN
TIM/FOS
x
x
x
2
CAZ
CAZ/TOB
CAZ/CIP
CAZ/MIN
CAZ/FOS
x
x
x
3
CIP
CIP/TOB
CIP/AZT
CIP/MIN
CIP/FOS
x
x
x
4
TAZ
TAZ/SXT
TAZ/AK
TAZ/TIM
TAZ/CAZ
TAZ/CIP
x
x
5
AK
AK/TIM
AK/CAZ
AK/CIP
AK/MIN
AK/FOS
x
x
6
MER
MER/CHL
MER/TAZ
MER/SXT
MER/AK
MER/TIM
MER/CAZ
MER/CIP
7
AZT
AZT/MER
AZT/CHL
AZT/TAZ
AZT/SXT
AZT/AK
AZT/TIM
AZ/CAZ
Tests performed in microtitre wells.
Final volume: 100µl.
8
TOB
TOB/AZT
TOB/MER
TOB/CHL
TOB/TAZ
TOB/SXT
TOB/AK
TOB/TIM
9
CHL
CHL/TAZ
CHL/SXT
CHL/AK
CHL/TIM
CHL/CAZ
CHL/CIP
x
10
MIN
MIN/MER
MIN/AZT
MIN/TOB
MIN/CHL
MIN/FOS
MIN/TAZ
x
11
FOS
FOS/MER
FOS/AZT
FOS/TOB
FOS/CHL
FOS/TAZ
Sterility
Sterility
12
SXT
SXT/AK
SXT/TIM
SXT/CAZ
SXT/CIP
SXT/MIN
SXT/FOS
Growth
Microtitre wells interpreted as :
‘growth’ or ‘no growth’ by
spectrophotometry.
Synergy testing (continued…)
• Clear or ‘no growth’ wells are
subcultured (50 µl) onto blood agar.
• Colony counts are then performed after
48 h incubation.
• Antibiotics are assessed as
bacteriostatic or bactericidal.
• Antimicrobial combinations that result in
complete kill are recommended for
therapy.
Best 10 antimicrobial combinations for kill of P.aeruginosa
(no. of strains = 42) from cases of CF.
Antibiotic combination
Piperacillin-tazobactam/Ciprofloxacin
Tobramycin/Piperacillin-tazobactam
Amikacin/Ceftazidime
Ceftazidime/Tobramycin
Ceftazidime/Ciprofloxacin
Tobramycin/Aztreonam
Tobramycin/Timentin
Piperacillin-tazobactam
Piperacillin-tazobactam/Amikacin
Fosfomycin/Tobramycin
% of strains killed
50
40
38
36
36
36
36
31
31
31
Best 10 antimicrobial combinations for kill of B. cepacia
(no. of strains = 36) from cases of CF.
Antibiotic combination
Piperacillin-tazobactam/Amikacin
Ceftazidime/Tobramycin
Minocycline/Piperacillin-tazobactam
Ceftazidime/Ciprofloxacin
Amikacin/Ceftazidime
Tobramycin/Piperacillin-tazobactam
Piperacillin-tazobactam/Ceftazidime
Aztreonam/Ceftazidime
Timentin/Ceftazidime
Chloramphenicol/Piperacillin-tazobactam
% of strains killed
33
28
28
25
25
25
22
22
19
19
Work performed by:
Larissa Laine, Susan Hughes,
Audrey Nicholson & John Perry.
Microbiology Department
Freeman Hospital
Newcastle upon Tyne