synergy testing (4)
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Extremely Drug-resistant
organisms:Synergy Testing
LIM TZE PENG
Principal Pharmacist
Singapore General Hospital
Acinetobacter baumannii& Pseudomonas aeruginosa
Emerging Gram-negative bacilli
Part of the ESKAPE group of organisms1
Enterococcus faecium
Staphylcoccus aureus
Klebsiella pneumoniae
Acinetobacter baumannii
Pseudomonas aerugionosa
Enterobacter spp.
Background
1. He len W. Boucher, Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious Diseases Society of America. Clin Infect Dis2009; 48:1-12
What is the difference between
MDR
XDR PDR
Definitions
1. Magiorakos, A. P., A. Srinivasan, e t al. (2011 ). "Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: aninternational e xpert proposal for interim standard def initions for acquired resistance." Clin Microbiol Infect .
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What is the difference between
Synergistic
Bactericidal
Inhibitory
Antagonistic
Definitions Typical MIC profile
Strain
Antibiotic18351 18352 27640 9447 11171
Ampicillin/Sulbactam 32/16 32/16 64/32 32/16 32/16Ciprofloxacin 16 16 16 16 16
Gentamicin 64 64 64 64 64
Imipenem 64 32 32 64 64
Meropenem 128 64 32 64 64
Aztreonam 128 64 32 128 128
Piperacillin/Tazobactam 256 256 256 256 256
Polymyxin B 64 32 128 16 16
Tigecycline 4 4 16 4 4Ceftazidime 128 128 128 128 128
Amikacin 128 128 128 128 128
Cefepime 128 128 128 128 64
Rifampicin 2 4 256 256 256
Strain
Antibiotic18351 18352 27640 9447 11171
Ampicillin/Sulbactam 32/16 32/16 64/32 32/16 32/16
Ciprofloxacin 16 16 16 16 16
Gentamicin 64 64 64 64 64
Imipenem 64 32 32 64 64
Meropenem 128 64 32 64 64
Aztreonam 128 64 32 128 128
Piperacillin/Tazobactam 256 256 256 256 256
Polymyxin B 64 32 128 16 16
Tigecycline 4 4 16 4 4
Ceftazidime 128 128 128 128 128
Amikacin 128 128 128 128 128
Cefepime 128 128 128 128 64
Rifampicin 2 4 256 256 256
Typical MIC profile
Decreasing new antibacterials approved for use
Only 2 new antibacterials in the last 6 years
Doripenem (2007) , Ceftaroline (2013)
Multi-pronged approach
Judicious use of existing agents
Efficient infection control
Antimicrobial Stewardship Program
Combination therapy
Potential solutions
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Often used in clinical practice TB & HIV
Increasingly used in MDRA. baumannii& P.aeruginosa
Enhanced pharmacodynamic effect(synergism)
Enhanced bactericidal effect Suppress emergence of resistance
Combination therapy
1. Maragakis LL, Perl TM. Acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options. Clin Infect Dis.2008 Apr 15;46(8):1254-63.
Various methodologies Checkerboard method
Time-kill studies
In-vitro pharmacodynamic models
Types of combination studies
1. Hsieh MH , Yu CM, Yu VL, Chow JW. Synergy assessed by checkerboard. A critical analysis. Diagn Microbiol Infect Dis. 1993 May-Jun;16(4):343-9.
Fractional Inhibitory ConcentrationIndex
Synergy: FIC index < 0.5
Additive: FIC index = 1
Antagonism: FIC index > 4
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Limitations of FIC index
Based on Loewe additivityAssume similar & linear concentration-time
relationship
Subjective endpoints
Cloudy vs Clear wells
Time-kill studies (TKS)
Antibiotic synergy:
2 log decrease in
cfu/ml
1. National Committe e for Clinical Laboratory Standards. 1999. Methods for Determining Bactericidal Activity of AntimicrobialAgents. Approved Guideline M26-A., vol. 19. N CCLS, Wayne, PA, USA
Limitations of TKS
Clinical relevance
24 hour endpoint
At least one of the drugs must be present in a concentration
which does not affe ct the growth curve of the tes t organism when
used alone.1
Pharmacokinetic factors ignored
Resource management
Labour & Time intensive
1. Antimicrobial Agents & Chemotherapy: Instructions to Authors
Hollow-fiber infection model allowing simulation of human PK
in vitro. (Tam, JID 2007)
bacteria
Drug
EliminationDistribution
Hollow-Fiber System
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Aims
To elucidate efficacious antibioticcombinations against PDRA. baumannii
Methods
Time-kill studies (TKS)
Maximal clinically achievable concentrations
Hollow Fiber Infection Model (HFIM)
In-vivo environment simulation
Time Kill Study (TKS)
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Susceptibility results
AntimicrobialTTSH 112
(mg/l)TTSH 105
(mg/l)SGH 8879
(mg/l)
Meropenem 32 (R) 64 (R) >32 (R)
Polymyxin B 1 (S) 1 (S) 2 (S)
Rifampicin1 4 4 2
Tigecycline 4 (I) 0.5 (S) 2 (S)
1There are current ly no international standards for rifampicin and susceptibility
testing against Acinetobacter baumannii
Pharmacokinetic Data
AntimicrobialSimulated
FREE drug
conc (mg/l)
Maximum clinicalachievable FREE
drug conc (mg/L)
Corresponding maximumclinical dose
Meropenem 64 64 (plasma) 2g q8h over 3h infusion
Polymyxin B 2 2 (plasma) At least 1 MU q12h
Rifampicin 2 2 (plasma) PO 600mg q12h
Tigecycline 2 2 (tissues) 100mg q12h
1. Jar uratanasirikul S, et al. Compar ison o f the pharmacodynamics ofmero penem in patients with ventilator-ass ociated pn eumonia following
administration b y 3-ho ur inf usion o r bolus injection. Antimicrob Agents Chemother . 2005 Apr;49(4):1337-9.2. Kwa AL, Lim TP, Low JG, Ho u J, Kurup A, Pr ince RA, Tam VH. Pharmacokinetics of po lymyxin B1 in patients with multidr ug-resistant Gram-
negative bacterial inf ections . Diagn Micro bio l In fect Dis. 2008 Feb ;60(2):163-7. Epub 2007 Oct 4.3. Gumbo T, Louie A, Deziel MR, Liu W, Par son s LM, Salfinger M, Drusano GL. Concen tration- dependent Mycobacterium tuberculosis killing and
pr evention of r esistance by rifam pin . Antimicro b Agents Chemo ther. 2007 Nov;51(11):3781-8. Epub 2007 Aug 27.
4. Rod vold KA, Gotfried MH, Cwik M, K orth- Bradley JM, Dukart G, Ellis-Gro sse EJ. Serum, tissue and bo dy fluid concen tration s of tigecycline after asingle 100 mg do se. J Antimicro b Chem oth er. 2006 Dec;58(6):1221-9.
TKS SGH AB 8879
Microbiological responses of AB againstvarious antibiotic combinations
Microbio log ical respon ses of ABagainst various antibiotics
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Objective
To evaluate the efficacy of :
Polymyxin B and Rifampicin or
PolymyxinB and Tigecycline or
Tigecycline and Rifampicin combined against PDR AB
from our local hospitals.
Methods
Bacteria
361 AB strains collected from National AntimicrobialResistance, Singapore
MIC testing (microtitre)
31 PDR AB with OXA-23, OXA-51 b-lactamases &
ISAba1OXA complex
Time-kill studies performed with the 31 PDR AB
strains
Baseline inoculums of 5 log10 CFU/ml
Pharmacokinetic Data
Antimicrobial
Simulated FREE
drug conc(mg/l)
Corresponding maximumclinical dose
Polymyxin B 2 (serum trough) At least 1 MU q12h
Rifampicin 2 (serum peak) PO 600mg q12h
Tigecycline 2 (tissue peak) 100mg q12h
Kwa AL, Lim TP, Low JG, Hou J, Kurup A, Prince RA, Tam VH. Pharmacokinet ics of poly myxin B1 in patient s with
multidrug-resistant Gram-neg ative bacterial infections. Diagn Microbiol Infect Dis. 2008 Feb;60(2) :163-7. Epub 2007 Oct 4.
Gumbo T, Louie A, Deziel MR, Liu W, Parsons LM, Salfinge r M, Drusano GL. Concentration-de pendent Mycobacterium
tube rculosis killing and prevention of re sistance by rifampin. Antimicrob Agents Chem other. 2007 Nov;51(11):3781-8. Epub
2007 Aug 27.
Rodvold KA, Gotfried MH, Cwik M, Korth-Bradley JM, Dukart G, Ellis-Grosse EJ. Serum, tissue and body fluid
conce ntrations of tigec ycline after a single 100 mg dose. J Antimicrob Chemother. 2006 Dec;58(6):1221-9.
Combination timekill
Antibiotic synergy:
2 log decrease in
cfu/ml
2 log decrease in
cfu/ml from original
inoculum
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MIC results (31 PDR AB strains)
Susceptibility (%)
Antib iotics MIC50 (mg/L) MIC90 (mg/L) Range (mg/L) R I S
Polymyxin B 1 2 0.5 2 100
Rifampicin 6 64 1 64
Tigecycline 4 32 0.5 32
Resistant to all ant ibiotics
Single TKS results24 hour mean bacteria burden after exposure to various antibiotics alone(1-2 log reduct ions strains denoted in red)
Tigecycline Polymyxin B Rifampicin
AB
strain
Starting
inocula Mean Mean Mean
8 5.22
12 5.27
16 5.23
17 5.03
23 5.35
25 5.44
28 5.30
32 5.30
41 5.38
59 5.25
60 5.36
69 5.25
70 5.26
88 5.28
91 5.20
3.50
3.74 3.29
3.40
4.02
3.12
3.00
97 5.33 6.94 4.85 5.62
7.13 8.07
8.60 5.72 7.77
7.72 8.69
9.14
7.94 4.94 5.99
6.86 5.72 7.83
8.08 8.61 8.73
5.11 7.91
7.99 8.04 5.56
8.08 5.13 9.17
7.97 8.46
8.16 5.48 8.97
8.33 8.60
7.17 4.84 6.11
7.14 8.58
8.53
Single TKS results (continued)24 hour mean bacteria burden after exposure to various ant ibiotics alone(1-2 log reduct ion strains denoted in red)
Tigecycline Polymyxin B Rifampicin
ABstrain
Startinginocula
Mean Mean Mean
98 5.54
102 5.21
104 5.16
126 5.20
128 5.42
129 5.19 4.07
138 5.38
170 5.37
174 5.26 3.57
112 5.18
8879 5.01
14101 5.40
3160 5.17
13631 5.43
48038 5.32
7.72 4.31 5.59
6.63 5.09 8.05
6.84 5.05 7.70
8.77 5.59 5.43
8.71 7.86 5.82
8.84 8.28
6.77 4.40 8.36
7.62 5.42 8.49
6.35 8.24
7.45 4.73 7.52
5.25 4.23 7.56
5.38 5.71 8.33
8.93 4.75 8.60
7.93 5.59 8.86
8.90 4.78 8.18
24 hour mean bacteria burden after exposure to various antibiotic combinations
(modified bacte ricidal combinations denoted in red, 1-2 log10 reduction in yellow)
Tigecycl ine + Rifampicin Polymyxin B + Rifampicin Polymyxin B + Tigecycline
ABstrain
Startinginocula
Mean Mean Mean
8 5.22 0.00
12 5.27 0.00 0.00
16 5.23 0.80
17 5.03 0.00 0.00 0.65
23 5.35
25 5.44 0.00
28 5.30 3.57
32 5.30 0.00 2.31
41 5.38
59 5.25 4.10 3.24
60 5.36 0.80
69 5.25 4.10
70 5.26 4.09
88 5.28 2.42 0.00
91 5.20 0.00 0.00
97 5.33 0.00 0.00
6.81 5.11
7.19
4.70 3.56
4.95 4.85 4.82
4.73 5.01
5.53 4.80
5.41
4.77 4.54 5.10
8.67
6.65 5.37
7.64 5.08
6.84 4.66
4.90
4.65
4.25
Combination TKS results
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Combination TKS results (continued)24 hour mean bacteria burden after exposure to various ant ibiotic combinations(modified bactericidal combinations denoted in red, 1-2 log10 reduction in yellow)
Tigecycl ine + Rifampicin Polymyxin B + Rifampicin Polymyxin B + Tigecycline
AB
strain
Starting
inocula Mean Mean Mean
98 5.54 0.00 0.00
102 5.21 0.00
104 5.16
126 5.20 2.60 2.48
128 5.42 2.69 0.00
129 5.19 2.28
138 5.38 3.86 4.15
170 5.37 3.79 3.66
174 5.26
112 5.18 0.00 0.00 0.00
8879 5.01 0.00 1.69 0.00
14101 5.40
3160 5.17
13631 5.43
48038 5.32
4.84
5.35 4.86
4.48 4.85 4.57
1.60
5.49
5.93 4.73
5.33
8.29
5.59 4.65 4.63
4.27 5.27 7.16
6.70 4.62 4.59
5.91 5.44 5.94
6.03 5.44 5.41
Time-Kill Results
Polymyxin B alone
6 out of 31 strains showed a reduction of 1-2 log10
CFU/mlin bacterial density compared to baseline at 24 hrs
25 out of 31 strains show insignificant reduction (< 1 log10CFU/ml) or higher inoculums (approx 8 log10 CFU/ml) at 24hrs
Tigecycline or rifampicin alone
Either < 2 log10 CFU/ml drop at 24 hrs from baseline
inoculums for 2 & 1 strain(s) in tigecycline & rifampicinrespectively
Or increase of > 2 log10 CFU/ml at 24 hrs from baselineinoculums
Combination Time-Kill Results
Polymyxin B+ rifampicin
14 out of 31 strains achieve > 2 log10 CFU/ml
decrease from baseline inoculum, at 24 hrs
Polymyxin B + tigecycline
10 out of 31 strains
Tigecycline + rifampicin
8 out of 31 strains
Time-Kill Results
None of the antibiotics combinations demonstratedmodified bactericidal activity against 14 out of 31
strains Polymyxin + rifampicin, polymyxin +tigecycline
demonstrated 1-2 log10 CFU/ml reduction in 5 & 4 strainsrespectively from baseline at 24hr. Total 6 (28,59, 69, 70,138, 170)
Tigecycline + rifampicin is at least additive in 7 strains (23,104, 174, 14101, 3160, 13631, 48038)
Polymyxin + rifampicin is additive to 1 strain (41)
Polymyxin alone demonstrated the lowest bacteria burden for5 strains (23, 174, 3160, 13631, 48038) at 24 hr ~ 3.5-5.6 log10CFU/ml
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TKS results24 hour mean bacteria burden after exposure to various ant ibiotic combinations(modified bactericidal combinations denoted in red, 1-2 log10 reduction in yellow)
Tigecycl ine + Rifampicin Polymyxin B + Rifampicin Polymyxin B + Tigecycline
AB
strain Mean Mean Mean
98 0.00 0.00 4.84
102 5.35 0.00 4.86
104 4.48 4.85 4.57
126 2.60 2.48 1.60
128 5.49 2.69 0.00
129 5.93 4.73 2.28
138 5.33 3.86 4.15
170 8.29 3.79 3.66
174 5.59 4.65 4.63
112 0.00 0.00 0.00
8879 0.00 1.69 0.00
14101 4.27 5.27 7.16
3160 6.70 4.62 4.59
13631 5.91 5.44 5.94
48038 6.03 5.44 5.41
Pharmacokinetic/Pharmacodynamic Modelling
of Polymyxin B, Rifampicin and Tigecyclineagainst Pandrug-resistantAcinetobacter
baumanniiin an In-vitro Model
T.P. Lim1, T.Y. Tan2, W. Lee1, Sasikala. S.2, T.T. Tan1, L.Y. Hsu3, A.L. Kwa1
1Singapore General Hospital, 2Changi General Hospital. 3National University Hospital
ECCMID 2010, Vienna, Austria
HFIM
2 representative strains used to validate the
results in hollow-fiber infection model (HFIM)
TTSH AB 112
SGH AB 8879
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HFIM results
0
2
4
6
8
10
0 1 2 3 4 5
LogCFU/ml
Days
Placebo
Polymyxin B 1MU q12h
Rifampicin 600mg q12h
Tigecycline 100mg q12h
Polymyxin B 1MU q12h +Rifampicin 600mg q12h
Polymyxin B 1MU q12h +Tigecycline 100mg q12h
Tigecycline 100mg q12h +Rifampicin 600mg q12h
HFIM results
Polymyxin B regimen simulated
Polymyxin B resistant isolates plated on drug-supplemented
media at 3X MIC.
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Antibiotic Combinations againstMDR Bacteria
Trial and Error
Countless permutations
Different combinationseffectiv e for differentstrains1
Certain combinations maylead to antagonism2,3
Guided by in-vitroTesting Avoid use of antagonistic
combinations
Identify effectiv ecombinations
1. Lim TP et al. (2009) I Antibiot (Tokyo).2. Aaro n SD et al,(2000) .Am J Respir Crit Care Med 161: 1206-1212.
3. Lang BJ et.al (2000). Am J Respir Crit Care Med 162: 2241-2245.
Methodology Advantages Limitations
Time-kill (TK) method Gold-standard
Measures bactericidal
activity
Describes extent of kill over24 hours
Time-consuming
Limits no. of combinat ions
tested
Need for repetitive samplingResult s likely retro spective in
nature
Multiple Combination
Bactericidal Testing(MCBT) method
Fast turn-around time
Large no. of antibioticcombinations tested
Nov el method
Limitations not f ullyelucidated
Inoculation of bacteria
Bac teria at s tandard concentration
added
More than 80 combinations tested
Day 0Isolates
received
TIME-LINE
Day -1
Day 1
Day 2
MCBT method
Preparation of microtiter plates Addition of one or two antibiotic(s) to
well
Prepared in bulk and stored till required
Sampling and plating of bacteria Preliminary results (based on turbid
wells)
Contents of wells sampled and plated
Bacteria Counts Counts enumerated based on growth
on plates
TK method
Preparation ofmicrotiter
plates
Inoculation of
bacteria
Sampling and
plating of bacteria
Bacteria Counts
Preparation of drugs /Inoculating bacteria Addition of one/two antibiotics to
flasks
Standard concentration of bacteriaadded
Up to 20 flasks tested
Sampling and plating of bacteria Contents of flasks sampled and
plated
Bacteria Counts Counts enumerated based on
growth on plates
Sampling and platingof bacteria
Bacteria Counts
Preparation of drugs/Inoculating bacteria
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ACI B AUMAN- - - - - - - - - - - -
POLYMYXIN + RIFAMPICIN
POLYMYXIN + TIGECYCLINE
POLYMYXIN + AZTREONAM
RIFAMPICIN + TIGECYCLINE
MEROPENEM +AZTREONAM
AZTREONAM + LEVOFLOXA CIN
POLYMYXIN + MEROPENEM
MEROPENEM + AZTREONAM
RIFAMPICIN + MEROPENEM
LEVOFLOXACIN + TIGECYCLINE
Legend
At least inhibitory No utility
Collaborators
Changi General Hospital
Dr Tan Thean Yen National University Hospital
A/P Hsu Li Yang
Tan Tock Seng Hospital
Dr David Lye
A*STAR (IBN)
A/P Yang Yi-Yan
Parkw ay Health
Dr AsokKurup
University of Houston
A/P Vincent Tam
Singapore General Hospital Dr Tan Thuan Tong
Dr Tan Ban Hock Dr Jenny Low
NUS (Pharmacy)
A/P Eric Chan
NUS (Chemical andBiomolecular Engineering ) A/P Xie Jianping
A/P Leong Tai
University of Monash
A/P Li Jian.
Thank You!
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