synergy testing (4)

7/28/2019 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)


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)


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!

synergy testing (4)

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