Extremely Drug-resistant organisms: Synergy Testing

LIM TZE PENG Principal Pharmacist Singapore General Hospital

Background
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.
1. Helen W . Boucher, Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious Diseases Society of America. Clin Infect Dis 2009; 48:1-12

Definitions
What is the difference between
MDR XDR PDR

1.

Magiorakos, A. P ., A. Srinivasan, et al. (2011). "Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance." Clin Microbiol Infect.

Definitions
What is the difference between

Synergistic Bactericidal Inhibitory Antagonistic

Typical MIC profile

Strain Antibiotic
Ampicillin/Sulbactam Ciprofloxacin Gentamicin Imipenem Meropenem Aztreonam Piperacillin/Tazobactam Polymyxin B Tigecycline Ceftazidime Amikacin Cefepime Rifampicin

18351
32/16 16 64 64 128 128 256 64 4 128 128 128 2

18352
32/16 16 64 32 64 64 256 32 4 128 128 128 4

27640
64/32 16 64 32 32 32 256 128 16 128 128 128 256

9447
32/16 16 64 64 64 128 256 16 4 128 128 128 256

11171
32/16 16 64 64 64 128 256 16 4 128 128 64 256

Typical MIC profile

Strain Antibiotic
Ampicillin/Sulbactam Ciprofloxacin Gentamicin Imipenem Meropenem Aztreonam Piperacillin/Tazobactam Polymyxin B Tigecycline Ceftazidime Amikacin Cefepime Rifampicin

18351
32/16 16 64 64 128 128 256 64 4 128 128 128 2

18352
32/16 16 64 32 64 64 256 32 4 128 128 128 4

27640
64/32 16 64 32 32 32 256 128 16 128 128 128 256

9447
32/16 16 64 64 64 128 256 16 4 128 128 128 256

11171
32/16 16 64 64 64 128 256 16 4 128 128 64 256

Potential solutions
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

Combination therapy
Often used in clinical practice
TB & HIV Increasingly used in MDR A. baumannii & P. aeruginosa

Enhanced pharmacodynamic effect (synergism)

Enhanced bactericidal effect Suppress emergence of resistance

1.

Maragakis LL, Perl TM. Acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options. Clin Infect Dis. 2008 Apr 15;46(8):1254-63.

Types of combination studies Various methodologies

Checkerboard method Time-kill studies In-vitro pharmacodynamic models

Fractional Inhibitory Concentration Index

Synergy: FIC index < 0.5 Additive: FIC index = 1 Antagonism: FIC index > 4

1.

Hsieh MH, Yu CM, Yu VL, Chow JW . Synergy assessed by checkerboard. A critical analysis. Diagn Microbiol Infect Dis. 1993 MayJun;16(4):343-9.

Limitations of FIC index

Based on Loewe additivity Assume similar & linear concentration-time relationship Subjective endpoints Cloudy vs Clear wells

Time-kill studies (TKS)

9 8 7
Log10 [cfu/ml]

Growth control Polymyxin B Rif ampicin Polymyxin B & Rifampicin

6 5 4 3 2 1 0 0 2 4 8 12 24
Time (Hours)

Antibiotic synergy: 2 log decrease in cfu/ml

1.

National Committee for Clinical Laboratory Standards. 1999. Methods for Determining Bactericidal Activity of Antimicrobial Agents. Approved Guideline M26-A., vol. 19. NCCLS, 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 affect the growth curve of the test organism when used alone. 1 Pharmacokinetic factors ignored

Resource management
Labour & Time intensive
1. Antimicrobial Agents & Chemotherapy: Instructions to Authors

Hollow-Fiber System
Drug

bacteria

Distribution

Elimination

Hollow-fiber infection model allowing simulation of human PK in vitro. (Tam, JID 2007)

Aims
To elucidate efficacious antibiotic combinations against PDR A. baumannii

Methods
Time-kill studies (TKS)
Maximal clinically achievable concentrations

Hollow Fiber Infection Model (HFIM)

In-vivo environment simulation

Time Kill Study (TKS)

Susceptibility results
Antimicrobial Meropenem Polymyxin B Rifampicin 1 Tigecycline
1 There

TTSH 112 (mg/l) 32 (R) 1 (S) 4 4 (I)

TTSH 105 (mg/l) 64 (R) 1 (S) 4 0.5 (S)

SGH 8879 (mg/l) >32 (R) 2 (S) 2 2 (S)

are currently no international standards for rifampicin and susceptibility testing against Acinetobacter baumannii

Pharmacokinetic Data
Antimicrobial Simulated FREE drug conc (mg/l) 64 Maximum clinical achievable FREE drug conc (mg/L) 64 (plasma) Corresponding maximum clinical dose 2g q8h over 3h infusion Meropenem

Polymyxin B

2 (plasma)

At least 1 MU q12h

Rifampicin

2 (plasma)

PO 600mg q12h

Tigecycline

2 (tissues)

100mg q12h

1. Jaruratanasirikul S, et al. Comparison of the pharmacodynamics of meropenem in patients with ventilator-associated pneumonia following administration by 3-hour infusion or bolus injection. Antimicrob Agents Chemother. 2005 Apr;49(4):1337-9. 2. Kwa AL, Lim TP, Low JG, Hou J, Kurup A, Prince RA, Tam VH. Pharmacokinetics of polymyxin B1 in patients with multidrug-resistant Gramnegative bacterial infections. Diagn Microbiol Infect Dis. 2008 Feb;60(2):163-7. Epub 2007 Oct 4. 3. Gumbo T, Louie A, Deziel MR, Liu W, Parsons LM, Salfinger M, Drusano GL. Concentration-dependent Mycobacterium tuberculosis killing and prevention of resistance by rifampin. Antimicrob Agents Chemother. 2007 Nov;51(11):3781-8. Epub 2007 Aug 27. 4. Rodvold KA, Gotfried MH, Cwik M, Korth-Bradley JM, Dukart G, Ellis-Grosse EJ. Serum, tissue and body fluid concentrations of tigecycline after a single 100 mg dose. J Antimicrob Chemother. 2006 Dec;58(6):1221-9.

TKS SGH AB 8879

10 8 Log10 C FU /m l

Microbiological responses of A B against various antibiotics

Pl acebo

Microbiological responses of AB against various antibiotic combinations 10

Pl ac e bo

8 L o g1 0 C F U/ml
PB

Ri f + PB Ri f + Me ro PB + Mero

6
Mero

6 4 2

4
Ri f

Tige + PB Tige + Ri f Tige + Me ro

2
Tige

0 0 4 8 12 Time (h) 16 20 24

0 0 4 8 12 T ime (h) 16 20 24

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 Antimicrobial Resistance, 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) 2 (serum trough) 2 (serum peak) 2 (tissue peak) Corresponding maximum clinical dose At least 1 MU q12h PO 600mg q12h 100mg q12h

Polymyxin B Rifampicin Tigecycline

Kwa AL, Lim TP , Low JG, Hou J, Kurup A, Prince RA, Tam VH. Pharmacokinetics of polymyxin B1 in patients with multidrug-resistant Gram-negative 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, Salfinger M, Drusano GL. Concentration-dependent Mycobacterium tuberculosis killing and prevention of resistance by rifampin. Antimicrob Agents Chemother. 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 concentrations of tigecycline after a single 100 mg dose. J Antimicrob Chemother. 2006 Dec;58(6):1221-9.

Combination timekill
9 8 7
Log10 [cfu/ml]

Growth control Polymyxin B Rif ampicin Polymyxin B & Rifampicin

6 5 4 3
Antibiotic synergy:
2 log decrease in cfu/ml from original inoculum

2 1 0 0 2 4 8 12 24
Time (Hours)

2 log decrease in cfu/ml

MIC results (31 PDR AB strains)

Susceptibility (%)

Antibiotics

MIC 50 (mg/L) MIC 90 (mg/L) Range (mg/L)

Polymyxin B Rifampicin Tigecycline

1 6 4

2 64 32

0.5 2 1 64 0.5 32

100

Resistant to all antibiotics

24 hour mean bacteria burden after exposure to various antibiotics alone (1-2 log reductions strains denoted in red)
Tigecycline AB strain 8 12 16 17 23 25 28 32 41 59 60 69 70 88 91 97 Starting inocula 5.22 5.27 5.23 5.03 5.35 5.44 5.30 5.30 5.38 5.25 5.36 5.25 5.26 5.28 5.20 5.33 Mean 8.53 8.60 7.72 3.74 7.94 6.86 8.08 3.40 7.99 8.08 7.97 8.16 8.33 7.17 7.14 6.94 Polymyxin B Mean 7.13 5.72 3.50 9.14 4.94 5.72 8.61 5.11 8.04 5.13 4.02 5.48 3.12 4.84 3.00 4.85 Rifampicin Mean 8.07 7.77 8.69 3.29 5.99 7.83 8.73 7.91 5.56 9.17 8.46 8.97 8.60 6.11 8.58 5.62

Single TKS results

24 hour mean bacteria burden after exposure to various antibiotics alone (1-2 log reduction strains denoted in red)
Tigecycline AB strain 98 102 104 126 128 129 138 170 174 112 8879 14101 3160 13631 48038 Starting inocula 5.54 5.21 5.16 5.20 5.42 5.19 5.38 5.37 5.26 5.18 5.01 5.40 5.17 5.43 5.32 Mean 7.72 6.63 6.84 8.77 8.71 8.84 6.77 7.62 6.35 7.45 5.25 5.38 8.93 7.93 8.90 Polymyxin B Mean 4.31 5.09 5.05 5.59 7.86 4.07 4.40 5.42 3.57 4.73 4.23 5.71 4.75 5.59 4.78

Single TKS results (continued)

Rifampicin Mean 5.59 8.05 7.70 5.43 5.82 8.28 8.36 8.49 8.24 7.52 7.56 8.33 8.60 8.86 8.18

Combination TKS results

24 hour mean bacteria burden after exposure to various antibiotic combinations (modified bactericidal combinations denoted in red, 1-2 log10 reduction in yellow)
Tigecycline + Rifampicin AB strain 8 12 16 17 23 25 28 32 41 59 60 69 70 88 91 97 Starting inocula 5.22 5.27 5.23 5.03 5.35 5.44 5.30 5.30 5.38 5.25 5.36 5.25 5.26 5.28 5.20 5.33 Mean 6.81 7.19 4.70 0.00 4.95 4.73 5.53 5.41 4.77 8.67 6.65 7.64 6.84 2.42 0.00 0.00 Polymyxin B + Rifampicin Mean 0.00 0.00 3.56 0.00 4.85 0.00 3.57 0.00 4.54 4.10 5.37 4.10 4.66 0.00 0.00 0.00 Polymyxin B + Tigecycline Mean 5.11 0.00 0.80 0.65 4.82 5.01 4.80 2.31 5.10 3.24 0.80 5.08 4.09 4.90 4.65 4.25

24 hour mean bacteria burden after exposure to various antibiotic combinations (modified bactericidal combinations denoted in red, 1-2 log10 reduction in yellow)
Tigecycline + Rifampicin AB strain 98 102 104 126 128 129 138 170 174 112 8879 14101 3160 13631 48038 Starting inocula 5.54 5.21 5.16 5.20 5.42 5.19 5.38 5.37 5.26 5.18 5.01 5.40 5.17 5.43 5.32 Mean 0.00 5.35 4.48 2.60 5.49 5.93 5.33 8.29 5.59 0.00 0.00 4.27 6.70 5.91 6.03 Polymyxin B + Rifampicin Mean 0.00 0.00 4.85 2.48 2.69 4.73 3.86 3.79 4.65 0.00 1.69 5.27 4.62 5.44 5.44

Combination TKS results (continued)

Polymyxin B + Tigecycline Mean 4.84 4.86 4.57 1.60 0.00 2.28 4.15 3.66 4.63 0.00 0.00 7.16 4.59 5.94 5.41

Time-Kill Results
Polymyxin B alone
6 out of 31 strains showed a reduction of 1-2 log10 CFU/ml in bacterial density compared to baseline at 24 hrs 25 out of 31 strains show insignificant reduction (< 1 log10 CFU/ml) or higher inoculums (approx 8 log10 CFU/ml) at 24 hrs

Tigecycline or rifampicin alone

Either < 2 log10 CFU/ml drop at 24 hrs from baseline inoculums for 2 & 1 strain(s) in tigecycline & rifampicin respectively Or increase of > 2 log10 CFU/ml at 24 hrs from baseline inoculums

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 demonstrated modified bactericidal activity against 14 out of 31 strains
Polymyxin + rifampicin, polymyxin +tigecycline demonstrated 1-2 log10 CFU/ml reduction in 5 & 4 strains respectively 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 for 5 strains (23, 174, 3160, 13631, 48038) at 24 hr ~ 3.5-5.6 log10 CFU/ml

24 hour mean bacteria burden after exposure to various antibiotic combinations (modified bactericidal combinations denoted in red, 1-2 log10 reduction in yellow)
Tigecycline + Rifampicin AB strain 98 102 104 126 128 129 138 170 174 112 8879 14101 3160 13631 48038 Mean 0.00 5.35 4.48 2.60 5.49 5.93 5.33 8.29 5.59 0.00 0.00 4.27 6.70 5.91 6.03 Polymyxin B + Rifampicin Mean 0.00 0.00 4.85 2.48 2.69 4.73 3.86 3.79 4.65 0.00 1.69 5.27 4.62 5.44 5.44 Polymyxin B + Tigecycline Mean 4.84 4.86 4.57 1.60 0.00 2.28 4.15 3.66 4.63 0.00 0.00 7.16 4.59 5.94 5.41

TKS results

Pharmacokinetic/Pharmacodynamic Modelling of Polymyxin B, Rifampicin and Tigecycline against Pandrug-resistant Acinetobacter baumannii in an In-vitro Model
T.P. Lim1, T.Y. Tan 2, W. Lee1, Sasikala. S.2, T.T. Tan 1, L.Y. Hsu 3, A.L. Kwa 1 1 Singapore General Hospital, 2 Changi General Hospital. 3 National 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

HFIM results

10 Placebo 8 Log CFU/ml Polymyxin B 1MU q12h Rifampicin 600mg q12h Tigecycline 100mg q12h 4 Polymyxin B 1MU q12h + Rifampicin 600mg q12h 2 Polymyxin B 1MU q12h + Tigecycline 100mg q12h Tigecycline 100mg q12h + Rifampicin 600mg q12h 0 1 2 Days 3 4 5

HFIM results
Polymyxin B regimen simulated
Polymyxin B resistant isolates plated on drug-supplemented media at 3X MIC.

10 Log C FU /m l 8 6 4 2 0 0 1 2 D ays 3 4 5
Tota l Res ist a nt

Antibiotic Combinations against MDR Bacteria

T rial and Error Countless permutations Different combinations effectiv e for different strains 1 Certain combinations may lead to antagonism2,3

Guided by in-vitro T esting Av oid use of antagonistic combinations Identify effectiv e combinations
1. Lim TP et al. (2009) I Antibiot (Tokyo). 2. Aaron 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 Time-kill (TK) method

Advantages Gold-standard Measures bactericidal activity Describes extent of kill over 24 hours

Limitations Time-consuming Limits no. of combinations tested Need for repetitive sampling Results likely retrospective in nature

Multiple Combination Bactericidal Testing (MCBT) method

Fast turn-around time Large no. of antibiotic combinations tested

Nov el method Limitations not f ully elucidated

MCBT method Preparation of microtiter plates Preparation of Addition of one or microtiter two antibiotic(s) to well plates Prepared in bulk and stored till required Inoculation of bacteria Inoculation of Bacteria at standard bacteria concentration added More than 80 combinations tested Sampling and Sampling and plating of bacteria plating of bacteria Preliminary results (based on turbid wells) Contents of wells sampled and plated Bacteria Counts Counts enumerated Bacteria based Counts on growth on plates

TK method

Day -1

Preparation of drugs /Inoculating bacteria Addition of one/two antibiotics to Day 0 Preparation of drugs/ flasks Isolates Inoculating bacteria Standard concentration of bacteria received added Up to 20 flasks tested and plating Day 1 Sampling and plating Sampling of bacteria bacteria Contents of flasks of sampled and plated Day 2

Bacteria Counts Counts enumerated Bacteria basedCounts on growth on plates

TIME-LINE

ACI B AUMAN -- ---- ---- -POLYMYXIN + RIFAMPICIN POLYMYXIN + TIG ECYCLINE POLYMYXIN + AZTREONAM RIFAMPICIN + TIG ECYCLINE MEROPENEM +AZTREONAM AZTREONAM + LEVOFLOXA CIN POLYMYXIN + MEROPENEM MEROPENEM + AZTREONAM RIFAMPICIN + MEROPENEM LEVOFLOXACIN + TIG ECYCLINE
Legend

At least inhibitory No utility

Collaborators
Changi General Hospital
Dr Tan Thean Yen Singapore General Hospital
Dr Tan Thuan Tong Dr Tan Ban Hock Dr Jenny Low

National University Hospital

A/P Hsu Li Yang

Tan Tock Seng Hospital

Dr David Lye

A*STAR (IBN)
A/P Yang Yi-Yan

NUS (Pharmacy) A/P Eric Chan NUS (Chemical and Biomolecular Engineering )
A/P Xie Jianping A/P Leong Tai

Parkw ay Health
Dr AsokKurup

University of Monash
A/P Li Jian .

University of Houston
A/P Vincent Tam

Thank You!