Evaluation of Polymyxin B-Based and Dual- Carbapenem Combinations Against Carbapenem-Resistant Enterobacteriaceae Co- Harbouring Mcr-1 and KPC-2

Home , Aztreonam, Doripenem, Ertapenem, Meropenem

Evaluation of Polymyxin B-based and Dual- Carbapenem Combinations against Carbapenem-Resistant Enterobacteriaceae co- harbouring mcr-1 and KPC-2

Jocelyn Teo1, Tze-Peng Lim1, Si-Xuan Tan1, Yiying Cai1, Winnie Lee1, Tse-Hsien Koh1, Thuan-Tong Tan1, Maciej Piotr Chlebicki1, Andrea Kwa1,2,3 1 Singapore General Hospital 2 National University of Singapore 3 Duke-NUS Medical School Disclosures

› This study was funded in part by the National Medical Research Council (Singapore) Centre Grant A serious public health threat CARBAPENEM-RESISTANT ENTEROBACTERIACEAE (CRE)

https://www.cdc.gov/drugresistance/pdf/ar-threats-2013-508.pdf Increasing carbapenem resistance in Singapore

15.0

- 13.0 11.0 9.0 7.0 5.0

3.0 susceptibility (%) susceptibility

% carbapenem non carbapenem % 1.0 -1.0 2011 2012 2013 2014 2015* Klebsiella spp. E. coli Enterobacter spp.

JQ Teo et al. Microorganisms 4 (1). 2016 Feb 16. Last resort antibiotic Polymyxin, an old antibiotic, is increasingly being used for multi-drug resistant infections POLYMYXIN RESISTANCE ANOTHER NAIL IN THE ANTIBIOTIC COFFIN…

http://www.nbcnews.com/health/health-news/nevada-woman- died-near-ultimate-superbug-n706641 S. Baron et al. International Journal of Antimicrobial Agents 48 (2016) 583–591 13% OF CRE ISOLATES WERE PB-RESISTANT

140 120 9% 100 17% 19% 80 11% 60

40 7%

No of CRE isolates, n isolates, CRE of No 20 0 2010 2011 2012 2013 2014 2015 2016 PB susceptible PB resistant J. Teo et al. 27th ECCMID (23rd Apr 2017) Poster P0711 Study objective

› To evaluate the in vitro activity of multiple antibiotics in combination with polymyxin B against mcr-1 -harbouring CRE through time-kill studies METHODS Study Isolates

› CRE prospective surveillance in Singapore General Hospital

› Retrospective screening for mcr-1 via WGS and/or PCR for the period 2013 - 2015 Susceptibility Testing (MIC)

› Microbroth dilution panels (Trek Diagnostics, West Sussex, UK) Study Antibiotics

Antibiotic Simulated Dosing Regimens Concentration (mg/L) Polymyxin B 30,000IU/kg/day 2 Ertapenem 1g every 24h 15 Aztreonam 8g every 24h (infused over 24h) 24 Cefepime 2g every 8h (infused over 4h) 50 Piperacillin/ tazobactam 4.5g every 6h (infused over 4h) 35/7 Meropenem 2g every 8h (infused over 3h) 20 Doripenem 2g every 8h (infused over 4h) 26 Imipenem 1g every 6h (infused over 1h) 12.5 Levofloxacin 750mg every 24h 8 Rifampicin 600mg every 12h 4 Tigecycline 100mg every 12h 2 Polymyxin B Combinations Time-kill Studies

5 /mL Bactericidal activity cfu 4

10 ≥ 3 log decrease in cfu/mL 3

Log from original inoculum 2 1 0 0 2 4 8 12 24 Time (Hours) RESULTS EC249 EC250 ENT702 Species E. coli E. coli E. aerogenes MLST ST2006 ST2006 N.A. Isolation site Rectal swab Rectal swab Urine Carbapenemase KPC-2 KPC-2 KPC-2 Other beta- TEM-1B, TEM-1A TEM-1A, AmpC1 lactamases CTX-M-15 strA, strB, aadA1, aadA2, aadA2, mph(A), aac(6')Ib-cr, Test Test Isolates Other resistance mph(A), cmlA1, cmlA1, sul3, aac(3)-IIa, qnrB66, determinants sul3, dfrA12 dfrA12 mph(A), catB3, sul2, tet(A), dfrA14 Porin loss No No Yes EC249 EC250 ENT702 Species E. coli E. coli E. aerogenes MLST ST2006 ST2006 N.A. Isolation site Rectal swab Rectal swab Urine Carbapenemase KPC-2 KPC-2 KPC-2 Other beta- TEM-1B, TEM-1A TEM-1A, AmpC1 lactamases CTX-M-15 strA, strB, aadA1, aadA2, aadA2, mph(A), aac(6')Ib-cr, Test Test Isolates Other resistance mph(A), cmlA1, cmlA1, sul3, aac(3)-IIa, qnrB66, determinants sul3, dfrA12 dfrA12 mph(A), catB3, sul2, tet(A), dfrA14 Porin loss No No Yes Highly similar → possibly clonal MIC, mg/L EC249 EC250 ENT702 Polymyxin B 4 4 8 Aztreonam ≥128 ≥128 ≥128 Cefepime ≥128 64 ≥128 Piperacillin/Tazobactam ≥256/4 ≥256/4 ≥256/4 Doripenem 8 8 8 Imipenem ≥32 ≥32 ≥32 Meropenem ≥32 ≥32 ≥32

Ertapenem ≥32 ≥32 ≥32 Susceptibilities Levofloxacin ≥64 ≥64 1 Tigecycline ≤0.25 ≤0.25 1 Rifampicin ≥64 ≥64 ≥64 MIC, mg/L EC249 EC250 ENT702 Polymyxin B 4 4 8 Aztreonam ≥128 ≥128 ≥128 Cefepime ≥128 64 ≥128 Piperacillin/Tazobactam ≥256/4 ≥256/4 ≥256/4 Doripenem 8 8 8 Imipenem ≥32 ≥32 ≥32 Meropenem ≥32 ≥32 ≥32

Ertapenem ≥32 ≥32 ≥32 Susceptibilities Levofloxacin ≥64 ≥64 1 Tigecycline ≤0.25 ≤0.25 1 Rifampicin ≥64 ≥64 ≥64 Polymyxin Time-kill Curves 10

8 Control 6

EC249 (PB MIC 4 mg/L) cfu/mL 10 4

EC250 (PB MIC 4 mg/L) Log 2 EC702 (PB MIC 8 mg/L) 0 0 4 8 12 16 20 24 Time/hr Doripenem Time-kill Curves

10 Control 8 EC249 6 cfu/m EC250 10 4

Log EC702 2

0 Doripenem MIC 0 4 8 12 16 20 24 = 8 mg/L Time/hr Levofloxacin Time-kill Curves

8 Control 6 ≥ cfu/mL EC249 (MIC 64 mg/L) 10 4 ≥ Log EC250 (MIC 64 mg/L) 2 EC702 (MIC 1 mg/L) 0 0 4 8 12 16 20 24 Time/hr Tigecycline time-kill curves

8 Control 6 ≤

cfu/mL EC249 (MIC 0.25 mg/L)

10 4 ≤

Log EC250 (MIC 0.25 mg/L) 2 EC702 (MIC 1 mg/L) 0 0 4 8 12 16 20 24 Time/hr EC249

kill Antibiotic

- Log CFU/mL at 24h Aztreonam 0.00 10 Control Cefepime 4.13 9 PT4 9.00 8 Polymyxin B 7 Doripenem 4.32 Polymyxin B + 6 Aztreonam Imipenem 4.05 5 Polymyxin B + Tigecycline

Polymyxin Meropenem 2.89 4 Log CFU/ml Log Ertapenem + Imipenem Rifampicin 2.78 3 Ertapenem + Tigecycline 2.34 2 Meropenem Levofloxacin 5.04 1 Ertapenem + Doripenem 0 Doripenem 0.00 0 4 8 12 16 20 24

Imipenem 0.00 Time (h) EC249 Combination Time Combination EC249 Ertapenem Meropenem 0.00 EC250

kill Antibiotic

- Log CFU/mL at 24h Control Aztreonam 4.81 10 Polymyxin B 9 Cefepime 0.65 Polymyxin B + 8 Cefepime PT4 9.00 Polymyxin B + 7 Doripenem Doripenem 0.00 6 Polymyxin B + Imipenem 5.50 Meropenem 5 Polymyxin B + Levofloxacin

Polymyxin Meropenem 2.03

Log CFU/ml Log 4 Polymyxin B + Rifampicin 0.00 3 Rifampicin Polymyxin B + Tigecycline 2.44 2 Tigecycline Ertapenem + Levofloxacin 0.00 1 Imipenem Ertapenem + 0.65 0 Doripenem 0 4 8 12 16 20 24 Meropenem Ertapenem +

Imipenem 1.80 Time (h) Doripenem EC250 Combination Time Combination EC250

Ertapenem Meropenem 0.65 EC720

kill Antibiotic

- Log CFU/mL at 24h Aztreonam 9.00 10 Control Cefepime 3.12 9 Polymyxin B PT4 9.00 8 7 Doripenem 9.00 Polymyxin B + Meropenem 6 Imipenem 9.00 Polymyxin B + 5 Levofloxacin

Polymyxin Meropenem 0.00 Polymyxin B +

Log CFU/ml Log 4 Rifampicin Rifampicin 0.00 3 Ertapenem + Tigecycline 4.76 Imipenem 2 Ertapenem + Levofloxacin 0.65 Meropenem 1 Ertapenem + Doripenem 0.00 0 Doripenem Imipenem 1.65 0 4 8 12 16 20 24

Time (h) EC720 Combination Time Combination EC720

Ertapenem Meropenem 0.00 Log CFU/mL at 24h Antibiotic EC249 EC250 ENT702