Multiple antibiotics in combination against extreme drug resistant acinetobacter baumannii in an in vitro pharmacokinetic pharmacodynamic model

MULTIPLE ANTIBIOTICS IN COMBINATION AGAINST EXTREME DRUG RESISTANT ACINETOBACTER BAUMANNII IN AN IN-VITRO PHARMACOKINETIC/PHARMACODYNAMIC MODEL LIM TZE PENG BSc (Pharm) (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MEDICINE YONG LOO LIN SCHOOL OF MEDICINE NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I HEREBY DECLARE THAT THE THESIS IS MY ORIGINAL WORK AND IT HAS BEEN WRITTEN BY ME IN ITS ENTIRETY I HAVE DULY ACKNOWLEDGED ALL THE SOURCES OF INFORMATION WHICH HAVE BEEN USED IN THE THESIS THIS THESIS HAS ALSO NOT BEEN SUBMITTED FOR ANY DEGREE IN ANY UNIVERSITY PREVIOUSLY _ Lim Tze Peng 21 Nov 2013 i Acknowledgement The dissertation presented before you is a piece of work that would not have been possible without the people who accompanied and supported me in these five rewarding years It is my utmost pleasure that I can express my gratitude to them at the beginning of this thesis, a work that is completed with their contributions First, I would like to express special thanks to my supervisor, Assistant Professor Li Yang HSU, for being the best mentor I have ever met and heard His relentless guidance and support has made this project as perfect as it can be By establishing the contacts between me and the right people, his directions certainly brought this work to another level Another important person I would like to thank is my co-supervisor, Prof OH Min Sen, Vernon for giving me a chance to work with this wonderful project I sincerely appreciate his support for my work Thirdly, I am very grateful to Dr Andrea KWA Apart from being a mentor, she is also a friend who walked by my side even in the most difficult moments of this project She constantly reminded me of my strengths and weaknesses, making me not only a better researcher, but a better person I am grateful to A/Prof Vincent H TAM and his team members, including Kimberly R LEDESMA and Dr Kai Tai CHANG, for the initial laboratory training and critical research analytical skills as well as their guidance and assistance in hollow-fibre infection model studies ii I am grateful to A/Prof Eric CHAN and his team members, especially Ms Lee Sun NEW, for the teaching, analysis and execution of the LC/MS/MS method studies; this project would not be complete without them I would like to thank A/Prof Mary NG for setting up the electron microscopy data Her efforts in explaining and analysing results marked the first step in understanding the resistant bacteria data I also thank Ms Pei Yun HON for spending many hours working on the clonality of the bacteria and preparing them for electron microscopy work Her experience and assistance are invaluable to this project I am also grateful to all of the co-workers of the Pharmacy research laboratory, in particular Ms Winnie Lee and Ms Sasikala D/O Suranthran Their help and support certainly made my studies more productive and enjoyable I thank Jocelyn TEO and Yiying CAI for their meticulous corrections and sincerity in proof-reading my thesis I also would like to thank the Department of Medicine, Yong Loo Lin School of Medicine, National University of Singapore, for giving me the opportunity for this study Finally, I am sincerely grateful to my family, friends and especially my wife, Cassandra CHANG for their understanding as well as endless support and encouragement in my life Thank you iii Formal communications arising from this thesis Publications Lim TP, Tan TY, Lee W, Sasikala S, Tan TT, Hsu LY, Kwa AL In-vitro activity of various combinations of antimicrobials against carbapenemresistant Acinetobacter species in Singapore J Antibiot (Tokyo) 2009 Dec;62(12):675-9 doi: 10.1038/ja.2009.99 Epub 2009 Oct 30 PubMed PMID: 19876075 Tan TY, Lim TP, Lee WH, Sasikala S, Hsu LY, Kwa AL In-vitro antibiotic synergy in extensively drug-resistant Acinetobacter baumannii: the effect of testing by time-kill, checkerboard, and Etest methods Antimicrob Agents Chemother 2011 Jan;55(1):436-8 doi: 10.1128/AAC.00850-10 Epub 2010 Oct 18 PubMed PMID: 20956606; PubMed Central PMCID: PMC3019682 Lim TP, Tan TY, Lee W, Sasikala S, Tan TT, Hsu LY, Kwa AL In-vitro activity of polymyxin B, rifampicin, tigecycline alone and in combination against carbapenem-resistant Acinetobacter baumannii in Singapore PLoS One 2011 Apr 21;6(4):e18485 doi: 10.1371/journal.pone.0018485 PubMed PMID: 21533030; PubMed Central PMCID: PMC3080872 New LS, Lim TP, Oh JW, Cheah GJ, Kwa AL, Chan EC Optimizing hollowfibre-based pharmacokinetic assay via chemical stability study to account for inaccurate simulated drug clearance of rifampicin Anal Bioanal Chem 2013 Feb;405(4):1407-15 doi: 10.1007/s00216-012-6549-7 Epub 2012 Nov 24 PubMed PMID: 23180085 Conferences abstracts Lim TP et al (Apr 2010) “Evaluation of antibiotic synergy against multidrugresistant Acinetobacter baumannii: comparison of three methods" 20th European Conference on Microbiology and Infectious Diseases Vienna, Austria Lim TP et al (Sep 2010) “In-vitro pharmacokinetic profiling of polymyxin B and rifampicin using ultra-performance liquid chromatography tandem mass spectrometry” 1st SingHealth-Duke NUS Scientific Congress Singapore Lim TP et al (Sep 2010) “The stability of polymyxin B (PB) components & rifampicin (R) in Muller-Hinton Broth” 1st SingHealth-Duke NUS Scientific Congress Singapore Oral presentations Lim TP et al (May 2010) “In-vitro activity of antimicrobials in combination against clinical strains of extreme drug-resistant Acinetobacter baumannii to all antibiotics including polymyxin B in Singapore” 20th European Conference on Microbiology and Infectious Diseases Vienna, Austria Lim TP et al (May 2010) “Pharmacokinetic/pharmacodynamic modelling of polymyxin B, rifampicin and tigecycline against pandrug-resistant Acinetobacter baumannii in an in-vitro model” 20th European Conference on Microbiology and Infectious Diseases Vienna, Austria iv Awards Best Oral Scientific Paper Presentation Singapore Pharmacy Congress 2007 Best Poster Scientific Paper Presentation Singapore Pharmacy Congress 2007 Young Investigator’s Award (Allied Health) 17th Annual Scientific Meeting 2008 Singapore General Hospital, SingHealth Best Oral Paper Award (Allied Health) 17th Annual Scientific Meeting 2008 Singapore General Hospital, SingHealth v Table of contents DECLARATION i Acknowledgement ii Formal communications arising from this thesis iv Table of contents vi Summary xi List of tables 13 List of figures 14 CHAPTER Introduction 17 1.1 Antimicrobial resistance 18 1.2 Acinetobacter - Taxonomy 19 1.3 Acinetobacter baumannii - Mechanisms of antibiotic resistance 20 1.4 Antibiotic Combinations - Clinical rationale 21 1.4.1 Achievement of a synergistic effect 22 1.4.2 Prevent emergence of resistance 23 1.4.3 Against highly resistant organisms 23 1.5 Methods for assessing drug interactions 26 1.5.1 Checkerboard method 27 1.5.2 Epsilometer test (Etest) method 29 1.5.3 Time-kill studies 30 1.5.4 In vitro pharmacodynamic models (IVPD) 32 1.6 Rationale for antibiotic combination testing 35 1.7 Hypothesis 36 1.8 Aims 37 CHAPTER Materials and methods 38 vi 2.1 Characterisation of XDR A baumannii isolates 39 2.1.1 Microorganism collection and selection 39 2.1.2 Susceptibility studies 39 2.1.3 Clonal relationship analysis 40 2.1.4 Determining resistance mechanism(s) of isolates 40 2.2 Antimicrobial agent selection 40 2.3 Time-kill studies (TKS) 41 2.3.1 Experimental setup 41 2.3.2 Pharmacodynamic endpoints 43 2.4 Synergy studies on the comparison of the checkerboard and Etest methods against time-kill results 45 2.5 Hollow-Fibre Infection Model (HFIM) studies 46 2.5.1 Experimental setup 47 2.5.2 Characterising the pharmacodynamic response in each HFIM 48 2.6 Pharmacodynamic validation studies on the HFIM 48 2.6.1 Characterising the emergence of resistance during antibiotic exposure in the HFIM 48 2.6.2 In vitro time-growth studies and modelling of bacteria growth kinetics 49 2.6.3 Electron microscopy studies 50 2.6.3.1 Sample preparation 51 2.6.3.2 Scanning electron microscopy (SEM) imaging 51 2.6.3.3 Transmission electron microscopy (TEM) imaging 51 2.7 Pharmacokinetic validation studies on the HFIM 52 2.7.1 Materials and reagents 52 2.7.2 Calibration and quality-control samples 53 2.7.3 Sample preparation 53 2.7.4 UPLC/MS/MS analysis 54 2.7.5 Fit-for-purpose qualification 55 vii 2.7.6 Chemical stability study 57 2.7.7 Quantification of PK samples 58 2.7.8 Analysis of PK samples 59 2.8 Multiple Combinations Bactericidal Testing (MCBT) 61 2.8.1 Validating the predictive ability of the MCBT to the HFIM for clinical use 61 2.8.2 Methods 61 CHAPTER Characterisation of A baumannii isolates 64 3.1 Susceptibility studies 65 3.2 Clonal relationship analysis 65 3.3 Confirmation of resistance 67 CHAPTER Demonstration of in vitro activity of various antibiotics against XDR A baumannii using TKS 68 4.1 Synopsis 69 4.2 Results 70 4.2.1 Single TKS 70 4.2.2 Combination TKS 72 4.2.3 Discussion 77 CHAPTER Examining the effect of synergy testing by various methods 82 5.1 Synopsis 83 5.2 Results 84 5.3 Discussion 85 viii CHAPTER 89 Pharmacokinetic validation studies on the Hollow Fibre Infection Model 89 6.1 Synopsis 90 6.2 UPLC/MS/MS assay- Results and Discussion 90 6.2.1 Method validation and optimisation 90 6.2.2 Matrix recovery 91 6.2.3 Accuracy and precision 92 6.2.4 Stability studies 92 6.3 Pharmacokinetic validation studies 99 6.4 Conclusions 101 CHAPTER Extending the time-kill approach and validating its predictive ability in a Hollow Fibre Infection Model (HFIM) 102 7.1 Synopsis 103 7.2 Pharmacodynamic assessment 103 7.3 Emergence of resistance studies 104 7.4 In vitro time-growth studies 107 7.5 Electron microscopy studies 107 7.5.1 TEM results 107 7.5.2 SEM results 108 7.6 Discussion 111 CHAPTER Multiple Combination Bactericidal Testing - Clinical applications of antibiotic combination testing 114 8.1 Synopsis 115 8.2 Results 115 8.3 Discussion 121 8.4 Conclusions 126 ix (2004) "National Nosocomial Infections Surveillance (NNIS) System Report, data summary from January 1992 through June 2004, issued October 2004." Am J Infect Control 32(8): 470-485 (2010) Tygacil® (tigecycline) Prescribing Information, Wyeth Pharmaceuticals Inc Aaron, S D., W Ferris, D A Henry, D P Speert and N E Macdonald (2000) "Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with Burkholderia cepacia." Am J Respir Crit Care Med 161(4 Pt 1): 1206-1212 Aaron, S D., K L Vandemheen, W Ferris, D Fergusson, E Tullis, D Haase, Y Berthiaume, N Brown, P Wilcox, V Yozghatlian, P Bye, S Bell, F Chan, B Rose, A Jeanneret, A Stephenson, M Noseworthy, A Freitag, N Paterson, S Doucette, C Harbour, M Ruel and N MacDonald (2005) "Combination antibiotic susceptibility testing to treat exacerbations of cystic fibrosis associated with multiresistant bacteria: a randomised, double-blind, controlled clinical trial." Lancet 366(9484): 463-471 Abdelraouf, K., S Kabbara, K R Ledesma, K Poole and V H Tam (2011) "Effect of multidrug resistance-conferring mutations on the fitness and virulence of Pseudomonas aeruginosa." J Antimicrob Chemother 66(6): 13111317 American Society for Microbiology (2011) Instructions to Authors Antimicrob Agents Chemother 55: 1-23 Balbao, M S., C Bertucci, M M Bergamaschi, R H Queiroz, W R Malfara, S A Dreossi, L de Paula Mello and M E Queiroz (2010) "Rifampicin determination in plasma by stir bar-sorptive extraction and liquid chromatography." J Pharm Biomed Anal 51(5): 1078-1083 Berenbaum, M C (1989) "What is synergy?" Pharmacol Rev 41(2): 93-141 Bergogne-Berezin, E and K J Towner (1996) "Acinetobacter spp as nosocomial pathogens: microbiological, clinical, and epidemiological features." Clin Microbiol Rev 9(2): 148-165 Bilello, J A., G Bauer, M N Dudley, G A Cole and G L Drusano (1994) "Effect of 2',3'-didehydro-3'-deoxythymidine in an in vitro hollow-fiber pharmacodynamic model system correlates with results of dose-ranging clinical studies." Antimicrob Agents Chemother 38(6): 1386-1391 Blaser, J (1985) "In-vitro model for simultaneous simulation of the serum kinetics of two drugs with different half-lives." J Antimicrob Chemother 15 Suppl A: 125-130 Bolmstrom, A., A Karlsson, U Nordstrom, and K Mills (1995) Etest for drug combination studies with mycobacteria, abstr 1142 19th International Congress of Chemotherapy, Oakville, Ontario, Canada 134 Bonapace, C R., R L White, L V Friedrich and J A Bosso (2000) "Evaluation of antibiotic synergy against Acinetobacter baumannii: a comparison with Etest, time-kill, and checkerboard methods." Diagn Microbiol Infect Dis 38(1): 43-50 Bonomo, R A and D Szabo (2006) "Mechanisms of multidrug resistance in Acinetobacter species and Pseudomonas aeruginosa." Clin Infect Dis 43 Suppl 2: S49-56 Boucher, H W., G H Talbot, J S Bradley, J E Edwards, D Gilbert, L B Rice, M Scheld, B Spellberg and J Bartlett (2009) "Bad bugs, no drugs: no ESKAPE! An update from the Infectious Diseases Society of America." Clin Infect Dis 48(1): 1-12 Brown, A N., J J McSharry, Q Weng, J R Adams, R Kulawy and G L Drusano (2011) "Zanamivir, at 600 milligrams twice daily, inhibits oseltamivir-resistant 2009 pandemic H1N1 influenza virus in an in vitro hollow-fiber infection model system." Antimicrob Agents Chemother 55(4): 1740-1746 Brown, S., H K Young and S G Amyes (2005) "Characterisation of OXA51, a novel class D carbapenemase found in genetically unrelated clinical strains of Acinetobacter baumannii from Argentina." Clin Microbiol Infect 11(1): 15-23 Caminero, J A., G Sotgiu, A Zumla and G B Migliori (2010) "Best drug treatment for multidrug-resistant and extensively drug-resistant tuberculosis." Lancet Infect Dis 10(9): 621-629 Centers for Disease Control and Prevention (CDC) (2004) "Acinetobacter baumannii infections among patients at military medical facilities treating injured U.S service members, 2002-2004." MMWR Morb Mortal Wkly Rep 53(45): 1063-1066 Chait, R., A Craney and R Kishony (2007) "Antibiotic interactions that select against resistance." Nature 446(7136): 668-671 Chen, T L., L K Siu, R C Wu, M F Shaio, L Y Huang, C P Fung, C M Lee and W L Cho (2007) "Comparison of one-tube multiplex PCR, automated ribotyping and intergenic spacer (ITS) sequencing for rapid identification of Acinetobacter baumannii." Clin Microbiol Infect 13(8): 801806 Chow, J W and V L Yu (1999) "Combination antibiotic therapy versus monotherapy for gram-negative bacteraemia: a commentary." Int J Antimicrob Agents 11(1): 7-12 Clinical and Laboratory Standards Institute (2010) Performance standards for antimicrobial testing: Seventeenth Informational Supplement M100-S20 CLSI Wayne, Pennsylvania, USA 30 135 D' Argenio DZ and S A, Eds (1997) ADAPT II user's guide: pharmacokinetic / pharmacodynamic systems analysis software Los Angeles: Biomedical simulations resource, University of Southern California Docquier, J D., M L Riccio, C Mugnaioli, F Luzzaro, A Endimiani, A Toniolo, G Amicosante and G M Rossolini (2003) "IMP-12, a new plasmidencoded metallo-beta-lactamase from a Pseudomonas putida clinical isolate." Antimicrob Agents Chemother 47(5): 1522-1528 Drusano, G L., N Sgambati, A Eichas, D Brown, R Kulawy and A Louie (2011) "Effect of administration of moxifloxacin plus rifampin against Mycobacterium tuberculosis for of days versus of days in an in vitro pharmacodynamic system." MBio 2(4): e00108-00111 Dufrene, Y F (2004) "Using nanotechniques to explore microbial surfaces." Nat Rev Microbiol 2(6): 451-460 Durante-Mangoni, E., R Utili and R Zarrilli (2014) "Combination therapy in severe Acinetobacter baumannii infections: an update on the evidence to date." Future Microbiol 9(6): 773-789 Elion, G B., S Singer, G H Hitchings, M E Balis and G B Drown (1953) "Effects of purine antagonists on a diaminopurine-resistant strain of Lactobacillus casei." J Biol Chem 202(2): 647-654 Fernandez-Reyes, M., M Rodriguez-Falcon, C Chiva, J Pachon, D Andreu and L Rivas (2009) "The cost of resistance to colistin in Acinetobacter baumannii: a proteomic perspective." Proteomics 9(6): 1632-1645 Foweraker, J E., C R Laughton, D F Brown and D Bilton (2009) "Comparison of methods to test antibiotic combinations against heterogeneous populations of multiresistant Pseudomonas aeruginosa from patients with acute infective exacerbations in cystic fibrosis." Antimicrob Agents Chemother 53(11): 4809-4815 Garrod, L P and P M Waterworth (1962) "Methods of testing combined antibiotic bactericidal action and the significance of the results." J Clin Pathol 15: 328-338 Gaynes, R and J R Edwards (2005) "Overview of nosocomial infections caused by gram-negative bacilli." Clin Infect Dis 41(6): 848-854 Gerner-Smidt, P (1992) "Ribotyping of the Acinetobacter calcoaceticusAcinetobacter baumannii complex." J Clin Microbiol 30(10): 2680-2685 Gerner-Smidt, P., I Tjernberg and J Ursing (1991) "Reliability of phenotypic tests for identification of Acinetobacter species." J Clin Microbiol 29(2): 277282 Glupczynski, Y., M Labbe, W Hansen, F Crokaert and E Yourassowsky (1991) "Evaluation of the E test for quantitative antimicrobial susceptibility testing of Helicobacter pylori." J Clin Microbiol 29(9): 2072-2075 136 Gosling, R D., L Okell, J Mosha and D Chandramohan (2011) "The role of antimalarial treatment in the elimination of malaria." Clin Microbiol Infect 17(11): 1617-1623 Govaerts, C., J Orwa, A Van Schepdael, E Roets and J Hoogmartens (2002) "Characterization of polypeptide antibiotics of the polymyxin series by liquid chromatography electrospray ionization ion trap tandem mass spectrometry." J Pept Sci 8(2): 45-55 Govaerts, C., J Rozenski, J Orwa, E Roets, A Van Schepdael and J Hoogmartens (2002) "Mass spectrometric fragmentation of cyclic peptides belonging to the polymyxin and colistin antibiotics studied by ion trap and quadrupole/orthogonal-acceleration time-of-flight technology." Rapid Commun Mass Spectrom 16(9): 823-833 Greco, W R., G Bravo and J C Parsons (1995) "The search for synergy: a critical review from a response surface perspective." Pharmacol Rev 47(2): 331-385 Grundmann, H J., K J Towner, L Dijkshoorn, P Gerner-Smidt, M Maher, H Seifert and M Vaneechoutte (1997) "Multicenter study using standardized protocols and reagents for evaluation of reproducibility of PCR-based fingerprinting of Acinetobacter spp." J Clin Microbiol 35(12): 3071-3077 Gumbo, T., A Louie, M R Deziel, W Liu, L M Parsons, M Salfinger and G L Drusano (2007) "Concentration-dependent Mycobacterium tuberculosis killing and prevention of resistance by rifampin." Antimicrob Agents Chemother 51(11): 3781-3788 Guo, B., K Abdelraouf, K R Ledesma, M Nikolaou and V H Tam (2012) "Predicting bacterial fitness cost associated with drug resistance." J Antimicrob Chemother 67(4): 928-932 Hartmann, M., M Berditsch, J Hawecker, M F Ardakani, D Gerthsen and A S Ulrich (2010) "Damage of the bacterial cell envelope by antimicrobial peptides gramicidin S and PGLa as revealed by transmission and scanning electron microscopy." Antimicrob Agents Chemother 54(8): 3132-3142 Hawley, J S., C K Murray and J H Jorgensen (2008) "Colistin heteroresistance in acinetobacter and its association with previous colistin therapy." Antimicrob Agents Chemother 52(1): 351-352 He, J., S Gao, M Hu, D S Chow and V H Tam (2013) "A validated ultraperformance liquid chromatography-tandem mass spectrometry method for the quantification of polymyxin B in mouse serum and epithelial lining fluid: application to pharmacokinetic studies." J Antimicrob Chemother Hoffner, S E., L Klintz, B Olsson-Liljequist, A Karlsson, and A Bolmstrom (1993) Rapid susceptibility testing of Mycobacterium chelonae and M.fortuitum to single and combined drugs using Etest Abstr # 939 18th International Congress of Chemotherapy, 137 , Stockholm Hogg, G M., J G Barr and C H Webb (1998) "In-vitro activity of the combination of colistin and rifampicin against multidrug-resistant strains of Acinetobacter baumannii." J Antimicrob Chemother 41(4): 494-495 Hsu, L Y., T Y Tan, V H Tam, A Kwa, D A Fisher and T H Koh (2010) "Surveillance and correlation of antibiotic prescription and resistance of Gram-negative bacteria in Singaporean hospitals." Antimicrob Agents Chemother 54(3): 1173-1178 Johnson, M D., C MacDougall, L Ostrosky-Zeichner, J R Perfect and J H Rex (2004) "Combination antifungal therapy." Antimicrob Agents Chemother 48(3): 693-715 Jorgensen, J H., A W Howell and L A Maher (1991) "Quantitative antimicrobial susceptibility testing of Haemophilus influenzae and Streptococcus pneumoniae by using the E-test." J Clin Microbiol 29(1): 109114 Kenny, M T and B Strates (1981) "Metabolism and pharmacokinetics of the antibiotic rifampin." Drug Metab Rev 12(1): 159-218 Kumar, A., D Roberts, K E Wood, B Light, J E Parrillo, S Sharma, R Suppes, D Feinstein, S Zanotti, L Taiberg, D Gurka and M Cheang (2006) "Duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock." Crit Care Med 34(6): 1589-1596 Kunin, C M., W A Craig, M Kornguth and R Monson (1973) "Influence of binding on the pharmacologic activity of antibiotics." Ann N Y Acad Sci 226: 214-224 Kuo, L C., C C Lai, C H Liao, C K Hsu, Y L Chang, C Y Chang and P R Hsueh (2007) "Multidrug-resistant Acinetobacter baumannii bacteraemia: clinical features, antimicrobial therapy and outcome." Clin Microbiol Infect 13(2): 196-198 Kwa, A L., T P Lim, J G Low, J Hou, A Kurup, R A Prince and V H Tam (2008) "Pharmacokinetics of polymyxin B1 in patients with multidrugresistant Gram-negative bacterial infections." Diagn Microbiol Infect Dis 60(2): 163-167 Kwa, A L., J G Low, E Lee, A Kurup, H L Chee and V H Tam (2007) "The impact of multidrug resistance on the outcomes of critically ill patients with Gram-negative bacterial pneumonia." Diagn Microbiol Infect Dis 58(1): 99-104 Kwa, A L., V H Tam and M E Falagas (2008) "Polymyxins: a review of the current status including recent developments." Ann Acad Med Singapore 37(10): 870-883 138 Landman, D., J M Quale, D Mayorga, A Adedeji, K Vangala, J Ravishankar, C Flores and S Brooks (2002) "Citywide clonal outbreak of multiresistant Acinetobacter baumannii and Pseudomonas aeruginosa in Brooklyn, NY: the preantibiotic era has returned." Arch Intern Med 162(13): 1515-1520 Lang, B J., S D Aaron, W Ferris, P C Hebert and N E MacDonald (2000) "Multiple combination bactericidal antibiotic testing for patients with cystic fibrosis infected with multiresistant strains of Pseudomonas aeruginosa." Am J Respir Crit Care Med 162(6): 2241-2245 Lange, J M (1995) "Triple combinations: present and future." J Acquir Immune Defic Syndr Hum Retrovirol 10 Suppl 1: S77-82 Leibovici, L., M Paul, O Poznanski, M Drucker, Z Samra, H Konigsberger and S D Pitlik (1997) "Monotherapy versus beta-lactam-aminoglycoside combination treatment for gram-negative bacteremia: a prospective, observational study." Antimicrob Agents Chemother 41(5): 1127-1133 Levin, A S., A A Barone, J Penco, M V Santos, I S Marinho, E A Arruda, E I Manrique and S F Costa (1999) "Intravenous colistin as therapy for nosocomial infections caused by multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii." Clin Infect Dis 28(5): 1008-1011 Li, J., C R Rayner, R L Nation, R J Owen, D Spelman, K E Tan and L Liolios (2006) "Heteroresistance to colistin in multidrug-resistant Acinetobacter baumannii." Antimicrob Agents Chemother 50(9): 2946-2950 Lim, T P., K R Ledesma, K T Chang, J G Hou, A L Kwa, M Nikolaou, J P Quinn, R A Prince and V H Tam (2008) "Quantitative assessment of combination antimicrobial therapy against multidrug-resistant Acinetobacter baumannii." Antimicrob Agents Chemother 52(8): 2898-2904 Lim, T P., T Y Tan, W Lee, S Sasikala, T T Tan, L Y Hsu and A L Kwa (2009) "In vitro activity of various combinations of antimicrobials against carbapenem-resistant Acinetobacter species in Singapore." J Antibiot (Tokyo) 62(12): 675-679 Lim, T P., T Y Tan, W Lee, S Sasikala, T T Tan, L Y Hsu and A L Kwa (2011) "In-vitro activity of polymyxin B, rifampicin, tigecycline alone and in combination against carbapenem-resistant Acinetobacter baumannii in Singapore." PLoS One 6(4): e18485 Lorian, V (2005) Antibiotics in laboratory medicine Philadelphia, Lippincott Williams & Wilkins Louie, A., B Vanscoy, W Liu, R Kulawy, D Brown, H S Heine and G L Drusano (2011) "Comparative efficacies of candidate antibiotics against Yersinia pestis in an in vitro pharmacodynamic model." Antimicrob Agents Chemother 55(6): 2623-2628 139 Magiorakos, A P., A Srinivasan, R B Carey, Y Carmeli, M E Falagas, C G Giske, S Harbarth, J F Hindler, G Kahlmeter, B Olsson-Liljequist, D L Paterson, L B Rice, J Stelling, M J Struelens, A Vatopoulos, J T Weber and D L Monnet (2011) "Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance." Clin Microbiol Infect Manikal, V M., D Landman, G Saurina, E Oydna, H Lal and J Quale (2000) "Endemic carbapenem-resistant Acinetobacter species in Brooklyn, New York: citywide prevalence, interinstitutional spread, and relation to antibiotic usage." Clin Infect Dis 31(1): 101-106 Maragakis, L L and T M Perl (2008) "Acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options." Clin Infect Dis 46(8): 1254-1263 Mendes, R E., T R Fritsche, H S Sader and R N Jones (2008) "Increased antimicrobial susceptibility profiles among polymyxin-resistant Acinetobacter baumannii clinical isolates." Clin Infect Dis 46(8): 1324-1326 Moffatt, J H., M Harper, P Harrison, J D Hale, E Vinogradov, T Seemann, R Henry, B Crane, F St Michael, A D Cox, B Adler, R L Nation, J Li and J D Boyce (2010) "Colistin resistance in Acinetobacter baumannii is mediated by complete loss of lipopolysaccharide production." Antimicrob Agents Chemother 54(12): 4971-4977 Montero, A., J Ariza, X Corbella, A Domenech, C Cabellos, J Ayats, F Tubau, C Borraz and F Gudiol (2004) "Antibiotic combinations for serious infections caused by carbapenem-resistant Acinetobacter baumannii in a mouse pneumonia model." J Antimicrob Chemother 54(6): 1085-1091 Moody, J (2004) Synergism testing: broth microdilution checkerboard and broth macrodilution methods, p 5.12.1–5.12.23 In H D Isenberg (ed.), Clinical microbiology procedures handbook, vol American Society for Microbiology, Washington, DC National Committee for Clinical Laboratory Standards (1999) Methods for Determining Bactericidal Activity of Antimicrobial Agents Approved Guideline M26-A, Vol 19 NCCLS Wayne, PA New, L S., S Saha, M M Ong, U A Boelsterli and E C Chan (2007) "Pharmacokinetic study of intraperitoneally administered troglitazone in mice using ultra-performance liquid chromatography/tandem mass spectrometry." Rapid Commun Mass Spectrom 21(6): 982-988 Nordmann, P and L Poirel (2002) "Emerging carbapenemases in Gramnegative aerobes." Clin Microbiol Infect 8(6): 321-331 Odds, F C (2003) "Synergy, antagonism, and what the chequerboard puts between them." J Antimicrob Chemother 52(1): 140 Pachon-Ibanez, M E., F Docobo-Perez, R Lopez-Rojas, J DominguezHerrera, M E Jimenez-Mejias, A Garcia-Curiel, C Pichardo, L Jimenez and J Pachon (2010) "Efficacy of rifampin and its combinations with imipenem, sulbactam, and colistin in experimental models of infection caused by imipenem-resistant Acinetobacter baumannii." Antimicrob Agents Chemother 54(3): 1165-1172 Pachon-Ibanez, M E., F Fernandez-Cuenca, F Docobo-Perez, J Pachon and A Pascual (2006) "Prevention of rifampicin resistance in Acinetobacter baumannii in an experimental pneumonia murine model, using rifampicin associated with imipenem or sulbactam." J Antimicrob Chemother 58(3): 689692 Pankey, G A and D S Ashcraft (2009) "The detection of synergy between meropenem and polymyxin B against meropenem-resistant Acinetobacter baumannii using Etest and time-kill assay." Diagn Microbiol Infect Dis 63(2): 228-232 Pantopoulou, A., E J Giamarellos-Bourboulis, M Raftogannis, T Tsaganos, I Dontas, P Koutoukas, F Baziaka, H Giamarellou and D Perrea (2007) "Colistin offers prolonged survival in experimental infection by multidrugresistant Acinetobacter baumannii: the significance of co-administration of rifampicin." Int J Antimicrob Agents 29(1): 51-55 Paterson, D L (2006) "The epidemiological profile of infections with multidrug-resistant Pseudomonas aeruginosa and Acinetobacter species." Clin Infect Dis 43 Suppl 2: S43-48 Paterson, D L and Y Doi (2007) "A step closer to extreme drug resistance (XDR) in gram-negative bacilli." Clin Infect Dis 45(9): 1179-1181 Peleg, A Y., H Seifert and D L Paterson (2008) "Acinetobacter baumannii: emergence of a successful pathogen." Clin Microbiol Rev 21(3): 538-582 Perez, F., A M Hujer, K M Hujer, B K Decker, P N Rather and R A Bonomo (2007) "Global challenge of multidrug-resistant Acinetobacter baumannii." Antimicrob Agents Chemother 51(10): 3471-3484 Poirel, L and P Nordmann (2006) "Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology." Clin Microbiol Infect 12(9): 826836 Poupard, J., R Langan, L Utrup, S Rittenhouse, and R B Clark (1993) Use of the AB BIODISK E-test as a screen for ticarcillin clavulanate (T/C)amikacin (Ak) synergy with isolates of Xanthomonas maltophilia Abstr # 366 18th International Congress of Chemotherapy, Stockholm Rahal, J J (2006) "Novel antibiotic combinations against infections with almost completely resistant Pseudomonas aeruginosa and Acinetobacter species." Clin Infect Dis 43 Suppl 2: S95-99 141 Rahal, J J (2008) "The role of carbapenems in initial therapy for serious Gram-negative infections." Crit Care 12 Suppl 4: S5 Rice, L B (2008) "Federal funding for the study of antimicrobial resistance in nosocomial pathogens: no ESKAPE." J Infect Dis 197(8): 1079-1081 Rodvold, K A., M H Gotfried, M Cwik, J M Korth-Bradley, G Dukart and E J Ellis-Grosse (2006) "Serum, tissue and body fluid concentrations of tigecycline after a single 100 mg dose." J Antimicrob Chemother 58(6): 12211229 Sabath, L D (1967) "Synergy of antibacterial substances by apparently known mechanisms." Antimicrob Agents Chemother (Bethesda) 7: 210-217 Sobieszczyk, M E., E Y Furuya, C M Hay, P Pancholi, P Della-Latta, S M Hammer and C J Kubin (2004) "Combination therapy with polymyxin B for the treatment of multidrug-resistant Gram-negative respiratory tract infections." J Antimicrob Chemother 54(2): 566-569 Song, J Y., S Y Kee, I S Hwang, Y B Seo, H W Jeong, W J Kim and H J Cheong (2007) "In vitro activities of carbapenem/sulbactam combination, colistin, colistin/rifampicin combination and tigecycline against carbapenemresistant Acinetobacter baumannii." J Antimicrob Chemother 60(2): 317-322 Soon, R L., R L Nation, M Harper, B Adler, J D Boyce, C H Tan, J Li and I Larson (2011) "Effect of colistin exposure and growth phase on the surface properties of live Acinetobacter baumannii cells examined by atomic force microscopy." Int J Antimicrob Agents 38(6): 493-501 Soon, R L., R L Nation, P G Hartley, I Larson and J Li (2009) "Atomic force microscopy investigation of the morphology and topography of colistinheteroresistant Acinetobacter baumannii strains as a function of growth phase and in response to colistin treatment." Antimicrob Agents Chemother 53(12): 4979-4986 Sopirala, M M., J E Mangino, W A Gebreyes, B Biller, T Bannerman, J M Balada-Llasat and P Pancholi (2010) "Synergy testing by Etest, microdilution checkerboard, and time-kill methods for pan-drug-resistant Acinetobacter baumannii." Antimicrob Agents Chemother 54(11): 4678-4683 Talbot, G H., J Bradley, J E Edwards, Jr., D Gilbert, M Scheld and J G Bartlett (2006) "Bad bugs need drugs: an update on the development pipeline from the Antimicrobial Availability Task Force of the Infectious Diseases Society of America." Clin Infect Dis 42(5): 657-668 Tam, V H., H Cao, K R Ledesma and M Hu (2011) "In vitro potency of various polymyxin B components." Antimicrob Agents Chemother 55(9): 4490-4491 Tam, V H., A N Schilling, R E Lewis, D A Melnick and A N Boucher (2004) "Novel approach to characterization of combined pharmacodynamic 142 effects of antimicrobial agents." Antimicrob Agents Chemother 48(11): 43154321 Tam, V H., A N Schilling and M Nikolaou (2005) "Modelling time-kill studies to discern the pharmacodynamics of meropenem." J Antimicrob Chemother 55(5): 699-706 Tamma, P D., S E Cosgrove and L L Maragakis (2012) "Combination therapy for treatment of infections with gram-negative bacteria." Clin Microbiol Rev 25(3): 450-470 Tascini, C., F Menichetti, S Bozza, A Del Favero and F Bistoni (1998) "Evaluation of the activities of two-drug combinations of rifampicin, polymyxin B and ampicillin/sulbactam against Acinetobacter baumannii." J Antimicrob Chemother 42(2): 270-271 Tilton, R C., L Lieberman and E H Gerlach (1973) "Microdilution antibiotic susceptibility test: examination of certain variables." Appl Microbiol 26(5): 658-665 Timurkaynak, F., F Can, O K Azap, M Demirbilek, H Arslan and S O Karaman (2006) "In vitro activities of non-traditional antimicrobials alone or in combination against multidrug-resistant strains of Pseudomonas aeruginosa and Acinetobacter baumannii isolated from intensive care units." Int J Antimicrob Agents 27(3): 224-228 Turton, J F., M E Ward, N Woodford, M E Kaufmann, R Pike, D M Livermore and T L Pitt (2006) "The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii." FEMS Microbiol Lett 258(1): 72-77 Waters, V and F Ratjen (2008) "Combination antimicrobial susceptibility testing for acute exacerbations in chronic infection of Pseudomonas aeruginosa in cystic fibrosis." Cochrane Database Syst Rev(3): CD006961 Wehrli, W (1983) "Rifampin: mechanisms of action and resistance." Rev Infect Dis Suppl 3: S407-411 White, R L., D S Burgess, M Manduru and J A Bosso (1996) "Comparison of three different in vitro methods of detecting synergy: timekill, checkerboard, and E test." Antimicrob Agents Chemother 40(8): 19141918 Woodford, N., M J Ellington, J M Coelho, J F Turton, M E Ward, S Brown, S G Amyes and D M Livermore (2006) "Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp." Int J Antimicrob Agents 27(4): 351-353 Yoon, J., C Urban, C Terzian, N Mariano and J J Rahal (2004) "In vitro double and triple synergistic activities of Polymyxin B, imipenem, and rifampin against multidrug-resistant Acinetobacter baumannii." Antimicrob Agents Chemother 48(3): 753-757 143 Zavascki, A P., L Z Goldani, G Cao, S V Superti, L Lutz, A L Barth, F Ramos, M M Boniatti, R L Nation and J Li (2008) "Pharmacokinetics of intravenous polymyxin B in critically ill patients." Clin Infect Dis 47(10): 1298-1304 144 Supplementary tables and figures 145 Supplementary Table Optimised compound dependent QTRAP MS parameters for the detection of rifampicin, rifabutin, and polymyxin B1, B2, B3, B5, and B6 Compound MRM Retention DP1, EP2, CE3, CXP4, Transition, Time, V V eV V m/z Rifabutin (IS) 847.9 -816.0 4.49 60 9.5 42 Rifampicin 823.5-91.5 4.59 44 9.0 28 Polymyxin B1 602.4-101.1 3.47 51 9.5 51 2.5 Polymyxin B2 595.4-101.1 2.99 51 9.5 50 2.0 Polymyxin B3 595.4-101.1 3.08 51 9.5 50 2.0 Polymyxin B5 602.4-101.1 3.28 51 9.5 51 2.5 Polymyxin B6 610.4-101.1 2.83 51 9.5 50 2.0 DP, declustering potential 2EP, entrance potential 3CE, collision energy, CXP, collision cell exit potential Supplementary Figure Time-kill graph of A baumannii 8879 against single antibiotics (A) and combination antibiotics (B) (A) 10 Log10 CFU/ml Control Polymyxin B Meropenem Rifampicin Tigecycline 0 12 16 20 24 Time (h) 146 (B) 10 Control Rifampicin + Polymyxin B Rifampicin + Meropenem Polymyxin B + Meropenem Log10 CFU/ml Tigecycline + Polymyxin B Tigecycline + Rifampicin 0 12 16 20 24 Tigecycline + Meropenem Time (h) Supplementary Figure Time-kill graph of Acinetobacter spp 105 against single antibiotics (A) and combination antibiotics (B) (A) 10 Log10 CFU/ml Control Polymyxin B Meropenem Rifampicin Tigecycline 0 12 Time (h) 16 20 24 147 (B) 10 Control Rifampicin + Polymyxin B Rifampicin + Meropenem Polymyxin B + Meropenem Log10 CFU/ml Tigecycline + Polymyxin B Tigecycline + Rifampicin 0 12 16 20 24 Tigecycline + Meropenem Time (h) Supplementary Figure Microbiologic response observed in the following infection models: polymyxin B + rifampicin, polymyxin + tigecycline and tigecycline + rifampicin against AB 8879 Placebo 10 Log CFU/ml Polymyxin B 1MU q12h Rifampicin 600mg q12h Tigecycline 100mg q12h 0 Days Polymyxin B 1MU q12h + Rifampicin 600mg q12h Polymyxin B 1MU q12h + Tigecycline 100mg q12h 148 ... in combination against clinical strains of extreme drug- resistant Acinetobacter baumannii to all antibiotics including polymyxin B in Singapore” 20th European Conference on Microbiology and Infectious... (May 2010) ? ?Pharmacokinetic/ pharmacodynamic modelling of polymyxin B, rifampicin and tigecycline against pandrug -resistant Acinetobacter baumannii in an in- vitro model? ?? 20th European Conference... Tan TY, Lee W, Sasikala S, Tan TT, Hsu LY, Kwa AL In- vitro activity of polymyxin B, rifampicin, tigecycline alone and in combination against carbapenem -resistant Acinetobacter baumannii in Singapore