METHODS. Imipenem Meropenem Colistin Polymyxin B Ampicillinsulbactam. Downloaded from by IP:

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1 Journal of Medical Microbiology (01), 1, DOI.99/jmm In vitro time-kill studies of antimicrobial agents against blood isolates of imipenem-resistant Acinetobacter baumannii, including colistin- or tigecycline-resistant isolates Kyong Ran Peck, 1 3 Min Ja Kim, 3 Ji Young Choi, 3 3 Hong Sun Kim, 3 Cheol-In Kang, 1 Yong Kyun Cho, Dae Won Park, 5 Hee Joo Lee, Mi Suk Lee 7 and Kwan Soo Ko 3, Correspondence Kwan Soo Ko ksko@skku.edu 1 Division of Infectious Diseases, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Republic of Korea Division of Infectious Diseases, Korea University Anam Hospital, Korea University, College of Medicine, Seoul, Republic of Korea 3 Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea Gachon University, Gil Hospital, Inchon, Republic of Korea 5 Division of Infectious Diseases, Korea University Ansan Hospital, Korea University, College of Medicine, Ansan, Republic of Korea Departments of Laboratory Medicine, School of Medicine, Kyung Hee University, Seoul, Republic of Korea 7 Department of Internal Medicine, School of Medicine, Kyung Hee University, Seoul, Republic of Korea Asia Pacific Foundation for Infectious Diseases (APFID), Seoul, Republic of Korea Received August 011 Accepted 17 October 011 The emergence of colistin or tigecycline resistance as well as imipenem resistance in Acinetobacter baumannii poses a great therapeutic challenge. The bactericidal and synergistic effects of several combinations of antimicrobial agents against imipenem-, colistin- or tigecyclineresistant A. baumannii isolates were investigated by in vitro time-kill experiments. Six imipenemresistant A. baumannii blood isolates were examined in this study, including colistin- and tigecycline-susceptible, colistin-resistant but tigecycline-susceptible, and colistin-susceptible but tigecycline-resistant isolates. Time-kill studies were performed using five antimicrobial agents singly or in combinations (imipenem plus colistin, imipenem plus ampicillin-sulbactam, colistin plus rifampicin, colistin plus tigecycline, and tigecycline plus rifampicin) at concentrations of 0.5¾ and 1¾ their MICs. Only imipenem was consistently effective as a single agent against all six A. baumannii isolates. Although the effectiveness of combinations of 0.5¾ MIC antimicrobial agents was inconsistent, combination regimens using 1¾ MIC of the antimicrobial agents displayed excellent bactericidal activities against all six A. baumannii isolates. Among the combinations of 0.5¾ MIC antimicrobial agents, the combination of colistin and tigecycline showed synergistic or bactericidal effects against four of the isolates. This in vitro time-kill analysis suggests that antimicrobial combinations are effective for killing imipenem-resistant A. baumannii isolates, even if they are simultaneously resistant to either colistin or tigecycline. However, the finding that the combinations of 0.5¾ MIC antimicrobial agents were effective on only some isolates may warrant further investigation of the doses of combination agents needed to kill resistant A. baumannii. 3These authors contributed equally to this work. Abbreviation: MDR, multidrug resistant G 01 SGM Printed in Great Britain IP: On: Mon, 7 Nov :03:5 353

2 K. R. Peck and others INTRODUCTION Acinetobacter baumannii has emerged as an important nosocomial pathogen, especially in intensive care units (Dijkshoorn et al., 007). A. baumannii infections may be difficult to treat due to the pathogen s multidrug resistance. Although carbapenems, including imipenem and meropenem, have been commonly used as the mainstay of treatment for severe A. baumannii infections, carbapenemresistant isolates have emerged and disseminated worldwide in recent years (Perez et al., 007). With the exception of polymyxins (such as polymyxin B and colistin) and tigecycline, few alternative therapeutic options are available (Munoz-Price & Weinstein, 00). However, polymyxinresistant isolates of A. baumannii have also developed (Li et al., 00a; Park et al., 009a), along with tigecyclineresistant isolates (Capone et al., 00; Park et al., 009b). Even pandrug-resistant (PDR) A. baumannii isolates, displaying resistance to all antimicrobial agents, including both polymyxins and tigecycline, have recently emerged (Doi et al., 009; Park et al., 009c). A. baumannii isolates cause bloodstream infection, nosocomial-acquired pneumonia or ventilator-associated pneumonia in critically ill patients. Especially those with inappropriate treatment are associated with higher mortality (Falagas et al., 00). However, the development of new antimicrobial agents to combat A. baumannii infections has been slow. Thus, the use of combinations of two or more agents has drawn attention as an option for treating multidrug-resistant (MDR) A. baumannii infections (Munoz-Price & Weinstein, 00; Peleg et al., 00), although the effectiveness of such combinations remains controversial (Moland et al., 00; Scheetz et al., 007). In addition to increasing eradication efficacy, combination therapy may also help to prevent the emergence of resistant populations (Pachón-Ibáñez et al., 00). So far, several combinations, such as imipenem and ampicillin-sulbactam, rifampicin and polymyxin B, imipenem and polymyxin B, and colistin and rifampicin, have been reported to be effective in vitro against carbapenem-resistant A. baumannii (Perez et al., 007; Peleg et al., 00). However, studies on the effects of these combinations against colistin- or tigecycline-resistant A. baumannii isolates are very limited (Moland et al., 00). In this study, we investigated the synergistic and bactericidal effects of combinations of antimicrobial agents against carbapenem-resistant A. baumannii blood isolates that were also resistant to either colistin or tigecycline by in vitro time-kill analysis using a microdilution method. METHODS Bacterial isolates. Six representative imipenem-resistant A. baumannii blood isolates that were isolated from intensive care unit patients at four university hospitals in South Korea were included in the present study (Table 1). All were also resistant to meropenem. Two isolates (KRU-A-3 and KHU-) were resistant to carbapenems, but susceptible to polymyxins and tigecycline (COL- S/TIG-S), two isolates (KCU- and SKKU-) were resistant to polymyxins but susceptible to tigecycline (COL-R/TIG-S), and two isolates (SKKU- and KCU-3) were resistant to tigecycline but susceptible to polymyxins (COL-S/TIG-R). All six isolates were resistant to ampicillin-sulbactam. While two isolates (KHU- and KCU-) were resistant to rifampicin, all the others were susceptible to rifampicin. Determination of MIC. In vitro antimicrobial susceptibility testing was performed by measuring MIC using the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI, 0). Fifteen antimicrobial agents were tested: imipenem, meropenem, polymyxin B, colistin, ciprofloxacin, rifampicin, amikacin, cefepime, ceftriaxone, cefoperazone-sulbactam, ceftazidime, piperacillin-tazobactam, ampicillinsulbactam, tetracycline and tigecycline. Fresh Mueller Hinton broth was used for all susceptibility testing. CLSI susceptibility interpretive criteria were used (CLSI, 0). No breakpoints for rifampicin and tigecycline are available in the CLSI guidelines; therefore CLSI criteria recommended for staphylococci were applied to rifampicin (resistant mgl 1 ), and the criteria of the United States Food and Drug Administration for Enterobacteriaceae were used for tigecycline (intermediate mg l 1 ;resistant mgl 1 ). Table 1. Antimicrobial resistance profiles of the A. baumannii isolates used in this study S, susceptible; R, resistant. Isolate MIC of antimicrobial agent (mg l 1 ) (resistance or susceptibility) Imipenem Meropenem Colistin Polymyxin B Ampicillinsulbactam Tigecycline Rifampicin KRU-A-3 1 (R). (R) 1 (S) 1 (S) 1/ (R) (S) (S) KHU- 1 (R). (R) 1 (S) 1 (S) 1/ (R) 1 (S) (R) KCU- 5 (R). (R) 3 (R) (R) 5/1 (R) 1 (S) (R) SKKU- 1 (R). (R) 1 (R) 1 (R) 1/ (R) (S) (S) SKKU- 1 (R). (R) 1 (S) 1 (S) /3 (R) 1 (R) (S) KCU-13 (R). (R) 1 (S) 1 (S) /3 (R) 1 (R) (S) 35 IP: Journal of Medical Microbiology 1 On: Mon, 7 Nov :03:5

3 In vitro combination therapy against A. baumannii Escherichia coli ATCC 59, Staphylococcus aureus ATCC 913, and Pseudomonas aeruginosa ATCC 753 were used as control strains. Time-kill analysis. Time-kill studies were performed on five antimicrobial agents (imipenem, colistin, ampicillin-sulbactam, rifampicin and tigecycline) and five combinations of these agents (imipenem plus colistin, imipenem plus ampicillin-sulbactam, colistin plus rifampicin, colistin plus tigecycline, and tigecycline plus rifampicin) according to a previously reported method (Petersen et al., 00). Time-kill assays were performed in duplicate using concentrations of 0.5 and 1 MIC in both single-agent and combination studies. Bacterial growth was quantified after 0,,,, 1 and h incubation at 37 uc by plating -fold dilutions on sheep blood agar. Antimicrobials were considered bactericidal when a 3 log decrease in c.f.u. ml 1 was reached compared with the initial inocula. Synergy of the antimicrobial combination was defined as a log decrease in c.f.u. ml 1 as compared to use of a single agent (Eliopoulos & Moellering, 199). RESULTS In vitro susceptibilities The MICs for imipenem, meropenem, colistin, polymyxin B, ampicillin-sulbactam, tigecycline and rifampicin of the six A. baumannii isolates are presented in Table 1. All isolates were resistant to the carbapenems imipenem and meropenem. Additionally, all were resistant to ciprofloxacin, cefepime, ceftriaxone, cefoperazone-sulbactam, ceftazidime, piperacillin-tazobactam, tetracycline and ampicillin-sulbactam. While KHU- was susceptible to amikacin (MIC mg l 1 ), the other isolates were resistant (MICs.1 mg l 1 ). Single-agent studies Only imipenem was bactericidal against all six A. baumannii isolates tested (Table ); even 0.5 MIC of imipenem resulted in bactericidal effects against two COL-S/TIG-S isolates and one COL-R/TIG-S isolate (KCU-). Colistin was bactericidal against KCU- only (Table ). Although 1 MIC of colistin initially decreased growth in all A. baumannii isolates after and h of incubation, regrowth was observed in five isolates. As observed with the colistin treatment, ampicillin-sulbactam treatment showed a bactericidal effect on KCU- and SKKU-. Even KCU- showed about log regrowth after h of incubation at 1 MIC of ampicillin-sulbactam, compared to that after 1 h of incubation with this agent. None of the A. baumannii isolates used in this study were completely killed by tigecycline as a single regimen at either 0.5 or 1 MIC. Rifampicin also had no bactericidal effect against any of the A. baumannii isolates tested. Combination studies Treatment with a combination of 1 MIC imipenem and colistin exerted bactericidal effects on all six A. baumannii isolates tested (Table 3, Fig. 1). However, treatment with 0.5 MIC imipenem plus colistin was bactericidal against only four isolates: the two COL-S/TIG-S isolates, a COL-R/ TIG-S isolate (KCU-) and a COL-S/TIG-R isolate (SKKU- ). This combination at 0.5 MIC was not effective against SKKU- and KCU-13. All A. baumannii isolates were also not detected in incubations with the combination of 1 MIC imipenem and ampicillin-sulbactam (Table 3). Compared with 1 imipenem alone, three A. baumannii isolates (KHU-, SKKU- and KCU-13) were killed earlier by the combination of 1 imipenem and ampicillin-sulbactam. Imipenem plus ampicillin-sulbactam at 0.5 MIC displayed bactericidal activities against all isolates. It is of note that the combination of 0.5 imipenem and ampicillin-sulbactam displayed synergistic and bactericidal effects even against the three isolates (SKKU-, SKKU- and KCU-13) that were not killed within h by 0.5 imipenem alone. Table. Bactericidal effects of single agents against imipenem-resistant A. baumannii isolates Resistance* Isolate Bactericidal effectd Imipenem Colistin Ampicillinsulbactam Rifampicin Tigecycline 0.5¾ MIC 1¾ MIC 0.5¾ MIC 1¾ MIC 0.5¾ MIC 1¾ MIC 0.5¾ MIC 1¾ MIC 0.5¾ MIC 1¾ MIC COL-S/TIG-S KRU-A-3 B B NB NB NB NB NB NB NB NB KHU- B B NB NB NB NB NB NB NB NB COL-R/TIG-S KCU- B B B B NB B NB NB NB NB SKKU- NB B NB NB NB NB NB NB NB NB COL-S/TIG-R SKKU- NB B NB NB NB B NB NB NB NB KCU-13 NB B NB NB NB NB NB NB NB B *COL-S, colistin susceptible; COL-R, colistin resistant; TIG-S, tigecycline susceptible; TIG-R, tigecycline resistant. DB, bactericidal (when 3 log decrease in c.f.u. ml 1 was reached compared with the initial inocula); NB, non-bactericidal IP: On: Mon, 7 Nov :03:5

4 K. R. Peck and others Table 3. Synergistic effects of antimicrobial combinations against imipenem-resistant A. baumannii isolates Resistance* Isolate Synergistic effectd Colistin+Rifampicin Colistin+Tigecycline Tigecycline+Rifampicin Imipenem+Colistin Imipenem+Ampicillinsulbactam 0.5¾ MIC 1¾ MIC 0.5¾ MIC 1¾ MIC 0.5¾ MIC 1¾ MIC 0.5¾ MIC 1¾ MIC 0.5¾ MIC 1¾ MIC COL-S/TIG-S KRU-A-3 S S S S NS S NS S NS S KHU- S S S S NS S S S NS S COL-R/TIG-S KCU- S S S S S S S S NS S SKKU- NS S S S NS S NS S NS S COL-S/TIG-R SKKU- NS S S S NS S S S S S KCU-13 NS S S S S S S S S S *COL-S, colistin-susceptible; COL-R, colistin-resistant; TIG-S, tigecycline-susceptible; TIG-R, tigecycline-resistant. DS, synergistic (when log decrease in c.f.u. ml 1 as compared to use of a single agent); NS, non-synergistic. All tested isolates of bacteria were not detected in incubations with the combination of 1 MIC colistin and rifampicin within h after incubation (Table 3). Although KRU-A-3 regrew temporarily after 1 h of incubation, it was eventually eliminated. However, treatment with 0.5 MIC colistin plus rifampicin was bactericidal against only two isolates: KCU- and KCU-13. Treatment with a combination of 1 MIC colistin and tigecycline was also bactericidal against all A. baumannii isolates (Table 3, Fig. ). Treatment with 0.5 MIC colistin plus tigecycline was bactericidal or synergistic against only four isolates: one COL-S/TIG-S (KRU-A-3), one COL-R/TIG-S (KCU-), and the two COL-S/ TIG-R isolates. However, SKKU- (a COL-S/TIG-R isolate) showed regrowth after 1 h of incubation with 0.5 colistin plus tigecycline. Treatment with the combination of tigecycline and rifampicin was the least effective against the A. baumannii isolates tested (Table 3). Although all isolates reached undetectable levels in incubations with 1 tigecycline plus rifampicin, removal took longer (1 h) than for the other combinations. The 0.5 MIC of tigecycline and rifampicin was synergistic against only two COL-S/TIG-R isolates. Therefore, only two COL-S/TIG-R isolates were not detected by the combination of 0.5 tigecycline and rifampicin within h of incubation. DISCUSSION A. baumannii infections have traditionally been treated with broad-spectrum cephalosporins, b-lactams and b-lactamase inhibitors, and carbapenems (Munoz-Price & Weinstein, 00). However, the emergence of and subsequent increase in MDR A. baumannii isolates, including carbapenemresistant isolates, have limited the treatment options. Thus, treatment with polymyxins such as polymyxin B and colistin, which had previously been abandoned due to problems of nephrotoxicity and neurotoxicity, is being used for these infections (Peleg et al., 00; Li et al., 00b). In addition, tigecycline, a new glycylcycline, has been introduced to treat MDR Gram-negative bacterial infections including A. baumannii (Peleg et al., 00). However, studies have reported development of resistance to colistin or tigecycline during the treatment (Hawley et al., 00; Liet al., 00a; Owen et al., 007; Peleg et al., 007). Monotherapy with colistin may be problematic for the treatment of colistinheteroresistant A. baumannii infections (Owen et al., 007). Even extreme drug-resistant (XDR) A. baumannii isolates, displaying resistance to all antimicrobials, including polymyxins and tigecycline, have emerged (Doi et al., 009; Park et al., 009d). Thus, combination therapy has been recommended not only to combat MDR A. baumannii infections but also to inhibit or reduce the emergence of resistance during treatment. Not a few studies have been performed on the in vitro activities of combination therapies against A. baumannii infections. Tripodi et al. (007) reported synergistic effects 35 IP: Journal of Medical Microbiology 1 On: Mon, 7 Nov :03:5

5 In vitro combination therapy against A. baumannii KRU-A-3 (COL-S/TIG-S) KHU- (COL-S/TIG-S) KCU- (COL-R/TIG-S) SKKU- (COL-S/TIG-R) SKKU- (COL-R/TIG-S) KCU-13 (COL-S/TIG-R) Fig. 1. Effects of imipenem (IMP), colistin (COL), and a combination of these agents on the viability of six imipenem-resistant A. baumannii isolates. COL-S, colistin-susceptible; COL-R, colistin-resistant; TIG-S, tigecycline-susceptible; TIG-R, tigecyclineresistant. e, 0.5¾ MIC IMP; h, 0.5¾ MIC COL; #, 0.5¾ MIC IMP+COL; X, 1¾ MIC IMP; &, 1¾ MIC COL; $, 1¾ MIC IMP+COL. The in vitro time-kill experiments were duplicated; mean values are plotted. In most duplicate experiments, similar time-kill results were obtained. for combinations of rifampicin plus imipenem or ampicillin-sulbactam against MDR A. baumannii isolates. Colistin plus minocycline, tigecycline plus amikacin, carbapenems (imipenem and meropenem) plus polymyxins (polymyxin B and colistin), imipenem plus tigecycline, colistin plus vancomycin, colistin plus teicoplanin, and rifampicin plus sulbactam have also been reported to have synergistic effects (Hornsey & Wareham, 011; Moland et al., 00; Pachón- Ibáñez et al., 00; Gordon et al., 0; Pankey & Ashcraft, 009; Sopirala et al., 0; Tan et al., 007). In addition, imipenem plus sulbactam and colistin plus rifampicin combinations were effective in vitro against carbapenemresistant A. baumannii isolates (Song et al., 007). However, other in vitro time-kill studies demonstrated that tigecycline is ineffective when used in combination with polymyxin B, minocycline, imipenem, levofloxacin, ampicillin-sulbactam and rifampicin against carbapenem-nonsusceptible A. baumannii isolates (Moland et al., 00; Scheetz et al., 007). Overall, many in vitro and a few in vivo time-kill studies have indicated that antibiotic combination therapy is effective against infections caused by imipenemresistant A. baumannii, although the specific regimens that were shown to be effective differ in each study. To our knowledge, few studies except that of Vila-Farres et al. (011) have been performed on colistin-resistant A. baumannii isolates. In the present study, the effectiveness of five antimicrobial agents singly and in combinations against imipenemresistant, colistin-resistant or tigecycline-resistant A. baumannii blood isolates were evaluated using an in vitro time-kill analysis. In single-agent studies, only imipenem exhibited consistent bactericidal activity against all A. baumannii isolates tested. Although a bacteridical effect of 1 imipenem might be anticipated, even 0.5 MIC imipenem was bactericidal against some A. baumannii isolates. The other agents used singly yielded inconsistent results against A. baumannii even at 1 MIC. In particular, colistin or tigecycline alone was not always bactericidal even against colistin-susceptible or tigecyclinesusceptible isolates, respectively (Table ). This suggests that either colistin or tigecycline monotherapy is not likely to be effective against infections with carbapenemresistant A. baumannii isolates, irrespective of antimicrobial resistance. IP: On: Mon, 7 Nov :03:5

6 K. R. Peck and others 1 KRU-A-3 (COL-S/TIG-S) 1 KHU- (COL-S/TIG-S) KCU- (COL-R/TIG-S) 1 SKKU- (COL-R/TIG-S) SKKU- (COL-S/TIG-R) KCU-13 (COL-S/TIG-R) Fig.. Effects of colistin (COL), tigecycline (TIG) and a combination of these agents on the viability of six imipenem-resistant A. baumannii isolates. COL-S, colistin-susceptible; COL-R, colistin-resistant; TIG-S, tigecycline-susceptible; TIG-R, tigecyclineresistant. e, 0.5¾ MIC COL; h, 0.5¾ MIC TIG; #, 0.5¾ MIC COL+TIG; X, 1¾ MIC COL; &, 1¾ MIC TIG; $, 1¾ MIC COL+TIG. The in vitro time-kill experiments were duplicated; mean values are plotted. In most duplicate experiments, similar time-kill results were obtained. In contrast to the single-agent experiments, combination regimens displayed excellent bactericidal activities. All imipenem-resistant A. baumannii isolates tested were not detected in incubations with all five combinations of antimicrobial agents at 1 MIC. Although treatment with combinations of antimicrobial agents at 0.5 MIC was effective against some isolates, the effects were not consistent. Only 0.5 MIC imipenem plus ampicillinsulbactam displayed bactericidal activity against all imipenem-resistant A. baumannii isolates. As both imipenem and ampicillin-sulbactam target the bacterial cell wall, their combination would be expected to kill the bacteria more rapidly, which may not have clinical implications. Excluding the combination imipenem and ampicillin-sulbactam, 0.5 MIC colistin plus tigecycline was the most effective, showing synergistic or bactericidal effects against four A. baumannii isolates. These results may indicate that increasing the dosage of antimicrobial agents sufficiently is required to achieve a bactericidal effect against resistant A. baumannii. However, high dosage of antimicrobial agents may lead to the further emergence of resistance and may increase the toxic effects of those agents. Thus, further investigation of the doses of combination agents that are sufficient to kill bacteria and to prevent the development of resistance is required. Although the imipenem-resistant A. baumannii isolates examined in this study belonged to different PFGE types, they have similar imipenem resistance mechanisms, bla OXA-51 and bla OXA-3, and all of them belonged to European clone II, an internationally disseminated clone (data not shown). While bla OXA-3 is the main mechanism of imipenem resistance in A. baumannii, especially in Korea (Park et al., 009d; Kim et al., 0), A. baumannii isolates possessing bla IMP and bla VIM are also important contributors to imipenem resistance, but such isolates were not included in this study. Our study did not analyse the colistin and tigecycline resistance mechanisms for the A. baumannii isolates tested. Thus, our results may not be generalizable to all imipenem-resistant, colistin-resistant or tigecyclineresistant A. baumannii isolates. However, our data suggest that some antimicrobial combinations may be effective for combating imipenem-resistant A. baumannii infections, including those due to colistin-resistant or tigecyclineresistant bacteria. 35 IP: Journal of Medical Microbiology 1 On: Mon, 7 Nov :03:5

7 In vitro combination therapy against A. baumannii ACKNOWLEDGEMENTS This study was supported by a grant from the Korea Health 1 R&D Project, Ministry of Health, Welfare, and Family Affairs, Republic of Korea (grant no. A05). REFERENCES Capone, A., D Arezzo, S., Visca, P. & Petrosillo, N. (00). In vitro activity of tigecycline against multidrug-resistant Acinetobacter baumannii. JAntimicrobChemother, 3. CLSI (0). Performance Standards for Antimicrobial Susceptibility Testing, 0th informational supplement, M0 S0. Wayne, PA: Clinical and Laboratory Standards Institute. Dijkshoorn, L., Nemec, A. & Seifert, H. (007). An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat Rev Microbiol 5, Doi, Y., Husain, S., Potoski, B. A., McCurry, K. R. & Paterson, D. L. (009). Extensively drug-resistant Acinetobacter baumannii. Emerg Infect Dis 15, Eliopoulos, G. M. & Moellering, R. C. (199). Antimicrobial combinations. In Antibiotics in Laboratory Medicine, th edn. 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