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1 Available online at Time-Kill Synergy Tests of Tigecycline Combined with Imipenem, Amikacin, and Ciprofloxacin against Clinical Isolates of Multidrug-Resistant Klebsiella pneumoniae and Escherichia coli 39 Haejun Yim, 1 Heungjeong Woo, 2 Wonkeun Song, 3 Min-Jeong Park, 3 Hyun Soo Kim, 3 Kyu Man Lee, 2 Jun Hur, 1 and Man-Seung Park 4 Departments of 1 Burn Surgery, 2 Internal Medicine, 3 Laboratory Medicine, and 4 Microbiology, Hallym University Medical Center and College of Medicine, Seoul and Chuncheon, Korea Abstract. This study evaluated the activity of tigecycline combined with imipenem, amikacin, and ciprofloxacin against clinical isolates of multidrug-resistant Klebsiella pneumoniae and Escherichia coli co-producing extendedspectrum β-lactamases and acquired AmpC β-lactamases. Broth microdilution tests were performed for cefotaxime, ceftazidime, cefepime, imipenem, amikacin, ciprofloxacin, and tigecycline. Time-kill synergy studies were tested for tigecycline plus imipenem, tigecycline plus amikacin, and tigecycline plus ciprofloxacin. Imipenem (MIC 90 = 1 µg/ ml for both K. pneumoniae and E. coli) and tigecycline (MIC 90 = 2 µg/ml for K. pneumoniae and 1 µg/ml for E. coli) were the most potent agents. Combination studies with tigecycline plus imipenem resulted in synergy against 18 K. pneumoniae and 3 E. coli isolates; tigecycline plus amikacin yielded synergy against 8 K. pneumoniae and 3 E. coli isolates; tigecycline plus ciprofloxacin yielded synergy against 7 K. pneumoniae and 2 E. coli isolates. No antagonism was observed with any combination. In the present study, imipenem, amikacin, and ciprofloxacin led to indifferent and some synergistic effects in combination with tigecycline, and none of them demonstrated antagonistic effects. Introduction b-lactamase production is the most common resistance mechanism to b-lactams in Klebsiella pneumoniae and Escherichia coli. K. pneumoniae and E. coli isolates producing extended-spectrum b-lactamases (ESBLs) and/or acquired AmpC b-lactamases have been prevalent worldwide. ESBL-producing isolates are considered resistant to all penicillins, cephalosporins, and aztreonam; carbapenems are the only b-lactams that remain consistently active for treatment in severe cases. The therapeutic options for severe infections caused by acquired AmpC-producing isolates are almost restricted to carbapenems and cefepime [1]. Since K. pneumoniae- and E. coli-resistant phenotypes, such as ESBLs, acquired AmpCs, and isolates with reduced susceptibility to carbapenems continue to spread worldwide, new therapeutic options are needed [2-4]. Tigecycline, the first class of glycyclines, exhibits expandedspectrum of activity against a wide variety of bacteria, including multidrug-resistant strains such as ESBL-producing Enterobacteriaceae, multidrug-resistant Acinetobacter baumannii, methicillinresistant Staphylococcus aureus, and vancomycin-resistant enterococci [5,6]. Tigecycline acts by binding to the 30S ribosomal subunit and prevents the binding of aminoacyl transfer RNA to the acceptor site on the messenger RNA-ribosome complex. Protein synthesis is inhibited, thereby exhibiting a bacteriostatic effect [7]. Tigecycline has potentially useful activity [8] but may not always be effective as monotherapy [9]. Therefore, a study was designed to investigate the in vitro activities of combinations of tigecycline and representative bactericidal agents including imipenem, amikacin, and ciprofloxacin. Antimicrobial combination therapy may provide clinicians an option in addition to imipenem for difficult infections due to ESBL- or AmpCproducing Enterobacteriaceae. Although ESBL- and/or acquired AmpC-producing K. pneumoniae and E. coli routinely express crossresistance to other drug classes, synergy from combination therapy may achieve successful outcomes. The purpose of this study was to evaluate the in vitro antimicrobial activity of tigecycline combined with other antimicrobials against clinical isolates of multidrug- Address correspondence to Wonkeun Song, M.D., Ph.D., Department of Laboratory Medicine, Kangnam Sacred Heart Hospital, Daelim-dong, Youngdeungpo-gu, Seoul , Republic of Korea; swonkeun@hallym.or.kr /11/ $ by the Association of Clinical Scientists, Inc.

2 40 resistant K. pneumoniae and E. coli co-producing ESBLs and acquired AmpC b-lactamases. Materials and Methods Bacterial isolates. This study included 35 clinical isolates of K. pneumoniae and 8 clinical isolates of E. coli known to co-produce ESBLs and AmpCs, which were obtained from two teaching hospitals in Korea during 2009 (Table 1). The isolates were identified with the Vitek 2 system (biomerieux Vitek, Hazelwood, MO). Searches for genes coding for the class A ESBLs were performed by PCR amplification as described previousely [10]. To identify isolates with ampc genes, AmpC multiplex PCR was performed by the method of Perez- Perez and Hanson [11]. Sequencing of ampc genes with primers DHA- 1, CMY-1, and CMY-2 was performed as described previously [12]. The PCR products were subjected to direct sequencing. Both strands of each PCR product were sequenced twice with an automatic sequencer (model 3730xl; Applied Biosystems, Weiterstadt, Germany). Sequence alignment and analysis was performed online using the BLAST program (National Center for Biotechnology Information (Bethesda, MD; Antimicrobial susceptibility testing. Broth microdilution MIC tests were performed for ceftazidime (Sigma- Aldrich, St. Louis, MO), cefotaxime (Sigma-Aldrich), cefepime (Boryung, Seoul, Korea), imipenem (LKT Laboratories, St. Paul, MN), amikacin (Sigma-Aldrich), ciprofloxacin (LKT Laboratories), and tigecycline (Wyeth, Pearl River, NY), according to the CLSI M7-A7 methods [13]. For quality control, E. coli ATCC and K. pneumoniae ATCC , were included in each set of tests. Time-kill synergy study. Thirtythree strains (28 K. pneumoniae and 5 E. coli) with tigecycline MICs of μg/ml were chosen for timekill experiments. The combination activities of tigecycline and either imipenem, amikacin, or ciprofloxacin were further evaluated in timekill experiments with concentrations at equal and one-fourth the MICs. Mueller-Hinton broth cultures were inoculated with 5 x 10 5 CFU/ml of each isolates, and killing was assessed at 0, 4, 8, and 24 hr. Aliquots (0.1 ml) were removed from 2-ml cultures at 0, 4, 8, and 24 hr and serially diluted in 0.85% sterile saline. Bacterial counts were determined by plating 0.1 ml of appropriate dilutions to enumerate CFU/ml. Plating was performed in duplicate and the blood agar plates Table 1. Bacterial isolates co-producing extended-spectrum b-lactamases and acquired AmpC b-lactamases used in this study Organism No. of isolates β-lactamase K. pneumoniae (n = 35) 23 SHV-12 plus DHA-1 1 SHV-12 plus CMY-2 1 SHV-12 and CTX-M-12 plus DHA-1 1 SHV-12 and CTX-M-15 plus DHA-1 2 CTX-M-14 plus DHA-1 7 CTX-M-15 plus DHA-1 E. coli (n = 8) 4 SHV-12 plus DHA-1 1 CTX-M-14 plus DHA-1 1 CTX-M-15 plus CMY-1 1 CTX-M-15 plus CMY-2 1 CTX-M-24 plus CMY-2 Table 2. MICs for 43 isolates of E. coli and K. pneumoniae co-producing extendedspectrum b-lactamases and acquired AmpC b-lactamases. Organism and Agent MIC (μg/ml) of: Range 50% 90% K. pneumoniae (n = 35) Ceftazidime Cefotaxime Cefepime Imipenem Amikacin Ciprofloxacin Tigecycline E. coli (n = 8) Ceftazidime Cefotaxime Cefepime Imipenem Amikacin Ciprofloxacin Tigecycline

3 Table 3. Results of time-kill synergy for 33 isolates of E. coli and K. pneumoniae co-producing extended-spectrum β-lactamases and acquired AmpC β-lactamases at equal to one-fourth the MICs for tigecycline, imipenem, amikacin, and ciprofloxacin. Isolate MIC (μg/ml) Time-kill synergy (μg/ml) Tigecycline against Enterobactericeae 41 TIG IPM AMK CIP TIG+IPM TIG+AMK TIG+CIP K. pneumoniae K S( ) I S(0.5+2) K > I NT S( ) K > S( ) NT I K35 2 < NT S(2+32, 2+8, , I 0.5+8) K >256 1 I NT 1 K >256 2 S(0.5+1, ) NT S(0.5+2, ) K S(0.5+1, ) S(0.5+16) I K > S(2+0.5) NT S(2+128) K >256 S(1+0.5) I NT K >256 2 S( ) NT I K >256 >256 I NT NT K < NT S(0.25+2) I K >256 S(4+1, , 1+1) S(4+8, 4+2) NT K >256 4 I NT I K S( ) S(0.5+2, ) S( ) K > S(0.5+1) NT I K I S( ) S( ) K >256 S(1+0.5) I NT K >256 I I NT K >256 2 S( ) NT I K > S(0.5+1) NT I K >256 S(1+1, ) S(1+8) NT K >256 >256 S(4+1, ) NT NT K >256 I I NT K > S(4+1) NT S(4+16, 4+4, 1+16) K I I I K > S(0.25+1, ) NT I K S(2+0.5) S(2+16) I E. coli E S( ) S( ) S( ) E >256 I I NT E > S( ) NT I E S(1+1, , S(1+64, , S(1+64, , ) ) ) E I S( ) I Abbreviations: TIG, tigecycline; IPM, imipenem; AMK, amikacin; CIP, ciprofloxacin; S, synergy; I, indifferent; NT, no tested. were incubated 19 to 24 hr at 35 C. Synergy was defined as a 2-log 10 CFU/ml diminution between the combination and the most active single agent at 24 hr. The number of surviving organisms in the presence of the combination was 2-log 10 CFU/ml and at least one of the drugs alone did not affect the growth curve of the tested organism. Indifferent effect and antagonism were defined at 24 hr as a ± 1 log 10 CFU/ml kill to <2-log 10 CFU/ml compared to the most active single agent and >1 log 10 CFU/ml growth compared to the least active single agent, respectively [14]. Results MICs results are listed in Table 2. The tigecycline and imipenem MIC 50 s ranged from to 0.5 μg/ml and 0.25 to 0.5 μg/ml, respectively, whereas MIC 90 values ranged from 1 to 2 μg/ml and 1 μg/ ml, respectively. Tigecycline MIC values of >2 μg/ml were detected in only three (8.6%) K. pneumoniae isolates. Based on the MIC 90 s, imipenem (MIC 90 = 1 μg/ml for both K. pneumoniae and E. coli) and tigecycline (MIC 90 = 2 μg/ml for K. pneumoniae and 1 μg/ml for E. coli)

4 42 were the most potent agents. Ceftazidime, cefotaxime, cefepime, amikacin, and ciprofloxacin were less active, exhibiting high MIC 90 ( 256 μg/ml for both K. pneumoniae and E. coli). Results of time-kill synergy studies are presented in Table 3. At levels equal the MIC, combination studies with tigecycline plus imipenem were synergistic against 18 K. pneumoniae and 3 E. coli isolates; tigecycline plus amikacin were synergistic against 8 K. pneumoniae and 3 E. coli isolates; tigecycline plus ciprofloxacin were synergistic against 7 K. pneumoniae and 2 E. coli isolates. At one-fourth the MIC, the combination studies with tigecycline plus imipenem were synergistic against 6 K. pneumoniae and 1 E. coli isolates; tigecycline plus amikacin were synergistic against 3 K. pneumoniae and 1 E. coli isolates; tigecycline plus ciprofloxacin were synergistic against 2 K. pneumoniae and 1 E. coli isolates. No antagonism was observed with any tigecycline combinations against all isolates. Discussion Tigecycline and imipenem showed excellent in vitro activity against Enterobacteriaceae regardless of the presence or absence of ESBLs and/or AmpCs [15,16]. In this study, tigecycline and imipenem were also shown to be the most active against K. pneumoniae and E. coli co-producing ESBL and acquired AmpC. Inappropriate antimicrobial therapy for patients with isolates harboring an ESBL and/or AmpC is correlated with increased mortality [17,18]. The complicated nature of infections associated with ESBL- or AmpC-producing Enterobacteriaceae often leads us to consider combination therapy. The use of antimicrobial combinations is one of the best options available to treat infections caused by multidrugresistant bacteria. Most patients with Enterobacteriaceae harboring an ESBL and/or AmpC can be treated with carbapenem monotherapy. However, combination therapy may also reduce the emergence of resistance and improve the spectrum of activity [19]. Our finding that drug interations between tigecycline and other compounds such as cefepime, imipenem, and gentamicin were essentially indifferent or synergistic is in accord with previous results [20]. The combination of one-fourth MIC of tigecycline and one-fourth MIC of imipenem, amikacin or ciprofloxacin was rarely synergistic and no antagonism was observed with any combinations. Overall, the present results indicate that the interaction of tigecycline with imipenem, amikacin, or ciprofloxacin against K. pneumoniae and E. coli co-producing ESBL and acquired AmpC was essentially indifferent or synergistic. Thus, tigecycline could be used safely with imipenem, amikacin or ciprofloxacin. Two K. pneumoniae isolates showed high MICs of imipenem (32 and 64 μg/ml). The isolates showed negative results in a modified Hodge test and EDTA-sodium mercaptoacetic acid double-disk synergy test [21] for screening of carbapenemases and metallo-b-lactamases, respectively (data not shown). Porin loss may have reduced susceptibility to carbapenems. However, no activity of tigecycline is affected by porin loss [15]. The two isolates were also susceptible to tigecycline, with MICs of 0.25 and 1 μg/ml each. In summary, in the present study, imipenem, amikacin, and ciprofloxacin led to indifferent and some synergistic effects in combination with tigecycline, and none of them demonstrated antagonistic effects. Therefore, they may be used often in clinical practice, especially treatment of infection due to multidrug-resistant Enterobacteriaceae. Further studies in animal models are needed to better understand the potential utility of tigecycline combination therapy for multidrug-resistant Enterobacteriaceae. Acknowledgments The authors are grateful to Tae-Jae Lee for excellent technical assistance. This study was supported by a grant of the Korea Healthcare Technology R & D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A084589). References 1. 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