JAC Journal of Antimicrobial Chemotherapy (2004) 54, 1130–1133 DOI: 10.1093/jac/dkh462 Advance Access publication 14 October 2004 Cefepime and the inoculum effect in tests with Klebsiella pneumoniae producing plasmid-mediated AmpC-type b-lactamase Cheol-In Kang1, Hyunjoo Pai2, Sung-Han Kim1, Hong-Bin Kim1, Eui-Chong Kim3,4, Departments of 1Internal Medicine and 3Laboratory Medicine, Seoul National University College of Medicine, Seoul; 2Department of Internal Medicine, Hanyang University College of Medicine, Seoul; 4 Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of Korea Received 10 July 2004; returned 19 August 2004; revised 5 September 2004; accepted 14 September 2004 Objective: In the past decade, a new problem in Klebsiella pneumoniae strains has emerged: plasmidmediated AmpC enzymes. This study was conducted to investigate the activity of cefepime against clinical isolates by determining the activities of cefepime and three other parenteral b-lactam agents in standard and high inoculum MIC tests. Methods: A total of 61 K. pneumoniae blood isolates, including 28 isolates producing AmpC-type b-lactamases (14 isolates of DHA-1 and 14 isolates of CMY-1-like) and 33 isolates producing extended-spectrum b-lactamases (ESBLs) (32 isolates of TEM- or SHV-related and one isolate of CTX-M-14-like), were included in the study. Antimicrobial susceptibilities were determined using broth microdilution MIC tests with standard and 100-fold-higher inocula. The inoculum effect was defined as an eight-fold or greater MIC increase on testing with the higher inoculum. Results: In tests with AmpC b-lactamase-producing K. pneumoniae isolates and their transconjugants, the inoculum effect was most consistently detected with cefepime, cefotaxime and ceftazidime, as inoculum effects were consistently detected in ESBL-producing isolates. However, the inoculum effect was least frequently detected with imipenem. Conclusion: Although the inoculum effect is an in vitro laboratory phenomenon, these results suggest that cefepime may be a less than reliable agent for therapy in cases of high inoculum infections caused by AmpC b-lactamase-producing K. pneumoniae. Keywords: K. pneumoniae, cephalosporins, b-lactam resistance Introduction In the past decade, a new problem in Klebsiella pneumoniae strains has emerged: plasmid-mediated AmpC enzymes. They are derived from the chromosomal AmpC genes of Gramnegative organisms, such as Citrobacter freundii, Enterobacter cloacae and Aeromonas species.1 Strains with plasmid-mediated AmpC enzymes are generally resistant to broad-spectrum penicillins, extended-spectrum cephalosporins and cephamycins. These strains are also resistant to aztreonam, but susceptible to cefepime, cefpirome and carbapenems.1 Previous studies, based on antimicrobial susceptibility data, suggested that cefepime might be effective for the treatment of infections caused by AmpC-producing K. pneumoniae.2,3 In vitro studies with high inocula show that the MICs of cefepime for extended-spectrum b-lactamase (ESBL)producing isolates of K. pneumoniae and Escherichia coli are often greatly elevated, suggesting that these agents are inactivated by ESBLs.4,5 Indeed, there are reports on cefepime failure in serious infections caused by ESBL producers.6 Similarly, in serious infections caused by AmpC b-lactamaseproducing K. pneumoniae, it has been proposed that a major .......................................................................................................................................................................................................................................................................................................................................................................................................................... *Correspondence address. Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong Chongno-gu, Seoul 110–744, Republic of Korea. Tel: +82-2-760-2945; Fax: +82-2-762-9662; E-mail: mdohmd@snu.ac.kr .......................................................................................................................................................................................................................................................................................................................................................................................................................... 1130 JAC vol.54 no.6 q The British Society for Antimicrobial Chemotherapy 2004; all rights reserved. Downloaded from https://academic.oup.com/jac/article-abstract/54/6/1130/856046 by guest on 22 August 2019 Myoung-don Oh1,4* and Kang-Won Choe1,4 C-I. Kang et al. inoculum effect for an antimicrobial agent precludes its use.7 Thus, this study was designed to further investigate the activity of cefepime against clinical isolates by determining the activities of this agent and three other parenteral b-lactam agents in standard and high inoculum MIC tests. Materials and methods Bacterial strains and the characterization of b-lactamases Antimicrobial susceptibility tests and the inoculum effect Antimicrobial susceptibilities were determined using a broth microdilution MIC method using inocula that differed 100-fold in density. The inocula comprised 105 (the standard inoculum) and 107 (the higher inoculum) cfu/mL suspended in Mueller – Hinton broth (Becton Dickinson, Sparks, MD, USA). Standard inoculum tests were conducted according to the National Committee for Clinical Laboratory Standards.9 An inoculum effect was defined as an eight-fold or greater increase in MIC on testing with the higher inoculum.4 Antimicrobials tested were cefotaxime (C.J. Corp. Pharmaceutical Division, Seoul, Korea), ceftazidime (Hanmi Pharmaceutical Co., Ltd., Seoul, Korea), cefepime (Boryung Pharm., Seoul, Korea) and imipenem (Choongwae Pharma Corp., Seoul, Korea). Inoculum effects in antimicrobial susceptibility tests with ESBL-producing K. pneumoniae isolates A total of 33 K. pneumoniae isolates, 32 isolates producing TEM- or SHV-related ESBLs and one isolate producing a CTX-M-14-like enzyme, were tested. Cefotaxime, ceftazidime and cefepime were found to be associated with inoculum effects in 100% of evaluable tests of ESBL-producing K. pneumoniae isolates (i.e. excluding those which could not be evaluated because of off-scale MICs) (Table 1). The MIC50 of cefepime was 8 mg/L in standard inoculum tests and >256 mg/L in higher inoculum tests. However, imipenem inhibited all isolates at a concentration of 0.5 mg/L in standard inoculum tests and also inhibited all isolates at 4 mg/L in higher inoculum tests. Inoculum effects in antimicrobial susceptibility tests with AmpC b-lactamase-producing K. pneumoniae isolates Fourteen isolates producing DHA-1-related b-lactamases and 14 isolates producing CMY-1-like enzyme were tested. As inoculum effects were consistently detected in ESBL-producing strains, cefotaxime, ceftazidime and cefepime were also associated with inoculum effects in 100% of evaluable tests with AmpC b-lactamase-producing K. pneumoniae isolates (i.e. excluding those which could not be evaluated because of offscale MICs) (Table 2). The MIC50 of cefepime was 1 mg/L in the standard inoculum tests and >256 mg/L in the higher inoculum tests. However, imipenem at 0.5 and 8 mg/L inhibited all isolates in standard and higher inoculum tests, respectively. For DHA-1-related AmpC-producing K. pneumoniae isolates, the MICs of antibiotics by the standard and higher inoculum tests are shown in Table 2. In addition, for CMY-1-like AmpCproducing K. pneumoniae isolates, the MICs of antibiotics by the standard and the higher inoculum tests are detailed in Table 2. As inoculum effects were consistently detected in CMY-1-like AmpC b-lactamases-producing isolates, cefotaxime, ceftazidime Table 1. MICs for ESBL- or AmpC-producing K. pneumoniae isolates according to inoculum MICa (mg/L) of antibiotics at inocula of 105 and 107 cfu/mL ESBL-producing K. pneumoniaeb (n = 33) 105 Antibiotics CTX CAZ FEP IPM AmpC-producing K. pneumoniaec (n = 28) 107 105 107 range 50% 90% range 50% 90% range 50% 90% range 50% 90% 1 – >256 1 – >256 1 – 128 < _ 0.25– 4 64 128 8 0.25 256 256 64 0.5 >256 >256 >256 0.5 –8 >256 >256 >256 1 >256 >256 >256 4 8 – >256 16 – >256 < _ 0.25 – 16 < _ 0.25 – 1 64 128 1 0.25 >256 >256 4 0.5 >256 >256 16 – >256 0.5– 8 >256 >256 256 4 >256 >256 >256 8 CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; IPM, imipenem. a 50% and 90%, MICs at which 50% and 90% of isolates are inhibited, respectively. b TEM, SHV or CTX-M-related b-lactamase-producing K. pneumoniae isolates. c DHA-1 or CMY-1-like b-lactamase-producing K. pneumoniae isolates. 1131 Downloaded from https://academic.oup.com/jac/article-abstract/54/6/1130/856046 by guest on 22 August 2019 As we previously reported,8 65 K. pneumoniae blood isolates producing ESBLs or AmpC b-lactamases were identified. b-Lactamases were characterized in 61 of 65 isolates. Twenty-eight isolates producing AmpC-type enzymes (14 DHA-1 and 14 CMY1-like), 32 isolates producing TEM- or SHV-related ESBLs and one strain producing a CTX-M-14-like enzyme were included in this study.8 The ESBL status of the AmpC-producing isolates was tested by the double-disc diffusion test, using cefepime and amoxicillin –clavulanic acid discs. After incubation, an enhanced zone of inhibition between the cefepime disc and clavulanic acid disc was interpreted as presumptive evidence for the presence of an ESBL. Two control organisms, E. coli ATCC 25922 and K. pneumoniae ATCC 700603, were inoculated in each set of tests for quality control. Results AmpC K. pneumoniae and the inoculum effect Table 2. Standard- and high-inoculum MICs for 28 K. pneumoniae isolates producing AmpC-type b-lactamasesa MIC (mg/L) of agent at inocula of 105 and 107 cfu/mL CTX Isolate no.b 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 7.7 5.4, 5.4, 7.7 7.6, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 5.4, 7.7 7.7 7.7 7.7 7.7 7.7 7.7 7.7, 8.2 7.6, 7.7 7.6, 7.7 7.7, 8.2 7.6, 7.7 7.6, 8.0 7.6, 8.0 7.6, 8.0 7.6, 8.0 7.6, 8.0 8.0 8.0 7.6, 8.0 8.0 8.0 8.0 8.0 7.6, 8.0 8.0 – FEP IPM Type of b-lactamases 105 107 105 107 105 107 105 107 DHA-1, TEM-1-like DHA-1, TEM-1-like DHA-1, TEM-1-like DHA-1, TEM-1-like DHA-1, TEM-1-like DHA-1, TEM-1-like DHA-1, TEM-1-like DHA-1, TEM-1-like, SHV-12-like DHA-1 DHA-1, SHV, TEM-1-like DHA-1, SHV, TEM-1-like DHA-1 DHA-1, SHV, SHV-12-like DHA-1, SHV, TEM-1-like TEM-1-like, SHV, CMY-1-like TEM-1-like, SHV, CMY-1-like TEM-1-like, SHV, CMY-1-like TEM-1-like, SHV, CMY-1-like TEM-1-like, SHV, CMY-1-like CMY-1-like, TEM-1-like CMY-1-like, TEM-1-like TEM-1-like, SHV, CMY-1-like CMY-1-like, TEM-1-like CMY-1-like, TEM-1-like CMY-1-like, TEM-1-like CMY-1-like, TEM-1-like TEM-1-like, SHV, CMY-1-like CMY-1-like, TEM-1-like SHV-18 64 32 64 128 8 32 16 64 64 16 32 32 32 8 64 64 128 64 64 256 256 256 >256 >256 >256 >256 >256 32 8 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 128 32 16 >256 >256 256 32 256 128 128 32 32 64 128 32 64 64 64 64 64 128 64 128 64 32 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 0.5 0.5 < _ 0.25 16 < _ 0.25 0.5 < _ 0.25 < _ 0.25 < _ 0.25 < _ 0.25 < _ 0.25 < _ 0.25 4 < _ 0.25 1 2 1 2 1 4 4 4 4 4 4 8 4 1 1 128 256 128 >256 128 >256 64 16 256 128 128 64 >256 128 >256 256 256 256 256 >256 >256 >256 >256 >256 >256 >256 >256 256 128 0.5 0.5 0.5 < _ 0.25 0.5 1 < _ 0.25 0.5 0.5 < _ 0.25 < _ 0.25 < _ 0.25 1 0.5 0.5 < _ 0.25 < _ 0.25 < _ 0.25 < _ 0.25 0.5 < _ 0.25 < _ 0.25 < _ 0.25 < _ 0.25 < _ 0.25 < _ 0.25 < _ 0.25 < _ 0.25 0.5 4 8 4 1 4 8 4 4 8 4 4 4 4 4 4 0.5 1 4 4 8 4 4 4 4 4 4 2 4 4 CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; IPM, imipenem. a Fourteen DHA-1 and 14 CMY-1-like b-lactamases-producing isolates were included. b KP21, 22, 28, 36, 37 and 57 showed a negative ESBL confirmatory test phenotypically. c Italics indicate that the b-lactamase with the indicated pI value is inhibited by 0.3 mM cloxacillin and thus represent AmpC enzymes.8 d ESBL reference strain, K. pneumoniae. and cefepime were also associated with inoculum effects in all conjugants of CMY-1-producing isolates. The increases in cefepime MIC on testing with the higher inoculum were more prominent in CMY-1-like AmpCproducing isolates than DHA-1 AmpC-producing isolates. In standard inoculum tests, all isolates were susceptible to imipenem, and cefepime was the next most active agent (96.4% susceptible). In higher inoculum tests, cefepime was dramatically affected, with susceptibility decreasing from 96.4% in standard inoculum tests to 0% of isolates inhibited by 8 mg/L in the higher inoculum tests. Inoculum effects with imipenem occurred in tests with 85.7% of these isolates (12 of 14 DHA1 isolates and 12 of 14 CMY-1 isolates). Even so, the higher inoculum MICs of imipenem exceeded 4 mg/L in only four strains (3 DHA-1 and 1 CMY). When the ESBL status of the AmpC-producing isolates was assessed phenotypically, all CMY-1-like AmpC-producing isolates and eight DHA-1 AmpC-producing isolates showed a positive ESBL confirmatory test. The remaining six isolates with a negative ESBL confirmatory test also showed an inoculum effect with cefepime. Discussion In this study of AmpC b-lactamase-producing K. pneumoniae blood isolates, the inoculum effect was found to be most pronounced in tests with cefepime, cefotaxime and ceftazidime, with cefepime being at least as affected as cefotaxime and ceftazidime. The inoculum effect was smallest and least common in tests with imipenem. An organism may appear susceptible to a drug when tested in vitro using a standard inoculum but may be ineffective in vivo due to a high inoculum.4 – 6 A high inoculum effect was recently reported for cefepime on ESBL-producing and AmpC-producing isolates of Enterobacteriaceae.3,4 However, no specific AmpC enzymes were identified,4 and only five (2 ACT-1 and 3 FOX-5) isolates were included.3 In our study, many relatively new isolates, harbouring DHA-1 or CMY-1-like b-lactamase, were 1132 Downloaded from https://academic.oup.com/jac/article-abstract/54/6/1130/856046 by guest on 22 August 2019 KP9 KP10 KP12 KP18 KP19 KP21 KP22 KP23 KP28 KP36 KP37 KP42 KP43 KP57 KP26 KP29 KP32 KP33 KP34 KP44 KP45 KP48 KP51 KP52 KP53 KP54 KP59 KP61 ATCC 700603d pI valuec CAZ C-I. Kang et al. by AmpC-type b-lactamase-producing K. pneumoniae to be prepared to monitor patients closely for signs of treatment failure. Acknowledgements The authors were not supported financially. References 1. Philippon, A. & Jacoby, G. A. (2002). Plasmid-determined AmpC-type b-lactamases. Antimicrobial Agents and Chemotherapy 46, 1 –11. 2. Yan, J. J., Ko, W. C., Jung, Y. C. et al. (2002). Emergence of Klebsiella pneumoniae isolates producing inducible DHA-1 b-lactamase in a university hospital in Taiwan. Journal of Clinical Microbiology 40, 3121– 6. 3. Coudron, P. E., Hanson, N. D. & Climo, M. W. (2003). Occurrence of extended-spectrum and AmpC b-lactamases in bloodstream isolates of Klebsiella pneumoniae: isolates harbor plasmidmediated FOX-5 and ACT-1 AmpC b-lactamases. Journal of Clinical Microbiology 41, 772 –7. 4. Thomson, K. S. & Moland, E. S. (2001). Cefepime, piperacillintazobactam, and the inoculum effect in tests with extended-spectrum b-lactamase-producing Enterobacteriaceae. Antimicrobial Agents and Chemotherapy 45, 3548–54. 5. Thauvin-Eliopoulos, C., Tripodi, M.-F., Moellering, R. C. et al. (1997). Efficacies of piperacillin-tazobactam and cefepime in rats with experimental intra-abdominal abscesses due to an extended-spectrum b-lactamase-producing strain of Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 41, 1053–7. 6. Paterson, D. L., Ko, W.-C., Gottberg, A. V. et al. (2001). Outcome of cephalosporin treatment for serious infections due to apparently susceptible organisms producing extended-spectrum b-lactamases: implications for the clinical microbiology laboratory. Journal of Clinical Microbiology 39, 2206–12. 7. Livermore, D. M. (1998). b-Lactamase-mediated resistance and opportunities for its control. Journal of Antimicrobial Chemotherapy 41, Suppl. D, 25– 41. 8. Pai, H., Kang, C. I., Byeon, J. H. et al. (2004). Epidemiology and clinical features of bloodstream infections caused by AmpC type b-lactamase-producing Klebsiella pneumoniae. Antimicrobial Agents and Chemotherapy 48, 3720– 8. 9. National Committee for Clinical Laboratory Standards. (2003). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically—Sixth Edition: Approved standard M7-A6. NCCLS, Wayne, PA, USA. 10. Rice, L. B., Yao, J. D. C., Klimm, K. et al. (1991). Efficacy of different b-lactams against an extended-spectrum b-lactamase-producing Klebsiella pneumoniae strain in the rat intra-abdominal abscess model. Antimicrobial Agents and Chemotherapy 35, 1243–4. 1133 Downloaded from https://academic.oup.com/jac/article-abstract/54/6/1130/856046 by guest on 22 August 2019 tested for inoculum effect. In the molecular epidemiological study including these isolates, DHA-1 enzyme-producing isolates showed seven types, and CMY-1-like enzyme-producing isolates revealed two types.8 In general, broad-spectrum cephalosporins are not recommended for the treatment of ESBL-producing organisms. Initial challenges of the notion that ESBL-producing organisms—for which MICs of broad-spectrum cephalosporins are in the susceptible range—may not be truly susceptible (when serious infections are considered) were posed by in vitro studies of the ‘inoculum effect’ and animal studies. In vitro, the MICs of cephalosporins increase as the inoculum of ESBL-producing organisms increases.4,5,10 Experience in experimental animal models of infections with ESBL-producing organisms has demonstrated ceftriaxone or cefotaxime failure, despite antibiotic serum levels far exceeding the MIC of the antibiotic when tested in a conventional inoculum of 105 organisms per mL.10 Studies on determining the therapeutic success or failure of third-generation cephalosporins in treating infections with plasmid-mediated inducible AmpC producers, such as our DHA1-producing K. pneumoniae isolates, are lacking. The drugs of choice for the treatment of infections caused by such organisms are also undetermined. Based on antimicrobial susceptibility data, a majority of the AmpC-producing K. pneumoniae isolates remained susceptible to cefepime and imipenem. Thus, Yan et al.2 suggested that cefepime and carbapenems could be better choices for the treatment of infections caused by AmpC producers. However, little information is available about the clinical effectiveness of cefepime for the treatment of infections caused by AmpC-type b-lactamase-producing K. pneumoniae. In our study, the inoculum effect was most consistently detected for cefepime, cefotaxime and ceftazidime. As previously reported by Livermore,7 it has been proposed that a major inoculum effect for an antimicrobial agent precludes its use in serious infections caused by the pathogen, and thus cefepime may be a less than reliable agent for therapy of serious infections caused by AmpC b-lactamase-producing K. pneumoniae. The inoculum effects with cefepime in this study might have arisen from co-production of ESBLs. However, cefepime was associated with inoculum effects in six isolates that showed a negative ESBL confirmatory test. To our knowledge, the clinical implications of the inoculum effect with respect to cefepime have not been evaluated.4 Therefore, as Thomson & Moland4 have suggested in their previous report, until reliable clinical data are available to resolve this issue, it might be prudent for clinicians who are considering using cefepime for therapy in cases of serious infections caused
