Original Article Antimicrobial resistance to cefotaxime and ertapenem in Enterobacteriaceae: the effects of altering clinical breakpoints Po-Yu Liu1,2,3*, Zhi-Yuan Shi1,4*, Kwong-Chung Tung5*, Ching-Lin Shyu5, Kun-Wei Chan6, Jai-Wen Liu7, Zong-Yen Wu5, Chih-Chuan Kao8, Yi-Ching Huang9, Chin-Fu Lin10,11 1 Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan 2 Center for General Education, Tunghai University, Taichung, Taiwan 3 Department of Pharmacy, Tajen University, Yanpu, Taiwan 4 National Yang-Ming University, Taipei, Taiwan 5 Department of Veterinary Medicine, College of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan 6 Department of Veterinary Medicine, National Chiayi University, Chiayi, Taiwan 7 Department of Emergency Medicine, Chung Shan Medical University Hospital, Taichung, Taiwan 8 Department of Internal Medicine, Dajia Lee's Hospital, Lee's Medical Corporation, Taichung, Taiwan 9 Department of Internal Medicine, Chiayi Branch, Taichung Veterans General Hospital, Taichung, Taiwan 10 Microbiology Section of the Medical Laboratory Department, Taichung Veterans General Hospital, Taichung, Taiwan 11Department of Laboratory Science and Biotechnology, Central Taiwan University of Science and Technology, Taichung, Taiwan. * authors contributed equally to this work Abstract Introduction: The Clinical and Laboratory Standards Institute (CLSI) updated its antimicrobial susceptibility testing interpretation criteria for Enterobacteriaceae. This study assessed the effects of clinical breakpoint changes in the CLSI 2009 to 2012 guidelines on antibiotic susceptibility testing reports. Methodology: In total, 2,076 non-duplicate clinical isolates of Enterobacteriaceae were analyzed. The disk diffusion method was used for susceptibility testing. The CLSI 2009-12 clinical breakpoints were applied to determine susceptibility of cefotaxime and ertapenem. Combined-disk testing was used for phenotypic confirmation of extended-spectrum beta–lactamase (ESBL) production. Results: In total, Enterobacteriaceae resistance rates to cefotaxime increased from 13.1% using the CLSI 2009 guidelines to 23.6% with the CLSI 2010-12 guidelines, and the resistance rates to ertapenem were 0.4%, 1.0% and 0.8% with CLSI 2009, 2011 and 2012, respectively. Based on the 2010-12 CLSI criteria, all ESBL-producing Escherichia coli and Klebsiella pneumoniae were resistant to cefotaxime. Marked differences in susceptibility to ertapenem between the 2009 CLSI criteria and 2012-12 CLSI criteria were noted in ESBL-producing K. pneumoniae. Conclusions: Breakpoints changes in the updated CLSI guidelines resulted in higher resistance rates to cefotaxime and ertapenem. In addition, the effects were different in individual Enterobacteriaceae species. As a result, clinicians may opt to use alternative antimicrobial agents. Upon implementation of the newer CLSI guidelines, laboratories should be aware of the possible consequences and closely monitor the effects. Key words: Drug Resistance; Enterobacteriaceae; microbial sensitivity tests; cefotaxime; ertapenem J Infect Dev Ctries 2014; 8(3):289-296. doi:10.3855/jidc.3335 (Received 24 January 2013 – Accepted 22 April 2013) Copyright © 2014 Liu et al. This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Introduction Hence, antimicrobial susceptibility testing should be Antimicrobial susceptibility testing is one of the performed according to generally accepted and most important tasks in clinical microbiology clinically relevant guidelines. laboratories. It is not only a valuable guide for Several laboratories, particularly in countries antibiotic therapy but also an important without a national antibiotic susceptibility testing epidemiological tool to monitor resistant organisms. system, have been using the Clinical and Laboratory Liu et al. – Susceptibility with New CLSI Breakpoints J Infect Dev Ctries 2014; 8(3):289-296. Standards Institute (CLSI) guidelines to interpret 1500-bed veterans hospital in central Taiwan. antibiotic-sensitivity testing. Recently, the CLSI Identification to the species level was performed by updated and included its recommendations into the routine laboratory methods including the Vitek 2 CLSI 2010, 2011 and 2012 guidelines, which include system (bioMérieux, Marcy l'Etoile, France). major changes in the antibiotic susceptibility testing for Enterobacteriaceae. Inhibition zone diameter Susceptibility testing breakpoints for Enterobacteriaceae defining The Kirby-Bauer disk diffusion test was used for susceptibility to third-generation cephalosporins and susceptibility testing. Cefotaxime and ertapenem were carbapenems are significantly higher in the CLSI tested during the study period. Antibiotics discs were 2010/11 version compared to the CLSI 2009 obtained from Becton Dickinson (BD Diagnostic breakpoints [1-3]. Recently, the CLSI recommended Systems, Cockeysville, USA). Susceptibility testing slightly lower zone diameter susceptibility breakpoints was done on Mueller-Hinton agar (BD Diagnostic for ertapenem in its 2012 updates, while breakpoints Systems, Cockeysville, USA) using McFarland 0.5 for third-generation cephalosporins remained from overnight cultures followed by incubation at unchanged [4]. In addition, with the increase of 35℃ for 16-18 hours. Inhibition zone diameters were cephalosporin breakpoints in the CLSI 2010-12 determined and recorded by two experienced guidelines, it is no longer necessary to detect microbiologists. extended-spectrum beta–lactamase (ESBL) producers and to adjust all cephalosporin sensitivity results to ESBL testing resistance, as previously recommended by CLSI 2009 In keeping with CLSI guideline, phenotypic guideline. identification of ESBL production in Escherichia coli, Nevertheless, a controversy arose with regard to Klebsiella pneumoniae, Klebsiella oxytoca and the appropriateness of current recommendations. Some Proteus mirabilis isolates was determined using investigators continue to support the importance of double disk diffusion method [4, 11]. Briefly, ESBL knowing the ESBL status in an isolate to ensure proper combined-disk testing was performed with therapy [5, 6]. Especially in ESBL-endemic areas, commercial disks (BD Diagnostic Systems, where therapeutic options are very limited due to Cockeysville, USA), according to the manufacturer’s associated co-resistance in other classes of instructions, using Mueller-Hinton II (BD Diagnostic antimicrobial agents [7, 8]. Moreover, in a survey on Systems, Cockeysville, USA) agar and an inoculation 144 infectious disease specialists, most respondents turbidity of 0.5 McFarland. The disks used were reported that they would not treat Enterobacteriaceae cefotaxime 30 μg, cefotaxime-clavulanic acid 30/10 infections based on the current breakpoints alone μg, ceftazidime 30 μg and ceftazidime-clavulanic acid without testing for ESBL [9]. Some experts even 30/10 μg. suggested that clinical laboratories should not use the most recent CLSI breakpoints [9, 10]. Comparison of CLSI 2009-2012 breakpoints Adoption of new guidelines and breakpoints can Inhibition zone diameters were interpreted have significant effects on antibiotic susceptibility according to the CLSI 2009-12 recommended zone testing reports, with concomitant changes in antibiotic diameter breakpoints for Enterobacteriaceae (Table prescriptions by clinicians. Prior to the implementation 1). The CLSI 2009 susceptibility breakpoint for of new guidelines in diagnostic microbiology cefotaxime was ≥ 23 mm which increased to ≥ 26 mm laboratories, the impact of changes in antibiotic based on the CLSI 2010-12 guidelines. The CLSI susceptibility testing reports needs to be evaluated. 2009 susceptibility breakpoint for ertapenem was ≥ 19 This study compared the new standards with mm using CLSI 2009, which increased to ≥ 23 mm respect to changes in cefotaxime and ertapenem using CLSI 2011 and reduced to ≥ 22 mm with CLSI among Enterobacteriaceae isolates. 2012. Rates of susceptibility to cefotaxime and ertapenem measured using 2009 CLSI breakpoints Methodology were compared with those obtained using 2010-12 Clinical isolates CLSI breakpoints [1, 3, 4]. Between March 2011 and June 2011, the study collected consecutive non-duplicate clinical isolates of Enterobacteriaceae in our clinical microbiological laboratory. Our clinical laboratory primarily serves a 290 Liu et al. – Susceptibility with New CLSI Breakpoints J Infect Dev Ctries 2014; 8(3):289-296. Table 1. CLSI 2009-12 susceptibility testing breakpoints of Enterobacteriaceae Clinical breakpoints (mm) CLSI 2009 CLSI 2010 CLSI 2011 CLSI 2012 Drug S I R S I R S I R S I R cefotaxime ≥23 15-22 ≤14 ≥26 23-25 ≤22 ertapenem ≥19 16-18 ≤15 ≥23 20-22 ≤19 ≥22 19-21 ≤18 Categories without value: interpretation guidelines were not changed compared to the previous version. S, susceptible; I, intermediate; R, resistant. Table 2. Susceptibility of Enterobacteriaceae clinical isolates according to CLSI 2009-12 Assignment of Enterobacteriaceae clinical isolates (%) to interpretative category CLSI 2009 CLSI 2010 CLSI 2011 CLSI 2012 Species/drug S I R S I R S I R S I R E.coli cefotaxime 663 120 125 646 17 245 % 73.0 13.2 13.8 71.1 1.9 27.0 ertapenem 907 1 0 898 10 0 904 4 0 % 99.9 0.1 0.0 98.9 1.1 0.0 99.6 0.4 0.0 Klebsiella pneumoniae cefotaxime 403 32 55 388 15 87 % 82.2 6.5 11.2 79.2 3.1 17.8 ertapenem 479 4