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Meropenem Resistance in Imipenem-Susceptible Meropenem-Resistant Klebsiella pneumoniae Isolates Not Detected by Rapid Automated Testing Systems Toshie Harino, Shizuo Kayama, Ryuichi Kuwahara, Seiya Kashiyama, Norifumi Shigemoto, Makoto Onodera, Michiya Yokozaki, Hiroki Ohge and Motoyuki Sugai J. Clin. Microbiol. 2013, 51(8):2735. DOI: 10.1128/JCM.02649-12. Published Ahead of Print 29 May 2013.

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Meropenem Resistance in Imipenem-Susceptible Meropenem-Resistant Klebsiella pneumoniae Isolates Not Detected by Rapid Automated Testing Systems Toshie Harino,a,b,c Shizuo Kayama,a,b Ryuichi Kuwahara,a,b,d Seiya Kashiyama,a,e Norifumi Shigemoto,a,b,f Makoto Onodera,a,g Michiya Yokozaki,a,g Hiroki Ohge,a,g,h Motoyuki Sugaia,b Project Research Center for Nosocomial Infectious Diseases, Hiroshima University, Minami-ku, Hiroshima City, Hiroshima, Japana; Department of Bacteriology, Hiroshima University Graduate School of Biomedical and Health Sciences, Minami-ku, Hiroshima City, Hiroshima, Japanb; Clinical Laboratory, Hiroshima City Asa Hospital, Asakita-ku, Hiroshima City, Hiroshima, Japanc; Clinical Laboratory, Hiroshima General Hospital of West Japan Railway Co., Higashi-ku, Hiroshima City, Hiroshima, Japand; Clinical Laboratory, Saiseikai Hiroshima Hospital, Saka-cho, Aki-gun, Hiroshima, Japane; Department of Surgery I, Hiroshima University Graduate School of Biomedical and Health Sciences, Minami-ku, Hiroshima, Japanf; Clinical Laboratory, Hiroshima university Hospital, Minami-ku, Hiroshima City, Hiroshima, Japang; Department of Infectious Diseases, Hiroshima University Hospital, Minami-ku, Hiroshima City, Hiroshima, Japanh

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e have recently identified an emergence of Klebsiella pneumoniae resistant to almost all ␤-lactams except imipenem in Hiroshima, Japan, and designated this strain as ISMRK (for imipenem-susceptible meropenem-resistant Klebsiella) (1). This unique susceptibility phenotype to ␤-lactams is due to the double production of a metallo-␤-lactamase, IMP-6, and the extended-spectrum ␤-lactamase (ESBL) CTX-M-2 by Klebsiella. ISMRK isolates continue to emerge sporadically in various hospitals in Hiroshima and also in the Kinki region of Japan (unpublished data). ISMRK was first discovered during a surveillance of ESBL-producing K. pneumoniae and E. coli strains in the Hiroshima region, and an unusual susceptibility pattern to carbapenems was found during surveillance of antimicrobial susceptibility of clinical isolates using the Microscan automated susceptibility testing system (1). According to the Microscan system, the MICs of imipenem and meropenem to the first five ISMRK isolates tested were 1 ␮g/ml (susceptible) and ⬎8 ␮g/ml (resistant), respectively. For the first screen of ESBL-producing bacteria, clinical laboratories involved in this surveillance were asked to submit strains that are resistant to one or more antimicrobial agents among cefpodoxime (CPDX), ceftazidime (CAZ), cefotetan (CTX), ceftriaxon (CTRX), and zidovudine according to the criteria of Clinical and Laboratory Standards Institute (CLSI) documents M07-A9 (published in 2012) and M100-s22 (published in 2012). During the surveillance, one of the laboratories employing the Vitek2 automated susceptibility testing system submitted a K. pneumoniae isolate described as an ESBL-producing strain. According to the system, it was reported as resistant to all ␤-lactams but susceptible to imipenem and meropenem. Later the isolate was shown to be susceptible to imipenem butresistanttomeropenembytheMicroscansystemandbrothmicrodilutionmethod(2),andmoleculartypingconfirmeditasbeingablaIMP-6and blaCTX-M-2-carrying isolate, i.e., ISMRK. We therefore sought to determine whether any automated susceptibility testing systems may miss the unusual resistance phenotype of ISMRK. We tested various susceptibility testing methods to measure the susceptibility of ISMRK isolates and evaluated the abilities of testing system to detect ISMRK phenotype. The tested ISMRK isolates were derived from different patients in eight general hospitals in Hiroshima region. All of the isolates were con-

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firmed to possess a plasmid carrying blaIMP-6 and blaCTX-M-2. We carried out genotyping of those clinical isolates (ca. 30 isolates) using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The isolates were classified into three groups according to PFGE pattern (groups I, II, and III): two of them (groups I and II, n ⫽ 24 isolates) appeared to possess fairly similar genotypes (similarity ⬎ 60%), and the group III isolates were different. MLST analysis indicated that the two groups with similar genotypes (groups I and II) belonged to ST37 and that group III belonged to ST23. We carefully picked 20 isolates representing these three groups (12 isolates from group I, 7 isolates from group II, and 1 isolate from group III). The broth microdilution method according to CLSI criteria was used as the reference method (2). The automated susceptibility testing systems and corresponding antibiotic panels were as follows: (i) MicroScan WalkAway (Siemens Healthcare Diagnostics, Tokyo, Japan), NMIC6.31J; (ii) dry plate Eiken (Eiken Chemical Co., Tokyo, Japan), DP-31; (iii) RAISUS (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan), NKNM3; (iv) Vitek2 (bioMérieux, Basingstoke, United Kingdom), AST-N124; and (v) RAISUS, PDCN2. Systems i to iii represent automated systems with overnight growth of bacteria (18 h), and systems iv and v can generate rapid (3.5 to 16 h) susceptibility test results. The MICs of imipenem and meropenem for 20 ISMRK isolates were tested according to the respective manufacturers’ recommendations. The susceptibilities to imipenem and meropenem determined by these systems were interpreted according to CLSI M100-S21 breakpoints (3). The susceptibility results are listed in Table 1. By the broth microdilution method, MICs against imipenem were 0.25 to 4 ␮g/ml, and those

Received 4 October 2012 Returned for modification 2 November 2012 Accepted 23 May 2013 Published ahead of print 29 May 2013 Address correspondence to Motoyuki Sugai, [email protected]. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.02649-12

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Klebsiella pneumoniae showing high resistance to all ␤-lactams except imipenem, designated as ISMRK (imipenem-susceptible meropenem-resistant Klebsiella) is emerging in Japan. The carbapenem resistance of ISMRK cannot be screened by the Vitek and the RAISUS rapid automated susceptibility test systems, which may lead to inappropriate antimicrobial therapy, resulting in compromised patient outcomes.

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MicroScan (NMIC6.31J)

Dry plate (DP31)

RAISUS (NKMN3)

32 32 32 32 64 64 64 64 32 64 64 64 64 32 32 32 32 32 32 32

⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8

⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8 ⬎8

⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1

ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1

Vitek2 (AST-N124)

MEPM

⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 2 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1 ⬍1

RAISUS (PDCN2)

2 2 1 1 2 2 2 2 1 1 2 2 2 1 2 1 1 1 1 2

Vitek2 (AST-N124)

a MIC breakpoint criteria for Enterobacteriaceae were interpreted according to CLSI M100-S21; these criteria for imipenem and meropenem are indicated as resistant (⬎4 ␮g/ml, gray shaded), intermediate (2 ␮g/ml, boldface type), and susceptible (⬍1 ␮g/ml). Isolates 1 to 12 belong to group I (ST37), isolates 13 to 19 belong to group II (ST37), and isolate 20 belongs to group III (ST23). MicroScan, dry plate, RAISUS, and Vitek2 are all automated susceptibility testing methods. For each method, an antibiotic panel (indicated in parentheses) was used for testing. IPM, imipenem; MEPM, meropenem-imipenem.

ⱕ1 ⱕ1 ⱕ1 ⱕ1 2 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 ⱕ1 2 ⱕ1 ⱕ1 ⱕ1

ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 ⱕ0.25 0.5 0.5 ⱕ0.25

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 2 4 1 4 2 2 1 2 2 4 1 1 2 0.25 0.5 0.5

Isolate

RAISUS (PDCN2)

IPM

MEPM Broth microdilution

RAISUS (NKMN3)

Dry plate (DP31)

Broth microdilution

MicroScan (NMIC6.31J)

IPM

MIC breakpoint (␮g/ml) (rapid growth)


2736

MIC breakpoint (␮g/ml) (overnight growth)

TABLE 1 Carbapenem susceptibility results for 20 ISMRK isolatesa

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in the presence of various concentrations of imipenem (a) or meropenem (b). Cell growth was periodically monitored by measuring the turbidity of each well at OD620. The dotted line in each graph indicates the timing when Vitek2 automated susceptibility testing system reported the data of the tested isolate.

against meropenem were 32 or 64 ␮g/ml. The Microscan automated susceptibility testing system was the original system demonstrated the unusual MIC patterns of ISMRK to carbapenems (1). By Microscan, as expected, the MIC values of the ISMRK isolates for imipenem and meropenem were pretty consistent and were 1 and ⬎8 ␮g/ml, respectively, i.e., imipenem susceptible and meropenem resistant (Table 1). Similar results were obtained with RAISUS using panel NKMN3. In case of dry plate Eiken (DP31), the MIC values for imipenem and meropenem were relatively lower than those of determined by Microscan and RAISUS (NKMN3), ⬍0.5 and 8 ␮g/ml, respectively, but the data clearly represented the ISMRK phenotype. On the other hand, Vitek2 (AST-N124) and RAISUS (PDCN2) were not able to correctly detect the carbapenem resistance of ISMRK. As shown in Table 1, the MICs for imipenem and meropenem of all the ISMRK strains reported by these systems were ⱕ1 ␮g/ml for imipenem and ⱕ2 ␮g/ml for meropenem, respectively. Of note, only one isolate was found to be intermediately resistant to meropenem, but others were totally susceptible to imipenem and meropenem. The Vitek2 Advanced Expert System (AES) using the parameter of global plus

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natural resistance (Japan) reported 19 in 20 isolates as strains producing ESBL, with decreased membrane permeability causing resistance to cefamycin, although the MICs for meropenem reported by AES were 2 ␮g/ml. One isolate was reported as resistant to both imipenem and meropenem, although the MICs reported by AES was ⬍1 and 2, respectively. These results clearly indicate that these rapid automated susceptibility testing systems may not be able to detect the meropenem-resistant phenotype of most ISMRK isolates. The Vitek2 system is able to carry out multiple turbidimetric monitoring of bacterial growth during a relatively short incubation period at an interval of 15 min (4). MICs of the antimicrobial agents are determined by the computer-assisted kinetic analysis of the growth based on the interpretive algorithms, and the system allowed us to test the susceptibility within 4 to 10 h, which is faster than aforementioned automated susceptibility test systems, which require ⱖ18 h of bacterial incubation in order to obtain results. RAISUS is a newly introduced rapid automated system for identification of the bacteria and antimicrobial susceptibility testing (5). Specially prepared plates of RAISUS contain redox in 96 wells,

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FIG 1 Turbidimetric analysis of ISMRK growth in the presence of carbapenem. ISMRK isolates (isolates 1, 2, 12, and 13) were grown in 96-well titer plate wells

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In conclusion, our data indicate that meropenem resistance of ISMRK cannot be screened by the current Vitek2 or the RAISUS rapid automated susceptibility test system, and thus ISMRK will be recognized as an ESBL-producing Klebsiella strain susceptible to carbapenems. Constant reassessment and improvement of algorithm of these testing devices is necessary, and development of a supplementary testing method may be required for the rapid detection of ISMRKs. ACKNOWLEDGMENTS We thank Jim Nelson and Larry Strand for editorial assistance.

REFERENCES 1. Shigemoto N, Kuwabara R, Kayama S, Shimizu W, Onodera M, Yokozaki M, Hisatsune J, Kato F, Ohge H, Sugai M. 2012. Emergence in Japan of an imipenem-susceptible, meropenem-resistant Klebsiella pneumoniae carrying blaIMP-6. Diagn. Microbiol. Infect. Dis. 72:109 –112. 2. Clinical and Laboratory Standards Institute. 2009. Methods for dilution antimicrobial susceptibility testing for bacteria that grew aerobically; approved standard M7-A10. Clinical and Laboratory Standards Institute, Wayne, PA. 3. Clinical and Laboratory Standards Institute. 2011. Performance standards for antimicrobial susceptibility testing; 21st informational supplement. CLSI document M100-S21. Clinical and Laboratory Standards Institute, Wayne, PA. 4. Jorgensen JH, Ferraro MJ. 2009. Antimicrobial susceptibility testing: a review of general principles and contemporary practices. Clin. Infect. Dis. 49:1749 –1755. 5. Kanemitsu K, Kunishima H, Inden K, Hatta M, Saga T, Ueno K, Harigae H, Ishizawa K, Kaku M. 2005. Assessment of RAISUS, a novel system for identification and antimicrobial susceptibility testing for enterococci. Diagn. Microbiol. Infect. Dis. 53:23–27. 6. Barenfanger J, Drake C, Karich G. 1999. Clinical and financial benefits of rapid bacterial identification and antimicrobial susceptibility testing. J. Clin. Microbiol. 37:1415–1418. 7. Doern GV, Vautour R, Gaudet M, Levy B. 1994. Clinical impact of rapid in vitro susceptibility testing and bacterial identification. J. Clin. Microbiol. 32:1757–1762. 8. Anderson KF, Lonsway DR, Rasheed JK, Biddle JW, Jensen B, McDougal LK, Carey RB, Thompson A, Stocker S, Limbago B, Patel JB. 2007. Evaluation of methods to identify the Klebsiella pneumoniae carbapenemase in Enterobacteriaceae. J. Clin. Microbiol. 45:2723–2725. 9. Bratu S, Mooty M, Nichani S, Landman D, Gullans C, Pettinato B, Karumudi U, Tolaney P, Quale J. 2005. Emergence of KPC-possessing Klebsiella pneumoniae in Brooklyn, New York: epidemiology and recommendations for detection. Antimicrob. Agents Chemother. 49:3018 – 3020. 10. Hirsch EB, Tam H. 2010. Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrugresistant infection. J. Antimicrob. Chemother. 65:1119 –1125. 11. Tenover FC, Kalsi RK, Williams PP, Carey RB, Stocker S, Lonsway D, Rasheed JK, Biddle JW, McGowan JE, Jr, Hanna B. 2006. Carbapenem resistance in Klebsiella pneumoniae not detected by automated susceptibility testing. Emerg. Infect. Dis. 12:1209 –1213. 12. Woodford N, Eastaway AT, Ford M, Leanord A, Keane C, Quayle RM, Steer JA, Zhang J, Livermore DM. 2010. Comparison of BD Phoenix, Vitek 2, and MicroScan automated systems for detection and inference of mechanisms responsible for carbapenem resistance in Enterobacteriaceae. J. Clin. Microbiol. 48:2999 –3002.



and the MICs of the antimicrobial agents are determined by the kinetic analysis of colorimetric signals generated from the redox upon bioreduction of viable bacteria at an interval of 15 min. This system indirectly evaluates the growth of the bacteria through measuring metabolic activity of the bacteria. We therefore tested the growth of ISMRKs in the presence of various concentration of imipenem or meropenem to mimic the automated susceptibility testing system. A bacterial cell suspension in the presence of antimicrobial agent was prepared according to the protocol by the supplier and inoculated 100 ␮l/well into 96-well flat-bottom plastic plate, and the turbidimetric change was monitored during culture at 37°C. The turbidity of cell suspension was periodically monitored at an optical density at 620 nm (OD620) by using a microplate reader. Preliminary investigation indicated a remarkable correlation between turbidimetry and viable cell counts. As shown in Fig. 1, significant retardation of growth was observed with the ISMRKs incubated with 0.5 ␮g imipenem/ml or 2 ␮g of meropenem/ml, respectively. At the time points at which the Vitek2 system reported the MIC, i.e., 3.75 to 4.25 h for imipenem and 4.75 h for meropenem, there was almost no growth of the ISMRKs incubated with 0.5 ␮g of imipenem/ml or 2 ␮g of meropenem/ml. These results strongly suggest that significant retardation of initial growth of ISMRKs in the presence of low concentration of carbapenem caused the misinterpretation of rapid automated susceptibility testing systems. Introduction of rapid automated susceptibility testing system has significantly contributed to shorten the timing to choose appropriate antimicrobial agents during empirical therapy (6, 7). The susceptibility test data could be critical information for the patient with a life-threatening infection, such as septicemia. In Japan, most clinical laboratories in hospitals use imipenem as a representative of carbapenem, and an imipenem-resistant strain in the first screen will be subjected to SMA test to check metallo␤-lactamase production. We therefore raised an alarm regarding the possible false diagnosis of ISMRK by a clinical laboratory technician (1). However, our finding in the present study suggests a more serious threat of the inability to identify ISMRK if the Vitek2 or the RAISUS rapid system is used for susceptibility screening. Detection of carbapenemases producing K. pneumoniae has been a challenging theme for automated susceptibility testing system. A previous study using K. pneumoniae producing carbapenemases indicated problems in detecting carbapenem resistance using several automated susceptibility testing systems, including MicroScan and Vitek2 (8–12). Fifteen K. pneumoniae strains producing carbapenemases used in that study were interpreted as resistant or intermediately resistant to imipenem-meropenem using the broth microdilution method (11). However, several strains were reported to be susceptible to imipenem and/or meropenem using several automated susceptibility testing systems. The variability of imipenem and meropenem resistance profile by these systems was also pointed out.