Abstract. Microbiology and Infectious Disease / Epidemiology of ESBLs in the Kinki Region of Japan

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1 Microbiology and Infectious Disease / Epidemiology of ESBLs in the Kinki Region of Japan Epidemiology of Escherichia coli, Klebsiella Species, and Proteus mirabilis Strains Producing Extended-Spectrum β-lactamases From Clinical Samples in the Kinki Region of Japan Tatsuya Nakamura, 1,2 Masaru Komatsu, PhD, 3 Katsutoshi Yamasaki, PhD, 4 Saori Fukuda, 5 Yugo Miyamoto, 6 Takeshi Higuchi, 7 Tamotsu Ono, 8 Hisaaki Nishio, 9 Noriyuki Sueyoshi, 1 Kenji Kida, 11 Kaori Satoh, 12 Hirofumi Toda, 12 Masahiro Toyokawa, PhD, 13 Isao Nishi, 13 Masako Sakamoto, 14 Masahiro Akagi, 15 Isako Nakai, 16 Tomomi Kofuku, 17 Tamaki Orita, 18 Yasunao Wada, 19 Takuya Zikimoto, 2 Chihiro Koike, 1 Shohiro Kinoshita, 2 Itaru Hirai, 2 Hakuo Takahashi, MD, 1 Nariaki Matsuura, MD, 2 and Yoshimasa Yamamoto, MD 2 Key Words: Extended-spectrum β-lactamases; Polymerase chain reaction; Surveillance; Replicon type Abstract In the present study, nonduplicate, clinical isolates of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, Klebsiella spp, and Proteus mirabilis were collected during a 1-year period from 2 to 29 at several hospitals in the Kinki region, Japan. The detection rate of E coli markedly increased from.24% to 7.25%. The detection rate of Klebsiella pneumoniae increased from % to 2.44% and that of P mirabilis from 6.97% to 12.85%. The most frequently detected genotypes were the CTX-M9 group for E coli, the CTX-M2 group for K pneumoniae, and the CTX-M2 group for P mirabilis. E coli clone O25:H4-ST131 producing CTX-M-15, which is spreading worldwide, was first detected in 27. The most common replicon type of E coli was the IncF type, particularly FIB, detected in 466 strains (69.7%). Of the K pneumoniae strains, 47 (55.3%) were of the IncN type; 77 P mirabilis strains (96.3%) were of the IncT type. In the future, the surveillance of various resistant bacteria, mainly ESBL-producing Enterobacteriaceae, should be expanded to prevent their spread. Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have been increasingly reported worldwide since their first description in Moreover, they have emerged worldwide as a significant cause of community and health care associated infections. 2 ESBLs are the major cause of resistance to oxyimino-cephalosporins in Enterobacteriaceae. ESBLs are mostly plasmid-mediated bacterial enzymes that can hydrolyze a wide variety of penicillins and cephalosporins. Most ESBLs have evolved by genetic mutation from native β-lactamases, particularly TEM-1, TEM-2, and SHV-1. These parent enzymes are commonly found in gram-negative bacteria, particularly in Enterobacteriaceae. 3 Until the 2s, most of the ESBLs were structurally related to the narrowspectrum TEM- and SHV-type β-lactamases, with one to several amino acid substitutions surrounding their active site. During the 199s, they were described mainly as members of the TEM- and SHV-β-lactamase families in Escherichia coli and Klebsiella pneumoniae causing nosocomial outbreaks. 3 Furthermore, in the late 199s, a novel type of ESBL, the CTX-M enzymes, emerged worldwide, mostly from E coli. 3,4 ESBL-producing E coli of the Toho-1-type were reported first in Japan in Nowadays, they are mostly found in E coli that cause community-acquired infections and, with increasing frequency, contain CTX-M enzymes. Moreover, E coli producing a CTX-M-type ESBL is an emerging cause of communityacquired urinary tract infection in young women in the United States, 6 Europe, 7 Hong Kong, 8 India, 9 and elsewhere. Increased community-acquired infection by ESBL-producing bacteria is complicating the selection of therapeutic drugs. More than 5 CTX-M enzymes reported thus far can be grouped into 5 main subgroups according to the similarity of their amino acid sequence (CTX-M-1, CTX-M-2, CTX-M-8, 62 Am J Clin Pathol 212;137: Downloaded 62 from

2 Microbiology and Infectious Disease / Original Article CTX-M-9, and CTX-M-25). 4 In particular, E coli clone O25:H4-ST131 producing CTX-M-15, which is resistant to many antibacterial agents, is spreading worldwide and causing serious problems. 2,1-16 In the present study, nonduplicate clinical isolates of ESBL-positive E coli, Klebsiella spp, and Proteus mirabilis were collected during a 1-year period from 1999 to 29 at several hospitals in the Kinki region, Japan. Our study examined the prevalence and type of β-lactamase genes and plasmid replicon type among the isolates. Moreover, susceptibilities to oral antimicrobial agents were determined. Materials and Methods Bacterial Isolates This laboratory surveillance was conducted with the cooperation of 18 institutions (17 clinical laboratories of various hospitals and 1 commercial laboratory) in the Kinki region, which is located in midwestern Japan. Specimens were collected from 2 to 29. A total of 4,522 isolates of gram-negative bacilli including E coli (25,32 isolates), K pneumoniae (11,582 isolates), Klebsiella oxytoca (2,933 isolates), and P mirabilis (1,187 isolates) were isolated from various clinical specimens, and antimicrobial sensitivity and genotype were tested. A single isolate was selected from each patient and identified by the clinical procedures routinely used in each laboratory. Screening for ESBL The cefpodoxime minimum inhibitory concentration (MIC) criterion of more than 2 μg/ml was used to initially screen isolates. Strains that met the cefpodoxime MIC criterion were investigated by the double-disk synergy (DDS) test with amoxicillin clavulanic acid (2 μg per disk/1 μg per disk), cefotaxime (3 μg per disk), ceftazidime (3 μg per disk), and cefepime (3 μg per disk), according to methods published previously. 17 DDS-positive strains were subjected to polymerase chain reaction (PCR) analyses for the detection of ESBL genes. PCR Amplification for the Detection of ESBL Genes All DDS-positive strains were screened for the resistance genes SHV, TEM, and CTX-M by using a single PCR assay. 18,19 Genetic detection and genotyping of TEM, SHV, and CTX-M were performed by using PCR with bacterial DNA, which was extracted from the isolates by boiling the bacterial suspensions. A solution with an extracted DNA concentration of.1 ng/ml was used as the template for PCR analysis. In the case of genotyping of CTX-M genes, 4 primer sets that amplify group-specific CTX-M genes were used, as described previously: the CTX-M1 group includes CTX-M-1, CTX-M-3, CTX-M-1 to CTX-M-12, CTX-M- 15, CTX-M-22, CTX-M-23, and CTX-M-28 to CTX-M-3; the CTX-M2 group, CTX-M-2, CTX-M-4 to CTX-M-7, CTX-M-2, and Toho-1; the CTX-M8 group, CTX-M-8; and the CTX-M9 group, CTX-M-9, CTX-M-13, CTX-M- 14, CTX-M-16 to CTX-M-19, CTX-M-21, CTX-M-27, and Toho-2. The PCR products were analyzed by using 2% agarose gel electrophoresis and visualized by staining with ethidium bromide. Detection of CTX-M-15 O25:H4-ST131 The serotyping of the CTX-M1 group was carried out by using E coli O and H antisera purchased from Denka Seiken (Tokyo, Japan), according to the manufacturer s instructions. The complete nucleotide sequences of CTX- M1 group O25:H4 E coli genes were determined on both strands by direct sequencing of the PCR products. Multilocus sequence typing (MLST) was performed on 17 strains of CTX-M-15 O25:H4 E coli by following the recommended procedure at the E coli MLST Web site ( dbs/ecoli). Plasmid Replicon Type Determination PCR-based replicon typing was performed on 837 strains as described by Carattoli et al. 2 Eighteen primer pairs targeting the FIA, FIB, FIC, HI1, HI2, I1-Ic, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, and FII replicons were used in separate PCR reactions. Susceptibility to Oral Antimicrobial Agents The susceptibilities to oral antimicrobial agents, amoxicillin-clavulanic acid (AMC), minocycline, levofloxacin, fosfomycin, colistin, and trimethoprim-sulfamethoxazole (SXT), were determined by the broth diffusion method on Mueller-Hinton agar (Eiken Chemical, Tokyo, Japan), according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI). 21 The quality control strains used in this study were E coli ATCC and E coli ATCC Throughout this study, the results were interpreted using CLSI criteria for broth dilution. 22 Results ESBL Detection Between 2 and 29, a total of 4,522 strains, including 25,32 E coli, 11,582 K pneumoniae, 2,933 K oxytoca, and 1,187 P mirabilis strains, were analyzed. ESBL isolation rates are shown in Figure 1 and Table 1. The detection of ESBL-producing E coli markedly increased Downloaded from Am J Clin Pathol 212;137:

3 Nakamura et al / Epidemiology of ESBLs in the Kinki Region of Japan No. of Isolates 3 1 P mirabilis 9 25 K oxytoca K pneumoniae 8 E coli 7 2 Detection rate (all strains) Figure 1 Distribution of detection among extended-spectrum β-lactamase (ESBL)-positive Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, and Proteus mirabilis in hospitals and associated health care facilities, from 2 strains (.24%) in 2 to 224 strains (7.25%) in 29. The detection of ESBL-producing K pneumoniae increased from (.%) to 3 strains (2.44%) and that of ESBL-producing K oxytoca increased from (.%) to 3 strains (1.18%). The detection of ESBL-producing P mirabilis increased from 14 (6.97%) to 23 strains (12.85%) in a survey conducted since 24. In the whole of the Kinki region, the detection rate increased from.42% (between 2 and 24) to 3.4% (between 25 and 29). Molecular Detection of ESBL The changes in the genotypes detected during the survey period are shown in Figure 2 for E coli and in Figure 3 for K pneumoniae. The most frequently detected genotypes in the 1 years were the CTX-M9 group for E coli (374 Detection Rate (%) No. of Isolates TEM group SHV group CTX-M9 group CTX-M8 group CTX-M2 group CTX-M1 group Figure 2 Distribution of different extended-spectrum β-lactamase (ESBL) genes among ESBL-positive Escherichia coli isolates detected in hospitals and associated health care facilities, The CTX-M1 group includes CTX-M-1, CTX-M-3, CTX-M-1 to CTX-M-12, CTX-M-15, CTX-M-22, CTX-M-23, and CTX-M-28 to CTX-M-3; the CTX-M2 group, CTX-M-2, CTX-M-4 to CTX-M-7, CTX-M-2, and Toho-1; the CTX-M8, group, CTX-M8; and the CTX-M9 group, CTX-M-9, CTX-M-13, CTX-M-14, CTX-M-16 to CTX-M-19, CTX-M-21, CTX-M-27, and Toho-2. strains [55.9%]), the CTX-M2 group for K pneumoniae (38 strains [44.7%]), and the CTX-M2 group for P mirabilis (79 strains [98.8%]). The detection number of E coli of the CTX-M9 group increased from 25 strains to 144 strains from 25. In addition, the detection number of E coli of the CTX-M1 group increased from 11 strains to 57 strains from 25. The detection number of K pneumoniae of the CTX-M9 group increased from 26. The detection rates Table 1 Number of Isolates of ESBL in Each Term * Organism Total Escherichia coli Collected strains 82 1,297 3,13 3,95 2,813 2,429 3,62 2,86 2,897 3,88 25,32 ESBL 2 (.24) 7 (.54) 15 (.5) 12 (.39) 15 (.53) 62 (2.55) 89 (2.91) 11 (3.6) 142 (4.9) 224 (7.25) 669 (2.64) Klebsiella pneumoniae Collected strains ,487 1,468 1,144 1, ,25 1,237 1,229 11,82 ESBL (.) 1 (.11) 7 (.47) 4 (.27) (.) 1 (.87) 5 (.7) 8 (.66) 2 (1.62) 3 (2.44) 85 (.77) Klebsiella oxytoca Collected strains ,933 ESBL (.) (.) (.) (.) (.) (.) (.) (.) (.) 3 (1.18) 3 (.1) Proteus mirabilis Collected strains ND ND ND ND ,187 ESBL ND ND ND ND 14 (6.97) 6 (2.8) 3 (2.54) 1 (3.98) 24 (1.71) 23 (12.85) 8 (6.74) Total Collected strains 1,516 2,431 4,9 4,919 4,541 4,18 4,99 4,63 4,655 4,75 4,522 ESBL 2 (.13) 8 (.33) 22 (.45) 16 (.33) 29 (.64) 78 (1.9) 97 (2.37) 119 (2.59) 186 (4.) 28 (5.89) 837 (2.7) ESBL, extended-spectrum β-lactamase; ND, not done. * Data are given as number or number (percentage). 622 Am J Clin Pathol 212;137: Downloaded 622 from

4 Microbiology and Infectious Disease / Original Article Prevalence of Plasmid Replicons The most common replicon type of E coli was the IncF type, particularly FIB, detected in 466 strains (69.7%), followed by FIA in 336 strains (5.2%), I1-1 in 83 strains (12.4%), and N in 65 strains (9.7%) Table 2. Of the K pneumoniae strains, 47 (55.3%) were of the N type. Most of the bacteria carried a single plasmid, but some bacteria carried multiple plasmid types. The average number of plasmids carried by the bacteria was from 1 to 1.5. Of the P mirabilis strains, 77 (96.3%) were of the IncT type. No. of Isolates TEM group SHV group CTX-M9 group CTX-M8 group CTX-M2 group CTX-M1 group Figure 3 Distribution of different extended-spectrum β-lactamase (ESBL) genes among ESBL-positive Klebsiella pneumoniae isolates detected in hospitals and associated health care facilities, The CTX-M1 group includes CTX-M-1, CTX-M-3, CTX-M-1 to CTX-M-12, CTX-M-15, CTX-M-22, CTX-M-23, and CTX-M-28 to CTX-M-3; the CTX-M2 group, CTX-M-2, CTX-M-4 to CTX-M-7, CTX-M-2, and Toho-1; the CTX-M8 group, CTX-M8; and the CTX-M9 group, CTX-M-9, CTX-M-13, CTX-M-14, CTX-M-16 to CTX-M- 19, CTX-M-21, CTX-M-27, and Toho-2. of the SHV/TEM groups were as follows: SHV group, 19 strains (2.8%) and TEM group, 6 strains (.9%) for E coli; SHV group, 14 strains (16.5%) and TEM group, 2 strains (2.4%) for K pneumoniae. The E coli clone O25:H4-ST131 producing CTX-M-15, which is spreading worldwide, was first detected in 27. In the 3 subsequent years, 17 strains (3 in 27, 1 in 28, and 4 in 29) were detected. Susceptibility for Oral Drug Oral drug susceptibility rates Table 3 for E coli were 96.% for colistin, 93.7% for fosfomycin, 62.6% for minocycline, 47.2% for AMC, 44.3% for SXT, and 32.2% for levofloxacin. The levofloxacin susceptibility rate was the highest for the CTX-M2 group genotype. The drug susceptibility rates for K pneumoniae were 86.9% for levofloxacin, 86.9% for colistin, 83.3% for fosfomycin, 51.2% for AMC, 46.4% for SXT, and 21.4% for minocycline. The SXT susceptibility rate was the lowest (%) for the TEM/SHV group genotype. Drug susceptibility rates for P mirabilis were 1% for AMC, 64.6% for SXT, 5.6% for fosfomycin, 15.2% for levofloxacin,.% for colistin, and.% for minocycline. The AMC susceptibility rate was 1%. Discussion In this study, long-term surveillance between 2 and 29 demonstrated that ESBL-producing E coli increased about 3 times from.24% to 7.25% in 1 years. According Table 2 Number of Replicons in Parental Strains of ESBL-Producing Strains * Replicon Type All Strain/Genotype Replicon Types Non- Group (No. of Isolates) A/C FII FIA FIB FIC H12 HI1 HI2 I1-1 K L/M N P T Y type Total Mean Escherichia coli CTX-M1 (174) CTX-M2 (95) CTX-M9 (374) SHV (19) TEM (6) Klebsiella pneumoniae CTX-M1 (16) CTX-M2 (38) CTX-M9 (14) Proteus mirabilis CTX-M2 (79) ESBL, extended-spectrum β-lactamase. * The CTX-M1 group included CTX-M-1, CTX-M-3, CTX-M-1 to CTX-M-12, CTX-M-15, CTX-M-22, CTX-M-23, and CTX-M-28 to CTX-M-3; the CTX-M2 group, included CTX-M-2, CTX-M-4 to CTX-M-7, CTX-M-2, and Toho-1; and the CTX-M9 group, CTX-M-9, CTX-M-13, CTX-M-14, CTX-M-16 to CTX-M-19, CTX-M-21, CTX-M-27, and Toho-2. Average plasmid-carrying rate. Downloaded from Am J Clin Pathol 212;137:

5 Nakamura et al / Epidemiology of ESBLs in the Kinki Region of Japan Table 3 Susceptibility of Oral Antibiotics in Each ESBL * Escherichia coli Klebsiella pneumoniae Proteus mirabilis Antibiotic CTX-M1 CTX-M2 CTX-M9 SHV/TEM Total CTX-M1 CTX-M2 CTX-M9 SHV/TEM Total CTX-M2 Levofloxacin Minocycline SXT Fosfomycin AMC Colistin AMC, amoxicillin clavulanic acid; ESBL, extended-spectrum β-lactamase; SXT, trimethoprim-sulfamethoxazole. * Susceptibility data are given as percentages according to the criteria of the Clinical and Laboratory Standards Institute (M1-S2) for Enterobacteriaceae. The CTX-M1 group includes CTX-M-1, CTX-M-3, CTX-M-1 to CTX-M-12, CTX-M-15, CTX-M-22, CTX-M-23, and CTX-M-28 to CTX-M-3; the CTX-M2 group, CTX-M-2, CTX-M-4 to CTX-M-7, CTX-M-2, and Toho-1; and the CTX-M9 group, CTX-M-9, CTX-M-13, CTX-M-14, CTX-M-16 to CTX-M-19, CTX-M-21, CTX-M-27, and Toho-2. to Fang et al, 22 ESBL-producing E coli increased by about 1 times between 21 and 26 in Sweden, a result comparable with that from a different survey conducted in the same period. Recently, many reports have been made on intestinal bacteria that acquired genes such as KPC 23 and NDM Until now, ESBL-producing E coli have rarely been detected in Japan. This study may be useful for the prediction of resistant bacteria in the future. Our study revealed the changes in the ESBL genotypes of ESBL-producing E coli, Klebsiella spp, and P mirabilis in the Kinki region of Japan. Shibata et al 25 investigated ESBLproducing intestinal bacteria of the CTX-M group in Japan. They reported that 89 of 168 E coli strains belonged to the CTX-M9 group. In particular, the CTX-M9 group, frequently detected in E coli, is presumably the most common genotype in Japan. Similarly, in this study, 374 of 669 E coli strains were of the CTX-M9 group. In particular, contrary to the decreased CTX-M2 group, the CTX-M9 group has increased since 27. The CTX-M2 group is frequently detected in food products such as meat. 26 It was thought that usage restrictions of the antimicrobial agent to domestic animals had influenced a decrease of CTX-M-2. On the other hand, the CTX-M1 group has slightly increased. In Japan, the trend of the E coli clone O25:H4-ST131 producing CTX-M-15, which causes problems worldwide, is unknown. Hawkey 1 reported trends of increase in Asia. In addition, detection in India and Pakistan were reported. The problem is that this strain is detected in hospital- and community-acquired urinary tract infections and develops multidrug resistance. This study demonstrated that the E coli clone O25:H4-ST131 producing CTX-M-15 was detected in 27 or later, suggesting the continuing existence of this strain in Japan. At present, community-acquired infection with this strain is uncommon. However, attention should be given to future trends. The study of plasmid replicon types provides information about the spread and risks of ESBL-producing bacteria. The genes responsible for CTX-M β-lactamases are encoded by plasmids belonging to the narrow host-range incompatibility types (ie, IncFI, IncFII, IncHI2, and IncI) or the broad host-range incompatibility types (ie, IncN, IncP-1-a, IncL/M, and IncA/C). 27 In this study, the IncF group predominated among the E coli strains, regardless of their genotypes. Many strains acquired multiple plasmids. Similar results have been obtained in other studies. 28 About 1% of the strains were of the I1-1- and N-types, suggesting the existence of various E coli clones. The spread of community-acquired infection indicates the potential spread of these various clones in different forms. On the other hand, the N-type predominated among K pneumoniae strains and the T-type among the P mirabilis strains, suggesting the spread of a single clone or plasmid. Many reports have been published particularly on these 2 strains in hospital-acquired infection In addition, in this study, both species were occasionally detected in the same facility and ward, suggesting an epidemiology different from that of E coli. Bacterial properties vary with species. Thus, measurements should be done carefully, according to the species. Recently, the spread of community-acquired infection by ESBL-producing strains of the CTX-M type is causing problems. Reportedly, more ESBL-producing strains have been detected in females with urinary tract infection. 6-9 The spread of community-acquired infection complicates the selection of antibacterial agents for outpatient care. Few oral antibacterial agents effective against ESBL-producing bacteria are available. According to recent reports, quinolone resistance is regarded as a serious problem. 32 In addition, in this study, quinolone-resistance rates were high among the E coli strains: about 7% of the strains were resistant. In particular, the resistance rate of the CTX-M1 group was the highest (about 85%). Thus, quinolones cannot be used for the treatment of those infectious diseases. The susceptibility of E coli to fosfomycin is being maintained. The reason for it will be that 3 g/d is recommended, as described in the Sanford guidelines. 33 On the other hand, 8% of the K pneumoniae strains are susceptible 624 Am J Clin Pathol 212;137: Downloaded 624 from

6 Microbiology and Infectious Disease / Original Article to quinolones. Thus, quinolones should be effective for K pneumoniae. Antibacterial susceptibility varies with strains and genotypes. Thus, antibacterial agents for areas should be selected on the basis of the results obtained by studies on the epidemiologic backgrounds of those areas. The detection rate of the ESBL-producing E coli in our institutions was low, in comparison with that in Western countries and other areas in Asia. One of the reasons for this may be the difference in the type of antibiotics used in these countries: carbapenems and oxacephems, in particular, have often been used in Japan. Because ESBL-producing bacteria are susceptible to these drugs, they might have suppressed the diffusion of these bacteria. However, recently, reduced use of these drugs is recommended in clinical settings to avoid the overall resistance to these antibiotics. This may lead to an increase of the ESBL-producing bacteria in the near future in Japan. This report described the epidemiologic trends of ESBLproducing E coli, Klebsiella spp, and P mirabilis in Japan. Various causative genes are known for β-lactam resistant intestinal bacteria. Many reports have been published on serious resistant bacteria (eg, KPC, NDM-1, CTX-M-15) worldwide. In the future, the surveillance of various resistant bacteria, mainly ESBL-producing bacteria, should be expanded to prevent their spread. From the Departments of Clinical Laboratory, 1 Kansai Medical University Hirakata Hospital, Osaka, Japan; 4 Wakayama Rosai Hospital, Wakayama, Japan; and 15 Osaka Police Hospital, Osaka; 2 Division of Biomedical Informatics, Course of Health Science, Graduate School of Medicine, Osaka University, Osaka; 3 Bacteriological Testing Section of Central Laboratory, FALCO Biosystems, Kyoto, Japan; 5 Department of Clinical Pathology, Tenri Hospital, Nara, Japan; Clinical Laboratory, 6 National Hospital Organization Minami Wakayama Medical Center, Wakayama, Japan; 8 Kyoto Second Red Cross Hospital, Kyoto; 9 Shiga Medical Center for Adults, Shiga, Japan; 1 Social Insurance Shiga Hospital, Shiga; 11 Japanese Red Cross Otsu Hospital, Shiga; 14 The Research Foundation for Microbial Diseases of Osaka University, Osaka; 16 Sumitomo Hospital, Osaka; 17 Hyogo Prefectural Amagasaki Hospital, Hyogo, Japan; 18 Takarazuka Municipal Hospital, Hyogo; 19 Hyogo Medical University Hospital, Hyogo; and 2 Kobe University Hospital, Hyogo; 7 Laboratory for Clinical Investigation, Kyoto University Hospital, Kyoto; 12 Department of Medical Technology, Kinki University School of Medicine, Osaka; and 13 Laboratory for Clinical Investigation, Osaka University Hospital, Osaka. Supported by the Study Group of Bacterial Resistance in the Kinki Region of Japan. Address reprint requests to Tatsuya Nakamura: Dept of Clinical Laboratory, Kansai Medical University Hirakata Hospital, Shinmachi, Hirakata City, Osaka, , Japan. Acknowledgments: We are grateful to K. Yamane and Y. Arakawa, Department of Bacterial and Blood Products, National Institute of Infectious Diseases, Tokyo, Japan. References 1. Knothe H, Shah P, Krcmery V, et al. Transferable resistance to cefotaxime, cefoxitin, cefamandole and cefuroxime in clinical isolates of Klebsiella pneumoniae and Serratia marcescens. Infection. 1983;11: Pitout JD, Hossain A, Hanson ND. Phenotypic and molecular detection of CTX-M-β-lactamases produced by Escherichia coli and Klebsiella spp. J Clin Microbiol. 24;42: Bradford PA. Extended-spectrum β-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev. 21;14: Bonnet R. 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