Received 3 April 2002; returned 19 August 2002; revised 4 November 2002; accepted 25 November 2002

Transcription

1 Journal of Antimicrobial Chemotherapy (2003) 51, DOI: /jac/dkg103 Advance Access publication 11 February 2003 Production of CTX-M-3 extended-spectrum β-lactamase and IMP-1 metallo β-lactamase by five Gram-negative bacilli: survey of clinical isolates from seven laboratories collected in 1998 and 2000, in the Kinki region of Japan Katsutoshi Yamasaki 1, Masaru Komatsu 2, Tomonari Yamashita 3, Koichi Shimakawa 2, Toshiro Ura 4, Hisaaki Nishio 5, Kaori Satoh 6, Ryoudou Washidu 3, Shohiro Kinoshita 7 and Masanori Aihara 2 * 1 Division of Clinical Microbiology, Department of Clinical Laboratory, Wakayama Rosai Hospital, 435 Koya, Wakayama; 2 Department of Clinical Pathology, Tenri Hospital, 200 Misima, Tenri, Nara ; 3 Bacteriological Testing Section of Central Laboratory, FALCO Biosystems Ltd, Central Laboratory, Kyoto; 4 Department of Clinical and Laboratory Medicine, National Cardiovascular Center, Osaka; 5 Department of Clinical Laboratory, Shiga Medical Center for Adults, Shiga; 6 Division of Clinical Microbiology, Department of Medical Technology, Kinki University School of Medicine, Osaka; 7 Department of Clinical Laboratory, Kobe University Hospital, Hyogo, Japan Received 3 April 2002; returned 19 August 2002; revised 4 November 2002; accepted 25 November 2002 The aim of this study was to research the distribution in the Kinki region of Japan of Enterobacteriaceae and Pseudomonas aeruginosa that produce extended-spectrum β-lactamase (ESBL) and metallo β-lactamase (MBL). One thousand isolates, 200 of each of four enterobacterial species (i.e. Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens) and 200 of P. aeruginosa, were collected from seven different laboratories during two 2 month periods, one in 1998 and one in A double-disc synergy test (DDST) and 2-mercaptopropionic acid inhibition test (2-MPAT) were used to confirm β-lactamase-producing isolates. The DDST was positive for one isolate of E. coli, five of K. pneumoniae, two of E. cloacae and 14 of S. marcescens. The 2-MPAT was positive for five isolates of S. marcescens and two of P. aeruginosa. We identified the β-lactamase type of each isolate by molecular confirmatory tests (isoelectric focusing, PCR and DNA sequencing): CTX-M-3 ESBLs (three isolates of K. pneumoniae, two of E. cloacae and 13 of S. marcescens), CTX-M-2 ESBL (one isolate of K. pneumoniae), SHV- 12 ESBLs (one isolate of E. coli and one of S. marcescens), CTX-M-3 and SHV-12 combination ESBL (one isolate of K. pneumoniae) and IMP-1 MBLs (five isolates of S. marcescens and two of P. aeruginosa). In conclusion, many species of Gram-negative bacilli that produce CTX-M-3 ESBLs and IMP-1 MBLs were disseminated widely in different hospitals of the Kinki region of Japan. Therefore, monitoring of laboratory bacterial ecology seems important to stop the spread of these strains through nosocomial outbreaks. Introduction Recently, Gram-negative bacilli that produce extendedspectrum β-lactamase (ESBL) 1 5 and metallo β-lactamase (MBL) 6 13 have been found widely in Japan. The recommendations of the NCCLS on detection of ESBL 14 are probably weak for the detection of all types of ESBL. Many scientific publications have reported the characteristics of such enzymes, whose presence may be suspected by a clear synergy when using clavulanate. An interesting review on this emerg ing group, CTX-M-type β-lactamase, was written by Tzouvelekis et al. 15 In this paper, we describe the results of a survey of... *Corresponding author. Tel: , ext. 8665; Fax: ; microbiology@tenriyorozu-hp.or.jp The British Society for Antimicrobial Chemotherapy

2 K. Yamasaki et al. aerobic Gram-negative bacilli that produce ESBL or MBL among clinical isolates collected in 1998 and 2000 in the Kinki region of Japan. Materials and methods Bacterial isolates We tested isolates of five species, four Enterobacteriaceae (i.e. Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Serratia marcescens) and Pseudomonas aeruginosa (200 isolates of each species; 1000 in total), which were collected by seven different laboratories during November and December 1998 (first study period) and January and February 2000 (second study period) and were non-repetitive. The isolates were identified with the MicroScan Neg BP Combo 3J Panel (Dade Behring, Tokyo, Japan). The seven laboratories included two in university hospitals ( beds), four in general hospitals ( beds) and one commercial laboratory that tests specimens for hospitals having <100 beds. The number of isolates of each organism that were collected by each laboratory is shown in Table 1. Susceptibility testing MICs were determined at each individual institution by a broth microdilution method with Dry Plate (Eiken Chemical Co., Ltd, Tokyo, Japan) with cation-adjusted Mueller Hinton broth (Eiken Chemical Co., Ltd) as described by the NCCLS. 16 The antimicrobial agents included ampicillin, piperacillin, co-amoxiclav combination (2:1 ratio), ampicillin/sulbactam combination (2:1 ratio), cefoperazone, cefoperazone/sulbactam combination (1:1 ratio), cefpodoxime, cefotaxime, ceftazidime, cefmetazole, imipenem, gentamicin and levofloxacin. E. coli ATCC and ATCC 35218, and P. aeruginosa ATCC were used as quality controls. Criteria of quality control for dilution method were used, and the cefoperazone/sulbactam combination was used for cefoperazone. Screening for ESBL and MBL producers The MIC criteria for detection of ESBL producers were as follows: (i) >1 mg/l cefpodoxime, cefotaxime or ceftazidime for E. coli and K. pneumoniae isolates, 14 and (ii) >8 mg/l cefotaxime or ceftazidime for E. cloacae and S. marcescens isolates. ESBLs were confirmed by a double-disc synergy test (DDST) that we conducted according to the method described by Jarlier et al. 17 with the modification of Tzelepi et al. 18 DDST involved a disc diffusion method, 19 with discs of co-amoxiclav, cefpodoxime, cefotaxime, ceftazidime, ceftriaxone and aztreonam in addition to cefepime. The MIC criterion for detection of MBL producers, >16 mg/l of both ceftazidime and the cefoperazone/sulbactam combination, was used for all isolates. MBLs were confirmed by a 2-mercaptopropionic acid inhibition test (2-MPAT). 20 Isoelectric focusing, PCR and DNA sequencing for characterization of β-lactamase type Strains that were positive for β-lactamases by the DDST or 2-MPAT were then evaluated for phenotype by isoelectric focusing (IEF), 5,9 inhibitor profile, 21 and PCR methods or DNA sequencing for genotyping. IEF for the β-lactamase analysis was carried out on 7% polyacrylamide gels containing Ampholine, a carrier ampholyte (ph ; Amersham Pharmacia Biotech, Uppsala, Sweden). Class A β-lactamases Table 1. Numbers of isolates of each organism that were collected by each laboratory No. of isolates for each organism E. coli K. pneumoniae E. cloacae S. marcescens P. aeruginosa Laboratory 1 a 2 b 1 a 2 b 1 a 2 b 1 a 2 b 1 a 2 b National Cardiovascular Center NC 15 NC 15 NC 14 NC 4 NC 15 Kobe University Hospital NC 20 NC 15 NC 15 NC 15 NC 20 Kinki University School of Medicine NC 11 NC 9 NC 3 NC 3 NC 15 Shiga Medical Center for Adults NC 9 NC 8 NC 5 NC 2 NC 15 Wakayama Rosai Hospital Tenri Hospital FALCO Biosystems Ltd Total NC, none collected. a First study period, November and December b Second study period, January and February

3 Survey of ESBLs and MBLs in Japan were distinguished with lithium clavulanate (SmithKline Beecham, Milan, Italy). 21 MBLs were distinguished with ZnSO 4. 9 PCR was carried out according to the method reported previously. 5,22 The primers were used to amplify the genes encoding TEM, 23 SHV, 23 CTX-M-2, 2 CTX-M-1, 24 CTX- M-9 25 and IMP-1 11 β-lactamases. DNA sequencing was carried out with consecutive primers specific for the bla CTX-M1 gene, 24 the bla CTX-M2 gene, 26 the bla SHV gene 27 and the bla IMP-1 gene, 28 according to the dideoxy method. 24 An automatic sequencer (ABI 1377; Perkin-Elmer/ Applied Biosystems, Inc.) was used. Pulsed-field gel electrophoresis (PFGE) analysis PFGE was carried out using the GenePath system (Bio-Rad Laboratories, Hercules, CA, USA). Briefly, chromosomal DNA was prepared using the GenePath Group 3 reagent kit (Bio-Rad Laboratories). After SpeI digestion, the DNA was electrophoresed through 1% agarose gel (Bio-Rad Laboratories) under the following conditions: 19.7 h at 6 V/cm (200 V), with a pulse time of s and non-linear of 21%. After electrophoresis, gels were stained with ethidium bromide. PFGE patterns were analysed based on a dendrogram, using Dendron (Bio-Rad Laboratories). Results MIC screening and confirmatory DDST for ESBL production The numbers of isolates that met the MIC criteria for ESBL production and were DDST positive in each species are shown in Table 2. Twenty (10.0%) of 200 E. coli isolates and eight (4.0%) of 200 K. pneumoniae isolates met the MIC criterion of >1 mg/l of cefpodoxime, cefotaxime or ceftazidime. Among them, one (0.5%) of 200 E. coli isolates and five (2.5%) of 200 K. pneumoniae isolates were confirmed by DDST to produce ESBL. Fifty-seven (27.9%) of 201 E. cloacae isolates and 86 (43.2%) of 199 S. marcescens isolates met the MIC criterion of >8 mg/l of cefotaxime or ceftazidime. Among them, two (1.0%) of 201 E. cloacae isolates and 14 (7.0%) of 199 S. marcescens isolates were confirmed by DDST to produce ESBL. ESBL typing by IEF, PCR method and DNA sequencing The MICs and β-lactamase types of ESBL-producing strains are shown in Table 3. ESBL type of each isolate detected by DDST was identified as either a CTX-M-3 (three isolates of K. pneumoniae, two of E. cloacae and 13 of S. marcescens), a CTX-M-2 (one isolate of K. pneumoniae), a SHV-12 (one isolate of E. coli and one of S. marcescens) or a CTX-M-3 and SHV-12 combination (one isolate of K. pneumoniae). Two isolates, one CTX-M-2-producing K. pneumoniae and one SHV-12-producing S. marcescens, were detected in Tenri Hospital; 13 isolates, one CTX-M-2-producing K. pneumoniae, one CTX-M-3-producing K. pneumoniae, two CTX-M-3-producing E. cloacae and nine CTX-M-3- producing S. marcescens, were detected by FALCO Biosystems Ltd; two isolates, one SHV-12-producing E. coli and one SHV-12 and CTX-M-3-producing K. pneumoniae, were detected in Kinki University School of Medicine; and five isolates, one CTX-M-3-producing K. pneumoniae and four CTX-M-3-producing S. marcescens, were detected in Kobe University Hospital. One of the CTX-M-2-producing K. pneumoniae isolates had a pi 8.0 enzyme, 19 of the CTX-M-3-producing bacilli had pi 8.2 enzymes, one of the SHV-12-producing bacilli had pi 8.2 enzyme and one of the SHV-12 and CTX-M-3- producing bacilli had pi 8.2 enzymes. MIC screening and confirmatory 2-MPAT for MBLs The numbers for each strain of isolates that fulfilled the MIC criteria for production of MBLs and the numbers that were 2-MPAT positive are shown in Table 4. Nineteen (9.5%) of 201 E. cloacae isolates, 38 (19.1%) of 199 S. marcescens Table 2. Number of isolates fulfilling MIC criteria for ESBL production and DDST-positive rates No. of isolates fulfilling MIC criteria for ESBL production (no. of DDST-positive isolates) Organism Total number first a second b total total % E. coli (0) 8 (1) 20 (1) 10.0 (0.5) K. pneumoniae (3) 3 (2) 8 (5) 4.0 (2.5) E. cloacae (1) 35 (1) 57 (2) 27.9 (1.0) S. marcescens (9) 42 (5) 86 (14) 43.2 (7.0) a First study period, November and December b Second study period, January and February

4 Table 3. The MICs and β-lactamase types of ESBL-producing strains MIC (mg/l) Strain no. Laboratory Organism AMP PIP AMC SAM CFP S/C CPD CTX CAZ CMZ IPM GEN LVX β-lactamase type 634 First study period: Nov. and Dec FL-KPN316 Falco K. pneumoniae > CTX-M-2 TH-KPN189 Tenri K. pneumoniae >64 > > CTX-M-3 FL-KPN391 Falco K. pneumoniae >64 > > CTX-M-3 FL-ECL203 Falco E. cloacae >64 64 >64 64 >32 8 >32 >32 8 > CTX-M-3 FL-SMA704 Falco S. marcescens >64 >64 >64 >64 >32 32 >32 >32 8 > CTX-M-3 FL-SMA747 Falco S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 32 > >8 CTX-M-3 FL-SMA774 Falco S. marcescens >64 >64 >64 >64 >32 32 >32 >32 8 > CTX-M-3 FL-SMA796 Falco S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 32 > >8 CTX-M-3 FL-SMA721 Falco S. marcescens >64 >64 >64 >64 >32 >32 32 >32 32 > >8 CTX-M-3 FL-SMA776 Falco S. marcescens >64 >64 >64 >64 >32 32 >32 >32 4 > CTX-M-3 FL-SMA777 Falco S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 16 > CTX-M-3 FL-SMA784 Falco S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 >32 > >8 CTX-M-3 FL-SMA785 Falco S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 >32 > >8 CTX-M-3 Second study period: Jan. and Feb KI-ECO0I5 Kinki Univ. E. coli >64 >64 >64 64 > > SHV-12 KI-KPN003 Kinki Univ. K. pneumoniae > > SHV-12, CTX-M-3 KO-KPN002 Kobe Univ. K. pneumoniae >64 > CTX-M-3 FL-ECL467 Falco E. cloacae >64 > >32 8 > > CTX-M-3 TH-SMA002 Tenri S. marcescens >64 64 > SHV-12 KO-SMA002 Kobe Univ. S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 8 > CTX-M-3 KO-SMA006 Kobe Univ. S. marcescens >64 >64 >64 >64 >32 16 >32 >32 4 > CTX-M-3 KO-SMA010 Kobe Univ. S. marcescens >64 32 >64 >64 >32 16 >32 >32 4 > CTX-M-3 KO-SMA013 Kobe Univ. S. marcescens >64 >64 >64 >64 >32 16 >32 >32 4 > CTX-M-3 K. Yamasaki et al. AMP, ampicillin; PIP, piperacillin; AMC, co-amoxiclav combination (2:1 ratio); SAM, ampicillin/sulbactam combination (2:1 ratio); CFP, cefoperazone; S/C, cefoperazone/sulbactam combination (1:1 ratio); CPD, cefpodoxime; CTX, cefotaxime; CAZ, ceftazidime; CMZ, cefmetazole; IPM, imipenem; GEN, gentamicin; LVX, levofloxacin; Falco, FALCO Biosystems Ltd; Tenri, Tenri Hospital; Kinki Univ., Kinki University School of Medicine; Kobe Univ., Kobe University Hospital. All strains from FALCO Biosystems were collected from 13 different hospitals having <100 beds.

5 Survey of ESBLs and MBLs in Japan Table 4. Number of isolates fulfilling MIC criteria for MBL production and 2-MPAT-positive rates Number of isolates fulfilling MIC criteria for metallo β-lactamase production (no. of 2-MPAT-positive isolates) Organism Total number first a second b total total % E. coli (0) 0 (0) 0 (0) 0 (0) K. pneumoniae (0) 0 (0) 0 (0) 0 (0) E. cloacae (0) 16 (0) 19 (0) 9.5 (0) S. marcescens (3) 19 (2) 38 (5) 19.1 (2.5) P. aeruginosa (2) 11 (0) 22 (2) 11.0 (1.0) a First study period, Nov. and Dec b Second study period, Jan. and Feb isolates and 22 (11.0%) of 200 P. aeruginosa isolates met the MIC criterion of >16 mg/l ceftazidime and the cefoperazone/sulbactam combination. Among them, five (2.5%) of 199 S. marcescens isolates and two (1.0%) of 200 P. aeruginosa isolates were confirmed by 2-MPAT to produce MBL. MBL typing by IEF, PCR method and DNA sequencing The MICs and β-lactamase types of MBL-producing strains are shown in Table 5. MBL types of each isolate detected by 2-MPAT were identified as five IMP-1-producing S. marcescens and two IMP-1-producing P. aeruginosa. Five IMP-1-producing S. marcescens and two IMP-1-producing P. aeruginosa were from FALCO Biosystems Ltd and had pi 9.5 enzymes detected with the use of ZnSO 4. PFGE analysis The four CTX-M-3-producing S. marcescens isolated in the second period in Kobe University Hospital belonged to different clones. Discussion The aim of this study was to research the distribution of ESBLs and MBLs in bacteria collected from various types of hospitals having a range of beds from 100 to A relatively large number of β-lactamase-producing strains obtained from FALCO Biosystems Ltd were either CTX-M- 3-producing isolates (S. marcescens) or IMP-1-producing isolates (S. marcescens and P. aeruginosa). These results indicate that these genotypes have been spreading among small hospitals with <100 beds. Two ESBL producers per institution were detected among strains collected at Tenri Hospital and at Kinki University School of Medicine. Five ESBL producers were detected among those collected at Kobe University Hospital. These hospitals have >900 beds; the hospitals in which no ESBL producers were found had beds. These results indicate that ESBL producers have been spreading among hospitals with >900 beds. A survey of P. aeruginosa isolates from throughout Japan between 1992 and 1994 found that 0.4% produced IMP Among isolates collected in 1996 and 1997 in Japan, 4.4% of S. marcescens isolates and 1.3% of P. aeruginosa isolates produced IMP During a survey in the Kanto region of Japan, which is 500 km from Kinki region, 0.09% of E. coli isolates and 0.35% of K. pneumoniae isolates were found to produce ESBLs. 4 ESBLs were present in 22 (2.8%) of 800 Enterobacteriaceae isolates evaluated in the present study. The overall prevalence of ESBL producers among E. coli and K. pneumoniae strains was greater than that reported by Yagi et al. 4 Our present data represent the first report of the prevalence of ESBL producers among E. cloacae and S. marcescens in an area of Japan. TEM- and SHV-type ESBLs have been described as the most prevalent types in Europe and the USA, 30 and CTX-M- 2-type ESBL has been noted as the most prevalent type in Japan. 4 However, in the present study, genotypes of ESBLs in Enterobacteriaceae were primarily the CTX-M-3 type (81.8%, 18 of 22). These results indicate that the CTX-M-3- type ESBL, which is different from the most common type reported by Yagi et al., 4 has been spreading among many groups of Enterobacteriaceae in the Kinki region of Japan. Plasmid-mediated AmpC β-lactamases of K. pneumoniae, which naturally lack chromosomal AmpC β-lactamases, were discovered in Japan, 31 and among some strains of K. pneumoniae have been reported the combination of ESBL and AmpC plasmid-encoded type. 32 In the present study, eight K. pneumoniae isolates were resistant using our criteria. However, MICs of cefmetazole for all isolates were 4 mg/l (data from three isolates with DDST negative, not shown), and it is not 635

6 K. Yamasaki et al. Table 5. The MICs and β-lactamase types of MBL-producing strains MIC (mg/l) Strain no. Laboratory Organism AMP PIP AMC SAM CFP S/C CPD CTX CAZ CMZ IPM GEN LVX β-lactamase type First study period: Nov. and Dec FL-PAE183 Falco P. aeruginosa >64 >64 >64 64 >32 32 >32 >32 >32 > IMP-1 FL-PAE111 Falco P. aeruginosa >64 32 >64 >64 >32 >32 >32 >32 >32 >16 >32 4 >8 IMP-1 FL-SMA706 Falco S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 >32 >16 > IMP-1 FL-SMA714 Falco S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 >32 >16 > IMP-1 FL-SMA722 Falco S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 >32 >16 > IMP-1 Second study period: Jan. and Feb FL-SMA606 Falco S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 >32 >16 > IMP-1 FL-SMA637 Falco S. marcescens >64 >64 >64 >64 >32 >32 >32 >32 >32 >16 > IMP-1 AMP, ampicillin; PIP, piperacillin; AMC, co-amoxiclav combination (2:1 ratio); SAM, ampicillin/sulbactam combination (2:1 ratio); CFP, cefoperazone; S/C, cefoperazone/sulbactam combination (1:1 ratio); CPD, cefpodoxime; CTX, cefotaxime; CAZ, ceftazidime; CMZ, cefmetazole; IPM, imipenem; GEN, gentamicin; LVX, levofloxacin; Falco, FALCO Biosystems Ltd. All strains from FALCO Biosystems were collected from eight different hospitals having <100 beds. possible that these β-lactamases will be present among these isolates. A strain of K. pneumoniae, producing the two types of ESBL that were detected in Kinki University School of Medicine, was rare in Japan. MBLs were present in only seven (0.7%) of 1000 isolates among the Enterobacteriaceae and P. aeruginosa strains evaluated in the present study. All seven strains produced IMP-1. The overall prevalence of MBLs among S. marcescens and P. aeruginosa was similar to that reported by Kurokawa et al. 29 The bla IMP gene, which was initially discovered in Japan, 6 has recently been reported outside Japan Thus it is quite possible that these β-lactamases will spread worldwide. The DDST, as described by Jarlier et al., 17 has poor performance in detecting the production of an ESBL in E. cloacae and S. marcescens: this is due in large part to the ability of clavulanate to induce the AmpC β-lactamase in these strains. Thus the real prevalence of ESBLs among E. cloacae and S. marcescens isolates might have been underestimated. ESBL-producing species of Enterobacteriaceae (especially bacilli producing chromosomal AmpC β-lactamase), except for E. coli and Klebsiella spp., are increasingly reported worldwide, 37,38 and nosocomial outbreaks have been reported Although NCCLS recommendations exist for screening and confirming ESBLs in E. coli and Klebsiella spp., 14 no recommendations exist for ESBLs in other bacilli or for MBLs in any organism. In the present study, ESBL-producing isolates among E. cloacae and S. marcescens and MBL-producing isolates among S. marcescens and P. aeruginosa were detected most often. Thus, we recommend that clinical laboratories adopt simple tests (e.g. DDST and 2-MPAT) for confirming all ESBL production in enterobacterial species among bacilli other than E. coli and Klebsiella spp. for which NCCLS recommendations exist, 14 and also for MBL production in any organism. In conclusion, species of Gram-negative bacilli that produce CTX-M-type ESBLs and IMP-1 MBLs were found to be widely disseminated among different hospitals in the Kinki region of Japan. Bacterial monitoring of the laboratory ecology seems important to stop the spread of these strains through nosocomial outbreaks. Acknowledgements This work was supported by the Bacterial Resistance Research Group in Kinki of Japan, and in part by the Japan β-lactamase Research Group (project of Pfizer Pharmaceutical Inc., Tokyo, Japan). The authors are all members of the Bacterial Resistance Research Group in Kinki, Japan. 636

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