Laboratory Surveillance for Prospective Plasmid-Mediated AmpC -Lactamases in the Kinki Region of Japan

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 2010, p Vol. 48, No /10/$12.00 doi: /jcm Copyright 2010, American Society for Microbiology. All Rights Reserved. Laboratory Surveillance for Prospective Plasmid-Mediated AmpC -Lactamases in the Kinki Region of Japan Katsutoshi Yamasaki, 1,2 * Masaru Komatsu, 3 Noriyuki Abe, 4 Saori Fukuda, 4 Yugo Miyamoto, 5 Takeshi Higuchi, 6 Tamotsu Ono, 7 Hisaaki Nishio, 8 Noriyuki Sueyoshi, 9 Kaneyuki Kida, 10 Kaori Satoh, 11 Masahiro Toyokawa, 12 Isao Nishi, 12 Masako Sakamoto, 13 Masahiro Akagi, 14 Isako Nakai, 15 Tomomi Kofuku, 16 Tamaki Orita, 17 Yasunao Wada, 18 Takumi Jikimoto, 19 Shohiro Kinoshita, 19 Kazuaki Miyamoto, 20 Itaru Hirai, 2 and Yoshimasa Yamamoto 2 Department of Clinical Laboratory, Wakayama Rosai Hospital, 1 Clinical Laboratory, National Hospital Organization Minami Wakayama Medical Center, 5 and Department of Microbiology, Wakayama Medical University, School of Medicine, 20 Wakayama, Department of Clinical Pathology, Tenri Hospital, Nara, 4 Department of Technical Section 3, Central Laboratory, FALCO biosystems Ltd., 3 Laboratory for Clinical Investigation, Kyoto University Hospital, 6 and Clinical Laboratory, Japanese Red Cross Kyoto Daini Hospital, 7 Kyoto, Clinical Laboratory, Shiga Medical Center for Adults, 8 Clinical Laboratory, Social Insurance Shiga Hospital, 9 and Clinical Laboratory, Japanese Red Cross Otsu Hospital, 10 Shiga, Department of Medical Technology, Kinki University Faculty of Medicine, 11 Laboratory for Clinical Investigation, Osaka University Hospital, 12 Clinical Laboratory, The Research Foundation for Microbial Diseases of Osaka University, 13 Department of Biomedical Informatics, Osaka University Graduate School of Medicine, 2 Department of Clinical Laboratory, Osaka Police Hospital, 14 and Clinical Laboratory, Sumitomo Hospital, 15 Osaka, Clinical Laboratory, Hyogo Prefectural Nishinomiya Hospital, 16 Clinical Laboratory, Takarazuka Municipal Hospital, 17 Clinical Laboratory, Hyogo Medical University Hospital, 18 and Clinical Laboratory, Kobe University Hospital, 19 Hyogo, Japan Received 29 October 2009/Returned for modification 18 January 2010/Accepted 14 June 2010 Extended-spectrum -lactamases, plasmid-mediated AmpC -lactamases (PABLs), and plasmid-mediated metallo- -lactamases confer resistance to many -lactams. In Japan, although several reports exist on the prevalence of extended-spectrum -lactamases and metallo- -lactamases, the prevalence and characteristics of PABLs remain unknown. To investigate the production of PABLs, a total of 22,869 strains of 4 enterobacterial species, Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, and Proteus mirabilis, were collected during six 6-month periods from 17 clinical laboratories in the Kinki region of Japan. PABLs were detected in 29 (0.13%) of 22,869 isolates by the 3-dimensional test, PCR analysis, and DNA sequencing analysis. PABL-positive isolates were detected among isolates from 13 laboratories. Seventeen of 13,995 (0.12%) E. coli isolates, 8 of 5,970 (0.13%) K. pneumoniae isolates, 3 of 1,722 (0.17%) K. oxytoca isolates, and 1 of 1,182 (0.08%) P. mirabilis isolates were positive for PABLs. Of these 29 PABL-positive strains, 20 (69.0%), 6 (20.7%), 2 (6.9%), and 1 (3.4%) carried the genes for CMY-2, DHA-1, CMY-8, and MOX-1 PABLs, respectively. Pattern analysis of randomly amplified polymorphic DNA and pulsed-field gel electrophoretic analysis revealed that the prevalence of CMY-2-producing E. coli strains was not due to epidemic strains and that 3 DHA-1-producing K. pneumoniae strains were identical, suggesting their clonal relatedness. In conclusion, the DHA-1 PABLs were predominantly present in K. pneumoniae strains, but CMY-2 PABLs were predominantly present in E. coli strains. The present findings will provide significant information to assist in preventing the emergence and further spread of PABL-producing bacteria. * Corresponding author. Mailing address: Department of Clinical Laboratory, Wakayama Rosai Hospital, 93-1 Kinomoto, Wakayama City, Wakayama , Japan. Phone: Fax: ktt-yamazaki@wakayamah.rofuku.go.jp. Published ahead of print on 7 July Lactamase production is the most important factor for -lactam resistance in Gram-negative rods (16). Plasmid-mediated -lactamases, such as extended-spectrum -lactamases (ESBLs), plasmid-mediated AmpC -lactamases (PABLs), and plasmid-mediated metallo- -lactamases (MBLs), hydrolyze broad-spectrum -lactams. Detection of these plasmidmediated -lactamase-producing isolates is important for epidemiological studies and hospital infection control, because plasmid-mediated genes can spread to other organisms. The Study of Bacterial Resistance in the Kinki Region of Japan (SBRK) Antimicrobial Surveillance Program was established in 1997 to monitor the predominant pathogens and antimicrobial resistance patterns of nosocomial and communityacquired infections via a broad network of clinical laboratories differing in geographic location and size. Our previously reported survey data from the Kinki region of Japan revealed the prevalence of ESBLs and plasmid-mediated MBLs (21, 30); however, the epidemiology of PABLs remains unknown. For this reason, a laboratory-based surveillance study was conducted to determine the presence and prevalence of PABLs among members of the family Enterobacteriaceae. 3267

2 3268 YAMASAKI ET AL. J. CLIN. MICROBIOL. TABLE 1. Primers used for multiplex PCR and sequencing of PABL genes Target Use Primer name Primer sequence (5 to 3 ) Nucleotide positions Product length (bp) Reference(s) CMY/MOX Amplification MOXMF GCT GCT CAA GGA GCA CAG GAT MOXMR CAC ATT GAC ATA GGT GTG GTG C Sequencing MOX-SQ-592F ATG GAG CAG ACC CTG CTG CC , 32 MOX-SQ-1170R TCT TTC TCC CGC CAT GTA AG CMY/LAT Amplification CITMF TGG CCA GAA CTG ACA GGC AAA CITMR TTT CTC CTG AAC GTG GCT GGC Sequencing CMY25F1 CAA TGT GTG AGA AGC AGT C , CMY2DR1 CGC ATG GGA TTT TGG TTG CTG DHA Amplification DHAMF AAC TTT CAC AGG TGT GCT GGG T DHAMR CCG TAC GCA TAC TGG CTT TGC Sequencing DHA-1A CTG ATG AAA AAA TCG TTA TC , DHA-1B ATT CCA GTG CAC TCA AAA TA ACC Amplification ACCMF AAC AGC CTC AGC AGC CGG TTA ACCMR TTC GCC GCA ATC ATC CCT AGC ACT-1/MIR-1 Amplification EBCMF TCG GTA AAG CCG ATG TTG CGG EBCMR CTT CCA CTG CGG CTG CCA GTT FOX Amplification FOXMF AAC ATG GGG TAT CAG GGA GAT G FOXMR CAA AGC GCG TAA CCG GAT TGG PABL CMY-1 was first found in a Klebsiella pneumoniae isolate in South Korea in 1989 (4, 5). Since then, additional organisms producing PABLs have been reported worldwide (25). PABLs are a heterogeneous group of enzymes that originated from the chromosomal genes of Enterobacter spp. (ACT-1/MIR-1 type), Citrobacter freundii (CMY/LAT type), Morganella morganii (DHA type), Hafnia alvei (ACC-1), and Aeromonas spp. (CMY/MOX type and FOX type). The most prevalent and most widely distributed PABLs are the CMY/ LAT-type enzymes (25). In addition to these enzyme types, DHA-type enzymes have been identified in Taiwan (31) and China (15). In Korea (14, 26), DHA-, CMY/MOX-, and ACT- 1/MIR-1-type enzymes have also been identified, while in the United States (1, 17), in addition to the types mentioned above, DHA-, ACT-1/MIR-1-, and FOX-type enzymes have been identified. To date, in Japan, MOX-1 (11), CMY-9 (9, 28), CMY-19 (28), CFE-1 (19), CMY-2 (18), and DHA-1 (18) have been found in clinical isolates. Muratani et al. (18) reported PABL producers among cephem-resistant K. pneumoniae isolates, but this report did not indicate the rate of occurrence of PABLs. For the present study, we collected 22,869 isolates from 17 clinical laboratories in the Kinki region of Japan, and we assessed the prevalence and types of PABL-positive bacteria. MATERIALS AND METHODS Bacterial isolates. This laboratory surveillance was conducted with the cooperation of 17 institutions (16 clinical laboratories of various hospitals and 1 commercial laboratory) in the Kinki region, which is located in Western Japan (21, 30). Specimens from inpatients and outpatients were collected during the following periods: November 2002 to April 2003 (first study period), November 2003 to April 2004 (second study period), November 2004 to April 2005 (third study period), November 2005 to April 2006 (fourth study period), November 2006 to April 2007 (fifth study period), and November 2007 to April 2008 (sixth study period). A total of 22,869 isolates of Gram-negative bacilli, including Escherichia coli (13,995 isolates), K. pneumoniae (5,970 isolates), Klebsiella oxytoca (1,722 isolates), and Proteus mirabilis (1,182 isolates), were isolated from various clinical specimens and were then tested. A single isolate was selected from each patient. These isolates were identified by the clinical procedures routinely used in each laboratory, and the identifications were confirmed using the WalkAway system (Siemens Healthcare Diagnostics, Tokyo, Japan). -Lactamase assays. A cefazolin (CFZ) MIC of 16 g/ml was used for the initial screening of isolates. Strains that met this criterion were tested using the three-dimensional (3D) test with cefoxitin (FOX) (30 g per disk) as previously described (8). In brief, the crude enzyme extracts were prepared by freezing and thawing cell pellets from centrifuged Trypticase soy broth cultures. A Mueller- Hinton agar plate (Becton Dickinson Co., Ltd., Tokyo, Japan) was inoculated with E. coli (ATCC 25922) according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (6), and a Sensi-Disk (Becton Dickinson Co., Ltd.) containing FOX was placed on the inoculated agar. A slit was cut in the agar with a sterile scalpel blade, and the crude enzyme preparation was dispensed into the slit. After overnight incubation at 35 C, the observation of enhanced bacterial growth at the location where the slit intersected the zone of inhibition was interpreted as a positive 3D test for that strain. Coexistence of AmpC and ESBLs was also investigated by the double-disk synergy (DDS) test with amoxicillin-clavulanic acid (20 and 10 g per disk, respectively), cefotaxime (CTX) (30 g per disk), ceftazidime (CAZ) (30 g per disk), and cefepime (FEP) (30 g per disk) according to previously published methods (12, 30). Multiplex PCR amplification and sequencing of PABLs. Strains positive for the 3D test were investigated further for genotype determination with a previously described multiplex PCR protocol (24). The template DNA was replaced with water in negative-control reactions. The positive-control templates, kindly provided by N. D. Hanson, were 6 DNA fragments of bla MOX-1, bla LAT-1, bla DHA-1, a gene native to Hafnia alvei, bla ACT-1, and bla FOX-1. DNA sequencing for PABLs was carried out by PCR amplification with consecutive primers for bla MOX-1 (11), bla CMY-8 (32), bla DHA-1 (33), and bla CMY-2 (10). The amplified products were sequenced in an automated DNA sequencer (ABI 3100; Applied Biosystems, Foster City, CA). The genotypes of strains found positive by the DDS test were also investigated using PCR methods, as described previously (2, 23, 30). The primers used for multiplex PCR or for the sequencing of PABL genes are listed in Table 1. Transfer of resistance. The transmissibility of PABLs was tested by conjugation analysis using an E. coli CSH2 strain that was resistant both to nalidixic acid and to rifampin (metb F Rif r Nal r ) as the recipient. The E. coli CSH2 recipient strain was kindly provided by Y. Arakawa. The donor-to-recipient ratio was 1:4, and mating plates were incubated at 35 C for 4 h. Transconjugants were selected on modified Drigalski agar (Eiken Chemical Co., Ltd., Tokyo, Japan) supplemented with 50 g/ml rifampin (Sigma-Aldrich, St. Louis, MO), 50 g/ml nali-

3 VOL. 48, 2010 SURVEY OF PABLs IN KINKI REGION OF JAPAN 3269 dixic acid (Sigma), and 50 g/ml ampicillin (AMP) (Sigma). The presence of CMY-2 genes in the transconjugants was confirmed by PCR. Plasmid DNAs extracted according to the procedure described by Kado and Liu (13) were used to transform E. coli strain DH5 (Nippon Gene Co., Ltd., Toyama, Japan). Transformants were selected on Mueller-Hinton agar (Becton Dickinson Co., Ltd.) supplemented with 50 g/ml AMP and were subjected to the conjugation experiment. Plasmid DNAs extracted from the transformants and from the resulting transconjugants were first electrophoresed on 1% agarose (Nippon Gene Co., Ltd.) at 4 C and then transferred to a nylon membrane (Roche Diagnostics, Tokyo, Japan). To detect CMY-2 gene-containing plasmids, the membrane was probed with digoxigenin (DIG)-labeled DNA probes prepared using PCR DIG Labeling Mix (Roche Diagnostics), with a DNA template prepared from a bla CMY-2 -producing E. coli strain and primers CITMF and CITMR. Testing for susceptibility to antimicrobial agents. MICs for strains with PABLs were determined by a broth microdilution method with the MicroScan Neg MIC 3.31E and Neg Combo 6.11J panels (Siemens Healthcare Diagnostics) as described by the NCCLS (now the CLSI) (20). The following concentrations of antimicrobial agents were used: piperacillin, 8 to 64 g/ml; CTX, 0.5 to 128 g/ml; CAZ, 0.5 to 128 g/ml; FEP, 1 to 32 g/ml; FOX, 2 to 32 g/ml; cefmetazole, 0.5 to 32 g/ml; imipenem (IPM), 1 to 8 g/ml; meropenem (MEM), 0.25 to 16 g/ml; amikacin (AMK), 4 to 32 g/ml; gentamicin (GEN), 1to8 g/ml; minocycline (MIN), 1 to 8 g/ml; levofloxacin (LVX), 1 to 4 g/ml; and sulfamethoxazole-trimethoprim (SXT), 38 and 2 g/ml. AMK, GEN, MIN, LVX, and SXT were used for antibiograms. E. coli ATCC and Pseudomonas aeruginosa ATCC were used as quality controls. The quality control criteria used for the dilution methods were those of the CLSI (7). RAPD pattern analysis. Isolates confirmed to be positive for the PABL gene by PCR were subjected to chromosomal DNA typing by random amplification of polymorphic DNA (RAPD) analysis in order to generate RAPD fingerprints (22). Genomic DNAs were prepared using the QIAamp DNA Mini kit (Qiagen, Tokyo, Japan). RAPD amplification was performed with primer ERIC2 (0.8 M) in a final volume of 50 l containing 20 mm Tris-HCl (ph 8.4), 50 mm KCl, 0.2 mm each deoxynucleoside triphosphate, 2.5 mm MgCl 2, 1.25 U of Taq DNA polymerase (Toyobo Co., Ltd., Osaka, Japan), and 4 l template DNA. The cycle conditions were as follows: initial denaturation at 94 C for 5 min; 35 cycles of denaturation for 1 min at 94 C, annealing for 1 min at 35 C, and extension for 1 min at 72 C; and a final extension at 72 C for 10 min. RAPD band patterns were compared visually, and samples differing in at least two bands were considered nonidentical. PFGE. Isolates showing identical RAPD patterns were analyzed by pulsedfield gel electrophoresis (PFGE) using the GenePath system (Bio-Rad Laboratories). Genomic DNAs were prepared using the CHEF genomic DNA plug kit (Bio-Rad Laboratories), digested with XbaI (Nippon Gene Co., Ltd.), and electrophoresed through 1% agarose (Bio-Rad Laboratories) under the following conditions: 19.7 h at 6 V/cm (200 V) and 14 C, with a pulse time of 5.3 to 49.9 s and a nonlinear ramp with a half-height of 21%. After electrophoresis, gels were stained with ethidium bromide. PFGE patterns were interpreted according to the criteria proposed by Tenover et al. (27). RESULTS Prevalence of PABL-positive isolates. Nine hundred (6.4%) of 13,995 E. coli isolates, 106 (1.8%) of 5,970 K. pneumoniae isolates, 140 (8.1%) of 1,722 K. oxytoca isolates, and 60 (5.1%) of 1,182 P. mirabilis isolates met the criterion of a CFZ MIC of 16 g/ml. Of these 1,206 isolates, 290 (2.1%) E. coli isolates, 13 (0.2%) K. pneumoniae isolates, 6 (0.3%) K. oxytoca isolates, and 1 (0.1%) P. mirabilis isolate were positive by the 3D test. The numbers of isolates positive for the PABL gene were 17 (0.13%) for E. coli, 8 (0.13%) for K. pneumoniae, 3 (0.17%) for K. oxytoca, and 1 (0.08%) for P. mirabilis. Of these 29 PABLpositive isolates, 1 E. coli and 1 K. pneumoniae isolate were positive for ESBLs by the DDS test. Characterization of PABL-producing isolates. Some characteristics and selected clinical associations of PABL-producing isolates are shown in Table 2. PABL-positive isolates were detected in 13 of 17 laboratories. With respect to the PABL genotypes, 20 (69.0%), 6 (20.7%), 2 (6.9%), and 1 (3.5%) isolate carried CMY-2, DHA-1, CMY-8, and MOX-1 genes, respectively. CMY-2 PABL genes were detected in 17 E. coli isolates from 8 hospitals in 5 prefectures, in 2 K. oxytoca isolates from 2 hospitals in 2 prefectures, and in 1 P. mirabilis isolate from 1 hospital. DHA-1 PABL genes were detected in 6 K. pneumoniae isolates from 4 hospitals in 3 prefectures. CMY-8 genes were detected in 2 K. pneumoniae isolates from 2 hospitals in 2 prefectures. The MOX-1 gene was detected in 1 K. oxytoca isolate from 1 hospital. The multiplex PCR results for PABLs are shown in Fig. 1. RAPD typing demonstrated that each of the 17 E. coli isolates (from 8 hospitals) carrying the genes for CMY-2 PABLs had a unique chromosomal DNA pattern. The RAPD patterns of the PABL-positive K. pneumoniae and K. oxytoca isolates are shown in Fig. 2. Three K. pneumoniae isolates from 1 hospital (strains F-1, F-2, and F-3) carrying a gene for PABL DHA-1 demonstrated identical RAPD patterns. Two K. pneumoniae isolates from 2 hospitals (strains I-1 and J-1) carrying a gene for PABL CMY-8 showed identical RAPD patterns. Three K. oxytoca isolates from 3 hospitals carrying genes for MOX-1 (strain M-1) or CMY-2 (strains C-1 and F-4) were distinct. The PFGE profiles of 3 DHA-1-producing K. pneumoniae isolates were identical or clearly related. However, 2 CMY-8-producing K. pneumoniae isolates were nonidentical. The results of PFGE analyses for PABL-positive K. pneumoniae isolates with identical RAPD patterns are shown in Fig. 3. Two PABL-positive isolates (strains C-2 and H-2) tested positive by the DDS test, and these isolates were positive for the CTX-M-type ESBL gene as determined by PCR. Transfer of resistance. Among the 17 CMY-2-producing E. coli strains, resistance was transferred from 12 isolates, and a CTX-M-type ESBL was cotransferred from the single strain expressing this resistance. One K. oxytoca strain and 1 P. mirabilis strain that produced CMY-2 transferred resistance. Resistance did not transfer from 1 CMY-2-producing K. oxytoca strain, 1 MOX-1-producing K. oxytoca strain, 2 CMY-8-producing K. pneumoniae strains, or 6 DHA-1-producing K. pneumoniae strains. Southern blotting was used to confirm the presence of CMY-2 genes on plasmids. In the conjugation experiment, one large plasmid was transferred to E. coli from each transformant. Southern blotting with the bla CMY-2 -specific probe showed the presence of bla CMY-2 on this plasmid (Fig. 4). Susceptibilities of PABL-producing isolates. The types of -lactamases produced by the 29 PABL-producing strains and the MICs of -lactams for these strains are shown in Table 3. The susceptibilities of these strains to several -lactams differed, with the exception of IPM and MEM. Although 2 PABL-positive strains coproducing CTX-M-type ESBLs demonstrated resistance to FEP, the remaining 27 PABL-positive strains were susceptible to FEP. The MICs for E. coli ATCC and P. aeruginosa ATCC fell within CLSI control ranges. DISCUSSION Optimal screening protocols for PABLs have yet to be defined. While some investigators (1, 8, 14, 15, 17, 26, 31) have used FOX for screening, the rare occurrence of FOX-susceptible strains harboring the -lactamase ACC-1 (3) makes this

4 3270 YAMASAKI ET AL. J. CLIN. MICROBIOL. Species (no. of strains) and strain no. RAPD type TABLE 2. Characteristics of the PABL-producing strains and selected clinical data PABL type Hospital Prefecture a Out/in b Ward c Source d Isolation date (mo/yr) Antibiogram e E. coli (17) A-1 a CMY-2 A H In Gastroenterology 6C Urine 2/08 GEN, MIN, LVX, SXT B-1 b CMY-2 B H In Gynecology 6W Pus 11/07 B-2 c CMY-2 B H In Emergency 8F Urine 11/07 MIN B-3 d CMY-2 B H Out Pediatrics Urine 2/08 B-4 e CMY-2 B H Out Urology Urine 2/08 GEN, SXT B-5 f CMY-2 B H Out Dermatology Pus 2/08 MIN, LVX, SXT C-2 g CMY-2 C K Out Surgery PTCD 1/07 MIN, SXT C-3 h CMY-2 C K In Surgery 4S Pus 1/07 LVX C-4 i CMY-2 C K In Hematology 7N Blood 11/07 SXT C-5 j CMY-2 C K In Surgery 3S Urine 11/07 SXT C-6 k CMY-2 C K In Urology 5S Urine 12/07 GEN D-1 l CMY-2 D O Out Internal medicine Urine 12/07 GEN, MIN, LVX, SXT E-1 m CMY-2 E O In Cardiac surgery 55 Artificial graft 2/08 SXT F-5 n CMY-2 F O In Surgery 5E Bile 1/09 LFX G-1 o CMY-2 G S In Surgery ICU Drain 4/08 MIN, LVX G-2 p CMY-2 G S In Neurosurgery 6W Urine 4/08 SXT H-1 q CMY-2 H W In Internal medicine 5H Urine 3/08 SXT K. pneumoniae (8) I-1 A CMY-8 I H Out Urology Urine 11/03 SXT J-1 A CMY-8 J N Out Internal medicine Joint liquid 4/05 SXT F-1 B DHA-1 F O In Respiratory medicine 6W Sputum 1/06 LVX F-2 B DHA-1 F O In Internal medicine 5S CVC 1/06 LVX F-3 B DHA-1 F O In Cardiology 3S Urine 2/07 LVX K-1 C DHA-1 K S In Urology 3B Urine 12/06 LVX, SXT L-2 D DHA-1 L S In Neurosurgery 9N Sputum 4/08 GEN, SXT H-2 E DHA-1 H W Out Urology Urine 2/08 GEN, LVX, SXT K. oxytoca (3) M-1 1 MOX-1 M O In Pediatric surgery 6S Throat 12/02 C-1 2 CMY-2 C K Out Surgery PTCD 1/07 F-4 3 CMY-2 F O In Cardiac surgery 55 Artificial graft 2/08 SXT P. mirabilis (1), L-1 CMY-2 L S In Cardiology 3W Urine 11/07 MIN, SXT a H, Hyogo; K, Kyoto; N, Nara; O, Osaka; S, Shiga; W, Wakayama. b Outpatient or inpatient. c ICU, intensive care unit. d CVC, central venous catheter; PTCD, extract from percutaneous transhepatic cholangial drainage. e AMK, amikacin (64 g/ml); GEN, gentamicin (16 g/ml); MIN, minocycline (16 g/ml); LVX, levofloxacin (8 g/ml); SXT, sulfamethoxazole-trimethoprim (76 and 4 g/ml, respectively). use of FOX unreliable. For this reason, our preliminary screen for PABL producers used as a criterion a MIC of 16 g/ml for the narrow-spectrum cephalosporin CFZ. However, no ACC-1 producers were detected in this study. We used the 3D test as the phenotypic test for PABL producers. The low proportion of positive 3D test results (25.7%; 310 of 1,206 strains) indicated that the preliminary CFZ screening method has low specificity. Plasmid-mediated class A -lactamases, such as TEM-1 and TEM-2, raise the MIC of CFZ, which could explain the low specificity obtained using CFZ. Although positive 3D test results were obtained for 290 (2.1%) of 13,995 E. coli isolates, only 17 of these E. coli isolates were PABL producers and only 2 were MBL producers (data not shown). The 271 remaining 3D test-positive E. coli isolates are probably hyperproducers of chromosomal AmpC. These data show that it is difficult to detect PABL-producing E. coli isolates by using phenotypic tests. On the other hand, 12 strains of K. pneumoniae, K. oxytoca, and P. mirabilis, which lack a native chromosomal ampc gene, gave positive 3D test results. Four of these strains were confirmed as PABL producers, and the remaining 8 strains were confirmed as MBL producers (data not shown). For 10 MBL producers, the addition of EDTA was inhibitory in the 3D test (data not shown), and this assessment was helpful in distinguishing between the AmpC and MBL phenotypes. In this study, we investigated the distribution and prevalence of PABL-producing Gram-negative rods with the cooperation of 16 clinical laboratories at large-scale general hospitals and one commercial clinical laboratory in the Kinki region of Japan. The prevalence of PABL producers in this study was found to be 0.13% overall (range, 0.02 to 0.5%). This rate of prevalence was much lower than that reported for other countries, such as Korea (3.1%) (26), China (2.8%) (14), and the United States (1.2%) (8). However, we isolated PABL producers from 13 of 17 laboratories in the Kinki region, suggesting that PABL producers may be on the increase. This is the first report on the prevalence of PABL producers among Enterobacteriaceae in the Kinki region of Japan. Nine of the 29 PABL-producing isolates were obtained from outpatients, but 7 of these 9 outpatients had been hospitalized

5 VOL. 48, 2010 SURVEY OF PABLs IN KINKI REGION OF JAPAN 3271 FIG. 2. RAPD fingerprinting of isolates investigated in this work, carried out with primer ERIC2. Lanes 1 and 2, the 2 CMY-8-producing K. pneumoniae isolates; lanes 3 to 8, the 6 DHA-1-producing K. pneumoniae isolates; lane 9, the single MOX-1-producing K. oxytoca isolate; lanes 10 and 11, the 2 CMY-2-producing K. oxytoca isolates; lanes M, 100-bp ladder. FIG. 1. Multiplex PCR results for PABL. (a) Lanes 1 to 6, positive controls for MOX-1, LAT-1, DHA-1, a gene native to H. alvei, ACT-1, and FOX-1, respectively; lane 7, negative control; lane 8, a CMY-2producing E. coli strain; lane 9, a DHA-1-producing K. pneumoniae strain; lane 10, a CMY-8-producing K. oxytoca strain; lanes M, 100-bp DNA ladder. (b and b ) Lanes 1 and 13, positive control for LAT-1; lanes 2 and 14, negative control; lanes 3 to 12 and 15 to 21, the 17 CMY-2-producing E. coli isolates; lanes 22 and 23, CMY-2-producing K. oxytoca strains; lane 24, a CMY-2-producing P. mirabilis strain; lanes M, 100-bp DNA ladder. (c) Lane 1, positive control for DHA-1; lane 2, negative control; lanes 3 to 8, the 6 DHA-1-producing K. pneumoniae isolates; lane 9, positive control for MOX-1; lane 10, negative control; lane 11, the single MOX-1-producing K. pneumoniae isolate; lanes 12 and 13, the 2 CMY-8-producing K. pneumoniae isolates; lanes M, 100-bp DNA ladder. within the past 2 years. Therefore, it is possible that PABL producers have disseminated from hospitals into the community. CMY-2 PABLs were detected in 17 (0.12%) of 13,995 E. coli isolates, 2 (0.12%) of 1,722 K. oxytoca isolates, and 1 (0.08%) of 1,182 P. mirabilis isolates. CMY-2 PABLs were detected in isolates from 5 prefectures and in isolates from 9 out of 17 laboratories, suggesting the spread of CMY-2 in this region. The conjugation experiment revealed that most CMY-2 PABL-producing isolates were transferable to E. coli. Southern blotting confirmed that these blacmy-2 genes were located on large, self-transmissible plasmids. These results may reflect the spread of CMY-2 genes among clinical isolates of E. coli. Multiple isolates of CMY-2-producing E. coli from 7 hospitals all yielded different RAPD patterns, revealing that each E. coli isolate carrying CMY-2 was genetically unrelated to the others. This finding suggests that the dissemination of resistance plasmids was responsible for the spread of CMY-2 among E. coli strains. DHA-1 PABLs were detected in 6 (0.10%) of 5,970 K. pneumoniae isolates; CMY-8 PABLs were detected in 2 (0.03%) of 5,970 K. pneumoniae isolates; and a MOX-1 PABL was detected in 1 (0.06%) of 1,722 K. oxytoca isolates. Despite multiple attempts, resistance could not be transferred by conjugation from DHA-1, CMY-8, or MOX-1 PABL producers to a FIG. 3. PFGE of PABL-positive K. pneumoniae isolates that had identical RAPD patterns. Lanes 1 and 2, the 2 CMY-8-producing K. pneumoniae isolates; lanes 3 to 5, the 3 DHA-1-producing K. pneumoniae isolates; lanes M, PFGE marker.

6 3272 YAMASAKI ET AL. J. CLIN. MICROBIOL. FIG. 4. Results of agarose gel electrophoresis and Southern blotting of plasmid DNAs from CMY-2-producing isolates. (A) Agarose gel electrophoresis. (B) Southern blotting with the bla CMY-2 -specific probe. Lanes 1, strain B-1, a CMY-2-producing E. coli isolate; lanes 2, its corresponding E. coli DH5 transformant; lanes 3, E. coli CSH2 transconjugant derived from the transformant; lanes 4, strain C-1, a CMY-2-producing K. oxytoca isolate; lanes 5, its corresponding E. coli DH5 transformant; lanes 6, E. coli CSH2 transconjugant derived from the lane 5 transformant; lanes M, 2.5-kbp DNA ladder. recipient E. coli strain. These results suggest that the PABL gene-containing plasmids of these strains might have reduced or no transmissibility. Three DHA-1-producing K. pneumoniae isolates from 1 hospital had the same RAPD and PFGE patterns, even though they were obtained from different wards. Thus, this strain was likely dispersed by nosocomial infection. No PABL producers resistant to AMK were detected in this study, although PABL producers resistant to GEN, MIN, LVX, and SXT were detected at frequencies of 6 (20.7%), 7 (24.1%), 11 (37.9%), and 17(58.6%), respectively. Coresistances in association with PABL-related -lactam resistance were noted. The MIC of CTX was 2 g/ml for one CMY-2- producing K. oxytoca isolate. For 1 CMY-8-producing K. pneumoniae isolate, 1 MOX-1-producing K. oxytoca isolate, and 1 CMY-2-producing P. mirabilis isolate, CAZ MICs were low ( 2 g/ml). These low MICs probably reflect low copy numbers of the PABL gene. Twenty-seven non-esbl-producing strains (86.2%) among 29 PABL producers were susceptible to FEP in this study. Although PABL-producing isolates that coproduce ESBL have been rare in Japan, it is noteworthy that Species (no. of strains) and strain no. TABLE 3. Types of -lactamases produced and MICs of -lactams for PABL-producing strains -Lactamase type(s) MIC ( g/ml) a PIP CTX CAZ FEP FOX CMZ IPM MEM E. coli (17) A-1 CMY B-1 CMY B-2 CMY B-3 CMY B-4 CMY B-5 CMY C-2 CMY-2, CTX-M-like C-3 CMY C-4 CMY C-5 CMY C-6 CMY D-1 CMY E-1 CMY F-5 CMY G-1 CMY G-2 CMY H-1 CMY K. pneumoniae (8) I-1 CMY J-1 CMY F-1 DHA F-2 DHA F-3 DHA K-1 DHA L-2 DHA H-2 DHA-1, CTX-M-like K. oxytoca (3) M-1 MOX C-1 CMY F-4 CMY P. mirabilis (1), L-1 CMY E. coli ATCC P. aeruginosa ATCC a Abbreviations: PIP, piperacillin; CTX, cefotaxime; CAZ, ceftazidime; FEP, cefepime; FOX, cefoxitin; CMZ, cefmetazole; IPM, imipenem; MEM, meropenem.

7 VOL. 48, 2010 SURVEY OF PABLs IN KINKI REGION OF JAPAN 3273 nosocomial outbreaks of such ESBL- and PABL-coproducing isolates have been reported (29). Therefore, it is important to identify PABL-producing isolates, and physicians may need to consider avoiding treatment with cephalosporins. In conclusion, the predominant type of PABL occurring in the Kinki region is CMY-2, followed in prevalence by DHA-1 and CMY-8. MOX-1 PABLs were found to be insignificant. Because the number of PABL producers detected in our study is small, further investigations will be necessary in order to monitor the spread of these antibiotic resistance genes. ACKNOWLEDGMENTS The 5 DNA samples from bla MOX-1, bla DHA-1, the gene native to H. alvei, bla ACT-1, and bla FOX-1 were kind gifts from N. D. Hanson (Department of Medical Microbiology and Immunology, Creighton University School of Medicine, Omaha, NE). E. coli strain CSH2 was the kind gift of Y. 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