J Med Microbiol 54 (2005), 1065-1070; DOI: 10.1099/jmm.0.46194-0
© 2005 Society for General Microbiology
ISSN 0022-2615
Metallo-ß-lactamase IMP-1 in Providencia rettgeri from two different hospitals in Japan
Katsuaki Shiroto1,
Yoshikazu Ishii1,
Soichiro Kimura1,
Jimena Alba1,
Kiwao Watanabe2,
Yoshiko Matsushima3 and
Keizo Yamaguchi1
1Department of Microbiology and Infectious Diseases, Toho University School of Medicine, 5-21-16 Omori-nishi, Ota-ku, 143-8540 Tokyo, Japan 2Institute of Tropical Medicine, Nagasaki University, Japan 3Department of Clinical Laboratory, Mie University Hospital, Japan
Correspondence Yoshikazu Ishii yoishii{at}med.toho-u.ac.jp
Received June 7, 2005
Accepted July 23, 2005
In 2002, 495 indole-positive proteae strains were isolated from patients at 60 hospitals in Japan. Nine indole-positive proteae strains had reduced susceptibility to imipenem (MIC 
8 µg ml–1) and were identified as Providencia rettgeri by BD Phoenix. Eight of the nine Prov. rettgeri isolates were confirmed as metallo-ß-lactamase producers by the double-disc synergy test. All the metallo-ß-lactamases were classified as IMP-1 by PCR and DNA sequence analysis. These blaIMP–1 genes were encoded in the integron structure on conjugative plasmids. These plasmids could transfer from Prov. rettgeri clinical isolates to Escherichia coli ML4903 at a frequency between 1.5 x 10–5 and 5.5 x 10–7. The eight blaIMP-positive strains were isolated from two hospitals, and showed two different PFGE patterns, two different integron structures and two different incompatibility groups, which corresponded to the two hospitals. These results strongly suggest the possibility of nosocomial infections by blaIMP–1-producing Prov. rettgeri isolates.
Abbreviations: DDST, double-disc synergy test; ESBL, extended-spectrum ß-lactamase; MßL, metallo-ß-lactamase.
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INTRODUCTION
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Proteae are normal inhabitants of the gut of animals including humans and are also found in the environment. Proteae were ranked as the fourth and fifth leading cause of urinary-tract infections in Europe and North America, respectively, in 1997 (Jones et al., 1999; Fluit et al., 2000). Normally, Proteus mirabilis, as the only indole-negative proteus, has a high susceptibility to antibiotics except for nitrofurantoin. Indole-positive proteae, such as Proteus vulgaris, Proteus penneri, Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii and Morganella morganii, are susceptible to expanded-spectrum cephalosporins, cefoxitin, cefepime, aztreonam, imipenem and aminoglycosides (Murray et al., 2003).
Nosocomial infections caused by extended-spectrum ß-lactamase (ESBL)-producing indole-positive proteae have been reported (Ma et al., 2002; Pagani et al., 2003; Tumbarello et al., 2004) in which the strains were resistant to most ß-lactams including expanded-spectrum cephalosporins. ESBL-producing Providencia species and Prot. vulgaris that produced CTX-M-type or SHV-type enzymes remained susceptible to carbapenems including imipenem. Tumbarello et al. (2004) described ESBL-producing multidrug-resistant Prov. stuartii. The strains were resistant to penicillins, cephalosporins, aminoglycosides and fluoroquinolones, but were susceptible to imipenem.
Shibata et al. (2003) reported metallo-ß-lactamase (MßL)-producing Prov. rettgeri and M. morganii that produced the IMP-1 enzyme. In the case of IMP-1-producing Pseudomonas aeruginosa and Serratia marcescens, the strains showed resistance to carbapenems such as imipenem, meropenem, panipenem, biapenem and doripenem; however, the results of drug susceptibility testing were not described. IMP-1 producers have no susceptibility to any ß-lactams except for aztreonam. The genes that encode these MßLs are located in an integron structure on a plasmid (Arakawa et al., 1995). An integron is one of the genetic elements capable of integrating gene cassettes by a site-specific recombination mechanism (Fluit & Schmitz, 2004). Therefore, horizontal spread of these resistance determinants can be anticipated.
In 2002, we conducted a surveillance programme involving 60 hospitals that were widely distributed geographically throughout Japan. The aim of the study was to gain a detailed understanding of ß-lactam antibiotic susceptibility data (Ishii et al., 2005). Of 495 indole-positive proteae isolates, nine, from two hospitals, showed reduced susceptibility to imipenem (MIC 8 µg ml–1). The aim of this study was to characterize the imipenem-resistance mechanism(s) and to investigate the possible clonal origins of the isolates.
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METHODS
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Bacterial strains.
In 2002 a total of 495 single strains of indole-positive proteae were isolated from patients at 60 hospitals in Japan that were widely distributed geographically (Ishii et al., 2005). Each participating laboratory performed its own identification tests. The species and number of isolated strains are listed in Table 1. Sources of the specimens isolated are described in Table 2.
Antimicrobial susceptibility testing.
Susceptibility testing of each isolate was performed by Etest strip (AB Biodisk) following the manufacturer's instruction manual. All clinical laboratories used Etest strips with the same lot number. The non-susceptible break point of indole-positive proteae against imipenem (MIC 8 µg ml–1) was based on that defined by the Clinical and Laboratory Standards Institute, formerly known as the National Committee for Clinical Laboratory Standards (NCCLS). Quality control of Etest strips was performed using the following reference strains: Staphylococcus aureus ATCC 21293, Escherichia coli ATCC 25922 and Ps. aeruginosa ATCC 27853. In addition, identification and susceptibility of all isolates collected were re-evaluated at the Department of Microbiology and Infectious Diseases, Toho University School of Medicine using the BD Phoenix system (Becton Dickinson).
Screening of MßL producers.
Strains selected by the criteria described above were subjected to a screening test for MßL production by using the double-disc synergy test (DDST) reported by Arakawa et al. (2000). The test was performed by placing a SMA (sodium mercaprotoacetic acid) disc containing 3 mg sodium mercaptoacetic acid (Eiken) and two commercially supplied Kirby–Bauer (KB) discs, each containing 30 µg ceftazidime or 10 µg imipenem (Eiken), on Mueller–Hinton agar plates. The distance between the two ceftazidime and imipenem discs was kept at about 3 cm, and the SMA disc was placed near one of the discs with a centre-to-centre distance of 1.5–2.0 cm. The plates were then incubated at 35 °C for 16–18 h. If the inhibition zone around the disc nearer to the SMA disc was bigger by more than 5 mm than that of ceftazidime or imipenem alone, the strain was considered to be an MßL producer.
PCR and sequencing of MßL genes.
The MßL gene cassettes and integrons were detected by a PCR method using previously described specific primer sets for blaIMP, blaVIM, blaSPM, intI1, intI2 and intI3 (Table 3). Template DNA from original strains and their transconjugants was used. PCR was performed in a GeneAmp PCR system 2400 thermal cycler (Applied Biosystem). The thermocycle protocol used was: an initial denaturation step at 94 °C for 2 min, followed by 25 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s and elongation at 72 °C for 90 s, plus a final extension step at 72 °C for 7 min. The resulting PCR product was purified with QIAquick PCR purification kit (Qiagen), prepared with ABI Prism Big Dye Terminator version 3.1 cycle sequencing ready reaction kit (Applied Biosystems) and sequenced with the automatic sequencer ABI Prism 310 genetic analyser (Applied Biosystems) using sequence specific primers for blaIMP and aacA4, one of the aminoglycoside acetyltransferases. A similarity search for the deduced amino acid sequences against sequence databases was done using the BLAST program at the DNA Database of Japan (Shizuoka, Japan).
Conjugation experiments.
Conjugation experiments were performed by the broth method using rifampicin-resistant E. coli ML4903 as a recipient (Ishii et al., 1995). A 0.9 ml : 0.1 ml mixture of exponentially growing donor and recipient isolates was incubated in 1 ml Mueller–Hinton broth at 35 °C for 1 h. E. coli transconjugants were selected on Drigalsky agar medium (BTB Agar, Eiken) containing ceftazidime (5 µg ml–1) and rifampicin (25 µg ml–1). Frequency of transfer was expressed as the number of transconjugants per number of donors. Susceptibility testing for donor, recipient and transconjugants was performed by the microdilution method according to the Clinical and Laboratory Standards Institute, formerly known as the NCCLS, document M7-A6 (NCCLS, 2003). Incompatibility tests were carried out as described in a previous report (Chabbert et al., 1972; Ishii et al., 1995). Briefly, the transconjugant was used as a donor and mixed with a recipient E. coli C600 that harboured plasmids of a known incompatibility group (Ishii et al., 1995). The conditions used were the same as for the conjugation experiments described above. Transconjugants were selected for resistance to ceftazidime and the resistance marker drugs of each plasmid.
Plasmid elimination.
Plasmid elimination was performed to prove that the resistance gene was on a transferable plasmid. The transconjugants of TUM1933 and TUM1936 were used as a recipient, and mixed with a donor E. coli C600 that harboured a plasmid with the same incompatibility group as the recipient. Conjugation conditions were the same as those mentioned above. Transconjugants were selected for resistance to rifampicin and the resistance marker drugs of each plasmid.
PFGE.
PFGE analysis was performed with a modified version of the instruction manual from Bio-Rad Laboratories. Agarose plugs containing genomic DNA were digested with SfiI (Bio-Rad Laboratories). Fragments were separated using SeaKem Gold Agarose gel (FMC Bioproducts) in 0.5 TBE buffer (0.089 M Tris base, 0.089 M boric acid and 0.002 M EDTA) at 14 °C for 20 h on a CHEF Mapper apparatus (Bio-Rad Laboratories). The banding patterns were evaluated by using Finger printing II DST software (Bio-Rad Laboratories) with Dice and UPGMA coefficients (Mariani-Kurkdjian et al., 2004). Isolates with a genetic similarity of > 80 % according to dendrogram results were considered to be from the same origin.
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RESULTS
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Nine of 495 indole-positive proteae clinical isolates were not susceptible to imipenem (MIC 8 µg ml–1) by Etest strip during evaluation at each medical centre, and confirmed at the Department of Microbiology and Infectious Diseases, Toho University School of Medicine. All nine isolates were identified as Prov. rettgeri by the BD Phoenix system. Eight of the nine isolates were positive on screening for MßL production by DDST. These isolates were from two hospitals, in Nagasaki prefecture and Mie prefecture. Six strains from one hospital were urine isolates from different patients. In the other hospital, the two IMP-1-producing Prov. rettgeri strains were isolated from sputum and blood from different patients (Table 4).
The types of MßL were assessed by PCR. Eight of the nine DDST-positive isolates were confirmed as carriers of IMP-1 type MßL, which was encoded by a gene located in a class 1 integron structure. The types of MßL were confirmed by sequencing analysis. PCR amplification of the variable region (located between the 5'- and 3'-conserved sequences) of the class 1 integron using the conjugative plasmids as the template yielded a 2.5 kb or 3.5 kb product. Sequence analysis of both the 2.5 kb and the 3.5 kb integrons revealed a structure with at least two gene cassettes containing blaIMP–1 and aacA4, which encodes resistance to tobramycin and amikacin. In the 2.5 kb product, the blaIMP–1 gene cassette was located immediately downstream of the 5'-CS and was followed by the aacA4 gene cassette. On the other hand, in the 3.5 kb product, the aacA4 gene cassette was located immediately downstream of the 5'-CS, and was followed by the blaIMP–1 gene cassette and another cassette, which did not code any protein.
The MßL genes of the eight isolates from two different hospitals were transferable to E. coli ML4903 at a frequency between 1.5 x 10–5 and 5.5 x 10–7. When production of MßL was checked by DDST for the E. coli transconjugants, all showed MßL production. Moreover, most ß-lactam MICs of the transconjugants were similar to those observed for Prov. rettgeri isolates, but the imipenem MICs (MIC 4 µg ml–1) tended to be lower than those of the donors (MIC 128 µg ml–1).
Incompatibility testing showed two groups. Group H1-harbouring strains were found in Nagasaki, and group T-harbouring strains were found in Mie. The transconjugants of TUM1933 and TUM1936 lost their plasmid when conjugated with E. coli C600. MICs of ß-lactam antibiotics for the products of this second conjugation were lower than those of the transconjugants of TUM1933 and TUM1936, which proved that the resistance gene was on a transferable plasmid. In addition, the MICs of the resistance marker drugs were the same as for E. coli C600 (Table 5). This result reflected the phenomenon of incompatibility. Two plasmids could not stably coexist in the same host when these plasmids had the same incompatibility groups.
The genetic similarity of the eight blaIMP–1-positive isolates was evaluated by using PFGE. Two types of PFGE patterns were observed (Fig. 1). The strains TUM1965, TUM1966, TUM1933, TUM1934, TUM1935 and TUM1967, isolated from the hospital of Nagasaki prefecture, and strains TUM1936 and TUM1937, from the hospital of Mie prefecture showed the same chromosomal DNA banding pattern.
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Fig. 1. PFGE profiles obtained with SfiI chromosomal digestion of Prov. rettgeri carrying the IMP-1 metallo-ß-lactamase.
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DISCUSSION
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The spread of nosocomial strains producing MßL has been reported around the world and is regarded as a serious clinical problem (Nordmann & Poirel, 2002; Walsh et al., 2005). MßL-encoding genes are located in an integron structure on a conjugative plasmid (Arakawa et al., 1995). These structures are one of the most important factors for multidrug-resistant bacteria because they can easily transfer from one strain to another, even to other species. Most MßL-producing strains show resistance not only to ß-lactams (except monobactams) but also to other antibacterial agents such as aminoglycosides, quaternary ammonium compounds, trimethoprim and sulfonamides because these resistance genes are located on the same integron structure (Laraki et al., 1999).
Recently, it has been reported that isolates of MßL-producing Ps. aeruginosa and Serratia marcescens probably have a high incidence (Kurokawa et al., 1999). The reported isolates were mainly isolated from immunocompromised hosts who had pre-existing conditions such as malignant diseases, and were caused by nosocomial spread (Hirakata et al., 1998). Among other Gram-negative bacteria, the isolation of MßL producers is also increasing. Shibata et al. (2003) reported that blaIMP–1, blaIMP–2 and blaVIM–2 as MßL genes were detected in Klebsiella oxytoca, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, M. morganii and Prov. rettgeri by PCR analysis. However, their study analysed only ceftazidime-resistant strains for ß-lactam resistance factors and integron structure.
In this study 495 indole-positive proteae strains were isolated. Among these, 54.9 % were M. morganii. It appears that this species is quite common in this part of the world, having a high incidence in Korea as well (Kim et al., 2003), even though it has been reported as rare in other places (Murray et al., 2003). Prot. vulgaris was second in frequency of isolation (25.7 %) in the current study and followed by Prov. rettgeri (12.5 %) (Table 1). Of the proteae, 43 % (213/495) were urinary-tract isolates (Table 2). Of the Prov. rettgeri isolates, 69.3 % (43/62) were isolates from the urinary tract. Proteae have been recognized as pathogens in urinary-tract infections, and the majority of these urinary-tract infections are a consequence of urinary-tract catheterization and instrumentation (Warren, 2001). Stickler et al. (1998) reported that Prov. rettgeri can form crystalline biofilms that rapidly encrust and block catheters. This study did not distinguish catheter specimens from other urine specimens. For future surveillance, it will be necessary to specify the origin and to evaluate biofilm formation as one of the pathogenic factors in this species.
The isolation frequency of MßL-producing Prov. rettgeri strains was 1.6 % (8/495). Kimura et al. (2005) reported an isolation frequency of MßL-producing Ps. aeruginosa of 1.9 % (11/594) in 2002 using strains from the same surveillance programme as the current study. It is of interest that no MßL-producing Prov. rettgeri were isolated from hospitals where MßL-producing Ps. aeruginosa were isolated. Eight MßL-producing Prov. rettgeri strains were isolated from only two hospitals, Mie and Nagasaki, which are separated by over 600 km. The genetic relatedness was evaluated by pulsed-field gel electrophoresis, integron structure and plasmid incompatibility group. These data show that the resistant Prov. rettgeri strains had two different origins, which coincided with the two different hospitals where they were isolated. The strains isolated in each hospital shared the same integron structure and also the same incompatibility group. These results strongly suggest that nosocomial infection by Prov. rettgeri occurred in the two different hospitals. Moreover, these blaIMP–1-encoding plasmids could transfer from Prov. rettgeri isolates to other species and their incompatibility groups could expand to other Enterobacteriaceae. This result suggests that the spread of this imipenem-resistance factor to other Enterobacteriaceae is not very difficult.
In conclusion, we report the finding of Prov. rettgeri isolates that harbour a conjugative plasmid containing an integron on which blaIMP–1 is encoded. Our results very strongly suggest that nosocomial infections by IMP-1-producing Prov. rettgeri occurred at two hospitals. IMP-1-producing Enterobacteriaceae could become a serious problem in the future. Thus, it is important to continue surveillance and monitoring of carbapenem resistance and reduced susceptibility Enterobacteriaceae including indole-positive proteae.
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ACKNOWLEDGEMENTS
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We thank the technical staff and directors in the 60 hospitals without whom this study could not have been performed. We thank Kenneth S. Thomson, Creighton University School of Medicine, for useful advice. This work was supported in part by a grant from Bristol-Myers K. K. and in part by grant from project research grant 16-3 and 16-6 from Toho University School of Medicine. This work was supported in part by two grants from the Ministry of Health, Labor and Welfare of Japan (H15-Shinkou-009 and H15-Shinkou-010). Jimena Alba is funded by the Japan Health Sciences Foundation. Soichiro Kimura is funded by the Japan Foundation for Emergency Medicine.
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INTRODUCTION
METHODS
