J Med Microbiol 57 (2008), 974-979; DOI: 10.1099/jmm.0.2008/001388-0
© 2008 Society for General Microbiology
ISSN 1473-5644
Potency of IMP-10 metallo-β-lactamase in hydrolysing various antipseudomonal β-lactams
Wei-Hua Zhao,
Zhi-Qing Hu and
Tadakatsu Shimamura
Department of Microbiology and Immunology, Showa University School of Medicine, Tokyo 142-8555, Japan
Correspondence
Wei-Hua Zhao
whzhao{at}med.showa-u.ac.jp
Received 19 February 2008
Accepted 31 March 2008
Limited β-lactams show antipseudomonal activity. The rapid spread of IMP-type metallo-β-lactamases (MBLs), which have a broad spectrum of substrates and a poor susceptibility to clinically available inhibitors, further restricts β-lactam use. In the present study, we evaluated the potency of IMP-10 MBL in hydrolysing antipseudomonal β-lactams currently available in the clinic. Crude IMP-10 MBL was prepared from two clinical isolates of Pseudomonas aeruginosa harbouring the blaIMP-10 gene. The sensitivity of β-lactams to hydrolysis by IMP-10 MBL was determined by comparing the MICs of 14 antipseudomonal β-lactams against a susceptible strain of P. aeruginosa in the presence and absence of IMP-10 MBL. Carbapenems (imipenem, meropenem and panipenem) and extended-spectrum cephems (ceftazidime, cefoperazone, cefsulodin and cefepime) were sensitive to the hydrolysing activity of IMP-10 MBL. By comparison, the fourth-generation cephem (cefpirome), the extended-spectrum penicillins (carbenicillin, ticarcillin, piperacillin and mezlocillin) and monobactams (aztreonam and carumonam) were relatively resistant to IMP-10 MBL. The sensitivity profile of antipseudomonal β-lactams to IMP-10 MBL generated in the present study provides a valuable reference for antibiotic selection by medical professionals.
Abbreviations: AZT, aztreonam; CAZ, ceftazidime; CBPC, carbenicillin; CFPM, cefepime; CFS, cefsulodin; CPR, cefpirome; CPZ, cefoperazone; CRMN, carumonam; IPM, imipenem; MBL, metallo-β-lactamase; MEPM, meropenem; MZPC, mezlocillin; PAPM, panipenem; PCase, penicillinase; PIPC, piperacillin; TIPC, ticarcillin.
 |
INTRODUCTION
|
|---|
Pseudomonas aeruginosa is intrinsically resistant to a large range of antibiotics and is one of the most common pathogens in nosocomial infections and microbial substitutions. Limited β-lactam antibiotics have antipseudomonal activity; these include the extended-spectrum penicillins (carbenicillin, ticarcillin, piperacillin, mezlocillin and azlocillin), extended-spectrum cephems (ceftazidime, cefoperazone, cefsulodin, cefepime and cefpirome), carbapenems (imipenem, meropenem and panipenem) and monobactams (aztreonam and carumonam) (Gilbert et al., 2005).
Carbapenems are highly resistant to most β-lactamases with the exception of the carbapenemases (Bush et al., 1995; Rasmussen & Bush, 1997; Livermore & Woodford, 2000). Two types of carbapenemases have been described: serine β-lactamases and metallo-β-lactamases (MBLs) (Frere, 1995; Livermore, 1998). MBLs have a poor susceptibility to clinically available inhibitors, such as clavulanate and sulbactam (Livermore, 1998). EDTA and mercaptoacetate are only used as specific inhibitors of MBL in vitro (Goto et al., 1997). Several types of MBLs including IMP, VIM, SPM-1 and GIM-1 have been identified (Walsh et al., 2005). Of these, the IMP-type MBLs are the most common and exhibit a worldwide distribution (Ito et al., 1995; Bush, 1998; Nordmann & Poirel, 2002; Zhao et al., 2007). IMP-1 MBL has been identified primarily in strains of P. aeruginosa and Serratia marcescens in Japan (Watanabe et al., 1991; Osano et al., 1994). Today, 18 types of blaIMP have been identified worldwide from a variety of clinical isolates in the Enterobacteriaceae (Walsh et al., 2005). In geographically diverse regions of Japan, approximately 1.9 % of clinical isolates of P. aeruginosa have acquired MBL, and 90 % of these are the IMP-1 type (Kimura et al., 2005). blaIMP-10 is a point mutation derivative of blaIMP-1 with a single base replacement of G by T at nucleotide 145, which leads to an amino acid alteration of Val49 to Phe (Iyobe et al., 2002). We identified 21 imipenem-resistant clinical isolates of P. aeruginosa from unrelated inpatients at our university hospital in 2007. Among the 21 isolates, seven strains (33 %) harboured blaIMP-10. We also identified two strains harbouring blaIMP-10 from seven IMP-type MBL-producing clinical isolates of S. marcescens collected at our university hospital between 1996 and 2002.
Here we present a profile of IMP-10 MBL potency in hydrolysing the antipseudomonal β-lactams currently available in the clinic.
|
METHODS
|
|---|
Bacterial strains and media.
Twenty-one imipenem-resistant P. aeruginosa isolates from unrelated inpatients at Showa University Hospital in 2007 were used. P. aeruginosa P11-2820, a clinical isolate susceptible to antipseudomonal β-lactams, and Escherichia coli ATCC 25922, a reference strain used in susceptibility tests recommended by the National Committee for Clinical Laboratory Standards (NCCLS, 2000), were used for MIC determination. Mueller–Hinton (MH) broth (Becton Dickinson) supplemented with 25 mg Ca2+ l–1 and 12.5 mg Mg2+ l–1 was used for bacterial cell culture and antibiotic susceptibility testing.
Reagents.
The following reagents were purchased from the indicated commercial sources: imipenem (IPM), mezlocillin (MZPC) and aztreonam (AZT) (US Pharmacopeia); piperacillin (PIPC), ceftazidime (CAZ) and meropenem (MEPM) (LKT Laboratories); panipenem (PAPM) (Sankyo Organic Chemicals); ticarcillin (TIPC), cefoperazone (CPZ) and penicillinase (PCase) from Bacillus cereus (Sigma); carbenicillin (CBPC), sodium mercaptoacetate and clavulanate (Wako Pure Chemical Industries); carumonam (CRMN) (Takeda Pharmaceutical); cefsulodin (CFS) (MP Biomedicals); cefepime (CFPM) (Bristol-Myers Squibb); cefpirome (CPR) (Sanofi-Aventis); and nitrocefin (Oxoid).
Gene detection by PCR amplification and DNA sequencing.
The presence of the blaIMP-1-like gene was identified by PCR analysis using the specific primers 1241 F 5'-CTA CCG CAG CAG AGT CTT TG-3' and 1828 R 5'-AAC CAG TTT TGC CTT ACC AT-3' (Arakawa et al., 1995), and a 588 bp fragment was amplified. The amplicons were purified using the QIAquick PCR purification kit and sequenced on an Applied Biosystems 3730xl DNA analyser (Applied Biosystems).
Preparation of IMP-10 MBL.
P. aeruginosa P0717 and P0706, which are positive for the blaIMP-10 gene but negative for blaVIM-1,2, blaTEM-1 and blaSHV-1, were used as a source of IMP-10 MBL. The two strains were cultured in MH broth until an OD600 of 0.5–0.7 units was reached. The bacterial cells were disrupted by sonication and insoluble cell debris was removed by centrifugation. The supernatants were collected, sterilized via filtration, and used as the crude IMP-10 MBL preparation. Enzyme activity was assayed using nitrocefin as a substrate, as described previously (O'Callaghan et al., 1972; Zhao et al., 2002).
Determination of IMP-10 MBL activity in hydrolysing β-lactams.
The MIC of the antibiotic was determined by the broth microdilution method based on the guidelines of the NCCLS (2000). The β-lactam hydrolysing activity of β-lactamases was evaluated by adding a crude IMP-10 MBL extract to MH broth containing twofold serial dilutions of β-lactam. Bacterial cells (5x105 ml–1) were then inoculated into the MH broth and cultured at 35 °C for 18 h. An increase in the MIC in the presence of IMP-10 MBL indicates β-lactamase hydrolysing activity. The hydrolysing activity of PCase was determined in parallel as a control.
Data presentation.
All experiments were performed three times or more to confirm repeatability.
|
RESULTS AND DISCUSSION
|
|---|
Sensitivity of carbapenems to IMP-10 MBL
The carbapenem hydrolysing activity of IMP-10 MBL was evaluated by adding a crude enzyme extract from P. aeruginosa P0717 to serial dilutions of the carbapenems IPM, MEPM and PAPM in MH broth. The MIC against P. aeruginosa P11-2820 was determined. The addition of IMP-10 MBL (5 U ml–1) increased the MIC of IPM, MEPM and PAPM from 2, 0.25 and 16 µg ml–1 to 32 (16-fold), 128 (512-fold) and 64 µg ml–1 (4-fold), respectively (Fig. 1a
). As a control, the addition of PCase (5 U ml–1) increased the MIC of IPM, MEPM and PAPM 4-, 16- and 2-fold, respectively. The increase in MIC over controls indicates that IPM, MEPM and PAPM are hydrolysed by IMP-10 MBL.
View larger version (16K):
[in this window]
[in a new window]
|
Fig. 1. Sensitivity of antipseudomonal β-lactams to IMP-10 MBL. (a) Carbapenems; (b) extended-spectrum cephems; (c) extended-spectrum penicillins; (d) monobactams. Symbols in (d) apply also to (a), (b) and (c). IMP-10 MBL at 5 U ml–1 was added in all the experiments; 5 U PCase ml–1 was added in the experiments of (a), (b) and (d), while 0.05 U PCase ml–1 was added in the experiments of (c). IPM, imipenem; MEPM, meropenem; PAPM, panipenem; CAZ, ceftazidime; CPZ, cefoperazone; CFS, cefsulodin; CFPM, cefepime; CPR, cefpirome; CBPC, carbenicillin; TIPC, ticarcillin; PIPC, piperacillin; MZPC, mezlocillin; AZT, aztreonam; CRMN, carumonam.
|
|
Sensitivity of extended-spectrum cephems to IMP-10 MBL
P. aeruginosa P11-2820 was susceptible to the third-generation (CAZ, CPZ and CFS) and fourth-generation (CFPM and CPR) cephems with MICs of 2–8 µg ml–1. The addition of IMP-10 MBL (5 U ml–1) increased the MIC to 16 (8-fold), 128 (16-fold), 64 (32-fold) and 32 µg ml–1 (4-fold) for CAZ, CPZ, CFS and CFPM, respectively, while the MIC of CPR remained unchanged (Fig. 1b
). As a control, the addition of PCase (5 U ml–1) increased the MIC of CPZ to 512 µg ml–1 (64-fold) and that of CFS to 8 µg ml–1 (4-fold), while the MIC of CAZ, CFPM and CPR remained unchanged. These data indicate that the third-generation cephems (CAZ, CPZ and CFS) and a fourth-generation cephem, CFPM, are sensitive to IMP-10 MBL, while another fourth-generation cephem, CPR, is resistant to IMP-10 MBL.
Sensitivity of extended-spectrum penicillins to IMP-10 MBL
The MIC of the penicillins CBPC, TIPC, PIPC and MZPC against P. aeruginosa P11-2820 was 4–32 µg ml–1. The addition of IMP-10 MBL (5 U ml–1) failed to increase the MIC of these penicillins (Fig. 1c). By comparison, PCase at 0.05 U ml–1 significantly increased the MIC of these penicillins to 256–1024 µg ml–1. These results suggest that the extended-spectrum penicillins CBPC, TIPC, PIPC and MZPC are resistant to IMP-10 MBL.
Sensitivity of monobactams to IMP-10 MBL
P. aeruginosa P11-2820 was also susceptible to the monobactams AZT and CRMN, with a MIC of 4 µg ml–1. Both IMP-10 MBL and PCase at 5 U ml–1 failed to increase the MIC of these monobactams, indicating that AZT and CRMN are resistant to IMP-10 MBL and PCase (Fig. 1d).
The outer-membrane barrier and the MexAB–OprM efflux system also affect the susceptibility of P. aeruginosa to β-lactams (Li et al., 2000). The effect of these factors on the MIC of β-lactams was assessed by testing E. coli ATCC 25922 in parallel (Table 1). The trends of the MIC changes in E. coli ATCC 25922 were consistent with those of P. aeruginosa P11-2820, indicating that IMP-10 MBL induces the observed changes in the MIC. Similar results were obtained using an additional IMP-10 MBL preparation, extracted from P. aeruginosa P0706.
View this table:
[in this window]
[in a new window]
|
Table 1. Sensitivity of antipseudomonal β-lactams to IMP-10 MBL
IPM, imipenem; MEPM, meropenem; PAPM, panipenem; CAZ, ceftazidime; CPZ, cefoperazone; CFS, cefsulodin; CFPM, cefepime; CPR, cefpirome; CBPC, carbenicillin; TIPC, ticarcillin; PIPC, piperacillin; MZPC, mezlocillin; AZT, aztreonam; CRMN, carumonam.
|
|
In this study, we used a crude preparation of IMP-10 MBL to evaluate the potency of IMP-10 MBL. To exclude the possibility of serine-type carbapenemases mixed in the crude preparation, inhibition testing of β-lactamase activity was performed. More than 90 % of the activity in the crude β-lactamase preparation (2.5 U ml–1) was inhibited by 3 µM sodium mercaptoacetate, a specific MBL inhibitor, while less than 0.5 % of the activity was inhibited by clavulanate, a specific serine-type β-lactamase inhibitor, indicating that possible effects of non-MBL in the preparation can be ignored. It is known that blaAmpC and blaOXA-50 are two naturally encoded β-lactamase genes in P. aeruginosa (Lodge et al., 1990; Stover et al., 2000). The expression of chromosomal ampC genes is mainly induced when the bacteria are exposed to some β-lactams as inducers (Hanson & Sanders, 1999). We did not add any inducers when the MBL was prepared. In addition, we used a strain of blaAmpC+/blaIMP– P. aeruginosa as a control to exclude the possible effect of AmpC. The effect of OXA-50 can also be excluded because PIPC is one of the restricted substrates of OXA-50 (Girlich et al., 2004), while the crude preparation in our experiment did not hydrolyse PIPC (Fig. 1c).
The efficiencies of IMP-10 MBL for hydrolysing some β-lactams have been reported by another group using purified IMP-10 MBL (Iyobe et al., 2002). The Kcat/Km values indicate that IPM, MEPM and CAZ are sensitive to IMP-10 MBL, while CBPC and PIPC are relatively resistant. These results are consistent with our data, which were obtained using a simple microbiological test, in the presence and absence of crude IMP-10 MBL.
The hydrolysing activities of IMP-10 for penicillins, except for carbenicillin, were very low compared to those of IMP-1, indicating that a single amino acid alteration, resulting from a point mutation, caused a decrease in penicillin-hydrolysing activity (Iyobe et al., 2002). Compared to the frequent reports on blaIMP-1, there are very limited reports on blaIMP-10. The possibility cannot be excluded that some blaIMP-10 strains might be misinterpreted as blaIMP-1 strains based on PCR analysis alone. Sequencing analyses of PCR products are essential to distinguish blaIMP-10 from blaIMP-1.
In conclusion, the carbapenems (IPM, MEPM and PAPM), the third-generation cephems (CAZ, CPZ and CFS) and a fourth-generation cephem, CFPM, are sensitive to IMP-10 MBL. By comparison, another fourth-generation cephem, CPR, the extended-spectrum penicillins (CBPC, TIPC, PIPC and MZPC) and the monobactams (AZT and CRMN) are relatively resistant to IMP-10 MBL. The present study has generated a comprehensive profile of antipseudomonal β-lactam sensitivity to IMP-10 MBL, thereby providing a valuable reference for antibiotic selection by medical professionals.
|
ACKNOWLEDGEMENTS
|
|---|
This study was partially supported by a grant from Showa University.
|
REFERENCES
|
|---|
Arakawa, Y., Murakami, M., Suzuki, K., Ito, H., Wacharotayankun, R., Ohsuka, S., Kato, N. & Ohta, M. (1995). A novel integron-like element carrying the metallo-β-lactamase gene blaIMP. Antimicrob Agents Chemother 39, 1612–1615.[Abstract]
Bush, K. (1998). Metallo-β-lactamases: a class apart. Clin Infect Dis 27 (Suppl. 1), S48–S53.[Medline]
Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for β-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 39, 1211–1233.[Medline]
Frere, J.-M. (1995). Beta-lactamases and bacterial resistance to antibiotics. Mol Microbiol 16, 385–395.[Medline]
Gilbert, D. N., Moellering, R. C., Eliopoulos, G. M. & Sande, M. A. (2005). The Sanford Guide to Antimicrobial Therapy 2005, 35th edn. Hyde Park, VT: Antimicrobial Therapy, Inc.
Girlich, D., Naas, T. & Nordmann, P. (2004). Biochemical characterization of the naturally occurring oxacillinase OXA-50 of Pseudomonas aeruginosa. Antimicrob Agents Chemother 48, 2043–2048.[Abstract/Free Full Text]
Goto, M., Takahashi, T., Yamashita, F., Koreeda, A., Mori, H., Ohta, M. & Arakawa, Y. (1997). Inhibition of the metallo-beta-lactamase produced from Serratia marcescens by thiol compounds. Biol Pharm Bull 20, 1136–1140.[Medline]
Hanson, N. D. & Sanders, C. C. (1999). Regulation of inducible AmpC beta-lactamase expression among Enterobacteriaceae. Curr Pharm Des 5, 881–894.[Medline]
Ito, H., Arakawa, Y., Ohsuka, S., Wacharotayankun, R., Kato, N. & Ohta, M. (1995). Plasmid-mediated dissemination of the metallo-β-lactamase gene blaIMP among clinically isolated strains of Serratia marcescens. Antimicrob Agents Chemother 39, 824–829.[Abstract]
Iyobe, S., Kusadokoro, H., Takahashi, A., Yomoda, S., Okubo, T., Nakamura, A. & O'Hara, K. (2002). Detection of a variant metallo-β-lactamase, IMP-10, from two unrelated strains of Pseudomonas aeruginosa and an Alcaligenes xylosoxidans strain. Antimicrob Agents Chemother 46, 2014–2016.[Abstract/Free Full Text]
Kimura, S., Alba, J., Shiroto, K., Sano, R., Niki, Y., Maesaki, S., Akizawa, K., Kaku, M., Watanuki, Y. & other authors (2005). Clonal diversity of metallo-β-lactamase-possessing Pseudomonas aeruginosa in geographically diverse regions of Japan. J Clin Microbiol 43, 458–461.[Abstract/Free Full Text]
Li, X.-Z., Zhang, L. & Poole, K. (2000). Interplay between the MexA–MexB–OprM multidrug efflux system and the outer membrane barrier in the multiple antibiotic resistance of Pseudomonas aeruginosa. J Antimicrob Chemother 45, 433–436.[Abstract/Free Full Text]
Livermore, D. M. (1998). β-Lactamase-mediated resistance and opportunities for its control. J Antimicrob Chemother 41 (Suppl. D), 25–41.[Abstract/Free Full Text]
Livermore, D. M. & Woodford, N. (2000). Carbapenemases: a problem in waiting? Curr Opin Microbiol 3, 489–495.[CrossRef][Medline]
Lodge, J. M., Minchin, S. D., Piddock, L. J. & Busby, J. W. (1990). Cloning, sequencing and analysis of the structural gene and regulatory region of the Pseudomonas aeruginosa chromosomal ampC beta-lactamase. Biochem J 272, 627–631.[Medline]
NCCLS (2000). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 5th edn. Document M7-A5. Wayne, PA: National Committee for Clinical Laboratory Standards.
Nordmann, P. & Poirel, L. (2002). Emerging carbapenemases in Gram-negative aerobes. Clin Microbiol Infect 8, 321–331.[CrossRef][Medline]
O'Callaghan, C. H., Morris, A., Kirby, S. M. & Shingler, A. H. (1972). Novel method for detection of β-lactamases by using a chromogenic cephalosporin substrate. Antimicrob Agents Chemother 1, 283–288.[Abstract/Free Full Text]
Osano, E., Arakawa, Y., Wacharotayankun, R., Ohta, M., Horii, T., Ito, H., Yoshimura, F. & Kato, N. (1994). Molecular characterization of an enterobacterial metallo β-lactamase found in a clinical isolate of Serratia marcescens that shows imipenem resistance. Antimicrob Agents Chemother 38, 71–78.[Abstract/Free Full Text]
Rasmussen, B. A. & Bush, K. (1997). Carbapenem-hydrolyzing β-lactamases. Antimicrob Agents Chemother 41, 223–232.[Medline]
Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S., Hufnagle, W. O., Kowalik, D. J. & other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964.[CrossRef][Medline]
Walsh, T. R., Toleman, M. A., Poirel, L. & Nordmann, P. (2005). Metallo-β-lactamases: the quiet before the storm? Clin Microbiol Rev 18, 306–325.[Abstract/Free Full Text]
Watanabe, M., Iyobe, S., Inoue, M. & Mitsuhashi, S. (1991). Transferable imipenem resistance in Pseudomonas aeruginosa. Antimicrob Agents Chemother 35, 147–151.[Abstract/Free Full Text]
Zhao, W.-H., Hu, Z.-Q., Hara, Y. & Shimamura, T. (2002). Inhibition of penicillinase by epigallocatechin gallate resulting in restoration of antibacterial activity of penicillin against penicillinase-producing Staphylococcus aureus. Antimicrob Agents Chemother 46, 2266–2268.[Abstract/Free Full Text]
Zhao, W.-H., Hu, Z.-Q., Chen, G., Matsushita, K., Fukuchi, K. & Shimamura, T. (2007). Characterization of imipenem-resistant Serratia marcescens producing IMP-type and TEM-type β-lactamases encoded on a single plasmid. Microbiol Res 162, 46–52.[CrossRef][Medline]
This article has been cited by other articles:
|
|
|
|
 
Z. Hu and W.-H. Zhao
Identification of plasmid- and integron-borne blaIMP-1 and blaIMP-10 in clinical isolates of Serratia marcescens
J. Med. Microbiol.,
February 1, 2009;
58(2):
217 - 221.
[Abstract]
[Full Text]
[PDF]
|
|
|
TOP
INTRODUCTION
METHODS
