ABSTRACT
A meropenem-resistant Pseudomonas aeruginosa isolate was obtained from a patient in a medical setting in Hanoi, Vietnam. The isolate was found to have a novel IMP-type metallo-β-lactamase, IMP-51, which differed from IMP-7 by an amino acid substitution (Ser262Gly). Escherichia coli expressing blaIMP-51 showed greater resistance to cefoxitin, meropenem, and moxalactam than E. coli expressing blaIMP-7. The amino acid residue at position 262 was located near the active site, proximal to the H263 Zn(II) ligand.
TEXT
Metallo-β-lactamases (MBLs) confer resistance to all β-lactams, except for monobactams, and are characterized by their efficient hydrolysis of carbapenems (1). Acquired MBLs are produced by Gram-negative bacteria, including Pseudomonas aeruginosa, Acinetobacer spp., and enterobacteria (1). The acquired MBLs are categorized by their amino acid sequences into various types (2–4), including AIM (5), DIM (6), FIM (7), GIM (8), IMPs (9), KHM (10), NDMs (11), SMB (12), SIM (13), SPM (14), TMBs (15) and VIMs (16). The most prevalent types of MBLs are the IMP-, VIM-, and NDM-type enzymes (1, 2, 17). We describe here a novel IMP-type MBL, IMP-51, produced by a clinical isolate of P. aeruginosa in a medical setting in Vietnam.
The P. aeruginosa clinical isolate NCGM3025 was obtained from a sputum sample of a patient in 2013 in an intensive care unit in a medical setting in Hanoi, Vietnam. MICs of various antibiotics were determined using the microdilution method, according to the guidelines of the Clinical and Laboratory Standards Institute (18). IMP-type MBLs and an aminoglycoside modification enzyme, AAC(6′)-Ib, were detected using immunochromatographic assay kits (19, 20). DNA was extracted from the isolate using DNeasy blood and tissue kits (Qiagen, Tokyo, Japan), and the entire genome was sequenced by MiSeq (Illumina, San Diego, CA). Sequence data were analyzed using CLC Genomics Workbench version 8.0 (CLC bio, Tokyo, Japan). Multilocus sequence typing (MLST) was deduced as described by the protocols of the PubMLST databases (http://pubmlst.org/paeruginosa/). Sequences of drug resistance genes, including β-lactamase-encoding genes at the Lahey Clinic website (www.lahey.org/studies), aminoglycoside, chloramphenicol, and fosfomycin resistance genes registered in GenBank (http://www.ncbi.nlm.nih.gov/nuccore/), and quinolone resistance genes (21), were determined using CLC Genomics Workbench version 8.0.
Escherichia coli transformants expressing blaIMP-7 and blaIMP-51 were produced, and the recombinant IMP-7 and IMP-51 were purified as previously described (22). During the purification process, β-lactamase activity was monitored using nitrocefin (Oxoid Ltd., Basingstoke, United Kingdom). The initial rate of hydrolysis in 50 mM Tris-HCl (pH 7.4), 0.3 M NaCl, and 10 μM Zn(NO3)2 at 37°C was determined by UV-visible spectrophotometry (V-530; Jasco, Tokyo, Japan), with the reaction initiated by the addition of substrate into spectrophotometer cells, and UV absorption measured during the initial phase of the reaction. Km, kcat, and the kcat/Km ratio were determined using a Hanes-Woolf plot. Wavelengths and extinction coefficients were used for the analysis of β-lactam substrates (23–25). Km and kcat were determined using triplicate analyses.
A DNA plug of NCGM3025, digested with I-CeuI, was prepared, separated by pulsed-field gel electrophoresis, and subjected to Southern hybridization (26) using 16S rRNA and blaIMP-51 probes (12, 27).
P. aeruginosa NCGM3025 was resistant to all antibiotics tested, except for amikacin, colistin, and tigecycline. The isolate was susceptible to amikacin and intermediate to colistin and tigecycline. The MICs of β-lactams in NCGM3025 are shown in Table 1; the MICs of other antibiotics were 16 μg/ml for arbekacin, 16 μg/ml for amikacin, 1 μg/ml for colistin, 64 μg/ml for gentamicin, 16 μg/ml for ciprofloxacin, >1,024 μg/ml for fosfomycin, and 4 μg/ml for tigecycline. NCGM3025 was positive for IMP-type MBLs and AAC(6′)-Ib. Whole-genome sequencing revealed that the isolate had a novel blaIMP variant, designated blaIMP-51. Its predicted amino acid sequence revealed that IMP-51 differed from IMP-7 by an amino acid substitution (Ser262Gly) and from IMP-43 by two amino acid substitutions (Phe67Val and Ser262Gly). A phylogenetic tree showed that IMP-51 belonged to an IMP-7-like clade (Fig. 1). In addition to blaIMP-51, NCGM3025 had several drug resistance genes, including aac(6′)-Ib-cr, aac(6′)-Ib, aph(3′)-IIb, blaPAO, blaOXA-246, blaOXA-50, cmlA1, catB7, and fosA. The isolate had a point mutation in the quinolone-resistance-determining region of gyrA with an amino acid substitution of Ser83Ile in GyrA. The MLST of NCGM3025 was sequence type 235 (ST235).
MICs of β-lactams for P. aeruginosa NCGM3025 and E. coli transformants containing blaIMP-7 and blaIMP-51
Dendrogram of 45 IMP-type MBLs for comparison with IMP-51. The dendrogram was calculated with the Clustal W2 program. Branch lengths correspond to the number of amino acid exchanges for IMP-type enzymes.
E. coli DH5α, expressing blaIMP-7 or blaIMP-51, showed a significant reduction in susceptibility to all tested β-lactams, except for aztreonam, compared with DH5α expressing a vector control (Table 1). E. coli DH5α expressing blaIMP-51 showed 4-fold higher MICs of cefoxitin, meropenem, and moxalactam, 4-fold lower MICs of ampicillin, ampicillin-sulbactam, penicillin G, cefpirome, and ceftazidime, and 8-fold lower MICs of cephradine than E. coli DH5α expressing blaIMP-7 (Table 1).
Recombinant IMP-7 and IMP-51 hydrolyzed all tested β-lactams, except for aztreonam (Table 2). IMP-51 showed markedly higher kcat/Km ratios for cefmetazole, cefotaxime, cefoxitin, doripenem, meropenem, and moxalactam and lower kcat/Km ratios for ampicillin, penicillin G, cefpirome, ceftazidime, cephradine, imipenem, and panipenem. In particular, the higher kcat values of IMP-51 than those of IMP-7 for doripenem and meropenem resulted in the higher kcat/Km ratios for IMP-51 (Table 2). The kcat/Km values of IMP-51 against cefepime were similar to those of IMP-7 (Table 2).
Kinetic parameters of IMP-7 and IMP-51 enzymesa
The differences of the kcat/Km values between IMP-7 and IMP-51 were well-correlated to those of the MICs of antibiotics between E. coli expressing blaIMP-7 and E. coli expressing blaIMP-51. Compared with IMP-7, IMP-51, which showed higher kcat/Km ratios for cefotaxime, cefoxitin, doripenem, meropenem, and moxalactam, conferred higher MICs for these antibiotics in E. coli, whereas IMP-51, which showed lower kcat/Km ratios for ampicillin, penicillin G, cefpirome, ceftazidime, and cephradine, conferred lower MICs for these antibiotics in E. coli (Table 1 and 2).
The sequence surrounding blaIMP-51 was determined to be tnpA-tnpR-intI1-blaIMP-51-aac(6′)-Ib-aac(6′)-cmlA1-blaOXA-246 (9,797 bp), which was obtained from a contig assembled by Genomic Workbench. The blaIMP-51 gene was located within a class I integron, of which the downstream region was not determined because it was not contained in the sequence of the contig. The genetic structure that included blaIMP-51 had a unique gene cassette array and was located on the chromosome by Southern hybridization (data not shown). In the structure, tnpA-tnpR (nucleotide 1 [nt 1] to nt 5,059) was identical to the sequence of the Tn1403-like transposon in a plasmid pOZ176 from P. aeruginosa PA96 isolated in China (28). The cmlA1-blaOXA-246 (nt 7,321 to nt 9,786) was similar to a part of the DK45-2 class 1 integron (nt 669 to nt 3,134) in P. aeruginosa DK45 isolated in South Korea (GenBank accession number GQ853420). The blaOXA-246 was first identified in a plasmid from P. aeruginosa pae943 isolated in China (GenBank accession number EU886980).
The Ser262Gly substitution in IMP-51 markedly affected the catalytic activities of the enzyme against β-lactams, especially against carbapenems. IMP-51 had higher kcat/Km ratios against doripenem and meropenem but lower kcat/Km ratios against imipenem and panipenem than those of IMP-7. These differences in catalytic activities may explain the high resistance of NCGM3025 against doripenem and meropenem (Table 1). Similarly, IMP-6 with a Ser262Gly substitution had higher activity against meropenem and panipenem than that of IMP-1 (29). Residue 262 is located near the Zn(II) binding site, which plays an important role in β-lactam turnover catalyzed by IMP-type MBLs (30). The Ser262Gly substitution in IMP-6 compared with that in IMP-1 (29) was found to stabilize the anionic intermediate of certain β-lactam substrates bound to IMP-6, enhancing catalysis (31).
In conclusion, a doripenem- and meropenem-resistant P. aeruginosa isolate producing IMP-51 has emerged in Vietnam. The Ser262Gly amino acid substitution in IMP-51 appeared to significantly increase its hydrolytic activity for doripenem and meropenem. This substitution may have arisen due to the selective pressure caused by the use of doripenem and meropenem.
Nucleotide sequence accession number.The genomic environment surrounding blaIMP-51 was identified and deposited in GenBank under the accession number LC031883.
ACKNOWLEDGMENTS
This study was approved by the Bach Mai Hospital institutional review board (approval no. 38) and the Biosafety Committee at the National Center for Global Health and Medicine.
The study was supported by grants from International Health Cooperation Research (no. 27-S-1102 and 26-A-103) and a grant from the Research Program on Emerging and Reemerging Infectious Diseases from Japan Agency for Medical Research and Development (AMED).
FOOTNOTES
- Received 8 July 2015.
- Returned for modification 31 July 2015.
- Accepted 10 August 2015.
- Accepted manuscript posted online 17 August 2015.
- Copyright © 2015, American Society for Microbiology. All Rights Reserved.
