IMP-51, a Novel IMP-Type Metallo-β-Lactamase with Increased Doripenem- and Meropenem-Hydrolyzing Activities, in a Carbapenem-Resistant Pseudomonas aeruginosa Clinical Isolate

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 bla_IMP-51 showed greater resistance to cefoxitin, meropenem, and moxalactam than E. coli expressing bla_IMP-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 bla_IMP-7 and bla_IMP-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. K_m, k_cat, and the k_cat/K_m ratio were determined using a Hanes-Woolf plot. Wavelengths and extinction coefficients were used for the analysis of β-lactam substrates (23–25). K_m and k_cat 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 bla_IMP-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 bla_IMP variant, designated bla_IMP-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 bla_IMP-51, NCGM3025 had several drug resistance genes, including aac(6′)-Ib-cr, aac(6′)-Ib, aph(3′)-IIb, bla_PAO, bla_OXA-246, bla_OXA-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).

TABLE 1.

MICs of β-lactams for P. aeruginosa NCGM3025 and E. coli transformants containing bla_IMP-7 and bla_IMP-51

Antibiotic(s)a	MIC (μg/ml) of antibiotic for:
	P. aeruginosa NCGM3025	E. coli DH5α(pHSG398/IMP-7)	E. coli DH5α(pHSG398/IMP-51)	E. coli DH5α(pHSG398)
Ampicillin	>1,024	128	32	8
Ampicillin-sulbactam	512	64	8	4
Penicillin G	>1,024	128	32	32
Aztreonam	32	0.063	0.063	0.063
Cefepime	256	8	8	0.063
Cefotaxime	1,024	32	64	0.031
Cefoxitin	>1,024	512	>2,048	16
Cefozopran	256	16	8	0.125
Cefpirome	32	2	0.5	≤0.007
Ceftazidime	256	512	128	0.5
Ceftriaxone	>1,024	64	128	0.031
Cephradine	>1,024	512	64	16
Doripenem	256	2	4	0.031
Imipenem	16	0.25	0.25	0.031
Meropenem	512	1	4	0.015
Panipenem	16	0.25	0.25	0.063
Moxalactam	>1,024	256	1,024	0.125

^aThe ratio of ampicillin to sulbactam was 2:1.

FIG 1.

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 bla_IMP-7 or bla_IMP-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 bla_IMP-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 bla_IMP-7 (Table 1).

Recombinant IMP-7 and IMP-51 hydrolyzed all tested β-lactams, except for aztreonam (Table 2). IMP-51 showed markedly higher k_cat/K_m ratios for cefmetazole, cefotaxime, cefoxitin, doripenem, meropenem, and moxalactam and lower k_cat/K_m ratios for ampicillin, penicillin G, cefpirome, ceftazidime, cephradine, imipenem, and panipenem. In particular, the higher k_cat values of IMP-51 than those of IMP-7 for doripenem and meropenem resulted in the higher k_cat/K_m ratios for IMP-51 (Table 2). The k_cat/K_m values of IMP-51 against cefepime were similar to those of IMP-7 (Table 2).

TABLE 2.

Kinetic parameters of IMP-7 and IMP-51 enzymes^a

β-Lactam	IMP-7			IMP-51
	Km (μM)b	kcat (s−1)b	kcat/Km (μM−1 s−1)	Km (μM)b	kcat (s−1)b	kcat/Km (μM−1 s−1)
Ampicillin	116 ± 18	8.5 ± 1.3	0.02	872 ± 153	3.5 ± 0.6	0.004
Penicillin G	212 ± 18	17.5 ± 1.3	0.081	976 ± 188	4.6 ± 0.7	0.0048
Aztreonam	NHc	NH	NH	NH	NH	NH
Cefepime	58 ± 3	1.2 ± 0.1	0.020	56 ± 4	1.4 ± 0.1	0.025
Cefmetazole	47 ± 5	3.7 ± 0.1	0.078	1.8 ± 0.4	2.78 ± 0.01	1.5
Cefotaxime	12 ± 2	1.7 ± 0.1	0.15	5.7 ± 1.8	4.4 ± 0.2	0.93
Cefoxitin	120 ± 13	4.9 ± 0.2	0.041	2.2 ± 0.6	1.91 ± 0.02	0.88
Cefpirome	57 ± 5	2.0 ± 0.1	0.035	182 ± 25	3.4 ± 0.4	0.019
Ceftazidime	19 ± 3	0.34 ± 0.01	0.018	35 ± 4	0.03 ± 0.01	0.0085
Cephradine	55 ± 8	12 ± 1	0.22	75 ± 21	0.80 ± 0.08	0.011
Doripenem	46 ± 7	2.7 ± 0.2	0.059	61 ± 7	10.7 ± 0.4	0.18
Imipenem	104 ± 13	5.0 ± 0.2	0.048	312 ± 29	5.5 ± 0.3	0.018
Meropenem	59 ± 8	0.99 ± 0.07	0.017	51 ± 8	2.7 ± 0.1	0.053
Panipenem	40 ± 5	4.0 ± 0.2	0.099	230 ± 7	10.6 ± 0.2	0.046
Moxalactam	57 ± 6	4.6 ± 0.2	0.081	24 ± 3	5.0 ± 0.1	0.21

^aThe proteins were initially modified by a His tag, which was removed after purification.

^bK_m and k_cat values represent the means ± standard deviations from three independent experiments.

^cNH, no hydrolysis was detected at substrate concentrations up to 1 mM and enzyme concentration up to 700 nM.

The differences of the k_cat/K_m values between IMP-7 and IMP-51 were well-correlated to those of the MICs of antibiotics between E. coli expressing bla_IMP-7 and E. coli expressing bla_IMP-51. Compared with IMP-7, IMP-51, which showed higher k_cat/K_m ratios for cefotaxime, cefoxitin, doripenem, meropenem, and moxalactam, conferred higher MICs for these antibiotics in E. coli, whereas IMP-51, which showed lower k_cat/K_m 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 bla_IMP-51 was determined to be tnpA-tnpR-intI1-bla_IMP-51-aac(6′)-Ib-aac(6′)-cmlA1-bla_OXA-246 (9,797 bp), which was obtained from a contig assembled by Genomic Workbench. The bla_IMP-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 bla_IMP-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-bla_OXA-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 bla_OXA-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 k_cat/K_m ratios against doripenem and meropenem but lower k_cat/K_m 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 bla_IMP-51 was identified and deposited in GenBank under the accession number LC031883.