Identification of a Novel NDM Variant, NDM-13, from a Multidrug-Resistant Escherichia coli Clinical Isolate in Nepal

Abstract

A novel New Delhi metallo-β-lactamase, NDM-13, was identified in a carbapenem-resistant Escherichia coli clinical isolate obtained from the urine of a patient in Nepal. The enzymatic activity of NDM-13 against β-lactams was similar to that of NDM-1. However, NDM-13 displayed significantly higher k_cat/K_m ratios for cefotaxime. The genetic environment of bla_NDM-13 was determined to be tnpA-IS30-bla_NDM-13-ble_MBL-trpF-dsbC-cutA-groES-groL, with bla_NDM-13 located within the chromosome.

TEXT

The emergence of metallo-β-lactamases (MBLs) and increased carbapenem resistance among Gram-negative pathogens has become a serious problem worldwide (1). MBLs, which are produced by many Gram-negative bacterial species (1) and by Gram-positive Bacillus spp. (2, 3), confer resistance or reduce bacterial susceptibility to carbapenems, cephalosporins, and penicillins, except for monobactams (1). NDM-1 was initially isolated in Sweden from Klebsiella pneumoniae and Escherichia coli in 2008 (4). Subsequently, 16 NDM variants (www.lahey.org/studies) have been reported in several countries (5–17).

E. coli IOMTU558 was isolated from a urine sample obtained from an inpatient at a university hospital in Nepal in 2013. The bacterial species was identified by biochemical analysis and confirmed by 16S rRNA sequencing (18). MICs were determined using the microdilution method (19). The MBL genes (bla_DIM, bla_GIM, bla_IMP, bla_NDM, bla_SIM, bla_SPM, and bla_VIM) were amplified by PCR (20, 21), and the sequence of the entire genome was determined by MiSeq (Illumina, San Diego, CA). Sequences of all drug resistance genes registered at the Lahey Clinic website (www.lahey.org/studies), including MBL genes, were determined using CLC genomics workbench version 5.5. Multilocus sequence typing (MLST) was performed according to protocols appropriate for E. coli sequences in the MLST database (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). E. coli transformants expressing bla_NDM genes were constructed as described previously (22), with bla_NDM-1, which originated from Pseudomonas aeruginosa IOMTU9 (17), used as the reference gene. Recombinant NDMs were purified as described previously (22), with β-lactamase activities during the purification process monitored using nitrocefin (Oxoid, Ltd., Basingstoke, United Kingdom). Initial rates of β-lactamase activities were determined as described previously (22), with some modifications. In the modified protocol, the substrate solutions were prewarmed to 37°C in a water bath to minimize fluctuations in the temperature of the reaction mixture. K_m values, k_cat values, and k_cat/K_m ratios were determined by analyzing β-lactam hydrolysis using Lineweaver-Burk plots. Previously reported wavelengths and extinction coefficients were used for analysis of β-lactam substrates (23–25). K_m and k_cat values were determined using triplicate analyses. A DNA plug, digested for 3 h with I-CeuI, was separated by pulsed-field gel electrophoresis and subjected to Southern hybridization as described previously (26). Probes for 16S rRNA and bla_NDM were prepared as described previously (27, 28).

The MICs of β-lactams for E. coli IOMTU558 are listed in Table 1. The MICs of the other antibiotics were as follows: amikacin, 16 μg/ml; arbekacin, 32 μg/ml; ciprofloxacin, >1,024 μg/ml; colistin, 1 μg/ml; fosfomycin, 8 μg/ml; gentamicin, 256 μg/ml; kanamycin, 256 μg/ml; levofloxacin, 32 μg/ml; minocycline, 4 μg/ml; tigecycline, 2 μg/ml; and tobramycin, 128 μg/ml. PCR showed that the isolate was positive for bla_NDM and negative for the other genes. Sequencing of the PCR product showed that it was a novel bla_NDM variant. This new variant was designated bla_NDM-13, and the sequence was deposited in GenBank. IOMTU558 belonged to sequence type 101 (ST101). Based on the predicted amino acid sequence, NDM-13 had two amino acid substitutions (D95N and M154L) compared to NDM-1 and one amino acid substitution each compared to NDM-3 (M154L) and NDM-4 (D95N). The isolate harbored bla_CTX-M-15, bla_TEM-166, and ampC. The promoter region of ampC (nucleotides [nt] −42 to −1) included a mutation at nt −18 (G to A), but there were no other mutations or nucleotide insertions compared with E. coli strain K-12. The isolate harbored ompC and ompF and their positive regulator, ompR. The ompR gene had no mutation compared with that of the K-12 strain. IOMTU588 harbored efflux pump genes (the acrAB-tolC operon), the marR repressor gene, and the gene (yedS) encoding the putative outer membrane protein. The marR gene had a mutation resulting in an amino acid substitution (Y137H).

TABLE 1.

MICs of various β-lactams for E. coli strain IOMTU558 and E. coli DH5α transformed with plasmids encoding NDM-1 or NDM-13

Antibiotic(s)	MIC (μg/ml) for:
	IOMTU558	DH5α(pHSG398/NDM-1)	DH5α(pHSG398/NDM-13)	DH5α(pHSG398)
Ampicillin	>1,024	256	512	4
Ampicillin-sulbactam	>1,024	256	256	4
Aztreonam	>1,024	≤0.063	≤0.063	≤0.063
Cefepime	>1,024	1	1	≤0.063
Cefoselis	>1,024	16	16	≤0.063
Cefotaxime	>1,024	16	64	≤0.063
Cefoxitin	>1,024	64	64	≤0.063
Cefpirome	>1,024	4	4	≤0.063
Ceftazidime	>1,024	256	512	≤0.063
Ceftriaxone	>1,024	16	32	≤0.063
Cephradine	>1,024	512	256	16
Doripenem	128	0.25	0.25	≤0.063
Imipenem	32	1	2	0.125
Meropenem	128	0.25	0.25	≤0.063
Moxalactam	>1,024	32	32	0.25
Penicillin G	>1,024	256	256	32

E. coli DH5α expressing bla_NDM-1 or bla_NDM-13 showed a significant reduction in susceptibility to all tested β-lactams, except for aztreonam, compared with the DH5α strain expressing a vector control (Table 1). E. coli DH5α expressing bla_NDM-13 showed a 4-fold-higher MIC of cefotaxime than E. coli DH5α expressing bla_NDM-1 (Table 1).

The recombinant NDM-1 and NDM-13 hydrolyzed all tested β-lactams, except for aztreonam (Table 2). The enzymatic activity profiles of NDM-13 against the tested β-lactams were similar to those of NDM-1; however, NDM-13 showed a higher k_cat/K_m ratio for cefotaxime, which may have been due to NDM-13 having a lower K_m value than NDM-1 (Table 2). The kinetic parameters for NDM-1 (Table 2) differed from those of our previous report (22). The modified protocol utilized in this study may have affected the results of the kinetic assays.

TABLE 2.

Kinetic parameters of NDM-1 and NDM-13 enzymes

β-Lactam	Result fora:
	NDM-1			NDM-13
	Km (μM)	kcat (s−1)	kcat/Km (μM−1 s−1)	Km (μM)	kcat (s−1)	kcat/Km (μM−1 s−1)
Ampicillin	76 ± 6	30 ± 0.6	0.40	140 ± 26	52 ± 3	0.38
Aztreonam	NHb	NH	NH	NH	NH	NH
Cefepime	129 ± 8	9.9 ± 0.4	0.077	83 ± 11	8.2 ± 0.6	0.099
Cefotaxime	49 ± 6	41 ± 2	0.85	22 ± 2	38 ± 1	1.7
Cefoxitin	13 ± 3	1.0 ± 0.03	0.076	12 ± 2	1.1 ± 0.1	0.093
Ceftazidime	54 ± 4	8.5 ± 0.2	0.16	30 ± 3	7.5 ± 0.3	0.25
Cephradine	14 ± 3	23 ± 1	1.8	28 ± 4	27 ± 1	0.98
Doripenem	39 ± 2	13 ± 0.3	0.34	49 ± 4	15 ± 1	0.31
Imipenem	56 ± 7	14 ± 1	0.26	71 ± 6	17 ± 1	0.24
Meropenem	21 ± 4	21 ± 1	1.1	37 ± 6	26 ± 1	0.73
Penicillin G	19 ± 6	19 ± 1	1.1	37 ± 5	49 ± 2	1.3

^aThe proteins were initially modified by a His tag, which was removed after purification. The K_m and k_cat values are means ± standard deviations from 3 independent experiments.

^bNH, no hydrolysis was detected under conditions with substrate concentrations up to 1 mM and enzyme concentrations up to 700 nM.

The genomic environment of bla_NDM-13 was tnpA-IS30-bla_NDM-13-ble_MBL-trpF-dsbC-cutA-groES-groL, and the sequence was deposited in GenBank. This genomic structure, except for bla_NDM-13, was identical to that of pPMK1 expressed by K. pneumoniae PMK1 (isolated in Nepal) (29), an Enterobacter hormaechei CCHB10892 plasmid (from Brazil) (30), pKPX-1 from K. pneumoniae KPX (from Taiwan) (31), and pNDM-MAR isolated from K. pneumoniae (from Morocco) (32, 33). However, the bla_NDM-13 gene was located on the chromosome (Fig. 1).

FIG 1.

Localization of the bla_NDM-13 gene on the chromosome of E. coli strain IOMTU558, separated by pulsed-field gel electrophoresis (PFGE). Lanes: A, contour-clamped homogeneous electric field (CHEF) DNA size marker (Saccharomyces cerevisiae; Bio-Rad, Hercules, CA); B, I-CeuI-digested total DNA of E. coli IOMTU558; C, lambda ladder pulsed-field gradient (PFG) marker (New England BioLabs, Ipswich, MA); D, hybridization of I-Ceu-I-digested total DNA of IOMTU558 with a 16S rRNA-specific probe; and E, hybridization of I-Ceu-I-digested total DNA of IOMTU558 using a bla_NDM-13-specific probe.

The two substitutions in NDM-13 compared to NDM-1 (i.e., D95N and M154L) increased the affinity of the enzyme for cefotaxime and affected the catalytic activity of the enzyme against this drug (Table 2). The D95N amino acid substitution was reported to reduce the k_cat values of NDM-3 compared with NDM-1 for all β-lactamases tested (28). An NDM-4 mutant containing M154L was found to have increased hydrolytic activity toward carbapenems and several cephalosporins compared with that of NDM-1 (12). NDM-13 with the D95N and M154L substitutions did not show increased hydrolytic activity against the tested carbapenems, cephalosporins, and penicillins, except for cefotaxime. Among the known NDM variants (NDM-1 to NDM-13), amino acid substitutions were observed at 13 amino acid positions: 28, 32, 36, 69, 74, 88, 95, 130, 152, 154, 200, 222, and 233. Positions 28, 32, and 36 are located in the signal peptide region. Positions 95, 130, and 154 have been reported to affect β-lactam hydrolyzing activity (12, 17, 28), although they are not located at the active site of NDM-1 or in amino acid residues that bind to zinc ions (34, 35). The effects of the remaining 10 substitutions on hydrolyzing activity have not yet been reported. Determination of the hydrolyzing activities of all NDM variants is critical to understanding the mechanisms underlying the molecular evolution of the NDMs.

The profile of β-lactam resistance in IOMTU558 (Table 1) could be explained by the presence of bla_NDM-13, as well as bla_CTX-M-15, bla_TEM-166, and ampC. Among these genes, bla_CTX-M-15 confers high resistance against most β-lactams (except carbapenems), including aztreonam, cefepime, cefotaxime, and ceftriaxone (36). ampC could not be overexpressed in the isolate. The β-lactam-susceptible E. coli wild-type strain has been found to express ampC at low levels due to degenerated promoter boxes upstream of ampC (37). Two mutations in the ampC promoter sequence, at positions −42 (C to T) and −18 (G to A), generate two alternative promoter boxes, resulting in high-level expression of ampC (37). IOMTU558 had a mutation at position −18 (G to A) but not at position −42. It is unclear whether the mutation at position −18 alone could affect the ampC expression level in IOMTU558. The other mechanisms of β-lactam resistance are decreased cell permeability due to loss or alteration of the ompC and ompF porins and activation of efflux systems (38). Loss or alteration of porins was not detected in IOMTU558. The marR repressor gene of marAB and the yedS gene encoding a putative outer membrane protein were reported to be involved in carbapenem resistance via downregulation of efflux pump genes (the acrAB-tolC operon). The marR mutation and yedS could contribute to carbapenem resistance in IOMTU558 (39).

NDM-producing E. coli belonging to ST101 seems to be an epidemic strain in several regions of the world, although ST101 E. coli strains are not recognized as a cause of pandemics, as is E. coli clone ST131 producing CTX-M types of β-lactamases (40). NDM-1-producing E. coli isolates belonging to ST101 have been reported in Bulgaria (41), Canada (42), England (43), India (44), Pakistan (43), South Korea (45), and the United States (46).

This is the first report describing an NDM-13-producing E. coli isolate. NDM-type MBLs have evolved under the pressure of antibiotic usage. Therefore, NDM-producing pathogens must be monitored.

Nucleotide sequence accession number.

The sequence of bla_NDM-13 has been deposited in GenBank under accession no. LC012596.