A novel VIM-type metallo-β-lactamase variant, VIM-60, was identified in multidrug-resistant Pseudomonas aeruginosa clinical isolates in Japan. Compared with VIM-2, VIM-60 had two amino acid substitutions (Arg228Leu and His252Arg) and higher catalytic activities against fourth-generation cephalosporins.
KEYWORDS: Pseudomonas aeruginosa, fourth-generation cephalosporins, metallo-β-lactamase
ABSTRACT
A novel VIM-type metallo-β-lactamase variant, VIM-60, was identified in multidrug-resistant Pseudomonas aeruginosa clinical isolates in Japan. Compared with VIM-2, VIM-60 had two amino acid substitutions (Arg228Leu and His252Arg) and higher catalytic activities against fourth-generation cephalosporins. The genetic context for blaVIM-60 was intI1-blaVIM-60-aadA1-aacA31-qacEdeltaI-sulI on the chromosome.
INTRODUCTION
The emergence of metallo-β-lactamases (MBLs) and the increased carbapenem resistance among Gram-negative pathogens have become serious problems 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). Since the identification in 1997 of Verona integron-encoded metallo-β-lactamase-1 (VIM-1) in a strain of Pseudomonas aeruginosa in northern Italy (4), at least 59 other VIM variants have been identified in Enterobacteriaceae, Acinetobacter baumannii, and P. aeruginosa in several countries (ftp://ftp.ncbi.nlm.nih.gov/pathogen/betalactamases/Allele.tab). This study describes a novel VIM-type MBL, VIM-60, produced by two clinical isolates of P. aeruginosa in a medical setting in Japan.
P. aeruginosa NCGM3661 and NCGM3750 were isolated in 2017 from the urine samples of two inpatients. MICs were determined using the broth microdilution method, as recommended by the Clinical and Laboratory Standards Institute. The genomic DNA of these isolates were extracted and sequenced by a next-generation sequencer (MiSeq; Illumina, San Diego, CA). Multilocus sequence typing (MLST) was deduced, as described by the protocols of the PubMLST database (http://pubmlst.org/paeruginosa/). Sequences of drug resistance genes, including genes encoding β-lactamases (www.lahey.org/studies); aminoglycosides, chloramphenicol, and fosfomycin resistance genes registered in GenBank (https://www.ncbi.nlm.nih.gov/nuccore/); and quinolone resistance genes, were determined using CLC Genomics Workbench version 9.5. The contig sequence constructed by CLC Genomics Workbench was used as the genetic environment surrounding blaVIM-60.
The blaVIM-2 and blaVIM-60 genes were amplified using the primer sets EcoRI-VIM-2-F (5′-ATGAATTCATGTTCAAACTTTTGAGTAAGT-3′) and PstI-VIM-2-R (5′-ATCTGCAGCTACTCAACGACTGAGCGATTT-3′). The PCR products ligated into pHSG398 (TaKaRa Bio, Shiga, Japan) were used to transform Escherichia coli DH5α (TaKaRa Bio, Shiga, Japan).
The open reading frames of VIM-2 and VIM-60 without signal peptide regions were cloned into the pET28a expression vector (Novagen, Inc., Madison, WI) using the primer sets BamHI-VIM-2 (TEV) 79F (5′-ATGGATCCGAAAACCTGTATTTCCAAGGCGTAGATTCTAGCGGTGAGTATCC-3′) and XhoI-VIM-2 R (5′-ATCTCGAGCTACTCAACGACTGAGCGATTT-3′), as previously described (5). The plasmids were transformed into E. coli BL-21-CodonPlus(DE3)-RIP (Agilent Technologies, Santa Clara, CA). Recombinant VIM proteins were purified using Ni-nitrilotriacetic acid (NTA) agarose. His tags were removed by digestion with TurboTEV protease (Accelagen, San Diego, CA), and untagged proteins were purified by an additional passage over the Ni-NTA agarose. The purities of VIM-2 and VIM-60 were >90%, as estimated by SDS-PAGE. The yields of VIM-2 and VIM-60 proteins were 1.105 and 1.209 mg/liter of culture, respectively. During the purification process, β-lactamase activity was monitored using nitrocefin (Oxoid Ltd., Basingstoke, UK). The initial rate of hydrolysis in 50 mM Tris-HCl (pH 7.4), 0.3 M NaCl, and 5 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 Lineweaver-Burk plot, with Km and kcat determined using triplicate analyses. To determine whether blaVIM-60 is located on the plasmids or chromosomes, the DNA plugs of the isolates digested with S1 nuclease were separated by pulsed-field gel electrophoresis (PFGE), followed by Southern blotting and hybridization with a labeled blaVIM-60 probe (6).
Both strains, P. aeruginosa NCGM3661 and NCGM3750, were resistant to all β-lactamases tested (Table 1). In NCGM3661 and NCGM3750 isolates, the MICs of other antibiotics were 64 and 16 μg/ml for amikacin, 32 and 16 μg/ml for arbekacin, 4 and 8 μg/ml for gentamicin, 1,024 and 512 μg/ml for kanamycin, 32 and 16 μg/ml for tobramycin, 64 and 16 μg/ml for ciprofloxacin, 32 and 32 μg/ml for levofloxacin, <0.25 and 0.25 μg/ml for colistin, and >1,024 and >1,024 μg/ml for fosfomycin, respectively.
TABLE 1.
MICs of β-lactams for P. aeruginosa NCGM3661 and NCGM3750 and E. coli transformants expressing VIM-2 and VIM-60
| Antibiotic | MIC (μg/ml) for: |
||||
|---|---|---|---|---|---|
|
P. aeruginosa |
E. coli transformant |
||||
| NCGM 3661 |
NCGM 3750 |
pHSG398/ VIM-2 |
pHSG398/ VIM-60 |
pHSG398 | |
| Ampicillin | >1,024 | >1,024 | 32 | 64 | 2 |
| Ampicillin- sulbactam |
>1,024 | >1,024 | 16 | 32 | 2 |
| Penicillin G | >1,024 | >1,024 | 64 | 128 | 32 |
| Aztreonam | 16 | 16 | 0.125 | 0.06 | 0.06 |
| Cefepime | 1,024 | 1,024 | 0.125 | 0.5 | 0.06 |
| Cefmetazole | >1,024 | >1,024 | 1 | 1 | 1 |
| Cefotaxime | >1,024 | >1,024 | 1 | 4 | ≤0.03 |
| Cefoxitin | 1,024 | 1,024 | 16 | 16 | 8 |
| Cefozopran | >1,024 | >1,024 | 0.5 | 4 | 0.06 |
| Cefpirome | >1,024 | 1,024 | 0.125 | 1 | 0.06 |
| Cefsulodin | >1,024 | >1,024 | 512 | 1024 | 256 |
| Ceftazidime | 256 | 128 | 1 | 4 | 0.125 |
| Ceftriaxone | 512 | 512 | 1 | 4 | ≤0.03 |
| Cefuroxime | >1,024 | >1,024 | 32 | 64 | 4 |
| Cephradine | >1,024 | >1,024 | 32 | 64 | 8 |
| Doripenem | 512 | 512 | ≤0.03 | 0.06 | ≤0.03 |
| Imipenem | 512 | 512 | 0.25 | 0.25 | 0.125 |
| Meropenem | 1,024 | 1,024 | ≤0.03 | 0.125 | ≤0.03 |
| Panipenem | 256 | 512 | 0.125 | 0.25 | 0.06 |
| Moxalactam | >1,024 | >1,024 | 8 | 16 | 0.125 |
Both isolates were positive for VIM-type MBL. Whole-genome sequencing revealed that these isolates had a novel blaVIM variant with two nucleotide substitutions (G614T and A686G) compared with blaVIM-2, and the novel blaVIM variant was designated blaVIM-60 (GenBank accession no. NG_061404). Its predicted amino acid sequence revealed that VIM-60 had two amino acid substitutions (Arg228Leu and His252Arg) compared with VIM-2. In addition to blaVIM-60, several other drug resistance genes were present in both isolates, including aadA1, aac(6')-Ia, aac(6')-31, blaPAO, blaOXA-50, cat, fosA, and sulI. Both NCGM3661 and NCGM3750 had amino acid substitutions in the quinolone resistance-determining regions (QRDR) of GyrA (T83I) and ParC (S87L) but did not have the plasmid-mediated quinolone resistance factors, such as AAC(6′)-Ib-cr, Qep, and Qnr (7–9). Moreover, the two isolates had efflux pump systems associated with ciprofloxacin resistance, including MexA, MexC, and MexE (10).
These isolates belonged to sequence type (ST) 1816 (allelic profile: 40, 5, 11, 3, 4, 40, 2) and were related to ST3002 (allelic profile: 40, 101, 11, 3, 4, 40, 2). P. aeruginosa ST1816 was first isolated in Mexico in 2011 (11). Until now, P. aeruginosa ST1816 was isolated in 2014 in Japan (id-2248 in P. aeruginosa MLST database [https://pubmlst.org/paeruginosa/]).
The contig of 5,865 bp, including blaVIM-60, was constructed after assembling the raw read data (GenBank accession no. DRA008130). The genetic environment surrounding blaVIM-60 was intI1-blaVIM-60-aadA1-aacA31-qacEdeltaI-sulI (GenBank accession no. LC434516). The novel class 1 integron structure was deposited in INTEGRALL (http://integrall.bio.ua.pt/) under the number In1610. PFGE and Southern blotting and hybridization revealed that the P. aeruginosa isolates NCGM3661 and NCGM3750 had no plasmids harboring blaVIM-60, indicating that blaVIM-60 is located on their chromosomes.
E. coli DH5α expressing blaVIM-60 was resistant to all cephalosporins, moxalactams, and penicillins and showed reduced susceptibility to carbapenems (Table 1). A comparison of the MICs of E. coli DH5α expressing blaVIM-2 and blaVIM-60 showed that blaVIM-60 was equally or more resistant to all antibiotics except aztreonam. In particular, the MICs to cefepime, cefotaxime, cefozopran, cefpirome, ceftazidime, and ceftriaxone of E. coli expressing blaVIM-60 were >4-fold higher than the MICs to bacteria expressing blaVIM-2.
Recombinant VIM-type enzymes hydrolyzed all β-lactams tested except aztreonam. VIM-60 had significantly greater enzymatic activities than VIM-2 toward cefepime, cefozopran, and cefpirome, due primarily to their differences in kcat values against these substrates (Table 2). In contrast, VIM-60 had significantly lower enzymatic activities against cephradine and panipenem than VIM-2, due primarily to differences in Km values. The amino acid substitutions Arg228Leu and His252Arg seem to have a significant impact on the ability of VIM-60 to efficiently hydrolyze cefepime, cefozopran, and cefpirome. The amino acid residue at position 228 is located in the L3 loop and is associated with the binding of substrates or inhibitors (12). VIM-24, with the amino acid substitution Arg228Leu, enhances resistance to ceftazidime and cefepime (13, 14). In contrast, the amino acid residue at position 252 is situated on the α4 helix, distant from the active site of the VIM enzyme. The His252Arg mutation may also contribute to tuning VIM activities and to resistance to fourth-generation cephalosporins.
TABLE 2.
Kinetic parameters of β-lactamases VIM-2 and VIM-60 with various substrates
| Substrate |
Km (μM)a
|
kcat (s−1)a
|
kcat/Km (μM s−1)a
|
|||
|---|---|---|---|---|---|---|
| VIM-2 | VIM-60 | VIM-2 | VIM-60 | VIM-2 | VIM-60 | |
| Ampicillin | 62 ± 6 | 160 ± 5 | 214 ± 8 | 345 ± 11 | 3.49 | 2.15 |
| Penicillin G | 72 ± 7 | 262 ± 9 | 252 ± 15 | 481 ± 10 | 3.51 | 1.83 |
| Aztreonam | NHb | NH | NH | NH | NH | NH |
| Cefepime | 200 ± 3 | 804 ± 83 | 7.9 ± 0.1 | 85 ± 8 | 0.040 | 0.11 |
| Cefmetazole | 51 ± 2 | 74 ± 7 | 1.9 ± 0.01 | 2.6 ± 0.2 | 0.037 | 0.035 |
| Cefotaxime | 41 ± 5 | 69 ± 1 | 121 ± 4 | 134 ± 2 | 3.00 | 1.93 |
| Cefoxitin | 67 ± 3 | 40 ± 3 | 4.1 ± 0.1 | 2.8 ± 0.02 | 0.061 | 0.069 |
| Cefozopran | 270 ± 8 | 133 ± 10 | 52 ± 1 | 195 ± 7 | 0.19 | 1.47 |
| Cefpirome | 246 ± 5 | 190 ± 2 | 88 ± 1 | 234 ± 2 | 0.36 | 1.23 |
| Ceftazidime | 71 ± 5 | 179 ± 6 | 2.1 ± 0.1 | 7.1 ± 0.2 | 0.030 | 0.040 |
| Ceftriaxone | 87 ± 2 | 54 ± 1 | 58 ± 1 | 33 ± 0.1 | 0.66 | 0.61 |
| Cephradine | 33 ± 0.5 | 94 ± 8 | 102 ± 1 | 68 ± 3 | 3.11 | 0.72 |
| Doripenem | 27 ± 1 | 27 ± 1 | 2.8 ± 0.01 | 2.3 ± 0.03 | 0.10 | 0.084 |
| Imipenem | 81 ± 7 | 40 ± 3 | 46 ± 1 | 13 ± 1 | 0.57 | 0.31 |
| Meropenem | 51 ± 2 | 68 ± 4 | 9.2 ± 0.1 | 16 ± 1 | 0.18 | 0.24 |
| Panipenem | 27 ± 3 | 39 ± 7 | 11 ± 0.2 | 3.6 ± 0.2 | 0.40 | 0.094 |
| Moxalactam | 79 ± 10 | 67 ± 1 | 87 ± 9 | 49 ± 0.2 | 1.10 | 0.73 |
Km and kcat were calculated as means ± SD from three independent experiments.
NH, no hydrolysis detected with substrate concentrations up to 1 mM and enzyme concentrations up to 700 nM.
To our knowledge, this is the first report describing VIM-60-producing Gram-negative pathogens in Japan. These findings indicate that VIMs have evolved in response to the use of fourth-generation cephalosporins, such as cefepime and cefpirome, in medical settings in Japan. Careful monitoring of VIM-producing pathogens is required.
Accession number(s).
The whole-genome sequences of NCGM3661 and NCGM3750 have been deposited in GenBank under accession number DRA008130. The genetic environment surrounding blaVIM-60 has been deposited in GenBank under accession number LC434516.
ACKNOWLEDGMENT
This study was supported by grants from the Japan Society for the Promotion of Science (grant number 18K07120) and the Research Program on Emerging and Re-emerging Infectious Diseases from the Japan Agency for Medical Research and Development (grant number 19fk0108061h0302).
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