A Carbapenem-Resistant Pseudomonas aeruginosa Isolate Harboring Two Copies of blaIMP-34 Encoding a Metallo-β-Lactamase

A carbapenem-resistant strain of Pseudomonas aeruginosa, NCGM1984, was isolated in 2012 from a hospitalized patient in Japan. Immunochromatographic assay showed that the isolate was positive for IMP-type metallo-β-lactamase. Complete genome sequencing revealed that NCGM1984 harbored two copies of blaIMP-34, located at different sites on the chromosome. Each blaIMP-34 was present in the same structures of the class 1 integrons, tnpA(ISPa7)-intI1-qacG-blaIMP-34-aac(6')-Ib-qacEdelta1-sul1-orf5-tniBdelta-tniA. The isolate belonged to multilocus sequence typing ST235, one of the international high-risk clones. IMP-34, with an amino acid substitution (Glu126Gly) compared with IMP-1, hydrolyzed all β-lactamases tested except aztreonam, and its catalytic activities were similar to IMP-1. This is the first report of a clinical isolate of an IMP-34-producing P. aeruginosa harboring two copies of blaIMP-34 on its chromosome.

The first gram-negative pathogen producing IMP-34 was a clinical strain of Klebsiella oxytoca showing intermediate resistance to imipenem, first isolated in 2013 in Japan [4]. The IMP-34 producer showed slightly decreased resistance to imipenem [4]. In this bacterium, bla IMP-34 was located on an 87343-bp plasmid, pKOI-34 (GenBank accession no. AB715422). There have been no previous reports regarding IMP-34-producing bacteria. At the amino acid sequence level, IMP-34 was found to have an amino acid substitution (Glu126Gly) compared with IMP-1, an amino acid substitution (Gly262Ser) compared with IMP-3, and two amino acid substitutions (Glu126Gly and Gly262Ser) compared with IMP-6 [4]. At the nucleotide sequence level, bla IMP-1 has two nucleotide sequence polymorphisms with four silent mutations at positions 189, 273, 496, and 702 (GenBank accession no. D50438 and AY250709). bla IMP-34 has two more nucleotide substitutions at positions 190 and 314. bla IMP-3 has an additional nucleotide substitution at position 640. Comparison of nucleotide sequence polymorphisms in these IMPs suggested that IMP-34 was evolutionarily close to IMP-1 [4].
Here, we describe a clinical isolate of carbapenem-resistant P. aeruginosa producing IMP-34, the complete genome sequence of the isolate, and the enzymatic properties of IMP-34.

Materials and Methods
Bacterial strains and drug susceptibility tests P. aeruginosa NCGM1984 was obtained in 2012 from a urine sample of a patient hospitalized in Hyogo prefecture, Japan. Escherichia coli DH5α (Takara Bio, Shiga, Japan) and E. coli BL21-CodonPlus (DE3)-RIP (Agilent Technologies, Santa Clara, CA) were used as hosts for recombinant plasmids and for expression of bla IMP-1 and bla IMP-34 .

Complete genome sequencing
The entire genome of P. aeruginosa NCGM1984 was extracted with cetyl-trimethylammonium bromide (CTAB), sequenced using PacBio RSII (Pacific Biosciences, Menlo Park, CA), and assembled using Minimus 2 to determine the complete genome sequence. Multilocus sequence typing (MLST) was determined according to the P. aeruginosa MLST Database website (http://pubmlst.org/paeruginosa/). RAST automated annotation servers (http://rast. nmpdr.org/) were used for primary coding sequence (CDS) extraction and initial functional assignment. CDS annotations were confirmed using In Silico Molecular Cloning software (In Silico Biology, Inc., Kanagawa, Japan), which assists in annotation with comparison to sequences registered in GenBank.

Comparative genome analysis
The genome sequences of P. aeruginosa PAO1 and NCGM2.S1 strains (accession no. AE004091 and AP012280, respectively) were used for comparative genome analysis with the sequence of NCGM1984. Genomic islands harboring a class 1 integron(s) were detected by comparison with the sequence of PAO1 strain. The sequence of NCGM2.S1 was used as the reference strain belonging to ST235.

Nucleotide sequence accession numbers
The complete genome sequence of NCGM1984 has been deposited in GenBank under the accession number AP014646.

Ethical statements
The study protocol was carefully reviewed and approved by the ethics committee of the National Center for Global Health and Medicine (No. 1268). Individual informed consent was waived by the ethics committee listed above because this study used currently existing samples collected during the course of routine medical care and did not pose any additional risks to the patients. Patient information was anonymized and de-identified prior to analysis. The study protocol was reviewed and approved by the Biosafety Committee, National Center for Global Health and Medicine (approval numbers: 26-D-088 and 26-D-089).

Identification of bla IMP-34 in P. aeruginosa NCGM1984
Immunochromatographic assays showed that P. aeruginosa NCGM1984 was positive for IMPs and AAC(6')-Ib, but negative for AAC(6')-Iae. The isolate harbored bla IMP-34 and aac(6')-Ib. Complete genome analysis of P. aeruginosa NCGM1984 The complete genome of P. aeruginosa NCGM1984 was obtained with 533-fold coverage. This genome consisted of a single circular chromosome, 6,850,954 bp in size, with an average GC content of 65.96%. The chromosome contained a total of 6,282 CDS, 66 tRNA genes, and 1 tmRNA for all amino acids. NCGM1984 had no plasmids. The chromosome contained three integrons, two of which were located close to each other in an area of lower GC content, whereas the other was not. The MLST of NCGM1984 was ST235. NCGM1984 had a gene associated with β-lactam resistance, bla IMP-34 , and three genes associated with aminoglycoside resistance, aac(6')-Ib, aacA7, and aadA6. In addition, the isolate had two point mutations in the quinolone resistance-determining regions of gyrA and parC, with amino acid substitutions of Thr83Ile in GyrA and Ser87Leu in ParC, which are associated with quinolone resistance [16,17], and had fosA, which is associated with fosfomycin resistance. NCGM1984 also had intrinsic β-lactamase encoding genes, bla OXA-50 and bla PDC-20 , which are not thought to be associated with β-lactam resistance [18].
bla IMP-34 was detected on the chromosome of NCGM1984. Unexpectedly, NCGM1984 harbored two copies of bla IMP-34 , located at different sites on the chromosome (nt 2,521,570 -2,522,310 and nt 4,467,459 -4,468,199). A copy of bla IMP-34 was located on a large genomic island of 65,600 bp between PA1984_2407 and PA1984_2462 (Fig 1A). The large genomic island contained two class 1 integrons, integrons A and B (Fig 1A). A copy of bla IMP-34 was located on integron A. Another copy of bla IMP-34 was located on a small genomic island of 9,408 bp between NCGM1984_4145 and NCGM1984_4155 (Fig 1B). This small genomic island was another class 1 integron, integron C, and contained no other CDS (Fig 1B). The small genetic island was located between putative sensor protein encoding gene, yegE, and twocomponent response regulator encoding gene, phoP. The genetic structures of integrons A and C containing bla IMP-34 were identical to each other ( Fig 1C). All three integrons had a pair of inverted repeats, IRi and IRt (Fig 1C).
Integron A (from IRi to IRt) was located on nt 2,516,107 -2,525,888 (9,782 bp) and integron C (from IRi to IRt) was located on nt 4,461,996 -4,471,777 (9,782 bp). The integron A was not flanked by 5 bp dupulication, whereas the integron C had made by a duplication (CAGGT in nt 4,461,991-4,461,995 and 4,471,778-4471782). The class 1 integrons A and C carried the qacG-blaIMP-aac(6')-Ib cassettes. In integrons A and C, the 5'-CS was interrupted by ISPa7 (Fig 1C). The class 1 integron B (from IRi to IRt) was located on nt 2,544,427 -2,553,926 (9,500 bp), and carried the aacA7-aadA6 cassette. The integron B was not flanked by 5 bp duplication.
Integrons A and B had a Pc promoter with TGGACA (-35 sequence) and TAAACT hexamers (-10 sequence) separated by a space of 17 bp within intI1, respectively; whereas integron C had another Pc promoter with TGGACA (-35 sequence) and TAAGCT hexamers (-10 sequence) separated by a space of the same size (17 bp) [19].

Drug susceptibility of E. coli DH5α expressing IMP-34 and enzymatic activities
The drug susceptibility profile of E. coli expressing IMP-34 was similar to that of E. coli expressing IMP-1, although the former showed slightly lower MICs for doripenem, imipenem, and meropenem compared with the latter (Table 1).

Discussion
The Glu126Gly substitution in IMP-34 slightly affected the catalytic efficiency of the enzyme for β-lactam hydrolysis compared with IMP-1 ( Table 2). The amino acid residue at position 126 is located in the same helix as position 120, which is a Zn 2+ binding site [20]. There have been no previous reports regarding the role of the amino acid residue at position 126 of IMPs.
IMPs in multidrug-resistant P. aeruginosa clinical isolates in Japan seem to have increased efficiency of catalytic activities against carbapenems due to the acquisition of various amino acid substitutions. For example, multidrug-resistant P. aeruginosa producing IMP-43 and IMP-44 showed greater catalytic activities against carbapenems than IMP-7 and IMP-11, respectively [6]. IMP-43 belonging to the IMP-7-like group has an amino acid substitution (Val67Phe) compared with IMP-7, and IMP-44 belonging to the IMP-11-like group has two substitutions (Val67Phe and Phe87Ser) compared with IMP-11. IMP-43 showed more efficient catalytic activities against doripenem, imipenem, and meropenem than IMP-7, while IMP-44 had more efficient catalytic activities against all carbapenems compared to IMP-11 [6].
The activity of the Pc promoter of integron A will be stronger than that of integron C [21][22][23]. There are four versions of Pc, designated as "weak," "strong," "hybrid 1," and "hybrid 2," which show differences in the -35 and /or -10 sequences, separated by 17 bases [21][22][23]. The Pc promoter of integron A was "hybrid 1" type, and that of integron C was "weak" type. It was reported that there are eight variants of the Pc promoter, which vary in their promoter activities [23]. The -35/-10 sequences of PcS (strong type) were TTGACA/TAAACT, whereas those of PcW (weak type) were TGGACA/TAAGCT. The promoter activity of the hybrid type 1 was 4.5-fold weaker than that of the strong type and 5.6-fold stronger than that of the weak type [22]. The Pc promoter of integron A and B was a hybrid type consisting of a weak type -35 sequence combined with a strong type-10 sequence (TGGACA/TAAACT). Two copies of bla IMP-34 in NCGM1984 will contribute to extremely high MICs of carbapenems, although these copies will express at different levels because of different Pc promoters of the integrons and the Pc promoter of integron A together with that of integron C may be the main determinant of the level of carbapenem resistance.  Emergence of IMP-34-Producing P. aeruginosa Multidrug-resistant P. aeruginosa belonging to ST235, including NCGM1984, is recognized as one of the international high-risk clones that spread in medical settings worldwide, which has acquired nearly 100 resistance elements, including resistance to 39 different β-lactamases [24]. Multidrug-resistant P. aeruginosa belonging to ST235 spread in Japan and often carried an In113-like integron, harboring bla IMP-1 , aac(6')-Iae, and aadA1a [25]. These horizontally acquired antibiotic resistance elements exist in various integrons in the multidrug-resistant isolates belonging to ST235 [24] as well as NCGM1984 (Fig 1C and 1D).
The large genomic island with two class 1 integrons found in NCGM1984 had a quite unique structure (Fig 1A) and seemed to originate from three different genomic islands. The large genomic island in NCGM1984 had only 56% similarity with P. aeruginosa C79 genomic island (accession no. JF826498) at the most; nevertheless, its partial structures divided by the two integrons had higher similarity with other P. aeruginosa genomes as follows: the upstream region of integron A (NCGM1984_2402 to _2410) had 99% identity with P. aeruginosa H47921 isolated in the United States; the region between integrons A and B (NCGM1984_2420 to _2436) had 100% similarity with P. aeruginosa VR-143/97 genomic island isolated in Italy (accession no. LK054503); the downstream region of integron B (NCGM1984_2446 to _2461) had 99% similarity with P aeruginosa C79 isolated in Australia (accession no. JF826498).
In addition to the acquisition of amino acid substitutions in IMPs, increases in the bla IMP copy number may represent an alternative pathway by which P. aeruginosa can acquire carbapenem resistance. That is, the extreme resistance of NCGM1984 against carbapenems may be associated with the presence of two copies of bla IMP-34 . Amplification of genes conferring bacterial resistance to antibiotics in bacteria may be associated with a gene dosage effect [26]. For example, a clinical isolate of P. aeruginosa carrying two copies of bla NDM-1 on the chromosome [27] was found to be extremely resistant to imipenem and meropenem (MIC > 32 μg/mL) [28].