PER-8, a Novel Extended-Spectrum β-Lactamase PER Variant, from an Acinetobacter baumannii Clinical Isolate in Nepal

ABSTRACT

A novel PER-type extended-spectrum β-lactamase, PER-8, was identified in an Acinetobacter baumannii clinical isolate obtained in Nepal. The amino acid sequence of PER-8 has a substitution at position 39 (Gly to Glu) compared with that of PER-7. The k_cat/K_m ratio of PER-8 for aztreonam was lower than that of PER-7, while the k_cat/K_m ratio of PER-8 for imipenem was higher than that of PER-7. The genomic environment surrounding bla_PER-8 was intI1 bla_PSE-1 qacEDI sulI ISCR1-bla_PER-8 gts sulI orfX on a 100-kb plasmid.

TEXT

The class A extended-spectrum β-lactamases (ESBLs) confer resistance to expanded-spectrum cephalosporins and are inhibited in vitro by clavulanic acid and tazobactam (1). Resistance to broad-spectrum cephalosporins in Acinetobacter baumannii mostly results from overexpression of the natural AmpC-type enzyme or from acquisition of ESBLs. To date, the following 5 types of ESBL genes have been reported in A. baumannii: bla_PER (2), bla_GES (3), bla_VEB (4), bla_TEM (5), and bla_CTX-M (6). The bla_PER-1 gene was first found in a Pseudomonas aeruginosa isolate (7). Since then, it has been reported worldwide in Enterobacteriaceae (8–11) and A. baumannii (2, 4). Until now, 7 types of PER variants have been reported in clinical isolates of Enterobacteriaceae (12–16) and A. baumannii (17) in various countries. The phylogenic tree based on amino acid sequences (Clustal W2) revealed two clusters in PER-type variants, one containing PER-1, PER-3, PER-4, PER-5, and PER-7 and the other containing PER-2 and PER-6.

Multidrug-resistant A. baumannii IOMTU442 and IOMTU448 were isolated from samples of wound swabs from hospitalized patients at a university hospital in 2013 in Nepal. The isolates were identified phenotypically, and species identification was confirmed by comparing sequences of 16S rRNA, gyrB, and bla_OXA-51-like genes. 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 expression of bla_PER genes, respectively.

MICs were determined using the broth microdilution method as recommended by CLSI (M100-S23). Whole genomes of IOMTU442 and IOMTU448 were extracted with DNeasy blood and tissue kits (Qiagen, Tokyo, Japan) and sequenced by MiSeq (Illumina, San Diego, CA). Multilocus sequence typing (MLST) was performed as described by the protocols of the Institut Pasteur MLST (http://pubmlst.org/perl/bigsdb/bigsdb.pl?db=pubmlst_abaumannii_pasteur_seqdef) databases.

The bla_PER-7 and bla_PER-8 were cloned into the corresponding sites of the pHSG398 vector plasmid (TaKaRa, Shiga, Japan) using the primer set EcoRI-PER-F (5′-GGGAATTCATGGAATTGCCCAATATTATG-3′) and PstI-PER-R (5′-AACTGCAGTCAGCGCAGCTTGTCGGCCAT-3′). E. coli DH5α was transformed with pHSG398-PERs, and the transformants were selected on chloramphenicol-containing plates (30 μg/ml).

The open reading frames of PER-7 and PER-8, without signal peptide regions, were cloned into the pET28a expression vector (Novagen, Inc., Madison, WI, USA) using the primer set BamHI-TEV-PER-F (5′-ATGGATCCGAAAACCTGTATTTCCAAGGCCAGCAAATGGAAACTGGCGAC-3′) and XhoI-PER-R (5′-ATCTCGAGTCAGCGCAGCTTGTCGGCCATG-3′). The plasmids were used to transform E. coli BL21-CodonPlus(DE3)-RIP (Agilent Technologies, Santa Clara, CA, USA). Recombinant PERs were purified, and initial hydrolysis rates were determined as previously described (18).

To determine the size of the plasmid harboring bla_PER-8, plasmid DNA in iOMTU442 was extracted and digested with S1 nuclease. Pulsed-field gel electrophoresis (PFGE) and Southern hybridization were performed. A probe for bla_PER from IOMTU442 was amplified by PCR using the EcoRI-PER-F and PstI-PER-R primer set. A DNA plug of IOMTU448, digested with I-CeuI, was prepared, separated by pulsed-field gel electrophoresis, and subjected to Southern hybridization using 16S rRNA and bla_PER-7 probes. Signal was detected using digoxigenin (DIG) High Prime DNA labeling and detection starter kit II (Roche Applied Science, Indianapolis, IN, USA).

IOMTU442 had bla_OXA-70, bla_PSE-1, and a novel bla_PER variant, bla_PER-8. IOMTU448 had bla_OXA-23, bla_OXA-371, bla_OXA-420 (bla_OXA-58-like), and bla_PER-7. The bla_OXA-371 gene in IOMTU 448 was grouped with the bla_OXA-69-type genes, whereas the bla_OXA-70 gene in IOMTU442 was not grouped with the bla_OXA-66-type genes, the bla_OXA-69-type genes, or the bla_OXA-71-type genes. Neither bla_OXA-70 nor bla_OXA-371 was flanked by ISAba1. The MICs for A. baumannii IOMTU442 and IOMTU448 are shown in Table 1. IOMTU442 and IOMTU448 were found to belong to ST103 and ST623, respectively. A. baumannii isolates belonging to ST103 have been found in Egypt (19) and Portugal (20), and ST623 belonged to CC1, which is known as international clone I, disseminated worldwide. The sequence of bla_PER-8 showed a nucleotide substitution compared with bla_PER-7. Similarly, analysis of their predicted amino acid sequences revealed that PER-8 had a substitution (Gly39Glu) compared with PER-7; therefore, PER-7 was used as a control for PER-8. The nucleotide sequences of bla_PER-8 and its flanking region have been deposited in GenBank under accession number AB985401.

TABLE 1.

MICs of various β-lactams for A. baumannii strains IOMTU442 and IOMTU448 and E. coli DH5α transformed with PER-7- or PER-8-encoding plasmids

Antibiotic(s)a	MIC (mg/liter)
	IOMTU442	IOMTU448	pHSG398/PER-7	pHSG398/PER-8	pHSG398
Amikacin	>1,024	8	NDb	ND	ND
Ampicillin	>1,024	>1,024	32	16	2
Ampicillin-sulbactam	64	64	2	2	2
Arbekacin	>1,024	2	ND	ND	ND
Aztreonam	>1,024	>1,024	8	4	≤0.063
Cefepime	512	512	0.125	0.25	≤0.063
Cefmetazole	256	>1,024	1	1	1
Cefotaxime	512	>1,024	16	8	≤0.063
Cefoxitin	512	>1,024	4	4	4
Cefpirome	256	256	≤0.063	≤0.063	≤0.063
Ceftazidime	>1,024	512	32	64	0.5
Cephradine	>1,024	>1,024	128	128	16
Ciprofloxacin	32	32	ND	ND	ND
Colistin	0.25	0.5	ND	ND	ND
Fosfomycin	128	256	ND	ND	ND
Gentamicin	>1,024	>1,024	ND	ND	ND
Imipenem	2	8	0.125	0.125	≤0.063
Kanamycin	>1,024	>1,024	ND	ND	ND
Levofloxacin	32	8	ND	ND	ND
Meropenem	1	16	≤0.063	≤0.063	≤0.063
Moxalactam	128	128	≤0.063	≤0.063	≤0.063
Penicillin G	>1,024	>1,024	64	64	32
Piperacillin	>1,024	512	4	4	2
Piperacillin-tazobactam	64	256	2	2	2
Tigecycline	0.25	0.5	ND	ND	ND

^aThe ratio of ampicillin to sulbactam was 2:1. The ratio of piperacillin to tazobactam was 4:1.

^bND, not determined.

Compared with E. coli DH5α harboring a pHSG398 control vector, DH5α harboring bla_PER-7 or bla_PER-8 showed significantly increased MICs of all penicillins and cephalosporins tested, except cefmetazole, cefpirome, cefoxitin, and piperacillin, as well as slightly increased MICs of imipenem (Table 1). DH5α harboring bla_PER-7 and bla_PER-8 had similar MICs of β-lactams (Table 1). Recombinant PER-7 and PER-8 hydrolyzed all β-lactams tested, except for cefmetazole, cefoxitin, and moxalactam (Table 2). PER-7 and PER-8 also hydrolyzed imipenem and meropenem, although their k_cat/K_m ratios against these substrates were quite low. The kinetic profiles of PER-8 against the β-lactams tested, except for imipenem, were similar to those of PER-7. The k_cat/K_m ratios of PER-8 were 2-fold higher for imipenem than those of PER-7 (Table 2).

TABLE 2.

Kinetic parameters of the PER-7 and PER-8 enzymes in hydrolyzing β-lactams^a

β-Lactam	PER-7			PER-8
	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	13 ± 4	52 ± 2	4.4	19 ± 3	54 ± 1	2.9
Penicillin G	13.7 ± 2.8	16.0 ± 0.2	1.2	13.7 ± 3.1	16.4 ± 0.3	1.2
Piperacillin	12.6 ± 3.6	0.60 ± 0.02	0.051	8.6 ± 1.8	0.65 ± 0.03	0.076
Cefepime	81 ± 7	10.2 ± 0.3	0.13	110 ± 16	12 ± 1	0.11
Cefmetazole	NHc	NH	NH	NH	NH	NH
Cefotaxime	138 ± 63	84 ± 20	0.65	114 ± 17	70 ± 4	0.62
Cefoxitin	NH	NH	NH	NH	NH	NH
Ceftazidime	258 ± 22	33 ± 2	0.13	212 ± 26	31 ± 2	0.15
Cephradine	54 ± 5	62 ± 2	1.2	48 ± 8	66 ± 2	1.4
Moxalactam	NH	NH	NH	NH	NH	NH
Aztreonam	17 ± 3	8.8 ± 0.2	0.54	14 ± 2	8.8 ± 0.2	0.64
Imipenem	172 ± 33	0.13 ± 0.01	0.00076	101 ± 10	0.16 ± 0.01	0.0016
Meropenem	28 ± 8	0.13 ± 0.01	0.0051	32 ± 3	0.17 ± 0.01	0.0053

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

^bK_m and k_cat values represent the means ± standard deviations of results of 3 independent experiments.

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

PFGE analysis showed that bla_PER-7 in IOMTU448 was located on the chromosome, whereas bla_PER-8 in IOMTU442 was located on a 100-kb plasmid whose replicon type was classified into the GR12 type of Acinetobacter plasmids (21). The genomic environments surrounding bla_PER-7 in IOMTU448 and bla_PER-8 in IOMTU442 are shown in Fig. 1. The genomic environments surrounding bla_PER-7 (nucleotide [nt] 1162 to nt 8196; GenBank accession no. LC020101) showed 99.4% nucleotide sequence identity with the region from nt 2172 to nt 9204 of the plasmid of P. aeruginosa RJ248 producing PER-1 in China (GenBank accession no. KU133340). The genomic environment surrounding bla_PER-8 (from nt 1994 to nt 8195; GenBank accession no. AB985401) showed more than 99.9% nucleotide sequence identity with the region from nt 3083 to nt 9284 of the plasmid from A. baumannii A068 producing PER-7 in Sweden (GenBank accession no. KT317086). bla_PER-7 and bla_PER-8 were both located downstream of ISCR1 and had identical genetic structures for the sequence between qacEDI and orfX (ofrX is a gene encoding a putative ABC transporter ATP-binding protein) in their respective plasmids (Fig. 1). In our previous study, we reported PER-7-producing A. baumannii IOMTU433 in 2015 in Nepal (22). The structures upstream of the 3′ coding sequence (CS) in IOMTU442 and IOMTU448 completely differed from the corresponding regions of a plasmid (pIOMTU433) in A. baumannii IOMTU433 (22) (Fig. 1).

FIG 1.

Genetic environments surrounding bla_PER genes in A. baumannii IOMTU442 (GenBank accession no. AB985401), IOMTU448 (GenBank accession no. LC020101), IOMTU433 (GenBank accession no. AP014650) (22), and AP2 (GenBank accession no. HQ713678) (17). The bla_PER-7 gene in A. baumannii AP2 was located on the chromosome, whereas the bla_PER-7 gene in A. baumannii IOMTU433 and the bla_PER-8 gene in A. baumannii IOMTU442 were located on plasmids.

A. baumannii harboring bla_PER genes, including bla_PER-7 and bla_PER-8, mediated by plasmids or chromosomes may be spreading in medical settings in Nepal, because our previous study showed that 49.2% of A. baumannii clinical isolates in Nepal harbored bla_PER genes, including bla_PER-7 and bla_PER-8 (22). The bla_PER-7 gene was first identified in A. baumannii AP2 (GenBank accession no. HQ713678) in France, and the gene was located on the chromosome (17). As shown in Fig. 1, the genetic structures surrounding bla_PER genes in IOMTU442 and IOMTU448 differ from that in AP2 because both IOMTU442 and IOMTU448 harbor intI1 in the region upstream of bla_PER-7 and bla_PER-8, respectively, but AP2 does not. The upstream region of bla_PER-7 in A. baumannii AP2, arr-2 cmlA7 qacED1 sulI ISCR1, had a structure identical to that in pIOMTU433 in A. baumannii IOMTU433 discovered in Nepal. The data from our present study suggest that PER-producing A. baumannii in Nepal probably has at least two types of genetic structures surrounding bla_PER genes.

The insertion element ISCR1 in the upstream region of bla_PER genes appears to be involved in the acquisition of bla_PER genes in A. baumannii in Nepal. The structure that includes 3′ CS-ISCR1 is commonly associated with the recent emergence of drug-resistant pathogens, including E. coli, Klebsiella pneumoniae, A. baumannii, and P. aeruginosa, which are linked to the drug resistance genes encoding not only metallo-β-lactamases but also 16S rRNA methylases (23). The ISCR1 may be associated with the genetic diversity of a β-lactamase-resistant factor in A. baumannii (24).

The bla_OXA-70 gene in IOMTU442 was first identified in A. baumannii clinical isolates in Hong Kong (25), whereas the bla_OXA-371 gene in IOMTU448 was first identified in A. baumannii clinical isolates in 2014 in Nepal (22). To date, bla_OXA-70 harboring A. baumannii was reported in 2014 in Canada (26). The bla_OXA-70 gene had 11, 17, and 17 nucleotide substitutions compared with bla_OXA-71, bla_OXA-66, and bla_OXA-69, respectively. The bla_OXA-371 gene had only one nucleotide substitution compared with bla_OXA-69.

In conclusion, this is the first report of A. baumannii isolates producing PER-7 and PER-8 in Nepal. The results of the present study indicate that plasmid- or chromosome-mediated PER-producing A. baumannii strains will spread in medical settings in Nepal.

Accession number(s).

The nucleotide sequences for bla_PER-8 and its flanking region in A. baumannii IOMTU442 and for bla_PER-7 and its flanking region in A. baumannii IOMTU448 have been deposited in the GenBank database under accession numbers AB985401 and LC020101, respectively.