A Novel Integron Gene Cassette Harboring VIM-38 Metallo-β-lactamase in a Clinical Pseudomonas aeruginosa Isolate.

Dear Editor,

Multidrug resistance of Pseudomonas aeruginosa has been attributed to both intrinsic and acquired antibiotic-resistance mechanisms. Multidrug-resistant (MDR) P. aeruginosa isolates have become a serious healthcare problem worldwide because they are resistant to almost all β-lactams, aminoglycosides, and quinolones. Production of zinc-dependent metallo-β-lactamases (MBLs) has been identified as the most significant mechanism among carbapenem-resistant P. aeruginosa isolates [1]. MBLs are of particular clinical concern because of their broad-spectrum activities, and Imipenemase (IMP)-, Verona Integron-Encoded Metallo-β-lactamase (VIM)-, Sao Paulo metallo-β-lactamase (SPM)-, Germany imipenemase (GIM)-, and New Delhi Metallo-β-lactamase (NDM)-type MBLs have been identified in P. aeruginosa worldwide [2]. Forty-six variants of VIM enzymes have been identified to date (http://www.lahey.org/Studies/other.asp). VIM-38 was recently identified in P. aeruginosa isolates in Turkey and was shown to differ from VIM-5 by a single substitution (Ala316Val) [3]. In P. aeruginosa, VIM-type MBLs have been reported within mobile genetic elements such as integrons, which contribute to the dissemination of antibiotic resistance [3].

We here report a new clinical P. aeruginosa strain isolated from a blood sample on January 2015 at Rize State Hospital in Turkey and identified by using the API 32GN system (bioMerieux, Marcy-l'Etoile, France). Minimal inhibitory concentrations were determined on a VITEK system for the following antibiotics: piperacillin/tazobactam, ceftazidime, cefepime, amikacin, netilmicin, ciprofloxacin, levofloxacin, imipenem, meropenem, cefoperazone-sulbactam, and inducible β-lactamase. 16S rDNA sequencing was used for molecular identification, performed according to Cicek et al [4].

The P. aeruginosa isolate was screened for β-lactamase-encoding genes and the class 1–class 2 integrases conserved region by PCR. The primers used for detection of β-lactamase-encoding genes and class 1 and class 2 integron gene cassettes are listed in Table 1 [45678].

Table 1

Primers used in the amplification of selected genes

Primers	5'→3'	Amplicon size	Tm (℃)
blaTEM	F: AGTATTCAACATTTYCGTGT	847	56
	R: TAATCAGTGAGGCACCTATCTC
blaSHV	F: ATGCGTTATATTCGCCTGTG	843	55
	R: TTAGCGTTGCCAGTGCTC
blaCTX-M1	F: GCGTGATACCACTTCACCTC	260
	R: TGAAGTAAGTGACCAGAATC
blaCTX-M2	F: TGATACCACCACGCCGCTC	341
	R: TATTGCATCAGAAACCGTGGG
blaGES	F: ATGCGCTTCATTCACGCAC	863	056
	R: CTATTTGTCCGTGCTCAGGA
blaVEB	F: ATTTCCCGATGCAAAGCGT	542	55
	R: TTATTCCGGAAGTCCCTGT
blaPER-2	F: ATGAATGTCATCACAAAATG	860	45
	R: ATAATAGCTTCATTGGTTC
blaKPC	F: ATGTCACTGTATCGCCGTCT	893	55
	R: TTTTCAGAGCCTTACTGCCC
blaIMP	F: CATGGTTTGGTGGTTCTTGT	488	56
	R: ATAATTTGGCGGACTTTGGC
blaVIM	F: ATTGGTCTATTTGACCGCGTC	780	58
	R: TGCTACTCAACGACTGAGCG
blaNDM	F: GAGATTGCCGAGCGACTTG	497	57
	R: CGAATGTCTGGCAGCACACTT
blaOXA-51	F: TAATGCTTTGATCGGCCTTG	353	52
	R: TGGATTGCACTTCATCTTGG
blaOXA-23	F: GATCGGATTGGAGAACCAGA	501
	R: ATTTCTGACCGCATTTCCAT
blaOXA-40	F: GGTTAGTTGGCCCCCTTAAA	246
	R: AGTTGAGCGAAAAGGGGATT
blaOXA-58	F: AAGTAT TGGGGCTTGTGCTG	599
	R: CCCCTCTGCGCTCTACATAC
blaGIM-1	F: TCGACACACCTTGGTCTG AA	477
	R: AACTTCCAACTTTGCCATGC
blaSPM-1	F: AAAATCTGGGTACGCAAACG	271
	R: ACATTATCCGCTGGAACAGG
blaSIM-1	F: TACAAGGGATTCGGCATC G	570
	R: TAATGGCCT GTTCCCATGTG
blaCMY	F: GACAGCCTCTTTCTCCACA	1,000	50
	R: TGGAACGAAGGCTA CGTA
5'-CS	F: GGCATCCAAGCAGCAAG		56
3'-CS	R: AAGCAGACTTGACCTGA
hep51	F: GATGCCATCGCAAGTACGAG		55
hep74	R: CGGGATCCCGGACGGATGCACGATTTGTA

Abbreviations: Tm, melting temperature; CS, conserved segment of class-I integron; hep51, forward primer of of class-II integron; hep75, reverse primer of of class-II integron; CMY cephalomycinase coding gene.

The positive PCR product of the class 1 integron was cloned into pGEM-T easy vector (Promega, Madison, WI, USA) and then sequenced by Macrogen (Amsterdam, The Netherlands). Sequencing results were analyzed by using the BLAST alignment search tool (http://www.ncbi.nlm.nih.gov/BLAST) and the multiple sequence alignment program CLUSTALW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

Transferability of antibiotic resistance was tested according to the previously defined protocol [9], by using the rifampin-resistant Escherichia coli K-12 strain J53-2 as a recipient [4]. Susceptibility testing of the MBL-producing integron-positive P. aeruginosa isolate showed that it was resistant to imipenem, meropenem, piperacillin/tazobactam, ceftazidime, cefepime, and cefoperazone-sulbactam. PCR analysis showed that the isolate did not harbor any of the antibiotic resistance genes listed in Table 1, except for blaVIM-type MBL. Sequence analysis of the blaVIM-variant identified it as blaVIM-38. The P. aeruginosa isolate contained a class 1 integron gene cassette, but not a class 2 integron gene cassette. The blaVIM-38-harboring class 1 integron gene cassette was sequenced and was found to be 3,239 bp long. DNA sequence analysis revealed that blaVIM-38 MBL was located on the class 1 integron gene cassette together with AAC(6´)-Ib/EmrE/aadA1 (Fig. 1). The conjugation assay revealed that the class 1 integron cassette is not transferable.

Fig. 1

Structure of the blaVIM-38-carrying class 1 integron gene cassette in Pseudomonas aeruginosa.

Abbreviations: Pint, integrase promoter; Pant, promoter of inserted gene(s); intI1, class 1 integrase; attI1, integron-associated recombination site; qacEΔ1, quaternary ammonium compound resistance gene cassette; ™, sulfonamide resistance gene; blaVIM-38, Verona Integron-Encoded Metallo-β-lactamase 38; CS, conserved segment.

The blaVIM-38 gene was identified in P. aeruginosa isolates in Turkey in 2014, and found to be located in a class 1 integron containing only two gene cassettes (blaVIM-38/orfD) [3]. This genetic structure has also been associated with the blaVIM-5 gene in a clinical isolate of Enterobacter cloaceae from Turkey [9]. Moreover, steady-state kinetic analyses in a study on the enzymatic properties of VIM-38 showed that VIM-38 hydrolyzed all of the tested penicillins, cephalosporins, and carbapenems [10].

In the present study, the class 1 integron included four gene cassettes with blaVIM-38 followed by AAC(6´)-Ib, EmrE (multi-drug transporter), and aadA1. This is the first report in Turkey of the blaVIM-38/AAC(6´)-Ib/EmrE/aadA1 gene cassette array. Therefore, we report a novel gene cassette array with an MBL gene in a P. aeruginosa clinical isolate, and this is the second report for the detection of VIM-38 in a P. aeruginosa isolate in Turkey with different hospitalization and isolation times.

In conclusion, the presence of class 1 integrons in P. aeruginosa leads to increased resistance to antibiotics. The present study demonstrates the emergence of VIM-producing MDR P. aeruginosa strains harboring class 1 integrons and a gene cassette in Turkey. In particular, the blaVIM-38 MBL gene appears to be spreading among P. aeruginosa isolates in Turkey.