Characterization of a novel metallo-β-lactamase variant, GIM-2, from a clinical isolate of Enterobacter cloacae in Germany.

The metallo-lactamase (MBL) GIM-1, first identified in Pseudomonas aeruginosa isolates in Germany in 2002 (1), has since been described sporadically and never outside Germany in Pseudomonas spp. (2, 3), Acinetobacter pittii (4), and a range of Enterobacteriaceae (3, 5, 6). No genetic variations of blaGIM-1 have been reported (7). The Enterobacter cloacae isolate described here was first identified in June 2014 from the rectal screening of a 49-year-old male patient from Saudi Arabia previously hospitalized in Germany and in Saudi Arabia. Results of phenotypic detection of a carbapenemase using a combined disk test (10 g meropenem with and without 930 g EDTA [8]) and a modified Hodge test (9) were both positive. The MICs of the following relevant antibiotics were determined by Etest (bioMérieux): piperacillin ( 256 mg/liter), piperacillin-tazobactam ( 256 mg/liter), ceftazidime ( 256 mg/liter), cefepime ( 12 mg/liter), imipenem (1 mg/liter), meropenem (4 mg/liter), aztreonam ( 256 mg/liter), gentamicin (4 mg/liter), amikacin (2 mg/liter), ciprofloxacin (0.5 mg/liter), and colistin (0.5 mg/liter). Multiplex PCRs were performed for the following -lactamase genes: blaIMP-1, blaVIM-1-type, blaVIM-2-type, blaGIM-1, and blaNDM-1 (3); blaKPC and blaOXA-48 (10); blaCTX-M groups 1, 2, 9, and 8/25 (11); and blaTEM and blaSHV (12). PCR detected blaGIM-1 and blaCTX-M group 9 genes. Sequencing of the blaGIM gene (GIM-Fflanking, 5=-TCCAGAACCTTGACCGAACG-3=, and GIM-Rflanking, 5=-GCCACTCATAGAGCATCGCA-3=) revealed a new variant of the metallo-lactamase blaGIM-1 gene (given the name blaGIM-2) with one nucleotide substitution, A290G, causing an amino acid substitution of glutamic acid to glycine at position 97. The sequenced class 1 integron, In1101, is identical in the order of the gene cassettes to integrons previously described in blaGIM-1positive E. cloacae (3, 5). The genes were located downstream of the attI1 recombination site in the following order: blaGIM-2 and aminoglycoside acetyltransferase gene aacA4 in one common (fused) gene cassette, aminoglycoside acetyltransferase gene aadA1, and -lactam resistance gene blaOXA-2. Genetic localization of the blaGIM-2-containing integron was determined by S1-nuclease digestion and in-gel hybridization with a P-labeled blaGIM probe as previously described (13). As a template, the amplicon of primers (5= to 3=) 5.1.R2 (CCAAGCA GCAAGCGCGTTAC) and GIMR (ACTCATGACTCCTCACG AGG) (1), which bind to blaGIM-2, was used. No blaGIM-2-containing plasmid was detected, and hybridization of the probe occurred only on chromosomal DNA. Conjugation experiments were carried out using the blaGIM-2 strain and sodium azide-resistant Escherichia coli J53 on sheep blood agar at a recipient/donor ratio of 1:10. The selective media contained 4 mg/liter ceftazidime and 100 mg/liter sodium azide. The blaGIM-2 gene was nonconjugative. Genotyping was carried out together with blaGIM-1-positive E. cloacae isolates previously described (3) using three methods: pulsed-field gel electrophoresis (PFGE) (XbaI, in accordance with the Tenover criteria [14]), repetitive sequence-based PCR (repPCR) (DiversiLab) (with a similarity cutoff of 95%), and multilocus sequence typing (MLST) (15). The blaGIM-2-positive strain, confirmed to be sequence type 108, was shown to be unrelated to the other isolates by all genotyping methods. In conclusion, the isolation of a new GIM-type MBL in Germany highlights the ongoing spread and evolution of this local metallo-lactamase gene. The isolate presented here may be easily missed in routine microbiology laboratories since isolates carrying the gene can show relatively low MICs for carbapenems; however, results of phenotypic tests for carbapenemases were positive. Nucleotide sequence accession number. The integron whose sequence was determined in this work has been allocated GenBank accession number KM659858.