First Detection of blaIMI-2 Gene in a Clinical Escherichia coli Strain

The dissemination of carbapenem-resistant Enterobacteriaceae is increasing worldwide in the last decade, mainly due to the acquisition of beta-lactamase genes encoding carbapenemases. Among class A carbapenemases, KPC is widespread (14); however, IMI enzymes have been described so far only in Enterobacter genus (1, 10, 15). The detected blaIMI genes are linked to a gene encoding a LysR-type transcriptional regulator, and as previously reported, the blaIMI-1 gene is located in the chromosome of Enterobacter cloacae (10), whereas blaIMI-2 is related to plasmids (1, 15). We report here the first description of a blaIMI-2-positive Escherichia coli strain. (Part of this study was presented at the 21st European Congress of Clinical Microbiology and Infectious Diseases Congress, Milan, Italy, 7 to 10 May 2011.) A carbapenem-resistant E. coli strain, W635, was recovered from a blood sample of an elderly oncologic patient (without any history of travel) who was admitted with sepsis and treated with piperacillin-tazobactam at a Spanish hospital in 2010. E. coli W635 was ascribed to a new sequence type registered as ST1998 (http: //mlst.ucc.ie/mlst/dbs/Ecoli) and showed resistance to imipenem (IPM), meropenem (MEM), ertapenem (ETP), doripenem, ampicillin (AMP), ticarcillin, amoxicillin-clavulanic acid (AMC), cephalothin, streptomycin, nalidixic acid, ciprofloxacin, norfloxacin, sulfonamides, and trimethoprim and intermediate resistance to aztreonam and chloramphenicol by the CLSI disk diffusion method (3). A class A carbapenemase phenotype was demonstrated in this strain by double-disk synergy test (5, 9). After multiplex PCR (8) and subsequent sequencing, a partial sequence of the blaIMI-2 gene was detected in E. coli W635. To gain insight into the blaIMI-2 gene and its surrounding structure, the flanking regions were amplified by PCR using specific primers designed in this work (according to GenBank accession number AY780889). The LysR-type regulator gene (blaIMI-2R) was found upstream of the blaIMI-2 gene, and their genetic environment was studied by inverse PCR using PvuII and BglII restriction enzymes. Sequence analysis revealed a total fragment of 6,184 bp that was deposited in GenBank database with the accession number JN412066. A new insertion sequence of 1,321 bp, designed ISEc36 by ISFinder (http://www-is.biotoul.fr/), was detected upstream of the blaIMI-2R gene. This IS belongs to the IS3 family and IS2 group, and it exhibits an identity of 92% with respect to the ISEc27 sequence (GenBank accession number AY857617). Testing for the presence of genes implicated in other antimicrobial resistances (beta-lactams, aminoglycosides, quinolones, trimethoprim, and sulfonamides) and the study of mutations in gyrA and parC genes, as well as the characterization of integrons and sul2 gene environment, were performed by PCR and sequencing (4, 11, 13). Table 1 shows the MICs and genotypic results. Two different transconjugants from W635 were obtained by mating experiments, using E. coli CSH26 as recipient and plates supplemented with either MEM (8 g/ml) or AMP (50 g/ml) and rifampin (100 g/ml). Detection and typing of plasmids of E. coli W635 and transconjugant strains were carried out by PCRbased replicon typing (2, 6). Plasmids of strain W635 and its transconjugants belonging to incompatibility group I1 (IncI1) and F (IncF) were subtyped by plasmid multilocus sequence typing (7, 12). E. coli W635 contained the following typeable plasmids: IncI1 (ST26, CC-26), IncF (F43:A :B ; Y2 variant:A :B ), and ColETP. The location of blaIMI-2 in strain W635 and its transconjugants was studied by pulsed-field gel electrophoresis (PFGE)-S1 nuclease and PFGE-XbaI Southern blotting and hybridization (4) with blaIMI-2, IncI1, IncF, and ColETP probes. The blaIMI-2 gene in E. coli W635 was detected in an IncF plasmid of approximately 48.5 kb. The size of this plasmid is smaller than found in previously reported results that described the blaIMI-2 gene located on transferable plasmids with sizes of 66 kb or 80 kb (1, 15). This is the first report of a carbapenem-resistant E. coli strain carrying the class A carbapenemase IMI-2. The blaIMI-2 gene located in a conjugative plasmid and linked to mobile elements might significantly spread between different members of the Enterobacteriaceae, and therefore this emerging resistance mechanism should be tracked in the future. Nucleotide sequence accession number. The nucleotide sequence of the novel genetic environment of blaIMI-2 gene determined in this study was included in the GenBank database with the accession number JN412066.