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

LETTER

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 bla_IMI genes are linked to a gene encoding a LysR-type transcriptional regulator, and as previously reported, the bla_IMI-1 gene is located in the chromosome of Enterobacter cloacae (10), whereas bla_IMI-2 is related to plasmids (1, 15). We report here the first description of a bla_IMI-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 bla_IMI-2 gene was detected in E. coli W635. To gain insight into the bla_IMI-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 (bla_IMI-2R) was found upstream of the bla_IMI-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 bla_IMI-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.

Table 1.

Resistance phenotype, genotype, and genetic elements of E. coli strain W635, transconjugants (TC), and recipient strain^a

Strain	MIC (μg/ml) ofb:						Resistance gene(s)				Amino acid change(s) detected		Class 1 integron	Incompatibility group detected (size of plasmid)
	AMP	ATM	IPM	MEM	STR	TMP					GyrA	ParC
W635 donor	>128	8	128	32	128	>128	blaTEM-1b	blaIMI-2	strA-strBc	sul1 sul2	S83L, D87N	S80I	dfrA1 aadA1	IncI1 (97 kb), IncF (48.5 kb), ColETP (<10 kb)
TC1	>128	8	128	16	>128	>128	blaTEM-1b	blaIMI-2	strA-strB	sul1 sul2	ND	ND	dfrA1 aadA1	IncI1/IncF (160 kb),d ColETP (<10 kb)
TC15	>128	0.125	0.25	0.032	>128	>128	blaTEM-1b	–	strA-strB	sul1 sul2	ND	ND	dfrA1 aadA1	IncI1 (97 kb)
CSH26 recipient	16	0.125	0.25	0.032	4	<0.125	–	–	–	–	ND	ND	ND	ND

^aND, not determined; –, not detected.

^bAMP, ampicillin; ATM, aztreonam; IPM, imipenem; MEM, meropenem; STR, streptomycin; TMP, trimethoprim.

^cstrA-strB genes were linked to the sul2 gene (repC-sul2-strA-strB-ISCR2).

^dIncI1 and IncF were detected by hybridization in a plasmid of approximately 160 kb in the bla_IMI-2-positive TC1 transconjugant.

Two different transconjugants from W635 were obtained by mating experiments, using E. coli CSH26 as recipient and plates supplemented with either MEM (8 μg/ml) or AMP (50 μg/ml) and rifampin (100 μg/ml). Detection and typing of plasmids of E. coli W635 and transconjugant strains were carried out by PCR-based 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 ColE_TP. The location of bla_IMI-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 bla_IMI-2, IncI1, IncF, and ColE_TP probes. The bla_IMI-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 bla_IMI-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 bla_IMI-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 bla_IMI-2 gene determined in this study was included in the GenBank database with the accession number JN412066.