VIM-1, VIM-34, and IMP-8 Carbapenemase-Producing Escherichia coli Strains Recovered from a Portuguese River

LETTER

The emergence of acquired carbapenemases is currently one of the most serious public health threats worldwide. These enzymes confer resistance to almost all β-lactams, including carbapenems leading to very few therapeutic options for treating patients infected by multidrug-resistant bacteria.

Here we report the identification of four environmental carbapenem-resistant Escherichia coli strains, E61, E201, E202, and E203. These strains were recovered in February 2015, from the Ave river, in the north of Portugal. Water samples of 100 ml were filtered through 0.45-μm-pore-size membrane filters (Millipore Corporation, USA), which were then placed on tryptone bile X-glucuronide agar (TBX) (BioKar Diagnostics, Beauvais, France) plates supplemented with imipenem (2 mg/liter). Strains growing on those selective plates were checked for carbapenemase activity by using the Carba NP test (1). Antimicrobial susceptibility testing was performed according to the standard disk diffusion method following CLSI recommendations (2) and using cation-adjusted Muller-Hinton plates (Bio-Rad, Cressier, Switzerland). The four isolates were resistant to all β-lactams, fluoroquinolones, and aminoglycosides (except amikacin), being susceptible only to tigecycline and colistin. Two out of the four isolates (E61 and E202) also remained susceptible to fosfomycin. MICs of carbapenems (imipenem, ertapenem, and meropenem) were performed using Etest strips (bioMérieux, La Balme-les-Grottes, France) (Table 1). Molecular investigations were then performed by PCR using specific primers for carbapenemase genes (3) followed by sequencing (Microsynth, Balgach, Switzerland). The phylogenetic group was determined as previously described (4) and showed that isolates E201 and E203 belonged to group E, strain E61 belonged to group A, and strain E202 belonged to group F. Multilocus sequence typing performed as described previously (5) confirmed that isolates E201 and E203 belonged to the same sequence type, whereas the two other strains were clonally unrelated. PCR and sequencing analysis revealed that strains E201 and E203 harbored the bla_IMP-8 gene and strains E61 and E202 harbored the bla_VIM-1 and bla_VIM-34 genes, respectively.

TABLE 1.

MICs of carbapenems for the environmental isolates and E. coli J53 harboring the carbapenemase genes

Carbapenem or parameter	MIC (μg/ml)a
	Environmental isolates				E. coli J53 transconjugants			E. coli J53
	E61	E201	E202	E203	E61TC	E201TC	E203TC
Imipenem	≥32	16	6	32	1	1.5	6	0.125
Meropenem	≥32	8	4	16	0.38	1	2	0.016
Ertapenem	≥32	16	4	32	0.38	0.5	3	0.012
Sequence type	167	2612	354	2612

^aMICs of carbapenems are shown unless specified otherwise.

Conjugation assays were performed in liquid medium using the azide-resistant E. coli J53 as the recipient strain. Transconjugants were selected onto Luria-Bertani agar plates supplemented with ertapenem (2 mg/liter) and azide (100 mg/liter) to detect the transfer of the carbapenemase genes. Transconjugants were obtained for three out of the four isolates, but not for strain E202 despite repeated attempts. Plasmid extractions were performed using the Kieser extraction method (6) using the environmental strains and transconjugants. Further analysis showed that the bla_VIM-1 and bla_IMP-8 genes were carried on a ca. 150-kb plasmid. Molecular typing of plasmids was performed using the PCR-based replicon typing (Diatheva, Fano, Italy) (7), revealing that all plasmids bearing the carbapenemase genes belonged to the IncFIB group.

The failure to obtain a transconjugant or transformant from strain E202 suggests a possible chromosomal location of the bla_VIM-34 gene. It is noteworthy that the bla_VIM-34 gene was previously identified from Klebsiella pneumoniae in Portugal in 2013 and was found to be located on the chromosome (8). PCR mapping followed by sequencing revealed that the bla_VIM-34 gene was embedded in class 1 integron In817 in strain E202, the exact same structure as identified in the K. pneumoniae isolate from Portugal (8).

The occurrence of carbapenemase-producing E. coli strains in the aquatic environment remains worrying. Interestingly, we first identified a KPC-producing E. coli in Portugal from the same river, as early as 2010 (9). This river may be a source of carbapenemase-producing E. coli or, conversely, humans might be the source of this environmental contamination. Note that IMP-8 producers have been reported in Portugal (10) in K. pneumoniae and Pseudomonas aeruginosa clinical isolates (11), suggesting a wide spread of this carbapenemase, which is not commonly identified in other European countries.

Finally, monitoring the occurrence of carbapenemase-producing members of the family Enterobacteriaceae in the environment can be of interest, since it may contribute to better control of the occurrence and spread of these bacteria. It is therefore mandatory to screen for carbapenemase producers not only among the hospitalized populations but also among healthy people and potential environmental cradles in order to identify possible hidden reservoirs.

Funding Statement

This study was financed by the University of Fribourg, Switzerland, and by the ANIHWA-ERA-NET project PRAHAD funded by the Federal Food Safety and Veterinary Office, Bern, Switzerland.