First Isolation of bla_VIM-2 in Latin America: Report from the SENTRY Antimicrobial Surveillance Program

Carbapenems, mainly imipenem and meropenem, are potent agents for the treatment of infections due to multiresistant Pseudomonas sp. clinical isolates. However, the prevalence of carbapenem resistance in this genus has been increasing worldwide. High-level resistance to carbapenems (>32 μg/ml) is still uncommon in Pseudomonas spp. but can be due to the presence of Ambler class B β-lactamases—metallo-β-lactamases (MBLs) (1). Three different clinically relevant types of mobile MBLs have been described in the literature: IMP, VIM, and SPM (2, 4; H. Kurokawa, T. Yagi, N. Shibata, K. Shibayama, and Y. Arakawa, Letter, Lancet 354:955, 1999). The VIM family has been reported mostly in European and Asian countries, although a distantly related MBL, VIM-7, has been characterized from the United States (5). We are not aware of VIM-type MBLs being reported from Latin America. In this work, we describe the presence of bla_VIM-2-producing Pseudomonas sp. isolates and characterize their genetic context among isolates obtained from Latin American medical centers representing Chile and Venezuela.

Four isolates of Pseudomonas spp., one of P. fluorescens (43-14926) from a blood culture isolated in December 2002 in Santiago, Chile, and three of P. aeruginosa (49-4583, 49-4596, and 49-4597) recovered from respiratory tract samples between August and September 2002 from the same hospital in Caracas, Venezuela, were obtained as part of the SENTRY Antimicrobial Surveillance Program. According to results of MIC tests performed as described by the National Committee for Clinical Laboratory Standards, the isolates were resistant to all β-lactams, aminoglycosides, quinolones, and other antimicrobial agents tested (Table 1). Only polymyxin B was active against all the isolates. MBL phenotypic tests were positive as judged by the disk approximation method (imipenem, meropenem, ceftazidime, EDTA, and 2-mercaptopropionic acid) and the Etest MBL strips (AB Biodisk, Solna, Sweden) (7). These results were confirmed by spectrophotometric assays measuring imipenem hydrolysis, as previously described (4). Imipenem hydrolysis was inhibited by 88 to 97% for each of the strains by EDTA (20 mM) (Table 2).

TABLE 1.

Antimicrobial susceptibility profiles of the bla_VIM-2-carrying Pseudomonas sp. isolates

Antimicrobial agent	MIC (μg/ml)
	P. fluorescens 43-14926	P. aeruginosa
		49-4583	49-4596	49-4597
β-Lactams
Cefazolin	>16	>16	>16	>16
Cefoxitin	>32	>32	>32	>32
Aztreonam	>16	>16	16	16
Cefuroxime	>16	>16	>16	>16
Ceftriaxone	>32	>32	>32	>32
Ceftazidime	>16	>16	>16	>16
Cefepime	16	>16	>16	>16
Imipenem	>8	>8	>8	>8
Meropenem	>8	>8	>8	>8
Piperacillin-tazobactam	>64	64	64	64
Quinolones
Ciprofloxacin	>4	>4	>4	>4
Gatifloxacin	>4	>4	>4	>4
Levofloxacin	>4	>4	>4	>4
Aminoglycosides
Amikacin	8	>32	>32	>32
Gentamicin	>8	>8	>8	>8
Netilmicin	>32	>32	>32	>32
Tobramycin	>16	>16	>16	>16
Others
Polymyxin B	≤1	≤1	≤1	≤1
Tetracycline	>8	>8	>8	>8
Trimethoprim-sulfamethoxazole	>2	>2	>2	>2

TABLE 2.

MICs by Etest MBL strips and imipenem hydrolytic activities (with and without EDTA) of the bla_VIM-2-carrying Pseudomonas sp. isolates

Strain	Etest MBL MIC (μg/ml)		Hydrolytic activity (absorbance/min)		% Inhibition
	Imipenem	Imipenem + EDTA	Imipenem	Imipenem + EDTA
43-14926	>256	≤1	0.07194	0.00384	94.6
49-4583	>256	4	0.04519	0.00251	94.5
49-4596	>256	6	0.10144	0.00253	97.5
49-4597	>256	8	0.04359	0.00500	88.5

Amplification with primers for the internal region of bla_VIM-like genes and 5′ conserved sequence and 3′ conserved sequence from class 1 integron and subsequent sequencing were performed as described earlier (3, 5). Primers used for amplification and sequencing of the bla_VIM gene were VIM-F (5′-AAAGTTATGCCGCACTCACC-3′) and VIM-R (5′-TGCAACTTCATGTTATGCCG-3′).

Sequencing results of the PCR amplicons revealed the presence of the bla_VIM-2 gene in the first position of a class 1 integron, which has qacEΔ/su1l downstream of the MBL gene. This integron gene arrangement has been previously reported for a P. aeruginosa isolate from a patient in France in 1996 (3). Other cases have been reported from P. aeruginosa in Greece in 2000 and from Serratia marcescens in Korea in 2002 (6, 8). Class 1 integrons carrying bla_VIM-type genes are now reported to contain genes encoding aminoglycoside-modifying enzymes, and it is uncertain whether the arrangement of the integron without the additional genes is the progenitor.

Despite repeated attempts, plasmid DNA analysis of the isolates did not show any plasmids, and transformation experiments were unsuccessful. Experiments in conjugation between the clinical isolates and Escherichia coli K-12 Rif^r did not yield any transconjugants. These data imply that the bla_VIM-2 found in these isolates is likely to be chromosomally carried. Automated ribotyping and pulsed-field gel electrophoresis showed that the three isolates from Venezuela are identical, suggesting clonal spread. The strain from Chile was unique and belonged to a different species from those of the other MBL-producing strains.

Dissemination of multiresistant bacteria coupled with the plasticity of class 1 integrons suggests that resistance to mainstay anti-Pseudomonas therapies, such as expanded-spectrum cephalosporins and carbapenems, will continue to increase. We urge greater local screening for MBL-producing strains with the disk approximation tests (8; Kurokawa et al., letter) and the Etest MBL strip (7).