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AAC Accepts, published online ahead of print on 30 January 2012

Antimicrob. Agents Chemother. doi:10.1128/AAC.06005-11

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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VIM-1-producing Pseudomonas mosselii isolated in Italy, pre-dating known VIM-producing

index strains

Acquired metallo-β-lactamases (MBLs) are emerging resistance determinants of great clinical

concern. They are found in Enterobacteriaceae, Pseudomonas aeruginosa and other gram-negative

nonfermenters, in which they can confer a very broad spectrum of β-lactam resistance, including the

expanded-spectrum cephalosporins and carbapenems, that cannot be reverted by β-lactamase

inhibitors currently available for clinical use (2).

The VIM-type enzymes were the second acquired MBLs to be detected, in the late 1990s in Europe

(5,10), and are currently the most widespread in terms of geographical distribution and range of

host bacterial species (2). VIM-1 and VIM-2 were the first reported VIM-type allelic variants, in P.

aeruginosa isolated in 1997 in Verona, Italy (index strain VR-143/97) (5) and in 1996 in Marseilles,

France, respectively (10).

We report here on the characterization of a Pseudomonas clinical isolate producing the VIM-1

MBL, isolated in Genoa, Italy, in 1994, which to our best knowledge represents the earliest known

VIM-producing strain.

Pseudomonas sp. AM/94 was isolated in November1994 from the lower respiratory tract of an

inpatient admitted in the general Intensive Care Unit of Genoa University Hospital. The isolate was

at the time identified as Pseudomonas fluorescens by the API 20E system (Bio-Merieux, Marcy

L’Etoile, France), with a low grade of discrimination (62%). Reidentification of the isolate by

Vitek-2 (Bio-Merieux) yielded a result of low discrimination between nosa (34%) P.

fluorescens (33%) and Pseudomonas putida (33%). MALDI-TOF analysis , using Microflex

TM

LT

bench-top MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) identified the

isolate as Pseudomonas montelii (score=2.03). Molecular identification by amplification and

sequencing of 16S rDNA, rpoD, rpoB and gyrB genes, unambiguously identified AM/94 as

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Pseudomonas mosselii, a recently recognized species of the P. putida group (3, 7).

Susceptibility testing, carried out with Etest (Bio-Merieux) or with broth microdilution (1) and

interpreted according to the EUCAST breakpoints for Pseudomonas spp. (document version 1.3,

2011-01-05, /clinical_breakpoints, accessed October, 2011) revealed that

AM/94 was resistant to all tested antibiotics except amikacin and colistin (Table 1).

MBL activity, assayed spectrophotometrically (5), was detected in a crude extract of AM/94

(specific imipenemase activity was 120±1 nmol/min/mg protein, inhibited by >80% after exposure

to 2 mM of EDTA).

Analysis of acquired MBL genes including bla

VIM

, bla

IMP

, and bla

NDM

using PCR and sequencing (4)

revealed the presence of bla

VIM-1

allele. PCR mapping and sequencing of the genetic context of the

bla

VIM-1

gene (12) revealed that the gene was carried on a gene cassette inserted into a class 1

integron whose variable region contained four cassettes (bla

VIM-1

, aacA4, aphA15 and aadA1) and

was identical to that of In70-type integrons previously described in other VIM-1-producing strains

from Italy (12). Mapping the region of the cognate Tn402-like transposon flanking the integrase

gene, however, revealed that the structure was identical to that flanking In70 detected in plasmid

pAX22 from Achromobacter xylosoxidans AX22 (11): it was lacking the ISPa7 insertion sequence

present downstream the intl1 gene in In70.2, the bla

VIM-1

-containing integron from the VR-143/97 P.

aeruginosa index strain (12) (Figure 1).

Altogether, present results indicate that influx of the bla

VIM-1

MBL gene in the clinical setting

started at least since the early 1990s, and that In70 might have been the original genetic element

involved in this influx.

P. mosselii is an environmental species detected in rhizospheric soil (8, 9), and an overall unusual

human opportunistic pathogen. In literature, only one report describes P. mosselii as a cause of

infection (a prosthetic valve endocarditis) in human (6). Similar environmental species occasionally