Nosocomial infections caused by multidrug-resistant isolates of pseudomonas putida producing VIM-1 metallo-beta-lactamase

Abstract

Successful carbapenem-based chemotherapy for the treatment of Pseudomonas infections has been seriously hindered by the recent appearance of IMP- and VIM-type metallo-beta-lactamases, which confer high-level resistance to carbapenems and most other beta-lactams. Recently, multidrug-resistant Pseudomonas putida isolates for which carbapenem MICs were >/=32 micro g/ml were recovered from cultures of urine from three inpatients in the general intensive care unit of the Ospedale di Circolo, Varese, Italy. Enzyme assays revealed production of a metallo-beta-lactamase activity, while molecular analysis detected in each isolate a bla(VIM-1) determinant carried by an apparently identical medium-sized plasmid. Conjugation experiments were unsuccessful in transferring the beta-lactamase determinant to Escherichia coli or Pseudomonas aeruginosa. Macrorestriction analysis by pulsed-field gel electrophoresis demonstrated that the isolates were of clonal origin. PCR mapping and sequencing of the variable region of the plasmid-borne class 1 integron carrying the bla(VIM-1) determinant (named In110) showed that the bla(VIM-1)-containing cassette was identical to that previously found in strains of different species from other Italian hospitals and that the cassette array of In110 was not identical but clearly related to that of In70 (a bla(VIM-1)-containing plasmid-borne integron from an Achromobacter xylosoxidans isolate), pointing to a common origin of this cassette and to a related evolutionary history of their cognate integrons.

FIG. 1.

(A) Agarose gel electrophoresis of the plasmid DNA preparation from isolate VA-304/99 after digestion with EcoRI. (B) Results of Southern blot hybridization of the sample shown in panel A with a bla_VIM-specific probe. Size standards (in kilobases) are indicated on the left.

FIG. 2.

Structure of the gene cassette array of integron In110, carried by plasmid pVA304, compared to that of In70, carried by plasmid pAX22 from A. xylosoxidans AX22 (24). Genes are indicated by arrows. The attC (also called the 59-base-element) recombination sites of gene cassettes are indicated by circles. The map is not drawn to scale.

FIG. 3.

PFGE banding patterns after SpeI digestion. Lanes 1 to 3, VIM-1-producing P. putida isolates from the general ICU (lane 1, isolate VA-304/99; lane 2, isolate VA-523/99; lane 3, isolate VA-420/00); lane 4, multidrug-resistant P. putida isolate from the nephrology department; lanes 5 and 6, two antibiotic-susceptible P. putida isolates obtained from the general ICU at the same hospital in 2000; lane L, λ phage DNA concatemers as a size marker.