Dynamics of a nosocomial outbreak of multidrug-resistant Pseudomonas aeruginosa producing the PER-1 extended-spectrum beta-lactamase

Abstract

From November 1998 to August 1999, a large outbreak occurred in the general intensive care unit of the Ospedale di Circolo in Varese (Italy), caused by Pseudomonas aeruginosa producing the PER-1 extended-spectrum beta-lactamase. A total of 108 clinical isolates of P. aeruginosa resistant to broad-spectrum cephalosporins were recovered from 18 patients. Epidemic isolates were characterized by synergy between clavulanic acid and ceftazidime, cefepime, and aztreonam. Isoelectric focusing of crude bacterial extracts detected two nitrocefin-positive bands with pI values of 8.0 and 5.3. PCR amplification and characterization of the amplicons by restriction analysis and direct sequencing indicated that the epidemic isolates carried a bla(PER-1) determinant. The outbreak was of clonal origin as shown by pulsed-field gel electrophoresis analysis. This technique also indicated that the epidemic strain was not related to three other PER-1-positive isolates obtained at the same hospital in 1997. Typing by enterobacterial repetitive intergenic consensus-PCR showed that minor genetic variations occurred during the outbreak. The epidemic strain was characterized by a multiple-drug-resistance phenotype that remained unchanged over the outbreak, including extended-spectrum cephalosporins, monobactams, aminoglycosides, and fluoroquinolones. Isolation of infected patients and appropriate carbapenem therapy were successful in ending the outbreak. Our report indicates that the bla(PER-1) resistance determinant may become an emerging therapeutic problem in Europe.

FIG. 1

(Top) Number of PER-1-positive P. aeruginosa isolates obtained each month from the 18 ICU patients involved in the outbreak (November 1998 to August 1999). Columns show the number of initial isolates recovered from every patient (solid columns) and the number of replicate isolates (open columns). (Bottom) Number of PER-1-negative P. aeruginosa isolates obtained per month from 69 of 560 patients hospitalized in the ICU (October 1998 to September 1999). Columns show the number of initial isolates from every patient (solid columns) and the number of replicate isolates (open columns).

FIG. 2

Amplification of the bla_PER gene from a representative epidemic P. aeruginosa isolate. Digestion of the amplicon (966 bp) (lane 1) with PvuII produced the expected restriction pattern of the bla_PER-1 gene (lane 2). Digestion with StuI (which cuts into bla_PER-2 but not into bla_PER-1) failed to digest the amplified product (lane 3). L, DNA size marker.

FIG. 3

PFGE banding patterns after SpeI digestion of representative PER-1-positive P. aeruginosa isolates. Epidemic isolates (lanes 1 to 8) show closely related patterns, with the exception of isolate VA-139/99 (lane 8), which differs from the other epidemic strains in 4 of 28 discernible bands. Lanes 9 to 11 show the different restriction patterns of nonepidemic PER-1-positive isolates obtained in 1997 before the beginning of the outbreak. L, λ-phage DNA pulse marker.

FIG. 4

ERIC-PCR analysis of representative epidemic PER-1-positive P. aeruginosa isolates obtained from November 1998 to February 1999 (lanes 1 to 4) and from April to August 1999 (lanes 5 to 8). Two clearly different banding patterns were seen, indicating that minor genetic rearrangements took place during the outbreak. L, DNA size marker.