Biochemical characterization of PER-2 and genetic environment of blaPER-2

Abstract

PER-2 was the first detected and the second most prevalent extended-spectrum beta-lactamase in clinical pathogens isolated in Argentina and was also reported only in other South American countries. Citrobacter freundii 33587 was isolated in 1999 in Buenos Aires and was resistant to all tested beta-lactams except cephamycins and carbapenems. The strain produced both plasmid-borne TEM-1 and PER-2 (pI 5.4), which could be transferred by conjugation. By PCR screening, thermal asymmetric interlaced PCR, and DNA sequencing, we detected an ISPa12/IS1387a insertion sequence upstream of bla(PER-2), previously reported as also being associated with bla(PER-1). The presence of similar structures upstream of bla(PER-1) and bla(PER-2) suggests a common origin and mobilization. Compared to bla(PER-1) genes, an additional putative promoter for bla(PER-2) was found. PER-2 kinetic analysis showed its high hydrolysis efficiencies toward both CTX and CAZ (k(cat)/K(m), 0.76 and 0.43 microM(-1).s(-1), respectively).

FIG. 1.

Schematic representation of the bla_PER-2 gene and neighboring sequences compared to those for bla_PER-1 genes. (A) C. freundii 33587 (plasmid borne). (B) Salmonella serovar Typhimurium and A. baumannii (plasmid borne). (C) P. aeruginosa, A. baumannii (chromosome encoded), and A. faecalis (plasmid borne). (D) P. stuartii (chromosome encoded). The patterns represent different genetic backgrounds. *, alternative names given in different references; nt, nucleotide.

FIG. 2.

Comparison of the upstream sequences of bla_PER-2 from C. freundii 33587 and bla_PER-1 from P. aeruginosa RNL-1 (AY779402), comprising the respective promoter regions. The +1 transcription initiation sites are indicated in large boldface letters, respective −10 and −35 boxes are in light-gray boxes, and entire promoters are marked with horizontal brackets. The IR of ISPa12/IS1387a is indicated in boldface letters in a shaded box, and the inverted black triangle indicates the ISPa12/IS1387a boundary.