Further Identification of CTX-M-2 Extended-Spectrum β-Lactamase in Pseudomonas aeruginosa

β-Lactamase production is the main mechanism for β-lactam resistance in gram-negative rods. The more threatening β-lactamases that have successfully emerged and are believed to spread only in Enterobacteriaceae are CTX-M-type extended-spectrum β-lactamases (ESBLs) (5). The acquired β-lactamases with wide activity spectra that are important in Pseudomonas aeruginosa include class B metallo-β-lactamases (mostly IMP and VIM types and especially SPM-1 in Brazil) and class A ESBLs, particularly VEB-, PER-, and GES-type enzymes (3, 8, 9). However, a single CTX-M-1-producing P. aeruginosa isolate has been reported from The Netherlands (1), as well as CTX-M-2- and CTX-M-43-positive P. aeruginosa isolates in Bolivia (2). This study reports the identification of a CTX-M-2-producing P. aeruginosa strain isolated from a Brazilian teaching hospital.

During June 2005, a 63-year-old male patient with a recent hospitalization history was admitted to the intensive care unit for suspicion of pneumonia. He received ceftriaxone and clindamycin as first-line therapy. Four days later, he presented with septic shock and died. A blood culture grew P. aeruginosa (isolate P6208).

Isolate P6208 was resistant to all β-lactams tested, except imipenem and ceftazidime. A double-disk synergy test was performed with ticarcillin-clavulanic acid- and cefotaxime-cefepime-containing disks. The production of an ESBL was evidenced only under unusual conditions (with a distance between the disks of 1.5 cm center to center) (Fig. 1). Isolate P6208 was also resistant to fluoroquinolones, amikacin, gentamicin, and tobramycin and was susceptible to colistin. The MICs of imipenem, ceftazidime, cefepime, and cefotaxime determined by using Etest strips (AB Biodisk, Solna, Sweden) were 1 μg/ml, 2 μg/ml, >256 μg/ml, and >32 μg/ml, respectively.

FIG. 1.

Double-disk synergy test with bla_CTX-M-2-positive P. aeruginosa clinical isolate P6208. Arrows indicate double-disk synergy. Abbreviations: CAZ, ceftazidime; TCC, ticarcillin-clavulanic acid; CTX, cefotaxime; FEP, cefepime.

HindIII-restricted total DNA from isolate P6208 as described previously (6) was used for cloning in pBK-CMV and was then transformed into Escherichia coli TOP10 and selected on agar plates containing ticarcillin (50 μg/ml) and kanamycin (30 μg/ml). The E. coli TOP10(p6208) recombinant strain displaying an ESBL phenotype was obtained. The sequencing of the 2,340-bp cloned DNA insert of recombinant plasmid p6208 identified a bla_CTX-M-2 gene. It was preceded by an ISCR1 element located 498-bp upstream and followed by the qacEΔ1 gene cassette. This ISCR1-bla_CTX-M-2 structure has already been identified in several enterobacterial species (7).

Plasmid extraction performed by the Kieser method (4) did not evidence any plasmid. In addition, repetitive attempts to transfer the bla_CTX-M-2 gene by electroporation failed, using both E. coli TOP10 and P. aeruginosa PAO1 as recipient strains. Thus, the bla_CTX-M-2 gene might be likely chromosomally located in isolate P6208.

This study identified a CTX-M-2-producing P. aeruginosa in Brazil. This finding is important since clinical laboratories may misidentify CTX-type enzymes in those nonfermenters, jeopardizing the choice of antimicrobial chemotherapy and the implementation of infection control measures. This report underlines that P. aeruginosa may become a hidden location for bla_CTX-M genes.