Characterization of In100, a New Integron Carrying a Metallo- -Lactamase and a Carbenicillinase, from Pseudomonas aeruginosa

Carbenicillin was introduced in therapeutics in 1967 and was especially useful, on its own or when associated with aminoglycosides, for the treatment of infections caused by Pseudomonas aeruginosa (23), a well-recognized nosocomial pathogen with intrinsic resistance to various antimicrobial agents and the ability to develop multidrug resistance (18). Nevertheless, an increasing frequency of P. aeruginosa isolates resistant to these antibiotics and to newer -lactams has been observed during the last decades, thus determining the use of the most potent -lactams, carbapenems (14). Intensive use of these antibiotics eased the emergence of carbapenem resistance in P. aeruginosa (7), among others, due to the production of metallo-lactamases, mainly IMP and VIM types (9, 10, 22). Initially reported in Japan (17), the new metallo-lactamases, have spread worldwide, nowadays being reported in gram-negative isolates from Europe (3, 7, 10, 20), Asia (9, 11), and, more recently, North America (26). Most IMPand VIM-like gene cassettes are found on class 1 integrons, with variable structures among isolates (11). In theory, gene cassettes can be incorporated at different positions in the integron (5); however, it seems that cassettes are preferentially integrated adjacent to the attI1 site (5, 19), with the order of the gene cassettes possibly reflecting antibiotic pressure (4). In this study, we report the genetic characterization of a novel class 1 integron carrying blaVIM-2 associated with carbenicillinase and aminoglycoside resistance genes, identified in a P. aeruginosa blood isolate (FF-PS2) from a patient located in the intensive care unit of a hospital in Coimbra, Portugal, in 2000. MICs of -lactam antibiotics were determined by the Etest method (AB Biodisk, Solna, Sweden), and the susceptibility to aminoglycosides and ciprofloxacin was determined by the disk diffusion method (16). Analytical isoelectric focusing (IEF) was performed with crude extracts on a pH 3-to-9 polyacrylamide gel (Phast gels; Amersham Biosciences, Uppsala, Sweden), and -lactamases were visualized with nitrocefin (100 M) (Oxoid, Basingstoke, United Kingdom). A bioassay (3) was employed in order to detect metallo-lactamase production. blaVIM and blaIMP genes were detected by a multiplex PCR assay with VIM/IMP primers (3, 24), and class 1 integrons were detected with previously described specific primers (12, 22). Extraction of plasmid DNA was carried out with the QIAGEN plasmid kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Transfer of resistance genes was screened by mating with rifampin-resistant P. aeruginosa strain PAO or rifampin-resistant Escherichia coli strain K802N. Transconjugant selection was performed in Mueller-Hinton agar plates containing rifampin (100 g/ml) (Sigma, St. Louis, Mo.) and amoxicillin (30 g/ml) (Sigma) or imipenem (2 g/ ml) (Merck Sharp & Dohme, Portugal) when Escherichia coli K802N and P. aeruginosa PAO were used as recipients, respectively. A possible chromosomal location of the blaVIM-2 gene was determined by hybridization of I-CeuI-digested genomic DNA with probes labeled with the ECL enhanced chemiluminescence kit (Amersham Biosciences Europe GmbH) for blaVIM-2 and the 16S and 23S rRNA genes, as previously described (8, 13). The PCR product obtained with the class1 integron primers INT/5 CS (22) and 3 CS (12) was purified with the StrataPrep PCR purification kit (Stratagene, Amsterdam, The Netherlands), and the insert was ligated into the pPCR-Script Cam SK( ) cloning vector (Stratagene, Amsterdam, The Netherlands) and transformed into Epicurian coli XL10-Gold Kan ultracompetent cells (Stratagene, Amsterdam, The Netherlands). Luria-Bertani agar plates supplemented with ampicillin (50 g/ml), chloramphenicol (30 g/ ml), X-Gal (5-bromo-4-chloro-3-indolyl-D-galactopyranoside), and IPTG (isopropyl-D-thiogalactopyranoside) were used as selection plates. The nucleotide sequence of the class 1 integron containing blaVIM-2 was determined in both directions through a primer-walking strategy using specific designed primers in both direct and cloned PCR products. P. aeruginosa FF-PS2 was detected in the context of the regular screening for metallo-lactamase producers among carbapenem-resistant nonfermenters from clinical and environmental sources. The isolate was highly resistant to pipera* Corresponding author. Mailing address: Laboratório de Microbiologia, Faculdade de Farmácia, Universidade do Porto, Rua Anı́bal Cunha, 164 4050-047 Porto, Portugal. Phone: 351-222078946. Fax: 351-222003977. E-mail: [email protected].