VEB-1-Like Extended-Spectrum β-Lactamases in Pseudomonas aeruginosa , Kuwait

Emerging Infectious Diseases Vol. 7, No. 3, May–June 2001 468 Pseudomonas aeruginosa has an inducible, naturally occurring cephalosporinase that confers low-level resistance to aminopenicillins and narrow-spectrum cephalosporins such as cephalothin and cefoxitin (1). Resistance to extendedspectrum cephalosporins may arise from overexpression of this cephalosporinase, acquired beta-lactamases, or both (1). The acquired beta-lactamases may be either clavulanic-acid inhibited (mostly Ambler class A enzymes) or clavulanic-acid resistant (class B and class D enzymes) (2). The class A extended-spectrum beta-lactamases (ESBLs) may derive from narrow-spectrum beta-lactamases of TEM and SHV types, as extensively reported for Enterobacteriaceae and rarely for P. aeruginosa (2). Other class A enzymes reported in P. aeruginosa include PER-1, which we first identified as chromosomally located and which is widespread in P. aeruginosa isolates in Turkey (11% of the hospital isolates) (3,4). Lately, another class A ESBL integron-located gene, blaVEB-1, has been identified from P. aeruginosa and enterobacterial isolates from Southeast Asia (5-7). We report on two novel VEB-1-like beta-lactamases from P. aeruginosa clinical isolates from Kuwait. This is the first report of extended-spectrum enzymes from nosocomial isolates from this part of the world.


The Study
P. aeruginosa KU-1 was isolated in January 1999 at Ibn Sina Hospital in Kuwait from endotracheal secretions of a 1day-old infant with respiratory tract infection, hospitalized in the intensive care unit (ICU) unit because of severe enterocolitis. He was first treated with cefotaxime, amikacin, and metronidazole. These antibiotics were discontinued, and he was given imipenem. He improved and was discharged 7 days later. As it was a single isolate of a multidrug-resistant strain, isolation precautions were carried out only in the neonatal ICU. The infant's mother could not remember whether she had received antibiotic therapy during her pregnancy (which was uneventful). She had not traveled outside Kuwait.
P. aeruginosa KU-2 was also isolated in June 1999 from the urine of a 73-year-old man admitted to the ICU of another Kuwaiti hospital, Mubarak Al-Kabeer, with ischemic chest pain and bronchectiasis. On day 2 of his hospitalization, fever developed, but blood and urine cultures were negative. He was treated with ceftazidime for 12 days beginning on day 2. During his hospital stay, hematuria developed, followed by urine retention. On day 28, pus from a urinary catheter infection after transurethral prostatitic surgery grew P. aeruginosa KU-2 that was susceptible to norfloxacin. P. aeruginosa KU-2 was the only P. aeruginosa strain isolated from this patient's clinical specimens. He was treated with norfloxacin. Repeated urine cultures did not yield any organism, and he was discharged 15 days later. He had no history of travel outside Kuwait.
Strains from both patients were identified by using an API-20 NE system (Biomerieux, Marcy-l'Etoile, France). Preliminary antibiotic susceptibility testing by disc diffusion (5) revealed a slight synergy between ceftazidime-and clavulanic acid-containing discs for two clinical isolates, P. aeruginosa KU-1 and KU-2. Susceptibility testing of betalactams for P. aeruginosa KU-1 and KU-2 was then performed by a Mueller-Hinton agar dilution method (8). Both strains showed decreased susceptibility to all beta-lactams except imipenem and piperacillin/tazobactam (Table). MICs of

VEB-1-Like Extended-Spectrum β β β β β-Lactamases in Pseudomonas aeruginosa, Kuwait
Two clinical Pseudomonas aeruginosa isolates from patients in intensive care units in Kuwait were resistant to expanded-spectrum cephalosporins and showed a synergistic effect between ceftazidime and clavulanic acid. This is the first report of extended-spectrum enzymes from nosocomial isolates from the Middle East. ceftazidime were decreased by addition of clavulanic acid and tazobactam, indicating the presence of a clavulanic-acid inhibited ESBL (Table). According to antibiotic susceptibility testing by Mueller-Hinton agar disc diffusion, P. aeruginosa KU-1 and KU-2 were also resistant to aminoglycosides (amikacin, gentamicin, netilmicin, tobramycin), chloramphenicol, tetracyclines, sulfonamides, and fluoroquinolones (except for KU-2). Beta-lactamase extracts from cultures of P. aeruginosa KU-1 and KU-2 were obtained (8). Isoelectric focusing analysis (8) revealed beta-lactamases with isoelectric points of 7.4 and 8-8.4, the latter likely corresponding to P. aeruginosa AmpC cephalosporinases. Whole-cell DNAs of P. aeruginosa KU-1 and KU-2 were then obtained (3). Preliminary polymerase chain reaction (PCR) experiments were performed with DNAs of P. aeruginosa KU-1 and KU-2 as templates and primers specific for the following class A beta-lactamases: TEM, SHV, CARB (PSE-1), GES-1, PER-1, and VEB-1 (3,5,(9)(10)(11). Only PCR using internal primers for bla VEB-1 gave a positive result with an identical 642-bp fragment. External bla VEB-1 specific primers gave 1,070-bp PCR fragments with DNAs of both P. aeruginosa strains as templates that were sequenced on both strands (6). The deduced amino acid sequences, obtained over the internet (8), identified VEB-1-like sequences that shared 99% amino acid identity with VEB-1 ( Figure). Compared with VEB-1, the amino acid changes in VEB-1a and VEB-1b from P. aeruginosa KU-1 and KU-2, respectively, occurred in the putative leader peptide sequence (Figure). Thus, the hydrolytic activity of VEB-1a and VEB-1b should be identical to that reported for VEB-1 beta-lactamase (5).
The genetic background of bla VEB-1a and bla VEB-1b was further characterized. Plasmid extraction, conjugation, and electroporation experiments were performed (8). A plasmid (pROT-1) of ca. 70 kb carrying bla VEB-1a gene was identified according to hybridization results by using an internal PCRobtained probe for bla VEB-1 (5). Plasmid pROT-1 was selfconjugative from P. aeruginosa KU-1 to in vitro obtained rifampin-resistant P. aeruginosa PU21 reference strain after selection of transconjugants onto Mueller-Hinton agar plates each containing 150 µg/mL rifampin and 200 µg/mL ticarcillin. As assessed by antibiotic susceptibility testing by disc diffusion, plasmid pROT-1 conferred additional resistance to gentamicin, netilmicin, sulfonamides, and tobramycin. MICs of beta-lactams for P. aeruginosa PU21 (pROT-1) mirrored those obtained for P. aeruginosa KU-1 (Table). While the plasmid location of bla VEB-1 gene is known only in Enterobacteriaceae (5-7), its report in P. aeruginosa may signal the evolution of its spread. The bla VEB-1b gene was not plasmid located, but a PCR-obtained 642-bp internal probe for bla VEB-1 hybridized at chromosomal position of whole-cell DNA of P. aeruginosa KU-2.
The bla VEB-1 gene is located on different structures of class 1 integrons (5-7). Integrons comprise two conserved regions (5'-CS and 3'-CS) flanking an internal variable region usually containing several gene cassettes (13). Integrons are in fact expression vectors for antibiotic resistance genes that are included as gene cassettes and are neighbored (13). By using primers located either in the 5'-CS sequence and the 5' end of bla VEB-1 or in the 3' end of bla VEB-1 and the 3'-CS sequence (5,14), PCR amplification experiments were performed with whole-cell DNAs of P. aeruginosa KU-1 and KU-2 as templates. In one case (strain KU-1), a PCR fragment was obtained by using bla VEB-1 and 5'-CS primers, indicating that bla VEB-1a was located downstream of a class 1 integrase gene. In this case, the 4-kb PCR fragment differed from those of known bla VEB-1 containing integrons identified in Escherichia coli and P. aeruginosa isolates (5-7). Amplimers of 1 kb were obtained for both strains using bla VEB-1 and 3'-CS primers, showing that the 3'-CS end was present in both cases and that the bla VEB-1 -like sequences were located next to the 3'-CS end within class 1 integrons. The attC (59-be) recombination sites (15) located downstream of gene cassettes were identical for bla VEB-1a and bla VEB-1b to those described for bla VEB-1 in P. aeruginosa and enterobacterial isolates identified so far from Southeast Asia (5-7). Therefore, an identical bla VEB-1 -like gene cassette may be located on different class 1 integrons. Using 5'-CS and 3'-CS primers, two additional PCR fragments were obtained for each P. aeruginosa strain, showing that both strains contained another bla VEB -negative class 1 integron. For P. aeruginosa KU-1, a 950-bp PCR fragment for an aadA1a gene coding for an aminoglycoside modifying enzyme was found to be plasmid-and integron-located in Salmonella enterica serotype Typhimurium (16). For P. aeruginosa KU-2, a 500-bp PCR fragment encoding a putative 95 amino acid protein of unknown function was PCR amplified. It shared 71% amino acid identity with an amino acid sequence from a gene that was Tn1696 transposon-located and In4 integronlocated in P. aeruginosa (17).
Finally, P. aeruginosa KU-1 and KU-2 isolates containing VEB-1-like beta-lactamases were compared with VEB-1 positive P. aerugionosa strain JES from Thailand by using random amplified polymorphic DNA technique (10,18). The isolates were not clonally related (data not shown). Although the patients had not traveled outside Kuwait, introduction of P. aeruginosa into Kuwaiti hospitals by travelers or patients from Southeast Asia cannot be ruled out.

Conclusions
The presence of clavulanic-acid inhibited ESBLs in P. aeruginosa isolates may account for part of the 50% resistance to ceftazidime of P. aeruginosa strains isolates from ICUs in Kuwait (19). ESBLs in P. aeruginosa in Kuwait and other Middle Eastern hospitals may be underestimated because routine detection with a double disc synergy test may be difficult. Identification of ESBLs is of interest since they confer resistance to all extended-spectrum cephalosporins and aztreonam, whatever their MICs. This has been confirmed by experimental data using a model of pneumonia in rats with the Ambler class A ESBL, PER-1 (20). This work underscores that very similar ESBLs may be identified in different parts of the world. It is the first report of ESBL genes characterized from P. aeruginosa isolates from the Middle East.