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Simultaneous Presence of bla<inf>TEM</inf> and bla<inf>SHV</inf> Genes on a Large Conjugative Plasmid Carried by Extended-Spectrum β -Lactamase-Producing Klebsiella pneumoniae

Abstract:

Background: Among members of the family Eaterobacteriaceae, the production of plasmid-mediated extended-spectrum β-lactamases (ESβLS) has emerged as an important mechanism of the resistance to β-lactams. Methods: The molecular basis of extended-spectrum β-lactamase in 48 strains of Klebsiella pneumoniae, recovered from neonatal patients with nosocomial septicemia during an outbreak that occurred in November 2000 at a Neonatal Intensive Care Unit of The Andes University Hospital in Venezuela, were investigated. Results: The isolates were resistant to expanded-spectrum cephalosporins, aztreonam, gentamicin, kanamycin, tetracycline, and chloramphenicol, but remained susceptible to cefoxitin, imipenem, amikacin, and tobramycin. Production of ESβL activity was confirmed by restoring susceptibility to ceftazidime in the presence of clavulanic acid. All isolates harbored an 87-kilobase plasmid. Analysis of the outer membrane protein patterns did not reveal relevant changes of the porins profile. All resistance markers were transferable to Escherichia coli by conjugation and were lost en bloc after treatment with acridine orange. Isoelectric focusing for β-lactamases was performed on transconjugants, obtaining 2 bands with isoelectric points of 5.4 and 8.2. Genes encoding both enzymes are located on the single, self-transferable 87-kilobase plasmid pKAM542. Analysis of the plasmid by hybridization revealed the presence of both blaTEM and blaSHV determinants. Cloning and sequencing identified them as blaTEM-1 and blaSHV-5, respectively; the latter was responsible for the ESβL activity among nosocomial isolates of K pneumoniae. Conclusions: Microbiologists, epidemiologists, and clinicians must be aware of potential ESβL-encoding organisms to assess proper antimicrobial managements and effective infection controls.