Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology

A screening technique for integrons in members of the family Enterobacteria ceae and nonfermenting gramnegative bacteria by real-time PCR is reported. A total of 226 isolates of gram-negative bacteria obtained from a variety o f clinical specimens were screened for class I integrons by real-time PCR p erformed on a LightCycler instrument. This technique used a primer pair spe cific for a 300-bp conserved region at the 5' ends of class 1 integrons. Th e screening assay was evaluated by comparison with results obtained by the conventional, thermal-block PCR (long PCR) by using established conditions and primers for the detection of class I integrons, and the real-time PCR t echnique was thus shown to be both sensitive and specific. DNA from 50 of 2 26 (22%) isolates screened was identified as containing an integron by the screening PCR, and sequence data were obtained across the integron for 34 o f 50 (68%) of these isolates. In an attempt to study the molecular epidemio logy of antimicrobial resistance genes carried within integrons, a comparis on of the types of gene cassettes carried by isolates from different patien ts was made. Adenyltransferase genes conferring resistance to streptomycin and spectinomycin were the predominant gene cassettes amplified in the stud y. Resistance to trimethoprim was also frequently found to be encoded withi n integrons. Furthermore, multiple bacterial isolates obtained from one pat ient over a 5-month period were all shown to carry an integron containing t he same single adenyltransferase gene cassette, suggesting that these eleme nts were relatively stable in this case.