Detection of gene cassettes in Tn402-like class 1 integrons.

Amplification of the gene cassettes in class 1 integrons by PCR using primers in the 5 conserved segment (5 -CS) and the 3 -CS (7, 8) has been used in hundreds of studies to identify integron-associated resistance genes (Fig. 1A). Equivalent PCR primers that detect cassettes in class 2 integrons, i.e., Tn7 family transposons (18), are also widely used. The approach used to detect integrons in antibiotic-resistant bacteria is to screen for the intI genes, using primers internal to these genes, and then amplify the cassettes in intI-positive strains by use of primers in the flanking conserved regions. Because different cassettes can have the same size and the arrays can include more than one gene (16), amplicon size alone cannot identify the cassettes, which are characterized by sequencing, PCR mapping (7), or restriction fragment polymorphisms (9, 10). However, for some intI1-positive strains, a cassette PCR amplicon is not observed, and to date these strains have been largely ignored, even though such isolates can represent a significant proportion of the isolates studied (3). For class 1 integrons, if the sul1 gene found in the 3 -CS in both of the main structural types (12, 13) is not present, the absence of a PCR product may indicate that the priming site in the 3 -CS is missing. This can occur because the integron is recombinant with the 5 -CS of class 1 and the tns module of class 2 (14). Additionally, the 3 -CS is not found in Tn402 (15), the likely