Influence of antibiotic concentration on the integration frequency of resistance cassette in the integron

Objective To observe the integration frequency of aadA2 resistance cassette at attI site of the integron under different concentration of streptomycin. Methods Class 1 integron with known gene sequence was cloned into plasmid pACYC184 to produce recombinant plasmid pACIDA, meanwhile the integrase gene was cloned into plasmid pET28a to construct recombinant plasmid pETINT. These two recombinant plasmids were consecutively transformed into E. coli BL(DE3). These transformed bacteria was cultured in the LB medium at 37 ℃ overnight with addition of different concentration of streptomycin. The copy number of total integrons and the copy number of integrated aadA2 at attI site of integrons were determined by using real-time PCR. and the integration frequency is the result of the former divided by the latter. Results The resulting frequencies were (1.97±0.24)×10-3, (3.23±1.77)×10-3, (3.27±0.67)×10-3, 0.45±0.13 and 1.32±0.11, with respective streptomycin concentrations of 0, 20, 30, 40 and 50 μg/ml. The background frequency of integration without integrase overexpression was less than (1.75±0.33)×10-7. Conclusion These findings indicate that antibiotic concentration significantly increase recombination frequency of aadA2 resistance cassette at attI site of the integron, catalyzed by integron integrase.(Chin J Lab Med, 2018, 41: 531-535) Key words: Integrons; Streptomycin; Drug resistance, microbial; Plasmids; Real-time polymerase chain reaction