A high-throughput sequencing determination method for upstream genetic structure (UGS) of ISEcp1-blaCTX-M transposition unit and application of the UGS to classification of bacterial isolates possessing blaCTX-M

Introduction: Because bla(CTX-M) is responsible for resistance of bacteria to the third generation cephalosporins, location of bla(CTX-M) could be a good indicator for classifying bacterial isolates harboring bla(CTX-M) in molecular epidemiology. However, determination of bla(CTX-M) location has been difficult when multiple copies of ISEcp1 were found on bacterial genome. We aimed to establish a high-throughput analytical method for upstream genetic structures (UGS) of ISEcp1 to facilitate determination of bla(CTX-M) location. Methods: Extracted DNA samples obtained from 168 Escherichia coli isolates possessing bla(CTX-M) were digested by restriction enzyme, HaeIII, and the digested DNA fragments were ligated with homemade barcode adaptors. Then, DNA fragments containing UGS of ISEcp1 were amplified and subjected to the Nanopore sequencer. Results: Nucleotide sequences and locations of 168 UGSs obtained from the examined E. coli isolates were determined. Among the 168 determined UGSs, 150 (89.3%) UGS were confirmed on plasmid and classified into eight types. Interestingly, coding sequence of ISEcp1 transposase gene in seven of the eight types were disrupted by IS26 insertion. The remaining 18 (10.7%) UGSs were observed in identical chromosomal region. The obtained nucleotide sequences the locations of UGSs were confirmed by conventional capillary sequencer and Southern blotting, respectively, and any discrepant result was not observed with these confirmation procedures. Conclusions: Our results indicated that the established method was efficient for simultaneously determining at least 100 different UGS, and suggested that the determined UGSs of ISEcp1-bla(CTX-M) transposition unit was useful for classification of bacterial isolates harboring bla(CTX-M). (C) 2021 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

Automated summary

By utilizing restriction enzyme digestion and Nanopore sequencer, we successfully determined the nucleotide sequences and locations of upstream genetic structures of ISEcp1 in 168 E. coli isolates. Among these isolates, 89.3% of UGS were confirmed on plasmids while 10.7% were observed in the identical chromosomal region.