Subtype Screening of bla_IMP Genes Using Bipartite Primers for DNA Sequencing

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of bla_IMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all bla_IMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for bla_IMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of bla_IMP, which is useful for the surveillance of bacteria with bla_IMP in clinical and public health settings or environmental fields.