New PCR-based open reading frame typing method for easy, rapid, and reliable identification of Acinetobacter baumannii international epidemic clones without performing multilocus sequence typing

Antimicrobial resistance issues have become a global health concern. The rapid identification of multidrug-resistant microbes, which depends on microbial genomic information, is essential for overcoming growing antimicrobial resistance challenges. However, genotyping methods, such as multilocus sequence typing (MLST), for identifying international epidemic clones of Acinetobacter baumannii are not easily performed as routine tests in ordinary clinical laboratories. In this study, we aimed to develop a novel genotyping method that can be performed in ordinary microbiology laboratories. Several open reading frames (ORFs) specific to certain bacterial genetic lineages or species, together with their unique distribution patterns on the chromosomes showing a good correlation with the results of MLST, were selected in A. baumannii and other Acinetobacter spp. by comparing their genomic data. The distribution patterns of the ORFs were visualized by agarose gel electrophoresis after multiplex PCR amplification and digitized. A. baumannii sequence types (STs) corresponding to international clones I and II were successfully discriminated from other STs and Acinetobacter species by detecting the distribution patterns of their ORFs using the multiplex PCR developed here. Since bacterial STs can be easily expressed as digitized numeric data with plus (+) expressed as 1 and minus (-) expressed as 0, the results of the method can be easily compared with those obtained by different tests or laboratories. This PCR-based ORF typing (POT) method can easily and rapidly identify international epidemic clones of A. baumannii and differentiate this microbe from other Acinetobacter spp. Since this POT method is easy enough to be performed even in ordinary clinical laboratories, it would also contribute to daily infection control measures and surveillance.