Abstract
The increasing prevalence of the mobile tigecycline resistance gene tet(X) poses a severe global health threat, and the genus Acinetobacter is a major reservoir. This study aimed to develop a rapid and specific multiplex PCR assay for detecting the predominant tet(X)-positive Acinetobacter species. Through pan-genome analyses of 390 tet(X)-positive Acinetobacter genomes, a total of 20 tet(X) variants were identified in 24 Acinetobacter species, including 17 published lineages and seven taxonomically unresolved Taxa. Acinetobacter indicus (30.8%), Acinetobacter amyesii (17.2%), and Acinetobacter towneri (16.1%) were the top three hosts of diverse tet(X) variants. Species-specific signature genes were identified and used for primer design, yielding amplicons of 267 bp (tet(X)), 424 bp (A. indicus), 690 bp (A. amyesii), and 990 bp (A. towneri). The assay was rigorously adjusted for an optimal annealing temperature of 52.8 °C and a primer ratio of 1:1:1:1, demonstrating high sensitivity with a detection limit of 0.3 ng/μL DNA and excellent stability under -20 °C, 4 °C, 20 °C storage conditions. Validation experiments on 151 bacterial strains showed high accuracy for DNA templates (≥97.8%) and bacterial suspensions (≥93.5%) within two hours. This cost-effective and highly accurate multiplex PCR provides a powerful tool for proactive surveillance and control of the critical Acinetobacter sp. pathogens.