Establishment of Specific Multiplex PCR Detection Methods for the Predominant tet(X)-Positive Acinetobacter Species

The increasing prevalence of the mobile tigecycline resistance gene <i>tet</i>(X) poses a severe global health threat, and the genus <i>Acinetobacter</i> is a major reservoir. This study aimed to develop a rapid and specific multiplex PCR assay for detecting the predominant <i>tet</i>(X)-positive <i>Acinetobacter</i> species. Through pan-genome analyses of 390 <i>tet</i>(X)-positive <i>Acinetobacter</i> genomes, a total of 20 <i>tet</i>(X) variants were identified in 24 <i>Acinetobacter</i> species, including 17 published lineages and seven taxonomically unresolved Taxa. <i>Acinetobacter indicus</i> (30.8%), <i>Acinetobacter amyesii</i> (17.2%), and <i>Acinetobacter towneri</i> (16.1%) were the top three hosts of diverse <i>tet</i>(X) variants. Species-specific signature genes were identified and used for primer design, yielding amplicons of 267 bp (<i>tet</i>(X)), 424 bp (<i>A. indicus</i>), 690 bp (<i>A. amyesii</i>), and 990 bp (<i>A. towneri</i>). 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 <i>Acinetobacter</i> sp. pathogens.