Research on the application of nanopore sequencing technology in investigating microbial data deviation.

This study aimed to address a microbial contamination incident in which Achromobacter xylosoxidans was detected in two batches of different sterile products at the quality control (QC) laboratory of a pharmaceutical company. Using nanopore sequencing combined with environmental source tracing, we conducted a microbial data deviation (MDD) investigation to rapidly identify the contamination source and provide a reference for future investigations. Strain isolation and biochemical identification were first performed on the two contaminated samples, both confirming the presence of A. xylosoxidans. An MDD investigation was initiated, including a preliminary assessment of the testing environment, facilities, personnel, testing procedures, and materials or consumables to evaluate the validity of the test results. No abnormalities were identified. Given the known ability of A. xylosoxidans to survive in disinfectants, attention was directed toward potential contamination sources among laboratory disinfectants. Two products, compound alcohol liquid disinfectant and waterless surgical hand sanitizer gel, were examined. Disinfection efficacy testing against A. xylosoxidans revealed that the bacterium exhibited strong resistance to the waterless surgical hand sanitizer gel. Subsequent sampling and testing of the gel yielded one isolate of A. xylosoxidans. Whole-genome sequencing of the three A. xylosoxidans isolates (including the newly isolated strain from the gel) was carried out using nanopore technology, followed by analyses of antimicrobial resistance genes, virulence factors, multilocus sequence typing (MLST), single-nucleotide polymorphisms (SNPs), and phylogenetic tree construction. By applying nanopore sequencing to rapidly pinpoint the contamination source, this study integrated genomic evidence with a targeted investigative approach, establishing a complete reasoning chain, from molecular characteristics to the identified pollution source. This approach shifts microbial source tracking from empirical speculation to scientific validation.