Pan-genome analysis identifies a Burkholderia cepacia-specific gene marker for rapid species-level identification

Abstract

Burkholderia cepacia is a representative member of the Burkholderia cepacia complex (Bcc) and is widely distributed in water and soil environments. It has a propensity to form biofilms in water-based products, making it a significant contaminant in the pharmaceutical, cosmetic, and food industries and posing threats to product quality control and public health. Therefore, rapid and accurate species-level identification of B. cepacia is of great importance. To achieve rapid and accurate species-level identification of B. cepacia , we performed a pan-genome analysis based on 138 high-quality genome sequences covering B. cepacia and its closely related Bcc species with taxonomic positions validated by average nucleotide identity (ANI) analysis. A conserved core gene encoding a glycosyl hydrolase family 18 (GH18) protein was identified as a species-specific molecular marker. A specific primer pair comprising SEQ ID NO.2 and SEQ ID NO.3 was designed based on this marker and validated with both genome sequences and bacterial pure cultures. The GH18-encoding gene is highly conserved across 115 B. cepacia strains (sequence identities ranging from 90.3 to 100.0%) and is absent in all other Bcc species examined. The primer pair exhibited high inclusivity and exclusivity for B. cepacia . The established PCR method achieved a detection limit of 5 pg/μL for pure genomic DNA and 10 3 CFU/mL in artificially contaminated water-based personal care and medical device products. The identification of this conserved core gene provides a promising basis for the rapid and accurate species-level detection of B. cepacia . Given the widespread occurrence of B. cepacia in water and soil environments and its ability to form biofilms in water-based products, such a species-specific molecular marker may help improve contamination monitoring and quality control in the pharmaceutical, cosmetic, and food industries, thereby contributing to public health protection.