Investigation of primary and relapse isolates of burkholderia pseudomallei through biofilm associated adaptation and proteomic analyses

Abstract

Melioidosis, an infectious disease found in tropical countries, is caused by Burkholderia pseudomallei. B. pseudomallei is present in contaminated soil and water and is known to produce several virulence factors, including biofilm, which enhances its ability to withstand antibiotic treatment. Although the treatment of melioidosis primarily depends on antibiotic therapy, the ability of B. pseudomallei to persist within the host contributes to chronic infection and the potential for relapse. This study investigates how variations in biofilm formation, biofilm-associated gene expression, and proteomic profiles among three isolates, primary infection (PS_UMC083; ST51), new infection (NI_UMC082; ST1644), and relapse (RS_UMC114; ST51), may underlie bacterial adaptation and persistence. This study aims to characterise differences in biofilm production, gene expression related to biofilm development, and proteomic signatures that could be linked to long-term survival and recurrent infection. Biofilm formation was quantified using the crystal violet assay, biofilm-associated gene expression was assessed by quantitative polymerase chain reaction (qPCR) targeting selected genes, and proteomic profiling was performed using liquid chromatography– mass spectrometry (LC-MS) followed by downstream analyses. Based on the bacterial growth curve, the mid-log phase for all three strains occurred at 16 hours. All strains demonstrated significant biofilm formation (p<0.05) at all time points (24, 48, and 72 hours), with PS_UMC083 showing the lowest biofilm formation across all intervals (24 hrs: 1.150±0.006; 48 hrs: 1.415±0.077; 72 hrs: 4.210±0.353). RS_UMC114 exhibited the highest biofilm quantity at 48 hrs (3.482±0.372) and 72 hrs (10.51±0.641) at OD570 compared to PS_UMC083 and NI_UMC082 (48 hrs: 2.075±0.201; 72 hrs: 7.133±0.195). qPCR analysis revealed that six biofilm-related genes (bpsl1607, bpsl1799, bpss0124, bpss0312, bpss1214, and bpss2016) were downregulated in NI_UMC082 and RS_UMC114 compared to PS_UMC083. Proteomic profiling revealed that the relapse isolate RS_UMC114 exhibited marked upregulation of virulence proteins (e.g., BipD), invasion regulators (e.g., BsaR), and oxidoreductase/stress response proteins (e.g., SodB, DnaK, DnaJ, GrpE), suggesting a persistence-oriented phenotype adapted to hostile host conditions. KEGG pathway enrichment and hub protein analysis further demonstrated that RS_UMC114 is enriched in biosynthetic, stress adaptation, and purine metabolism pathways, consistent with long-term survival strategies. In contrast, NI_UMC082 was characterised by motility and chemotaxis pathways, reflecting an acute infection profile. Together, these findings indicate that virulence, oxidoreductase, and stress response proteins may be central to relapse in B. pseudomallei, providing new insight into the molecular adaptations underpinning chronic infection.