Laboratory strategies for the identification of Burkholderia species: From classical phenotyping to advanced genomic and proteomic approaches

The genus Burkholderia, particularly the Burkholderia cepacia complex (Bcc), encompasses Gram-negative bacteria of recognized ecological, biotechnological, and clinical relevance. Accurate identification of species within this complex is challenging due to high phenotypic and genetic similarity, impacting clinical diagnostics and epidemiological surveillance. This review addresses phenotypic, proteomic, and molecular methods for species- and clone-level identification, highlighting their advantages and limitations. Classical methods, including selective media, Burkholderia cepacia selective agar (BCSA), Oxidation-Fermentation Polymyxin Bacitracin Lactose agar (OFPBLA), and Pseudomonas cepacia agar (PCA), and biochemical tests are useful for initial screening but have limited resolution for differentiating closely related Bcc species. Automated systems, such as VITEK® 2, provide rapid genus-level identification, whereas Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) and Fourier Transform Infrared Spectroscopy (FTIR) improve accuracy for species differentiation and clone typing, depending on the quality and currency of reference databases. Molecular methods, including Multilocus Sequence Typing (MLST), which sequences housekeeping genes such as gyrB and recA, and Whole-Genome Sequencing (WGS), offer high-resolution species- and clone-level identification, enabling detailed epidemiological monitoring. WGS represents the current gold standard, providing comprehensive information on identification, antimicrobial resistance, virulence factors, and clone typing. A tiered strategy is recommended, combining accessible methods for initial screening with advanced techniques for research centers and genomic surveillance. This study emphasizes the critical need for methodological standardization to enhance clinical diagnosis, epidemiological surveillance, and management of infections caused by species of the genus Burkholderia.