Use of recA gene sequencing as a complementary tool for identification of Burkholderia spp. isolated from an immunobiological industry facility

Introduction:The contamination of pharmaceuticals by Burkholderia cepacia complex (Bcc) is often associated with aqueous solutions, representing a significant risk in the production of immunobiologics.The accurate identification of microorganisms is essential to ensure the safety of parenteral products.However, the guidelines and regulations published by the Brazilian Health Regulatory Agency have yet to establish specific methodologies for the identification of Bcc.The 16S rRNA gene has limitations when applied to the differentiation of Bcc due to the high similarity between strains.To overcome these limitations, other molecular markers, such as the recA gene, has proven to be more effective.Objectives: To identify Burkholderia strains in samples collected at different stages of the production chain of an immunobiological industry, using molecular techniques.Methodology: A total of 354 Bcc strains isolated between 2008 and 2022 were evaluated.Initially, the strains were characterized as belonging to the Bcc using the VITEK 2. Further, 40 strains were selected and submitted to identification using MALDI Biotyper and 16S rRNA sequencing by Sanger method.Species-level identification was determined by comparing 16S rRNA sequences with the EZBioCloud database, considering a similarity percentage of 98.7%.Subsequently, the sequencing of the recA gene, a housekeeping gene used in MLST protocol, was carried out.Results: MALDI Biotyper identified the 40 strains with four possible species: B. cepacia (75.0%),B. cenocepacia (15.4%),B. lata (5.8%), and B. pyrrocinia (3.8%).Full 16S rRNA gene sequencing revealed 27 possible species, including B. contaminans.Two strains showed a similarity percentage lower than 98.7%, suggesting they are new species.The recA sequencing identified them as B. contaminans, distributed among the following alleles: 89 (n=25; 62.5%), 217 (n=7; 17.5%), 249 (n=4; 10.0%), 152 (n=2; 5.0%), and 243 (n=2; 5.0%). Conclusion:Considering the recA gene sequencing as "gold standard", MALDI Biotyper incorrectly identified the species of all the strains.16S rRNA gene sequencing included B. contaminans species as possibility but could not identify it at species level.So, expansion of MALDI Biotyper database, including the spectrum of these strains, can be a strategy for a faster and cheaper identification of B. contaminans in pharmaceutical industry.