Selective enumeration strategies for Brevundimonas diminuta from drinking water

Abstract

Brevundimonas diminuta is used as a control organism for validating the efficiency of water filtration systems. Since these protocols use nonselective growth media, heterotrophic plate count bacteria (HPCs) indigenous to the water distribution system may interfere with B. diminuta enumeration, thus leading to inaccurate assessment of the filter's microbial reduction capability. This could negatively impact public health as unsafe drinking water may be produced. This study was conducted to evaluate different potential routes for selective enumeration of B. diminuta in drinking water. B. diminuta's biochemical and molecular relationships to HPCs recovered from a laboratory drinking-water system were investigated. Of the 24 HPC morphotypes recovered, members of the Alpha- and Betaproteobacteria were most commonly identified. Based on comparisons of catabolic profiles (generated by the Biolog system) using principal component analysis, B. diminuta possessed similar metabolic patterns to several of the Alphaproteobacteria (Sphingomonas and Caulobacter), indicating that development of a selective medium based solely on carbon source was not feasible. Antibiotic susceptibility profiles revealed that the HPCs were least resistant to kanamycin, making it a candidate for future selective applications. Sequence comparisons of partial 16S rRNA sequences did not reveal any distinct similarities. However, basic local alignment search tool (BLAST) alignments of the gyrB and rpoD sequences for B. diminuta did show uniqueness, with the next closest match being to Caulobacter (88% and 79% similarity, respectively). Future investigation will focus on applying molecular assays, such as fluorescent in situ hybridization and quantitative real-time polymerase chain reaction (PCR), and incorporating an antibiotic marker or expressed fluorescent protein into the wild-type strain of B. diminuta for selective enumeration of B. diminuta.