Identification of environmental aquatic bacteria by mass spectrometry supported by biochemical differentiation

In this study, the purposefulness of using the API20E biochemical identification system as a supportive tool for enhancing the discrimination of environmental bacteria by MALDI-TOF MS method was evaluated. The identification results of MALDI-TOF MS and API20E for 321 Gram-negative strains isolated from the riverine freshwater and its sediment, and from the tissues of fish from the same water body were compared. Of 190 isolates identified with probable to highly probable species-level identification, and secure genus to probable species identification, 14 isolates (7.37%) had identification score over 2.300, and from the same group 19 isolates (10%) had excellent or very good identification to the genus by API20E system. With regard to agreement at genus level, out of 231 strains with genus designation available by API20E at any level of identification reliability, MALDI-TOF MS genus identification agreed in 163 (70.6%) strains. Of these, 135 (82.8%) were Aeromonas species and the remaining isolates belonged to 7 different genera. Although API20E resulted in frequent misidentification due to a limited profile index, its individual biochemical reactions might contribute to overall characterization of isolates. For example, for all reliable A. hydrophila strain identifications with MALDI-TOF MS, ONPG, GLU and OX reactions were unarguably positive for all fish and water/sediment isolates, whereas only fish isolates yielded additional 100% positive TDA and VP reactions. Thus, after initial identification with MALDI-TOF MS, environmental isolates with lower identification scores should be further analyzed. Before commencing confirmatory testing with nucleic acid-based methods, biochemical API20E tests could be applied as a purposeful and inexpensive identification support in targeting better identification accuracy. In this study, this was particularly evident with A. hydrophila, Chryseobacterium sp. and Pseudomonas sp. This identification strategy could significantly resolve methodological and cost-related shortcomings frequently occurring with large number of environmental isolates.