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Acinetobacter species are common inhabitants of freshwater and marine ecosystems with a capacity to induce disease in affected fish. To facilitate their rapid and reliable identification, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was applied to Acinetobacter from fish. The aims of the study were, thus, to identify and profile the Acinetobacter species from tissues of fish living in a vulnerable environment impacted by wastewaters, and to assess the potential of MALDI-TOF MS as a method for discriminating these acinetobacters. Fish were sampled from waterways impacted by the activity of a wastewater treatment plant. Samples of gills, spleen, kidney and liver were streaked onto general-purpose media to purity. The profiling and identification of acinetobacters was conducted with MALDI-TOF MS, with the samples prepared by ethanol/formic acid extraction.The identified acinetobacters were retrieved from gills (68.96 %), kidney (13.79 %), liver (10.34 %), and spleen (6.89 %). The Acinetobacter species isolated from all tested fish tissues were A. johnsonii (79.31 %), A. pittii (10.34 %), A. tandoii (3.44 %), A. guilouiae (3.44 %), and A. gerneri (3.44 %). Highly probable and probable species identifications were obtained for 48.27 % of all acinetobacters tested, indicating fully reliable identification. MALDI‐TOF MS gave excellent identification and profiling results for piscine Acinetobacter species from the wastewater-affected waterways. It is a recommendable technique for future Acinetobacter species discrimination, as accurate and rapid identification of these bacteria is critical in both environmental pollution management and human/veterinary clinical diagnostics.
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