Matrix-Assisted Laser Desorption Ionisation–Time of Flight Mass Spectrometry (MALDI–TOF MS) has increasingly been used for rapid and reliable identification of clinically relevant micro-organisms. To establish the applicability of this technique in (drinking) water quality analysis, the MALDI–TOF MS identification results for Enterococcus spp. isolated from various water environments were compared with those obtained using the commercial Rapid 32 ID Strep system. One hundred and one bacterial isolates were isolated from various types of water and determined as enterococci on the basis of their growth on Slanetz-Bartley agar in typical colonies. The isolates were identified by MALDI–TOF MS and the commercial biochemical test Rapid 32 ID Strep. Isolates yielding in discrepant identifications were genotyped using 16S rRNA gene sequence analysis. For 86 isolates (85%), the results of Rapid ID 32 Strep were identical to those obtained with MALDI–TOF MS. Six isolates were impossible to be classified by means of the Rapid 32 ID Strep test. And for eight out of a total of nine discrepant results (89%), the 16S analyses confirmed the MALDI–TOF MS identification. MALDI–TOF MS produced highly reproducible results. These results indicated that the use of two different culture media had no effect on the identification. In addition, no significant differences (p = 0.32; n = 20) were evident between the scores obtained from a 20-fold measurement of the same isolate. The results of this study showed that MALDI–TOF MS identification (Bruker) is a reliable method for identifying E. faecium, E. faecalis, E. durans, E. hirae and E. casseliflavus isolated from water samples. E. mundtii and E. moraviensis were not included in the Rapid 32 ID Strep database and could therefore not be identified using that test set. However, MALDI–TOF MS and 16S identified all six isolates as members of these species.

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