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False-positive enterococci counts in seawater with the IDEXX Enterolert-E most probable number technique caused by Bacillus licheniformis

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Abstract

Enterolert-E is an easy-to-use method for the enumeration of enterococci in water samples as an indicator of fecal pollution. This most probable number technique replaced the laborious and more time-consuming MEA-BEA plating method, and it is used extensively in ballast water testing and monitoring. In spring 2018, the Control Union Water ballast water test facility measured high enterococci concentrations in Wadden Sea water without any correlation with polluted freshwater input. By isolating bacteria from samples incubated in Enterolert-E culture medium, followed by analyses of colony morphology and DNA, it is shown that these erroneously high concentrations were caused by Bacillus licheniformis, a gram-positive rod-shaped chlorine-resistant bacterium. It is concluded that control analyses or the MEA-BEA method or dilution to reduce salinity must be performed when high enterococci concentrations are measured in water samples that are not suspected to be polluted.

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Data availability

Bacillus strains are available at LP; sequences have been deposited in GenBank® (legend Fig. 2).

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Acknowledgments

Ms. Z. Kulbieda and Mr. M. van Harten (CUW) carried out the Enterolert-E and MEA-BEA laboratory tests. Dr. K. Kaag (WMR, Den Helder, The Netherlands) and Dr. S. Gollasch (GoConsult, Hamburg, Germany) commented on a draft manuscript. The comments of several anonymous reviewers helped to improve the manuscript.

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Authors and Affiliations

  1. Control Union Water B.V., NIOZ Royal Institute for Sea Research and Utrecht University, Landsdiep 4, NL-1797, SZ, Den Hoorn, The Netherlands

    Louis Peperzak

  2. Department Marine Microbiology and Biogeochemistry (MMB), NIOZ Royal Institute for Sea Research and Utrecht University, PO Box 59, NL-1790, AB, Den Burg, The Netherlands

    Louis Peperzak & Judith van Bleijswijk

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  1. Louis Peperzak
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  2. Judith van Bleijswijk
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LP draw outline of study, performed enterococci/Bacillus experiments, and made first draft. JB performed DNA analyses and calculations and provided text and comments to first draft.

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Correspondence to Louis Peperzak.

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Peperzak, L., van Bleijswijk, J. False-positive enterococci counts in seawater with the IDEXX Enterolert-E most probable number technique caused by Bacillus licheniformis. Environ Sci Pollut Res 28, 10654–10660 (2021). https://doi.org/10.1007/s11356-020-11342-6

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  • DOI: https://doi.org/10.1007/s11356-020-11342-6

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