False-positive enterococci counts in seawater with the IDEXX Enterolert-E most probable number technique caused by Bacil
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RESEARCH ARTICLE
False-positive enterococci counts in seawater with the IDEXX Enterolert-E most probable number technique caused by Bacillus licheniformis Louis Peperzak 1,2
&
Judith van Bleijswijk 2
Received: 10 August 2020 / Accepted: 20 October 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
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 chlorineresistant 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. Keywords Ballast water . Enterococci . False positive . IDEXX Enterolert-E . Bacillus licheniformis . Wadden Sea
Introduction Enterococci are widely used as an indicator of fecal pollution in water samples (Leclerc et al. 1996). In the case of testing Ballast Water Treatment Systems (BWTSs) for type approval by the International Maritime Organization (IMO 2018) and United States Coast Guard (USCG, EPA 2010), enterococci are one of the several groups of organisms that are enumerated before and after water treatment, which is done predominantly chemically or by UV radiation, in order to determine treatment efficacy. The ballast water of ships is to be treated to prevent the global spread of potentially harmful microorganisms. Once type-approved and installed on board of ships, BWTSs
Responsible Editor: Diane Purchase * Louis Peperzak [email protected]; [email protected] 1
Control Union Water B.V., NIOZ Royal Institute for Sea Research and Utrecht University, Landsdiep 4, NL-1797, SZ Den Hoorn, The Netherlands
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
should reduce the risk of transferring harmful aquatic organisms and pathogens (HAOPs) by ballast water discharges, and enterococci are used as an indicator microbe. The IMO and USCG have regulated the minimum concentrations of organisms that are needed before treatment and the maximum concentrations of organisms that are allowed after treatment (Table 1). The three indicator microbes, Escherichia coli, enterococci, and Vibrio cholerae, have no minimum required concentrations before treatment in BWTS land-based type approval tests. In other wo
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