Abstract
Detection and quantification of pathogenic free-living amoebae (FLA) in water samples is critical for assessing water quality and for disease management issues. The most probable number (MPN) is commonly used to account for FLA in water. Nevertheless, this requires a high number of water replicates and working volumes, and a consequent number of non-nutrient agar (NNA)-plates seeded with Escherichia coli. Herein, we aimed at optimizing this difficult method, taking also into account key factors such as (i) the counting method, (ii) the delay between sample collection and sample processing, and (iii) the temperature during water sample transportation. To simplify the MPN method, we filtrated 1 × 1000 and 1 × 100 mL water samples, and cellulose acetate filters were cut in 10 parts and inverted on NNA-plates overlaid with E. coli. The comparison between the classical and our optimized MPN method showed that the final counts were similar, therefore validating the use of the optimized method. Our results also showed that for thermophilic FLA (such as Naegleria fowleri), water samples can be kept at around +30°C and processed within 24 h. This improved MPN method is now routinely used in our laboratory to control Naegleria sp. in the water samples in Guadeloupe.
Highlights
Free-living amoebae (FLA) are ubiquitous unicellular organisms, being found in water, soil, dust, and air samples
The results show that for FLA, the percentage of samples with equal or higher counts obtained with the mMPN method compared to those obtained with the classic method are 84% (16/19 samples) for both 100 mL boxes and 10 mL boxes
Only 2 samples tested positive for N. fowleri using the classical MPN method (cMPN), while the optimized method revealed the presence of this amoeba in six water samples
Summary
Free-living amoebae (FLA) are ubiquitous unicellular organisms, being found in water, soil, dust, and air samples. Some FLA are thermophilic, being able to survive and/or replicate at temperatures equal or above 37 ◦ C [1,2,3]. The thermophilic FLA Naegleria fowleri is frequently found in freshwater and soil [11,12], and it may cause primary amoebic meningoencephalitis (PAM), a rare but often fatal disease of the central nervous system [13]. It is generally acquired while swimming or during other recreational activities in freshwater lakes and ponds. Infection of the brain occurs after N. fowleri reaches the nasal cavity
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