Occurrence of Uncultured Legionella spp. in Treated Wastewater Effluent and Its Impact on Human Health (SCA.Re.S Project)

Abstract

Wastewater treatment plants (WWTPs) provide optimal conditions for the environmental spread of Legionella. As part of the Evaluation of Sanitary Risk Related to the Discharge of Wastewater to the Ground (SCA.Re.S) project, this study was conducted to evaluate the presence of Legionella in WWTP effluent and in groundwater samples collected from two wells located downstream from the plant. The samples were analyzed to determine the concentrations of Legionella spp using the standard culture-based method and molecular techniques, followed by genomic sequencing analysis. Legionella was detected only with the molecular methods (except in one sample of effluent positive for L. pneumophila serogroup 6), which showed viable Legionella pneumophila and L. non-pneumophila through the use of free DNA removal solution in both the effluent and groundwater, with concentrations that progressively decreased downstream from the plant. Viable L. pneumophila appeared to be slightly more concentrated in warm months. However, no significant differences (p ≥ 0.05) in concentrations between cold and warm months were observed. A genotypic analysis characterized the species present in the samples and found that uncultured Legionella spp, as yet undefined, constituted the prevalent species in all the samples (range 77.15-83.17%). WWTPs play an important role in the hygienic and sanitary quality of groundwater for different uses. The application of Legionella control systems during the purification of effluents is warranted to prevent possible outbreaks of legionellosis.

Keywords: Legionella non-pneumophila; detection methods; groundwater; viability; wastewater treatment plant.

Figure 1

The study area, including the wastewater treatment plant and monitoring wells, on the Salento peninsula, Apulia, Italy.

Figure 2

Schematic representation of the wastewater discharge path from the wastewater treatment plant into draining trenches and then to downgradient monitoring wells W1 (400 m) and W2 (1000 m).

Figure 3

Rate (%) of positive samples and viable cell concentrations of Legionella spp. and L. pneumophila, collected from the effluent of WWTP, W1 (well 1), and W2 (well 2).

Figure 4

Average concentrations ± SE of viable Legionella spp. and L. pneumophila in WWTP effluent W1 (well 1) and W2 (well 2) recorded in cold months (November 2022–April 2023) and warm months (May–October 2022).

Figure 5

Legionella spp.’s composition in water samples assessed by sequencing 16S rRNA gene V3–V4 domain amplicon with Illumina MiSeq (similarity > 97%).

Figure 6

(a) NCBI BLASTN phylogenetic tree showing the similarities (>97%) between the uncultured Legionella sp. clone BFI-A19-1 (in yellow) and known Legionella species (in green), determined with 16S rDNA sequencing. The scale bar corresponds to 0.02 substitutions per nucleotide position. (b) NCBI BLASTN phylogenetic tree showing the similarities (>97%) between uncultured Legionella sp. clone Tag 4-1 (in yellow) and known Legionella species (in green), determined with 16S rRNA sequencing. The scale bar corresponds to 0.02 substitutions per nucleotide position.

Figure 6

(a) NCBI BLASTN phylogenetic tree showing the similarities (>97%) between the uncultured Legionella sp. clone BFI-A19-1 (in yellow) and known Legionella species (in green), determined with 16S rDNA sequencing. The scale bar corresponds to 0.02 substitutions per nucleotide position. (b) NCBI BLASTN phylogenetic tree showing the similarities (>97%) between uncultured Legionella sp. clone Tag 4-1 (in yellow) and known Legionella species (in green), determined with 16S rRNA sequencing. The scale bar corresponds to 0.02 substitutions per nucleotide position.