Effects of sulfadiazine and ciprofloxacin on the occurrence of free-living and particle-associated opportunistic pathogens in bulk water of simulated drinking water distribution systems (DWDSs) were investigated. It was found that sulfadiazine and ciprofloxacin greatly promoted the occurrence of opportunistic pathogens including Pseudomonas aeruginosa, Legionella pneumophila, Mycobacterium avium and its broader genus Mycobacterium spp., as well as the amoebae Acanthamoeba spp. and Hartmanella vermiformis, in bulk water of DWDSs. Moreover, sulfadiazine and ciprofloxacin exhibited much stronger combined effects on the increase of these opportunistic pathogens. Based on the analysis of the antibiotic resistance genes (ARGs) and extracellular polymeric substances (EPS), it was verified that EPS production was increased by the antibiotic resistant bacteria arising from the effects of sulfadiazine/ciprofloxacin. The combined effects of sulfadiazine and ciprofloxacin induced the greatest increase of EPS production in DWDSs. Furthermore, the increased EPS with higher contents of proteins and secondary structure β-sheet led to greater bacterial aggregation and adsorption. Meanwhile, large numbers of suspended particles were formed, increasing the chlorine-resistance capability, which was responsible for the enhancement of the particle-associated opportunistic pathogens in bulk water of DWDSs with sulfadiazine/ciprofloxacin. Therefore, sulfadiazine and ciprofloxacin promoted the occurrence of particle-associated opportunistic pathogens in bulk water of DWDSs due to the role of EPS produced by the bacteria with ARGs.

The incidence of Legionella and Acanthamoeba spp. was correlated to microbial indicator analysis and physico-chemical characteristics of rainwater harvested from catchment areas constructed from galvanized zinc, Chromadek®, and asbestos, respectively. Quantitative PCR (qPCR) analysis indicated that no significant difference (p > 0.05) in copy numbers of Legionella spp. and Acanthamoeba spp. was recorded in tank water samples collected from the respective roofing materials. However, significant positive Spearman (ρ) correlations were recorded between the occurrences of Legionella spp. gene copies vs. nitrites and nitrates (p = 0.05) in all tank water samples. Significant positive correlations were also established between Acanthamoeba spp. vs. barium (p = 0.03), magnesium (p = 0.02), sodium (p = 0.02), silicon (p = 0.05), arsenic (p = 0.03), and phosphate (p = 0.01), respectively. Additionally, while no significant correlations were observed between Legionella spp. vs. the indicator bacteria (p > 0.05), positive correlations were observed between Acanthamoeba spp. vs. total coliforms (p = 0.01) and Acanthamoeba spp. vs. Escherichia coli (p = 0.02), respectively. Results obtained in the current study thus indicate that the incidence of Acanthamoeba and Legionella spp. in harvested rainwater was not influenced by the roofing material utilized. Moreover, it is essential that the microbial quality of rainwater be assessed before this water source is implemented for potable and domestic uses as untreated harvested rainwater may lead to legionellosis and amoebae infections.

Opportunistic pathogens (OPs) in drinking water, like Legionella spp., mycobacteria, Pseudomonas aeruginosa, and free-living amobae (FLA) are a risk to human health, due to their post-treatment growth in water systems. To assess and manage these risks, it is necessary to understand their variations and environmental conditions for the water routinely used. We sampled premise tap (N cₒₗd = 26, N ₕₒₜ = 26) and shower (N ₛₕₒwₑᵣ = 26) waters in a bathroom and compared water temperatures to levels of OPs via qPCR and identified Legionella spp. by 16S ribosomal RNA (rRNA) gene sequencing. The overall occurrence and cell equivalent quantities (CE L⁻¹) of Mycobacterium spp. were highest (100 %, 1.4 × 10⁵), followed by Vermamoeba vermiformis (91 %, 493), Legionella spp. (59 %, 146), P. aeruginosa (14 %, 10), and Acanthamoeba spp. (5 %, 6). There were significant variations of OP’s occurrence and quantities, and water temperatures were associated with their variations, especially for Mycobacterium spp., Legionella spp., and V. vermiformis. The peaks observed for Legionella, mainly consisted of Legionella pneumophila sg1 or Legionella anisa, occurred in the temperature ranged from 19 to 49 °C, while Mycobacterium spp. and V. vermiformis not only co-occurred with Legionella spp. but also trended to increase with increasing temperatures. There were higher densities of Mycobacterium in first than second draw water samples, indicating their release from faucet/showerhead biofilm. Legionella spp. were mostly at detectable levels and mainly consisted of L. pneumophila, L. anisa, Legionella donaldsonii, Legionella tunisiensis, and an unknown drinking water isolate based on sequence analysis. Results from this study suggested potential health risks caused by opportunistic pathogens when exposed to warm shower water with low chlorine residue and the use of Mycobacterium spp. as an indicator of premise pipe biofilm and the control management of those potential pathogens.

In recent decades, free-living protozoa (FLP) have gained prominence as the focus of research studies due to their pathogenicity to humans and their close relationship with the survival and growth of pathogenic amoeba-resisting bacteria. In the present work, we studied the presence of FLP in operational man-made water systems, i.e. cooling towers (CT) and hot sanitary water systems (HSWS), related to a high risk of Legionella spp. outbreaks, as well as the effect of the biocides used, i.e. chlorine in CT and high temperature in HSWS, on FLP. In CT samples, high-chlorine concentrations (7.5 ± 1.5 mg chlorine L⁻¹) reduced the presence of FLP by 63.8 % compared to samples with low-chlorine concentrations (0.04 ± 0.08 mg chlorine L⁻¹). Flagellates and amoebae were observed in samples collected with a level of 8 mg chlorine L⁻¹, which would indicate that some FLP, including the free-living amoeba (FLA) Acanthamoeba spp., are resistant to the discontinuous chlorine disinfection method used in the CT studied. Regarding HSWS samples, the amount of FLP detected in high-temperatures samples (53.1 ± 5.7 °C) was 38 % lower than in low-temperature samples (27.8 ± 5.8 °C). The effect of high temperature on FLP was chiefly observed in the results obtained by the culture method, in which there was a clear reduction in the presence of FLP at temperatures higher than 50 °C, but not in those obtained by PCR. The findings presented here show that the presence of FLP in operational man-made water systems should be taken into account in future regulations.

In this study, 18S rRNA high-throughput sequencing was applied to investigate the eukaryotic community in a full-scale drinking water treatment plant. Eukaryotic species and microbial functions in raw water and filter biofilms were identified by metagenomic sequencing. The eukaryotic species richness and diversity presented declining trends throughout the treatment process. The lowest eukaryotic species richness was observed in disinfected water. Arthropoda, Ciliophora, Ochrophyta, and Rotifera were the dominant eukaryotic phyla and exhibited high variations in relative abundance among the different treatment units. Sedimentation significantly decreased the abundance of all eukaryotes except Arthropoda. Biological activated carbon (BAC) filtration and chlorine disinfection exerted strong effects on community composition. The eukaryotic communities in water were distinct from those in filter biofilms, as were the communities of different filter biofilms from each other. In contrast, communities were functionally similar among different filter biofilms, with the category metabolism being the dominant category represented, within which amino acid transport and metabolism (E) and energy production and conversion (C) dominated among subcategories. Seventy-one eukaryotic species pathogenic to humans were identified in raw water and filter biofilms. Quantitative PCR (qPCR) results showed that Acanthamoeba spp. and Vermamoeba vermiformis were present during some treatment processes, with concentrations of 12–1.2 × 10⁵ copies/mL and 1 copy/mL, respectively. Neither of the two pathogenic amoebae was found in disinfected water. Canonical correspondence analysis (CCA) showed that pH was the most important environmental factor affecting eukaryotic community composition. Overall, the results provide insights into the eukaryotic community diversity in drinking water treatment plants and the potential eukaryotic hazards involved in drinking water production.