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Antibiotic resistance poses a significant threat to human health. While most studies focus on bacteria, interactions between antibiotics and other crucial microbial groups like protists remain uncertain. This study investigates how protists interact with antibiotics and examines how these interactions impact the fate of resistance genes. It reveals that amoebae exhibit high resistance to eight high-risk environmental antibiotics, accumulating significant quantities within their cells. Wild amoeboid strains from distant locations carry substantial antibiotic resistance genes (ARGs) and metal resistance genes (MRGs), with significant heterogeneity within a single species. Amoeboid symbionts and pathogens predominantly carry these genes. Paraburkholderia symbionts have reduced genomes and fewer resistance genes compared to free-living strains, while amoeba-endogenous Stenotrophomonas maltophilia does not exhibit a significantly reduced genome size. This suggests that the amoeboid hosts serve as a temporary medium facilitating its transmission. In summary, the study unveils that soil amoebae represent unexpected hotspots for antibiotics and resistance genes. Future research should assess the effects of antibiotics on often-overlooked protists and explore their role in spreading ARGs and MRGs in ecosystems. Incorporating protists into broader antibiotic resistance research is recommended, highlighting their significance within a One Health perspective.

Illustrative distribution system operation and management practices shaped the occurrence and persistence of Legionella spp., nontuberculous mycobacteria (NTM), Pseudomonas aeruginosa, and two amoebae host (Acanthamoeba spp., Vermamoeba vermiformis) gene markers in the effluent of standardized simulated household water heaters (SWHs). The interplay between disinfectant type (chlorine or chloramine), water age (2.3-5.7 days) and materials (polyvinyl chloride (PVC), cement or iron) in upstream simulated distribution systems (SDSs) profoundly influenced levels of pathogen gene markers in corresponding SWH bulk waters. For example, Legionella spp. were 3-4 log higher in SWHs receiving water from chloraminated vs chlorinated SDSs, because of disinfectant decay from nitrification. By contrast, SWHs fed with chlorinated PVC SDS water not only harbored the lowest levels of all pathogen markers, but effluent from the chlorinated SWHs were even lower than influent levels in several instances (e.g., 2 log less Legionella spp. and NTM for PVC and 3-5 log less P. aeruginosa for cement). However, pathogen gene marker influent levels correlated positively to effluent levels in the SWHs (P < 0.05). Likewise, microbial community structures were similar between SWHs and the corresponding SDS feed waters. This study highlights the importance and challenges of distribution system management/operation to help control opportunistic pathogens.

Free-living amoebae are protozoa ubiquitously found in water systems. They mainly feed on bacteria by phagocytosis, but some bacterial species are able to resist or even escape this lethal process. Among these amoeba resistant bacteria are numerous members of the genus Mycobacterium. Nontuberculous Mycobacteria (NTM) are opportunistic pathogens that share the same ecological niches as amoebae. While several studies have demonstrated the ability of these bacteria to colonise and persist within drinking water networks, there is also strong suspicion that mycobacteria could use amoebae as a vehicle for protection and even replication. We investigated here the presence of NTM and FLA on a drinking water network during an all year round sampling campaign. We observed that 87.6% of recovered amoebal cultures carried high numbers of NTM. Identification of these amoeba and mycobacteria strains indicated that the main genera found in drinking water networks, that is, Acanthamoeba, Vermamoeba, Echinamoeba, and Protacanthamoeba are able to carry and likely to allow replication of several environmental and potentially pathogenic mycobacteria including M. llatzerense and M. chelonae. Direct Sanger sequencing as well as pyrosequencing of environmental isolates demonstrated the frequent association of mycobacteria and FLA, as they are part of the most represented genera composing amoebae's microbiome. This is the first time that an association between FLA and NTM is observed in water networks, highlighting the importance of FLA in the ecology of NTM.

Common garden hoses may generate aerosols of inhalable size (≤10 μm) during use. If humans inhale aerosols containing Legionella bacteria, Legionnaires' disease or Pontiac fever may result. Clinical cases of these illnesses have been linked to garden hose use. The hose environment is ideal for the growth and interaction of Legionella and free-living amoebae (FLA) due to biofilm formation, elevated temperatures, and stagnation of water. However, the microbial densities and hose conditions necessary to quantify the human health risks have not been reported. Here we present data on FLA and Legionella spp. detected in water and biofilm from two types of garden hoses over 18 months. By culturing and qPCR, two genera of FLA were introduced via the drinking water supply and reached mean densities of 2.5 log10 amoebae·mL(-1) in garden hose water. Legionella spp. densities (likely including pathogenic L. pneumophila) were significantly higher in one type of hose (3.8 log10 cells·mL(-1), p < 0.0001). A positive correlation existed between Vermamoebae vermiformis densities and Legionella spp. densities (r = 0.83, p < 0.028). The densities of Legionella spp. identified in the hoses were similar to those reported during legionellosis outbreaks in other situations. Therefore, we conclude that there is a health risk to susceptible users from the inhalation of garden hose aerosols.

The occurrence of free-living amoebae (FLA) was investigated in 83 water samples from reservoirs and water treatment plants, with culture positive in 64 of them (77.1%). Polymerase chain reaction (PCR) of partial 18S rRNA gene and ITS region was performed in order to identify amoeba isolates, and the presence of Legionella pneumophila , Mycobacterium spp., Pseudomonas spp., and Microcystis aeruginosa was investigated in 43 isolates of amoebae by multiplex PCR. Of the isolated amoebae, 31 were Acanthamoeba spp., 21 were Hartmannella vermiformis, 13 were Naegleria spp., and one was Vanella spp. T2, T4, and T5 genotypes of Acanthamoeba have been identified, and T4 isolates were grouped into five subgenotypes and graphically represented with a Weblog application. Inside amoebae, L. pneumophila was detected in 13.9% (6/43) of the isolates, and Pseudomonas spp. and Mycobacterium spp. were detected in 32.6% (14/43) and 41.9% (18/43), respectively. No statistical correlation was demonstrated between FLA isolation and seasonality, but the presence of intracellular bacteria was associated with warm water temperatures, and also the intracellular presence of Mycobacterium spp. and Pseudomonas spp. were associated. These results highlight the importance of amoebae in natural waters as reservoirs of potential pathogens and its possible role in the spread of bacterial genera with interest in public and environmental health.

Opportunistic pathogens represent a unique challenge because they establish and grow within drinking water systems, yet the factors stimulating their proliferation are largely unknown. The purpose of this study was to examine the influence of pipe materials, disinfectant type, and water age on occurrence and persistence of three opportunistic pathogens (Legionella pneumophila, Mycobacterium avium, and Pseudomonas aeruginosa), broader genera (Legionella and mycobacteria), and two amoeba hosts (Acanthamoeba spp. and Hartmanella vermiformis). Triplicate simulated distribution systems (SDSs) compared iron, cement, and PVC pipe materials fed either chlorinated or chloraminated tap water and were sampled at water ages ranging from 1 day to 5.7 days. Quantitative polymerase chain reaction quantified gene copies of target microorganisms in both biofilm and bulk water. Legionella, mycobacteria, P. aeruginosa, and both amoebas naturally colonized the six SDSs, but L. pneumophila and M. avium were not detected. Disinfectant type and dose was observed to have the strongest influence on the microbiota. Disinfectant decay was noted with water age, particularly in chloraminated SDSs (due to nitrification), generally resulting in increased microbial detection frequencies and densities with water age. The influence of pipe material became apparent at water ages corresponding to low disinfectant residual. Each target microbe appeared to display a distinct response to disinfectant type, pipe materials, water age, and their interactions. Differences between the first and the second samplings (e.g., appearance of Legionella, reduction in P. aeruginosa and Acanthamoeba) suggest a temporally dynamic drinking water microbial community.