Beyond cyanotoxins: increased Legionella, antibiotic resistance genes in western Lake Erie water and disinfection-byproducts in their finished water

Background Western Lake Erie is suffering from harmful cyanobacterial blooms, primarily toxic Microcystis spp., affecting the ecosystem, water safety, and the regional economy. Continued bloom occurrence has raised concerns about public health implications. However, there has been no investigation regarding the potential increase of Legionella and antibiotic resistance genes in source water, and disinfection byproducts in municipal treated drinking water caused by these bloom events. Methods Over 2 years, source water (total n = 118) and finished water (total n = 118) samples were collected from drinking water plants situated in western Lake Erie (bloom site) and central Lake Erie (control site). Bloom-related parameters were determined, such as microcystin (MC), toxic Microcystis, total organic carbon, N, and P. Disinfection byproducts (DBPs) [total trihalomethanes (THMs) and haloacetic acids (HAAs)] were assessed in finished water. Genetic markers for Legionella, antibiotic resistance genes, and mobile genetic elements were quantified in source and finished waters. Results Significantly higher levels of MC-producing Microcystis were observed in the western Lake Erie site compared to the control site. Analysis of DBPs revealed significantly elevated THMs concentrations at the bloom site, while HAAs concentrations remained similar between the two sites. Legionella spp. levels were significantly higher in the bloom site, showing a significant relationship with total cyanobacteria. Abundance of ARGs (tetQ and sul1) and mobile genetic elements (MGEs) were also significantly higher at the bloom site. Discussion Although overall abundance decreased in finished water, relative abundance of ARGs and MGE among total bacteria increased after treatment, particularly at the bloom site. The findings underscore the need for ongoing efforts to mitigate bloom frequency and intensity in the lake. Moreover, optimizing water treatment processes during bloom episodes is crucial to maintain water quality. The associations observed between bloom conditions, ARGs, and Legionella, necessitate future investigations into the potential enhancement of antibiotic-resistant bacteria and Legionella spp. due to blooms, both in lake environments and drinking water distribution systems.


Introduction
Changes in the global climate have been linked to increasing eutrophication and an increase in frequency, duration and intensity of harmful algae blooms (Glibert, 2020).Among these blooms, freshwater blooms are dominated by cyanobacterial blooms, in which most are associated with the production of cyanotoxins (Loftin et al., 2016).These blooms, pose health risks by compromising drinking water sources, food production, aquatic ecosystems, and recreational waters (Brooks et al., 2016;Lee J. et al., 2017;Mrdjen et al., 2018).Microcystins (MCs), prevalent cyanotoxins, are commonly detected in freshwater used for drinking water supplies, irrigation, recreation, and fish farms (Loftin et al., 2016;Lee S. et al., 2017;Mrdjen et al., 2018).Epidemiological and animal studies have linked exposures to bloom-affected water to an increased frequency of non-alcoholic liver diseases, liver cancer, neurodegenerative diseases, gut microbiome disturbance, and more progressed liver cancer (Zhang et al., 2015;Lee et al., 2019Lee et al., , 2020Lee et al., , 2022;;Gorham et al., 2020).
Lake Erie in the United States has witnessed recurring severe cyanobacteria blooms, particularly the massive 2011 event spanning 5,000 km 2 (Stumpf et al., 2016;Zhang et al., 2017;Jankowiak et al., 2019).Elevated MCs exceeding 1 μg/L in finished drinking water in 2013-2014 prompted "Do Not Drink" advisories (Stumpf et al., 2016).Effective drinking water treatment is pivotal for safeguarding public health from cyanotoxins, necessitating robust strategies for MCs and cyanotoxin removal in bloom-impacted source water beyond source protection measures.While MCs are effectively removed using disinfection with free chlorine or ozone followed by additional activated carbon treatment (Cheung et al., 2013;Gonsior et al., 2019) in most cases, little is known about disinfection by-products (DBPs) that can form via chlorination in the Lake Erie region from MCs and other organics associated with blooms.Previous studies have demonstrated that bloom conditions are associated with an increase in DBP pre-cursors and DBPs (Zamyadi et al., 2012;Zong et al., 2015;Foreman et al., 2021).Disinfectants and chlorine react easily with all types of organic matter, including dissolved and algal organic matter to produce genotoxic and carcinogenic DBPs including trihalomethanes (THMs) and haloacetic acids (HAAs) (Liu et al., 2018).Therefore, conventional water treatment using activated carbon can be useful for removing cyanotoxins, and dissolved and algal organic compounds (Ho et al., 2011;U.S. EPA, 2016;Cermakova et al., 2017).
While activated carbon and rest procedures reduce unwanted contaminants, biofilms can become established in the system and contribute to promoting antibiotic resistance genes (ARGs) (Guo et al., 2018).Specifically, activated carbon filtration has been linked to increasing the abundance of antibiotic resistance bacteria (ARB) and antibiotic resistance genes (ARGs) (Xu et al., 2016(Xu et al., , 2020a)).Furthermore, recent studies report that cyanobacteria, especially Microcystis, harbor significant amounts of ARGs and may play a significant role as a reservoir and source for ARGs in bloom seasons whereby cyanobacterial blooms promote conjugative transfer of ARGs (Wang et al., 2020(Wang et al., , 2021)).More studies recently reported that cyanobacteria may promote the spread of ARGs in bacteria due to the significant contribution of mobile genetic elements (MGEs) located in genera, such as Microcystis (Volk and Lee, 2022).The interest in Legionella spp. is heightened in bloom-affected source waters as biofilm-loving Legionella spp.including L. pneumophila have long been known to grow when cyanobacteria densities are great and biofilm masses exist (Tison et al., 1980;Fliermans et al., 1981;Pope et al., 1982;Declerck, 2010;Cai et al., 2014).Additionally, biofilms can support the growth and persistence of Legionella spp. in drinking water systems (Berjeaud et al., 2016) including where activated carbon is used (Li et al., 2017).
While drinking water treatment plants (DWTPs) in bloomaffected areas give necessary and considerable attention to removing cyanotoxins and cyanobacteria during bloom seasons, there are emerging issues not yet studied in the Lake Erie region.We hypothesize that water from heavy bloom areas have (1) higher DBP levels in finished water due to increased interactions between bloom-associated organic matter and chlorine during treatment, (2) higher ARG from using activated carbon to absorb toxins as well as from the higher abundance of Microcystis in the source water, and (3) bloom-related aggregated biomass provides a niche for promoting Legionella in the lake water and is thus positively associated with Microcystis in Lake Erie.In the present study, we investigated two water treatment plants in the Lake Erie region (western Lake Erie for greater bloom frequency and intensity, and central Lake Erie for low bloom frequency and intensity) for 2 years.Specifically, we (1) examined the dynamics of Microcystis and MCs in the source water, (2) determined the association between cyanobacteria and Legionella in the source water, and (3) monitored both DBPs and ARGs in their finished water.The study outcome provides more holistic understanding about the water quality issues beyond bloom toxicity and cyanotoxin where cyanobacterial blooms are chronically prevalent.

Study sites and water samples
Two drinking water treatment plants in Ohio, United States, were selected based on annual cyanobacterial bloom intensity.The first was the Collins Park Water Treatment Plant in Toledo, which is a historically high-bloom area in western Lake Erie (Ohio Department Health et al., 2020) and the second was the City of Painesville Water Treatment Plant, a low-or no-bloom area in central Lake Erie (Supplementary Figure S1).Water samples were collected once weekly during the summer and fall (May-November) of 2013 and 2014 from the Toledo and Painesville sites, totaling 58 and 60 samples, respectively.Samples were collected from the source water (sampled at the drinking water intakes within Lake Erie) and from the finished drinking water at each plant.Collections were done in sterile bottles and shipped in a cooler on ice.For toxin measurements, amber glass vials were used.The samples reached The Ohio State University (Columbus, Ohio, United States) for laboratory analysis within 15 h of sample collection.In the lab, the collected source water (200 mL) was filtered using the polycarbonate membrane (0.45 μm pore size, Millipore, Burlington, MA, United States) for molecular analyses, and filters were kept at −20°C and further analyses were performed as soon as possible.

Water quality parameters
Conventional water quality parameters (water temperature, turbidity, hardness, and pH) and total chlorine in finished water were measured routinely on-site according to the Standard Methods (Rice et al., 2012) by the two water treatment plants and the data were obtained.Chlorophyll-a and phycocyanin were measured using the AquaFluor ® Handheld fluorometer (Turner Designs, California, United States).Total phosphorus (TP) and soluble reactive phosphorus were measured using the USEPA-accepted Method 8190 (Hach PhosVer 3 with Acid Persulfate Digestion).Total nitrogen (TN) in the higher range (0.23-13.50 mg/L NO 3 − -N) and the lower range (0.01-0.5 mg/L NO 3 − -N) were analyzed using Method 10206 (Hach dimethylphenol method) and Method 8192, respectively.Total organic carbon (TOC) was analyzed at Alloway Laboratory (Marion, Ohio, United States) using the USEPA 451.3 (U.S. EPA, 2005).

MC measurement
Total MCs in water samples were measured using enzyme-linked immunosorbent assay (ELISA) kits (Abraxis, Warminster, PA, United States) with MC-LR (0.15-5 μg/L) as a working standard.The absorbance was read at 450 nm on the Dynex MRX microplate reader (Dynex Technologies.Inc., Chantilly, VA, United States).The detection range of the assay is 0.15-5.0ng/mL with a detection limit of 0.10 ng/mL.All the ELISA assays were performed in triplicate.

Microcystis abundance
We quantified concentrations of total and MC-producing Microcystis by targeting the Microcystis phycocyanin intergenic spacer (PC-IGS) and microcystin synthetase gene B (mcyB), using published approaches with slight modifications (Klase et al., 2019).For Microcystis PC-IGS, the PCR mixture (20 μL) in duplicate contained TaqMan universal PCR master mix (10 μL) (Life Technologies, United States), each primer (188F/254R, 300 nM), PC-IGS probe (100 nM), additional MgCl 2 (1.25 mM) and DNA template (2 μL).The PCR profile followed an initial cycle of 50°C for 2 min, 95°C for 10 min, 50 cycles at 95°C for 30 s, 56°C for 1 min, and 72°C for 30 s.For Microcystis mcyB, the PCR protocol was identical to that for PC-IGS, except we used 900 nM of each mcyB-specific-primer (30F/108R) and 250 nm of mcyB probe.The standard working curve was generated for each assay according to the standard Microcystis PC-IGS and mcyB DNA standard (pGEMT-PC-IGS and mcyB), respectively.The PC-IGS probe had a 5′ end of the fluorescent reporter, 6FAM, and a 3′ end of the non-fluorescent quencher (NFQ) that was attached with a minor groove binder (MGB) moiety (MGB-NFQ).In contrast, the mcyB probe contained a 5′ end of the fluorescent reporter, instead.We estimated the proportion of the potentially MC-producing genotype in the Microcystis population according to the ratio between Microcystis mcyB and PC-IGS in percentage unit (%).

Statistical analysis
Data were explored using scatterplots, time-series plots, box plots and bar charts.Means and standard deviations (SD) were calculated

Source water
The severity of blooms in the source water from western (Toledo) and central (Painesville) Lake Erie was examined (Figures 1-3) as were source water chemical parameters and water temperatures.The mean concentrations of chlorophyll-a and phycocyanin in the source water from western Lake Erie (bloom site) were 8.32 μg/L and 37.4 μg/L, respectively (Figure 1).The mean concentrations of chlorophyll-a and phycocyanin in the source water from central Lake Erie (control site) were 2.92 μg/L and 14.8 μg/L, respectively (Figure 1).The mean concentrations of two bloom indicators in the bloom site were significantly higher than that from the control site (p < 0.05).Similarly, significantly higher concentrations of total Microcystis (PC-IGS gene) and toxin-producing Microcystis (mcyB gene) were observed in the bloom site source water (p < 0.05) (Figure 2).Specifically, the mean concentration of the PC-IGS gene (gene copies/ mL) was 5.57 × 10 4 in Toledo source water (bloom site) vs. 8.79 × 10 1 in the Painesville source water (control site).The mean concentration of the mcyB gene (gene copies/mL) was 1.48 × 10 4 in the bloom site vs. 1.32 × 10 1 in the control site.In regard to the cyanotoxin measurement, the mean concentration of microcystin in the bloom site source water was 1.65 μg/L which was also higher than the control site (not detected) (Figure 3).These results show that the intensity and frequency of blooms at the bloom site (Toledo) was obviously higher than the control site (Painesville).
Among water chemistry parameters, concentrations were consistently higher at the bloom sites; whereby mean total phosphorus levels for the two-year study period were 140 μg/L and 90 μg/L for the Toledo and Painesville DWTP source water samples.The mean total organic carbon was 0.36 mg/L (Toledo) and 0.24 mg/L (Painesville) and the mean total nitrate was 0.64 mg/L and 0.33 mg/L in the Toledo and Painesville source water samples, respectively.In addition to chemistry parameters, the mean temperatures of source water samples were 19.6°C and 16.1°C for Toledo and Painesville, respectively.
To evaluate the potential association between cyanobacteria and Legionella, the concentrations of Legionella were examined in the source water from the western Lake Erie and the control site for 2 years.The concentrations of Legionella (gene copies/ mL) were 2.49 × 10 2 (ranging from 5.10 × 10 0 to 5.09 × 10 3 ) in the bloom source water and 1.63 × 10 2 (ranging from 4.66 × 10 1 to

Finished water
The concentrations of DBPs (TTHMs and HAA5) in the finished water from the bloom vs. control sites are summarized in Figure 5.The mean concentrations of TTHMs were 22.43 μg/L from the bloom site and 14.03 μg/L from the control site, and the mean TTHMs concentration from the bloom site was significantly higher than the control site (p < 0.05).The mean concentrations of HAA5 were 8.86 μg/L from the bloom site and 9.06 μg/L from the control site, and the mean HAA5 concentrations between the two locations were not significantly different (p > 0.05).Figure 6 shows that TOC levels in finished water were not statistically different between the bloom vs. control sites (p > 0.05).The mean concentrations of TOC were 1.42 mg/L from the bloom site and 1.72 mg/L from the control site.Furthermore, microcystin was not detected in finished water samples from either location.

Concentrations of MGE (class 1 integron-integrase gene [intI1]
) and the total bacteria gene (16S rRNA) were also significantly reduced by drinking water treatment.After water treatment processing, the mean concentrations of the MGE (gene copies/100 mL of intI1) in the finished water were 4.19 × 10 0 in Toledo and 0.82 × 10 0 in Painesville which represented significant (p < 0.01) reductions from their source water (Figure 8).Similarly, concentrations of the total bacterial gene in the finished water of the bloom and control sites (4.56 × 10 3 in Toledo, 3.55 × 10 4 in Painesville) were significantly lower (p < 0.01) than in their Temporal change of concentration of Microcystis (green) and Legionella (purple) in source water from the bloom site (western Lake Erie, Toledo) (A) and the control site (central Lake Erie, Painesville) (B).Statistical analysis for relationship between total cyanobacteria and Legionella species in the source water.Temporal change of concentration of total organic carbon in the finished water of the bloom site (Toledo) (dark pink) and the control site (Painesville) (blue). 10.3389/fmicb.2023.1233327 Frontiers in Microbiology 08 frontiersin.org

Discussion
Bloom conditions at the western Lake Erie site Two drinking water treatment plants, drawing from Lake Erie as their source water, were chosen in Toledo (a high-bloom region in western Lake Erie) and Painesville (a low-or no-bloom area in central Lake Erie) to examine water quality challenges beyond cyanotoxins when utilizing bloom-impacted source water.In assessing blooms, with an emphasis on cyanobacteria, multiple water quality parameters were employed to evaluate source and finished water quality.Upon characterizing bloom conditions, as expected, there were significant differences in chlorophyll-a, phycocyanin, total Microcystis, MC-producing Microcystis, and MC between the two locations.These differences were supported by the underlying water chemistry and temperatures enabling greater primary productivity by cyanobacteria as warmer water temperatures and higher concentrations of nutrients (P and N) are linked to cyanobacteria bloom formation, particularly when water temperatures approach or exceeds 20°C (Paerl and Huisman, 2008;Stumpf et al., 2012).
As predicted, bloom conditions manifested in source water during the warm months, mainly from July, August and September at the Toledo water intake (bloom site).Figures 1-4 show corroboration between bloom indicators (chlorophyll-a and phycocyanin), Microcystis densities, and ultimately cyanotoxins.Three of the source water MC results from Toledo exceeded the provisional 2015 Ohio contact recreation advisory threshold (6 μg/L) and one sample exceeded the revised and current (post-2019) Ohio and U.S. EPA contact recreation advisory threshold (8 μg/L standard) (Kasich et al., 2015;U.S. EPA, 2019).Notably, although MC levels were elevated in Toledo's source water, they remained undetectable in finished water samples.Of note, the Microcystis concentrations indicated by PC-IGS genetic marker measurement in the source water in Toledo were similar to those in a Japanese Lake Mikata where Microcystis blooms occurred (Yoshida et al., 2007).

Elevated DBP formation potential and DBPs from bloom conditions
Once cyanobacterial blooms occur, blooms can impair surface water supplies used for drinking water.Blooms are associated with increases in TOC, turbidity, taste and odor compounds, and precursors for DBP formation (Nguyen et al., 2005).As hypothesized, TOC and turbidity levels were elevated in the bloom site source water relative to the control site (Painesville).Algal-derived organic carbon can be a significant source of DBP precursors for drinking water treatment facilities (Nguyen et al., 2005).
While DBP precursors can be elevated in source water, the presence of DBPs in finished drinking water varies according to the source water characteristics (e.g., temperature, pH, natural organic matter, etc.) and the processes used in water treatment.TOC concentrations are often used to predict DBP formation because TOC is a precursor to DBP formation.In this study, we investigated not only temporal variation in TOC (DBP formation precursor), but also the concentration of two commonly regulated DBPs, TTHMs and HAA5, in drinking water.TTHMs and HAA5, considered potentially carcinogenic, are the most important groups of DBPs (Rodriguez et al., 2004).The primary drinking water regulations for the U.S. mandates that the maximum acceptable levels of DBPs are 80 μg/L for TTHMs and 60 μg/L for HAA5 (U.S. EPA, 2023).Our results show that TOC concentrations in Toledo source water were higher than Painesville source water, and it is likely that the increase in TOC is related to cyanobacteria densities (Cory et al., 2016).As TOC levels were higher in the source water at Toledo, so were TTHMs and HAA5 in the finished water relative to the Painesville finished water.While DBPs were quantifiable, none of the samples exceeded primary standards set by the U.S. EPA, indicating that the current TOC removal processes at the two DWTPs can effectively control common DBPs.

Relationship between Legionella and Microcystis in bloom site source water
Beyond altering source and finished water quality, cyanobacteria blooms in western Lake Erie occur in the context of a complex microbial ecosystem and can alter microbial communities.
One type of change was the observed apparent positive relationship between Legionella spp.abundance and Microcystis in the source water for the Toledo DWTP (bloom site), which was present in both study years, but non-existent in the source water of the Painesville DWTP that served as a control site.Recent research from western Lake Erie (near Toledo [bloom site]) focusing on bacterial diversity from non-cyanobacteria showed clear differential responses among non-cyanobacteria to cyanobacteria abundance (estimated by chlorophyll-a) (Berry et al., 2017).Among bacteria that are linked to an increase in abundance during bloom conditions, there is evidence that the density of Legionella is correlated with Microcystis and eutrophication (Numberger et al., 2022).When looking at similar warm season water samples from three inland Ohio lakes, which like Western Lake Erie exhibit eutrophic conditions, Lee et al. (2016) documented Legionella spp.and the Legionellacea as being among the most abundant and ubiquitous bacteria present in Ohio inland lakes.
While Legionella species are common in various natural and human-made aquatic environments, some species, mostly L. pneumophila, may cause legionellosis, which is a serious pulmonary infection established in persons following the inhalation of particles of contaminated aerosolized water.While there is some evidence that certain cyanobacteria can stimulate the growth of Legionella (Świątecki and Zdanowski, 2007;Bergeron et al., 2015), there are few studies primarily focusing on Legionella and cyanobacteria densities.Tison The relative abundances (gene copies/16S rRNA gene) of ARGS [tetracycline (A), sulfonamide (B), carbapenem resistance genes (C)] and mobile genetic element (D) in source water and finished water from the bloom site (Toledo) and the control site (Painesville).b Represents a significant difference from "a" (p < 0.01).c Represents a significant difference from "a" (p < 0.01) and from "b" (p < 0.05).et al. (1980) suggested an intimate association between Legionella spp.and cyanobacteria (Fisherella).Their observation indicated that Legionella could use algal extracellular products as its carbon and energy sources (Tison et al., 1980;Berendt, 1981).A previous study also reported that symbiotic interactions between Legionella and cyanobacteria may help the colonization of aquatic environments (Carvalho et al., 2007).Several factors (e.g., temperature, pH, and concentrations of nutrients and ions) and possibly products produced by other non-cyanobacterial bacteria, such as earlier arriving Betaproteobacteriales (van der Kooij et al., 2018), which are also abundant in Lake Erie during blooms (Berry et al., 2017), may all contribute to Legionella growth in aquatic environments as part of an ecological succession linked to bloom conditions of certain cyanobacteria species, like Microcystis.
While the potential for an ongoing increased global incidence of legionellosis due to a warmer climate has been described (Walker, 2018), the role of environmental waters warrants additional study.In Ohio, the incidence rate of legionellosis has consistently been 1.9 to 2.6 times greater than the U.S. average in recent years (Han, 2021).Recently, aerosols associated with roadway exposures have been considered as part of the increased incidence (Han, 2021), but of particular interest here is how anticipated future increases in eutrophic waters experiencing further eutrophication and warming globally may contribute to increased aerosol-related Legionella exposures, which could occur from boat wake (water skiing), fountains, etc.Beyond warmer water, there may be possible synergisms occurring in warming eutrophic waters as high concentrations of phosphates can enable substantial growth of cyanobacteria and increased turbidity and/or aggregated biofilm materials which can prompt or enhance the growth of Legionella and other Gammaproteobacteria (Cai et al., 2013;Lee et al., 2016;Srivastava et al., 2016).While the Gammaproteobacteria include many biofilm formers that may benefit from increased turbidity, these bacteria are also associated with an abundance of antibiotic resistance genes (Zhang et al., 2021).

Relative abundance of ARGs increases in finished water from Lake Erie
Adding to the complexity of bloom ecology beyond Legionella densities are other broader emergent concerns regarding the establishment, maintenance, or promotion of antibiotic resistance bacteria (ARB) and ARGs in the source water (Volk and Lee, 2022), finished water (Li et al., 2016), and the distribution system (Xi et al., 2009;Xu et al., 2016).When the source water is impacted by bloom conditions, a convergence of numerous selective pressures emerge in the microbial community, including interspecies competition in the source water coupled with impacts from the water treatment processes, such as activated carbon use and disinfection.Xu et al. (2020b) demonstrated that when MCs were present an increase in ARGs was observed in the DWTP.
In this study, while hypothesized there would be differences in the densities of MGEs and ARGs in the source water samples from bloom and control sites, no differences were observed for MGEs or the ARGs for tetracycline, sulfonamide, and carbapenem resistance (Figure 7).At both study locations, the DWTP processes significantly reduced the amount of MGEs and ARGs from source water to finished water.The differences that emerged were specific to relative abundances of ARGs and MGEs.At both DWTPs, while reduced in overall density, the total bacterial density was reduced even more greatly by the treatment processes.In comparison of the two DWTPs, the MGEs increased in relative abundance more in the Toledo DWTP than the Painesville DWTP.
Previous studies have revealed that commonly used disinfection technology can enrich ARB and spread ARGs (Shi et al., 2013).Water processing, including filtration and chlorination, remove most bacteria; however, extracellular stress can promote the replication of plasmids in bacteria.For example, chlorination might increase the copy number of plasmids in the surviving bacteria cells, resulting in the higher relative abundance of ARGs in treated water (Shi et al., 2013).While densities of ARGs and MGE were higher in the source water than the finished water, a limitation of this study was that it did not study the water distribution system, which can have higher levels of bacteria than the finished water due to regrowth potential of bacteria in the distribution system (Xi et al., 2009).If the source water selected for greater survival of biofilm formers preferring higher phosphate levels, corrosion control measures for the distribution system using phosphate may play a role in the regrowth of antibiotic resistant bacteria (Kappell et al., 2019;Kimbell et al., 2020).As expected, the finished water which included corrosion control had higher total P levels than the source water at the bloom site and control site (Supplementary Table S1).

Future research needs
The main scope of the present study was source water and finished water in Lake Erie region, thus tap samples or samples from within the distribution system were not included in this study, but we suggest that future study includes examining the distribution systems of bloom-impacted community water systems since it can contribute great knowledge regarding the public health implications of biofilm formation, ARGs, MGEs, and potential bacterial regrowth.This study adds to the body of evidence that some efforts should be made to monitor ARG concentrations before, during, and after drinking water treatments.As part of monitoring for ARGs and Legionella, methods reliant on culturable Legionella or other bacteria (e.g., Pseudomonas spp., Mycobacterium spp.) may provide benefits, but may underreport densities of some bacteria resistant to disinfection when hosted inside free-living Amoeba, which warrants a need to use a PCR-based approach (Calvo et al., 2013).
Complicating future studies on ARGs and MGEs in finished water and distribution systems are not only disinfectants but also disinfection byproducts.While this study illustrated that chlorinerelated DBPs (THMs and HAA5) were elevated during bloom conditions, yet meeting regulatory thresholds, we suggest to characterize other DBPs that are likely elevated in cyanobacteriaimpacted waters.Since there is considerable evidence that THMs and HAAs are linked to ARGs in the finished water and distribution system (Lv et al., 2014;Li and Gu, 2019;Zhang et al., 2023), we recommend to assess unregulated N-DBPs which likely are less abundant than THMs and HAAs, but are more common in bloomimpacted waters, and able to elicit greater cytotoxicity (Fang et al., 2010;Liu et al., 2020) in a future study.In addition, emerging literature indicates that future studies on DBPs ought to consider a broader range of DBPs for also assessing potential human health risks (Li and Mitch, 2018;Kali et al., 2021).Thus, in bloom-impacted waters there would likely be benefits from assessing N-DBPs which are associated with the chlorination of Microcystis aeruginosa (Fang et al., 2010).
These same N-DBPs may also of interest for improving understanding of antibiotic resistance phenomena in treated waters.

Conclusion
In this study, a compelling and statistically significant correlation emerged between Legionella and cyanobacteria within the water of the bloom site.Increasing levels of Microcystis were also associated with disinfection byproducts (THMs and HAA5) in the water of the bloom site, but did not exceed primary regulatory standards.At both the bloom site and control site, the DWTPs reduced the density of ARGs and MGEs; however, their relative abundance increased in finished water.This study not only fills existing gaps in the understanding of cyanobacteria and Legionella ecology but also underscores several pivotal areas of needed future research for aquatic environments with human exposure potential.Furthermore, our findings indicate a potential health risk of Legionellarelated disease in proximity to areas impacted by blooms.
10.3389/fmicb.2023.1233327Frontiers in Microbiology 04 frontiersin.organd data are presented as the mean ± SD.A one-way analysis of variance (ANOVA) was used for the spatial difference in the variables with a significance level set at p < 0.05.After ANOVA, Tukey Honest Significant Difference tests were used to determine if the means were different (p < 0.05) between the groups.All analyses were conducted using SPSS 24.0 (SPSS, Chicago, IL, United States).

FIGURE 1
FIGURE 1Temporal changes of concentration (pg/L) of bloom-related parameters, chlorophyll-a (A) and phycocyanin (B), in source water from the bloom site (Toledo) (dark pink) and the control site (Painesville) (blue).

FIGURE 2
FIGURE 2Temporal changes of population of total Microcystis (A) and toxin-producing Microcystis (B) in source water from the bloom site (Toledo) (dark pink) and the control site (Painesville) (blue).

FIGURE 3
FIGURE 3Temporal changes of concentration of microcystin in source water from the bloom site (Toledo) (dark pink) and the control site (Painesville) (blue).

FIGURE 4
FIGURE 4 source water (6.48 × 10 8 in Toledo, 9.00 × 10 9 in Painesville).While drinking water treatment resulted in significantly lower concentrations of total bacteria, ARGs, and MGE, the relative abundance of ARGs among total bacteria (16S rRNA) increased significantly (p < 0.05) at both the bloom and control sites (Figure9).

FIGURE 5
FIGURE 5Temporal change of concentration of disinfection by-products in finished water from the bloom site (A) and the control site (B).

FIGURE 7
FIGURE 7 Concentrations of antibiotic resistance genes [tetQ (A), sull (B), and blak PC (C)] in source and finished waters from the bloom site (Toledo) (dark pink) and the control site (Painesville) (blue).*p < 0.05.

FIGURE 8
FIGURE 8 Concentrations of mobile genetic elements [MGES (A)] and 16S rRNA [total bacteria (B)] in source and finished waters from the bloom site (Toledo) (dark pink) and the control site (Painesville) (blue).**p < 0.01.