Metagenomic study of acanthamoeba endosymbionts in recreational waters: role in ocular and systemic infections

Metagenomic study of acanthamoeba endosymbionts in recreational waters: role in ocular and systemic infections | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Metagenomic study of acanthamoeba endosymbionts in recreational waters: role in ocular and systemic infections Shayaan Kaleem, Zhuochen Yuan, Christi Le Mahieu, Tanja Morariu, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7554315/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Acanthamoeba , a free-living amoeba found in recreational waters, is both a pathogen and a host for microbial endosymbionts implicated in human infections, particularly in the eye. This study used metagenomic sequencing to characterize known endosymbionts associated with Acanthamoeba infection in recreational water sites across Miami. Over a three-week sampling period, 51 unique microbial species were identified, with the highest diversity observed at sites with high human activity. Vibrio parahaemolyticus and Pseudomonas aeruginosa - known ocular pathogens and Acanthamoeba endosymbionts - were among the most abundant. Overall, 51% of species were associated with eye infections and 43% were linked to both keratitis and endophthalmitis. As well, 53% of the endosymbionts were associated with skin infections and 47% with respiratory infections. Sites with greater microbial diversity also demonstrated higher numbers of disease-associated species. The frequent detection of Acanthamoeba endosymbionts in popular aquatic settings raises public health concerns, especially for contact lens users who may be at increased risk of ocular infections. Metagenomic surveillance of recreational waters may help inform prevention strategies and reduce the burden of vision-threatening and systemic infections. Health sciences/Diseases Biological sciences/Microbiology ocular microbiology acanthamoeba endosymbionts metagenomics Figures Figure 1 Figure 2 Figure 3 Introduction Acanthamoeba is a genus of free-living amoebae widely distributed in soil, air, and water, including chlorinated and non-chlorinated recreational water sources. While typically non-pathogenic in healthy individuals, Acanthamoeba species have been increasingly recognized as agents of severe ocular infections, particularly Acanthamoeba keratitis (AK) and, less commonly, endophthalmitis. 1 – 3 These infections, often associated with contact lens use and water exposure, can lead to significant visual impairment or blindness if not diagnosed and managed early. 4 , 5 Beyond acting as primary pathogens, Acanthamoeba species also serve as environmental reservoirs for a wide variety of microbial endosymbionts, including bacteria, fungi, and viruses. 6 , 7 These endosymbionts are protected within the amoeba’s cysts and trophozoites, enhancing their resistance to environmental stressors and disinfectants. This intracellular shelter may increase the virulence and persistence of pathogens, many of which have been linked to eye infections. 8 , 9 Among the most clinically relevant are Pseudomonas aeruginosa and Vibrio parahaemolyticus , which have been associated with both keratitis and endophthalmitis. 10 , 11 The role of Acanthamoeba as both a pathogen and a microbial host raises important public health concerns in the context of recreational water use. Activities such as swimming, kayaking, or paddleboarding can expose individuals - particularly contact lens users - to contaminated water sources harboring these organisms. 4 , 12 Yet, few studies have comprehensively characterized the broader microbial ecosystem within recreational waters, particularly regarding pathogens known to be associated with Acanthamoeba infection. This study aimed to characterize the microbial diversity within recreational waters in Miami and assess the disease associations of species previously reported as Acanthamoeba endosymbionts, with a focus on ocular infections. Using metagenomic sequencing, we evaluated the presence of known Acanthamoeba endosymbionts linked to keratitis, endophthalmitis, any eye infection, respiratory illnesses, gastrointestinal illnesses, skin infections, and human feces presence. These findings provide insight into the environmental reservoirs of Acanthamoeba endosymbionts in the eye and other body systems, supporting the development of targeted surveillance strategies to mitigate vision-threatening as well as systemic infections. Methods Study design and sample collection. This serial cross-sectional study was conducted over a three-week period between April 12 and April 26, 2024. Water samples were collected from eight recreational water sites located throughout Miami, Florida. Sites included Purdy Kayak Launch (Maurice Gibb Park), Key Biscayne Crandon Park South, Little River Pocket Park, 1st Street Beach, 53rd Street Beach, Key Biscayne Beach Club, Parkview Kayak Launch, and Surfside 93rd Street Beach. At each site, samples were collected in triplicate once per week. DNA extraction and metagenomic sequencing. Sequencing was performed using the Oxford Nanopore Technologies MinION system. 13 Libraries were prepared using the Ligation Sequencing Kit (SQK-LSK114), and samples were run on R10.4 flow cells. Basecalling was conducted using Guppy software, and taxonomic classification was performed using Kraken2 with the NCBI RefSeq database. Microbial identification and data analysis. A structured literature review was conducted to determine whether each species has previously been documented as part of the Acanthamoeba microbiome. Species recovered from the recreational waters with at least one reported co-infection with Acanthamoeba in any human disease were included in this analysis. Disease associations were categorized based on documented links to specific organ systems or fluids, including any eye infection, keratitis, endophthalmitis, respiratory infection, gastrointestinal infection, skin infection, and human feces. Statistical analysis. Descriptive statistics were used to summarize species frequency, diversity, and disease associations. Weekly and site-specific variations were analyzed using count-based comparisons. All data analysis and visualization were performed using STATA (version 18.5). Results Across the three-week sampling period, a total of 51 unique microbial species were recovered from recreational waters across eight Miami locations (Fig. 1 ). Week 1 yielded the highest diversity with 19 species, followed by 21 in week 3 and 11 in week 2. Figure 2 and Supplementary Table S1 shows the temporal distribution of unique species per site and sampling week. The most microbially diverse location overall was Purdy Kayak Launch at Maurice Gibb Park, with 15 unique species detected, followed by Key Biscayne Crandon Park South (13 species), and Little River Pocket Park (10 species). Other locations including 1st Street Beach, 53rd Street Beach, Key Biscayne Beach Club, Parkview Kayak Launch, and Surfside 93rd Street Beach had between 2–4 unique species across all three weeks. Among all species identified, Vibrio parahaemolyticus and Pseudomonas aeruginosa were the most consistently abundant, detected across multiple sites and weeks, with 3,039,033 and 1,573,011 total sequence reads, respectively (Table 1 ). These organisms are both known ocular pathogens implicated in keratitis and endophthalmitis and are also well-documented endosymbionts of Acanthamoeba . Table 1 Most common microbial species identified by weekly sequence reads Species Week 1 Week 2 Week 3 Total Count Vibrio parahaemolyticus 9,157 6,645 3,023,231 3,039,033 Pseudomonas aeruginosa 3,183 116,665 1,453,163 1,573,011 Pseudomonas (other) 27,769 27,569 453,121 508,459 Pseudomonas otitidis 0 317 342,846 343,163 Pseudomonas mendocina 4,229 1,233 325,420 330,882 Escherichia coli (other) 14,235 9,880 205,951 230,066 Pseudomonas stutzeri 5,515 9,072 57,959 72,546 Pseudomonas entomophila L48 441 0 63,535 63,976 Pseudomonas fragi 18,328 676 2,898 21,902 Pseudomonas fluorescens 2,612 2,679 9,605 14,896 Pseudomonas chlororaphis 225 1,053 13,457 14,735 Pseudomonas koreensis 514 484 5,985 6,983 Mycobacterium 0 0 5,511 5,511 Escherichia coli O139:H28 str. E24377A 0 434 3,884 4,318 Mycobacteriaceae 784 169 3,304 4,257 Despite the detection of several endosymbionts known from the literature to be associated with Acanthamoeba , conventional PCR testing did not detect the presence of Acanthamoeba DNA in any collected water samples. Thus, the association between detected microbial species and Acanthamoeba is inferred from existing literature, as co-presence was not confirmed through PCR in our methodology. When examining what coinfections with Acanthamoeba the endosymbionts were associated with, 53% of the recovered species were linked to skin infections, 51% to eye infections, and 47% to respiratory illnesses. Notably, 43% were associated with both keratitis and endophthalmitis, highlighting their ophthalmic relevance. Additional findings revealed that 37% of species had previously been isolated from human feces, and 33% were associated with gastrointestinal disease. The distribution of disease associations by source is depicted in Fig. 3 and Supplementary Table S2. Little River Pocket Park demonstrated the highest number of total disease associations (n = 107), followed by Key Biscayne Crandon Park South (n = 105), Purdy Kayak Launch, Maurice Gibb Park (n = 97), and Parkview Kayak Launch (n = 94). These findings suggest a correlation between microbial diversity and potential pathogenicity, particularly at locations with high human recreational activity. Discussion This study underscores the potential health risks posed by Acanthamoeba -associated microbes in recreational waters, particularly in the eye. Nearly half of all species identified across sites were linked to eye infections, with 43% specifically associated with both keratitis and endophthalmitis. While Acanthamoeba was not detected in our PCR samples, the presence of known ocular pathogens with potential to survive within protozoan hosts raises concerns about environmental vectors contributing to serious ocular complications. 12 , 14 The frequent detection of Pseudomonas aeruginosa and Vibrio parahaemolyticus - both previously implicated in microbial keratitis and endophthalmitis - adds further concern. 10 , 11 These organisms are known to survive intracellularly within Acanthamoeba , which protects them from environmental stress and disinfection. 6 , 7 This protective niche may enhance their persistence in aquatic environments and their virulence when introduced to the eye. Sites with high microbial diversity and ocular disease associations, such as Key Biscayne Crandon Park South and Maurice Gibb Park, represent areas of potential risk, particularly for contact lens wearers. Swimming while wearing lenses has been strongly linked to Acanthamoeba keratitis and may also facilitate exposure to other ocular pathogens. 12 , 14 Although keratitis is the more frequently discussed outcome, post-infectious or secondary endophthalmitis remains a devastating, albeit rare, possibility with polymicrobial infections. The intracellular protection offered by protozoans and the potential synergy between co-resident microbes may contribute to chronic, recurrent, or treatment-resistant ocular infections. 14 Standard antimicrobial regimens may fail to adequately penetrate cyst walls or address secondary pathogens, resulting in poor visual outcomes. Understanding the diversity of these microbial communities, particularly their ocular relevance, is critical for early intervention and tailored management. This study also demonstrates the utility of metagenomic sequencing via the Oxford Nanopore MinION platform to rapidly identify microbial species in complex environmental samples. 14 Long-read sequencing can detect co-infections and rare species missed by culture or short-read techniques, offering a promising tool for environmental surveillance in high-risk recreational settings. Limitations include the short sampling duration, absence of environmental metadata (e.g., pH, salinity), inability to detect Acanthamoeba through PCR, and the lack of direct clinical correlation. Future research should aim to connect environmental isolates to clinical cases of keratitis, endophthalmitis, other eye infections, and Acanthamoeba infections in other body systems to confirm co-occurrence with protozoan hosts and to better understand the pathogenic potential of specific endosymbionts. Ultimately, this study highlights a substantial ocular risk associated with exposure to recreational waters. Public health strategies should target education for contact lens users, reinforce safe water practices, and incorporate targeted surveillance in high-risk aquatic environments to reduce the burden of sight-threatening infections. Declarations Author Contributions: SK, ZY, and DM were involved in conceptualizing the study, analyzing the data, and interpreting the results. CL, TM, and AD were involved in collecting and sequencing the data. GA, HF, and DM were involved in overseeing the project. All authors contributed to the writing and review of the manuscript and have approved the final version. Acknowledgements: None Competing Interests: The authors declare no competing interests. Funding: The authors declare no funding. Ethics Declaration: None Consent to participate/publish: NA Data Availability: The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. References Lorenzo-Morales, J. et al. Acanthamoeba keratitis: an emerging disease gathering importance worldwide? Trends Parasitol. 29 , 181–187 (2013). Francine, M. C. & Guy, C. Acanthamoeba spp. as Agents of Disease in Humans. Clin. Microbiol. Rev. 16 , 273–307 (2003). Khan, N. A. & Acanthamoeba (eds) : Biology and Pathogenesis. (Second edition. Norfolk, England: Caister Academic Press, [2015] ©2015). Seal, D. V. Acanthamoeba keratitis update—incidence, molecular epidemiology and new drugs for treatment. Eye 17 , 893–905 (2003). Carnt, N., Minassian, D. C. & Dart, J. K. G. Acanthamoeba Keratitis Risk Factors for Daily Wear Contact Lens Users: A CaseControl Study. Ophthalmology 130 , 48–55 (2023). Gilbert, G. & Didier, R. Microorganisms Resistant to Free-Living Amoebae. Clin. Microbiol. Rev. 17 , 413–433 (2004). Corsaro, D., Pages, G. S., Catalan, V., Loret, J. F. & Greub, G. Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants. Int. J. Hyg. Environ. Health . 213 , 158–166 (2010). Linder Ewert, J. W. K. Free-living Amoebae Protecting Legionella in Water: The Tip of an Iceberg? Scand. J. Infect. Dis. 31 , 383–385 (1999). Scheid, P. Relevance of free-living amoebae as hosts for phylogenetically diverse microorganisms. Parasitol. Res. 113 , 2407–2414 (2014). Letchumanan, V., Chan, K. G. & Lee, L. H. Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques. Front Microbiol 5 , (2014). Pang, Z., Raudonis, R., Glick, B. R., Lin, T. J. & Cheng, Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. Biotechnol. Adv. 37 , 177–192 (2019). Maycock, N. J. R. & Jayaswal, R. Update on Acanthamoeba Keratitis: Diagnosis, Treatment, and Outcomes. Cornea 35, (2016). Jain, M., Olsen, H. E., Paten, B. & Akeson, M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. Genome Biol. 17 , 239 (2016). Siddiqui, R. & Khan, N. A. Biology and pathogenesis of Acanthamoeba. Parasit. Vectors . 5 , 6 (2012). Additional Declarations No competing interests reported. Supplementary Files AcanthamoebaSupplementalMaterial.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7554315","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":553708238,"identity":"c46d3e31-7d90-493b-a103-86a8346397f1","order_by":0,"name":"Shayaan Kaleem","email":"","orcid":"","institution":"University of Toronto Temerty Faculty of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Shayaan","middleName":"","lastName":"Kaleem","suffix":""},{"id":553708239,"identity":"4e3e5460-9aa6-4235-95bc-1bb9bffd6ff4","order_by":1,"name":"Zhuochen Yuan","email":"","orcid":"","institution":"University of Miami Miler School of 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15:27:33","extension":"html","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":60225,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7554315/v1/16737fae7acbab6731a02559.html"},{"id":97365281,"identity":"a5afb7bf-4dfd-4fba-8909-2d54d5e7f5a1","added_by":"auto","created_at":"2025-12-03 15:27:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":205967,"visible":true,"origin":"","legend":"\u003cp\u003eLocation of eight water sample collection sites across Miami Florida\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7554315/v1/ce187858d70c0068a021401f.png"},{"id":97370398,"identity":"7349a617-09b8-49fa-ad67-4e006ad7139e","added_by":"auto","created_at":"2025-12-03 16:27:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig. 2 \u003c/strong\u003eUnique Acanthamoeba endosymbiont species count per week for each source\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7554315/v1/7d1b3edea893420ce905f050.png"},{"id":97365284,"identity":"92f18a7d-3632-42ce-bb6e-855f1136779e","added_by":"auto","created_at":"2025-12-03 15:27:33","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1676985,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of associated diseases by source\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7554315/v1/39a80cd162815bedb9856cde.png"},{"id":105191824,"identity":"c6169102-fa50-4a13-8393-d434d4789e83","added_by":"auto","created_at":"2026-03-23 09:29:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1945225,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7554315/v1/c99de24b-adc5-4bc2-b856-da4b4372776d.pdf"},{"id":97365285,"identity":"ec7f5053-a19a-49ac-9d32-2d4ad9816a2a","added_by":"auto","created_at":"2025-12-03 15:27:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":107642,"visible":true,"origin":"","legend":"","description":"","filename":"AcanthamoebaSupplementalMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7554315/v1/908ce1d8b8940b7aa3bb1fa2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Metagenomic study of acanthamoeba endosymbionts in recreational waters: role in ocular and systemic infections","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eAcanthamoeba\u003c/em\u003e is a genus of free-living amoebae widely distributed in soil, air, and water, including chlorinated and non-chlorinated recreational water sources. While typically non-pathogenic in healthy individuals, \u003cem\u003eAcanthamoeba\u003c/em\u003e species have been increasingly recognized as agents of severe ocular infections, particularly \u003cem\u003eAcanthamoeba\u003c/em\u003e keratitis (AK) and, less commonly, endophthalmitis.\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e These infections, often associated with contact lens use and water exposure, can lead to significant visual impairment or blindness if not diagnosed and managed early.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eBeyond acting as primary pathogens, \u003cem\u003eAcanthamoeba\u003c/em\u003e species also serve as environmental reservoirs for a wide variety of microbial endosymbionts, including bacteria, fungi, and viruses.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e These endosymbionts are protected within the amoeba\u0026rsquo;s cysts and trophozoites, enhancing their resistance to environmental stressors and disinfectants. This intracellular shelter may increase the virulence and persistence of pathogens, many of which have been linked to eye infections.\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e Among the most clinically relevant are \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e, which have been associated with both keratitis and endophthalmitis.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe role of \u003cem\u003eAcanthamoeba\u003c/em\u003e as both a pathogen and a microbial host raises important public health concerns in the context of recreational water use. Activities such as swimming, kayaking, or paddleboarding can expose individuals - particularly contact lens users - to contaminated water sources harboring these organisms.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e Yet, few studies have comprehensively characterized the broader microbial ecosystem within recreational waters, particularly regarding pathogens known to be associated with \u003cem\u003eAcanthamoeba\u003c/em\u003e infection.\u003c/p\u003e\u003cp\u003eThis study aimed to characterize the microbial diversity within recreational waters in Miami and assess the disease associations of species previously reported as \u003cem\u003eAcanthamoeba\u003c/em\u003e endosymbionts, with a focus on ocular infections. Using metagenomic sequencing, we evaluated the presence of known \u003cem\u003eAcanthamoeba\u003c/em\u003e endosymbionts linked to keratitis, endophthalmitis, any eye infection, respiratory illnesses, gastrointestinal illnesses, skin infections, and human feces presence. These findings provide insight into the environmental reservoirs of \u003cem\u003eAcanthamoeba\u003c/em\u003e endosymbionts in the eye and other body systems, supporting the development of targeted surveillance strategies to mitigate vision-threatening as well as systemic infections.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003eStudy design and sample collection.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThis serial cross-sectional study was conducted over a three-week period between April 12 and April 26, 2024. Water samples were collected from eight recreational water sites located throughout Miami, Florida. Sites included Purdy Kayak Launch (Maurice Gibb Park), Key Biscayne Crandon Park South, Little River Pocket Park, 1st Street Beach, 53rd Street Beach, Key Biscayne Beach Club, Parkview Kayak Launch, and Surfside 93rd Street Beach. At each site, samples were collected in triplicate once per week.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDNA extraction and metagenomic sequencing.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSequencing was performed using the Oxford Nanopore Technologies MinION system.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e Libraries were prepared using the Ligation Sequencing Kit (SQK-LSK114), and samples were run on R10.4 flow cells. Basecalling was conducted using Guppy software, and taxonomic classification was performed using Kraken2 with the NCBI RefSeq database.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMicrobial identification and data analysis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA structured literature review was conducted to determine whether each species has previously been documented as part of the \u003cem\u003eAcanthamoeba\u003c/em\u003e microbiome. Species recovered from the recreational waters with at least one reported co-infection with \u003cem\u003eAcanthamoeba\u003c/em\u003e in any human disease were included in this analysis.\u003c/p\u003e\u003cp\u003eDisease associations were categorized based on documented links to specific organ systems or fluids, including any eye infection, keratitis, endophthalmitis, respiratory infection, gastrointestinal infection, skin infection, and human feces.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis.\u003c/h2\u003e\u003cp\u003eDescriptive statistics were used to summarize species frequency, diversity, and disease associations. Weekly and site-specific variations were analyzed using count-based comparisons. All data analysis and visualization were performed using STATA (version 18.5).\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAcross the three-week sampling period, a total of 51 unique microbial species were recovered from recreational waters across eight Miami locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Week 1 yielded the highest diversity with 19 species, followed by 21 in week 3 and 11 in week 2. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e shows the temporal distribution of unique species per site and sampling week.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe most microbially diverse location overall was Purdy Kayak Launch at Maurice Gibb Park, with 15 unique species detected, followed by Key Biscayne Crandon Park South (13 species), and Little River Pocket Park (10 species). Other locations including 1st Street Beach, 53rd Street Beach, Key Biscayne Beach Club, Parkview Kayak Launch, and Surfside 93rd Street Beach had between 2\u0026ndash;4 unique species across all three weeks.\u003c/p\u003e\u003cp\u003eAmong all species identified, \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e were the most consistently abundant, detected across multiple sites and weeks, with 3,039,033 and 1,573,011 total sequence reads, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These organisms are both known ocular pathogens implicated in keratitis and endophthalmitis and are also well-documented endosymbionts of \u003cem\u003eAcanthamoeba\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMost common microbial species identified by weekly sequence reads\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSpecies\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eWeek 1\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWeek 2\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eWeek 3\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTotal Count\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9,157\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6,645\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3,023,231\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e3,039,033\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3,183\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e116,665\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1,453,163\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e1,573,011\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas\u003c/em\u003e (other)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27,769\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e27,569\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e453,121\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e508,459\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas otitidis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e317\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e342,846\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e343,163\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas mendocina\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4,229\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1,233\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e325,420\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e330,882\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e (other)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e14,235\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9,880\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e205,951\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e230,066\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas stutzeri\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e5,515\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e9,072\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57,959\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e72,546\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas entomophila\u003c/em\u003e L48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e441\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e63,535\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e63,976\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas fragi\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e18,328\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e676\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2,898\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e21,902\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas fluorescens\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2,612\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2,679\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9,605\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e14,896\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e225\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1,053\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e13,457\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e14,735\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003ePseudomonas koreensis\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e514\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e484\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5,985\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e6,983\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMycobacterium\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5,511\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e5,511\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eEscherichia coli\u003c/em\u003e O139:H28 str. E24377A\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e434\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3,884\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e4,318\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cem\u003eMycobacteriaceae\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e784\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e169\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3,304\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e\u003cb\u003e4,257\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eDespite the detection of several endosymbionts known from the literature to be associated with \u003cem\u003eAcanthamoeba\u003c/em\u003e, conventional PCR testing did not detect the presence of \u003cem\u003eAcanthamoeba\u003c/em\u003e DNA in any collected water samples. Thus, the association between detected microbial species and \u003cem\u003eAcanthamoeba\u003c/em\u003e is inferred from existing literature, as co-presence was not confirmed through PCR in our methodology.\u003c/p\u003e\u003cp\u003eWhen examining what coinfections with \u003cem\u003eAcanthamoeba\u003c/em\u003e the endosymbionts were associated with, 53% of the recovered species were linked to skin infections, 51% to eye infections, and 47% to respiratory illnesses. Notably, 43% were associated with both keratitis and endophthalmitis, highlighting their ophthalmic relevance. Additional findings revealed that 37% of species had previously been isolated from human feces, and 33% were associated with gastrointestinal disease. The distribution of disease associations by source is depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Supplementary Table S2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLittle River Pocket Park demonstrated the highest number of total disease associations (n\u0026thinsp;=\u0026thinsp;107), followed by Key Biscayne Crandon Park South (n\u0026thinsp;=\u0026thinsp;105), Purdy Kayak Launch, Maurice Gibb Park (n\u0026thinsp;=\u0026thinsp;97), and Parkview Kayak Launch (n\u0026thinsp;=\u0026thinsp;94). These findings suggest a correlation between microbial diversity and potential pathogenicity, particularly at locations with high human recreational activity.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study underscores the potential health risks posed by \u003cem\u003eAcanthamoeba\u003c/em\u003e-associated microbes in recreational waters, particularly in the eye. Nearly half of all species identified across sites were linked to eye infections, with 43% specifically associated with both keratitis and endophthalmitis. While \u003cem\u003eAcanthamoeba\u003c/em\u003e was not detected in our PCR samples, the presence of known ocular pathogens with potential to survive within protozoan hosts raises concerns about environmental vectors contributing to serious ocular complications.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\u003cp\u003eThe frequent detection of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e - both previously implicated in microbial keratitis and endophthalmitis - adds further concern.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e These organisms are known to survive intracellularly within \u003cem\u003eAcanthamoeba\u003c/em\u003e, which protects them from environmental stress and disinfection.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e This protective niche may enhance their persistence in aquatic environments and their virulence when introduced to the eye.\u003c/p\u003e\u003cp\u003eSites with high microbial diversity and ocular disease associations, such as Key Biscayne Crandon Park South and Maurice Gibb Park, represent areas of potential risk, particularly for contact lens wearers. Swimming while wearing lenses has been strongly linked to \u003cem\u003eAcanthamoeba\u003c/em\u003e keratitis and may also facilitate exposure to other ocular pathogens.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Although keratitis is the more frequently discussed outcome, post-infectious or secondary endophthalmitis remains a devastating, albeit rare, possibility with polymicrobial infections.\u003c/p\u003e\u003cp\u003eThe intracellular protection offered by protozoans and the potential synergy between co-resident microbes may contribute to chronic, recurrent, or treatment-resistant ocular infections.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Standard antimicrobial regimens may fail to adequately penetrate cyst walls or address secondary pathogens, resulting in poor visual outcomes. Understanding the diversity of these microbial communities, particularly their ocular relevance, is critical for early intervention and tailored management.\u003c/p\u003e\u003cp\u003eThis study also demonstrates the utility of metagenomic sequencing via the Oxford Nanopore MinION platform to rapidly identify microbial species in complex environmental samples.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e Long-read sequencing can detect co-infections and rare species missed by culture or short-read techniques, offering a promising tool for environmental surveillance in high-risk recreational settings.\u003c/p\u003e\u003cp\u003eLimitations include the short sampling duration, absence of environmental metadata (e.g., pH, salinity), inability to detect \u003cem\u003eAcanthamoeba\u003c/em\u003e through PCR, and the lack of direct clinical correlation. Future research should aim to connect environmental isolates to clinical cases of keratitis, endophthalmitis, other eye infections, and \u003cem\u003eAcanthamoeba\u003c/em\u003e infections in other body systems to confirm co-occurrence with protozoan hosts and to better understand the pathogenic potential of specific endosymbionts.\u003c/p\u003e\u003cp\u003eUltimately, this study highlights a substantial ocular risk associated with exposure to recreational waters. Public health strategies should target education for contact lens users, reinforce safe water practices, and incorporate targeted surveillance in high-risk aquatic environments to reduce the burden of sight-threatening infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e SK, ZY, and DM were involved in conceptualizing the study, analyzing the data, and interpreting the results. CL, TM, and AD were involved in collecting and sequencing the data. GA, HF, and DM were involved in overseeing the project. All authors contributed to the writing and review of the manuscript and have approved the final version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThe authors declare no funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Declaration:\u0026nbsp;\u003c/strong\u003eNone\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate/publish:\u0026nbsp;\u003c/strong\u003eNA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLorenzo-Morales, J. et al. Acanthamoeba keratitis: an emerging disease gathering importance worldwide? \u003cem\u003eTrends Parasitol.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e, 181\u0026ndash;187 (2013).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrancine, M. C. \u0026amp; Guy, C. Acanthamoeba spp. as Agents of Disease in Humans. \u003cem\u003eClin. Microbiol. Rev.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e, 273\u0026ndash;307 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKhan, N. A. \u0026amp; Acanthamoeba (eds) : Biology and Pathogenesis. (Second edition. Norfolk, England: Caister Academic Press, [2015] \u0026copy;2015).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSeal, D. V. Acanthamoeba keratitis update\u0026mdash;incidence, molecular epidemiology and new drugs for treatment. \u003cem\u003eEye\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 893\u0026ndash;905 (2003).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarnt, N., Minassian, D. C. \u0026amp; Dart, J. K. G. Acanthamoeba Keratitis Risk Factors for Daily Wear Contact Lens Users: A CaseControl Study. \u003cem\u003eOphthalmology\u003c/em\u003e \u003cb\u003e130\u003c/b\u003e, 48\u0026ndash;55 (2023).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGilbert, G. \u0026amp; Didier, R. Microorganisms Resistant to Free-Living Amoebae. \u003cem\u003eClin. Microbiol. Rev.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 413\u0026ndash;433 (2004).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCorsaro, D., Pages, G. S., Catalan, V., Loret, J. F. \u0026amp; Greub, G. Biodiversity of amoebae and amoeba-associated bacteria in water treatment plants. \u003cem\u003eInt. J. Hyg. Environ. Health\u003c/em\u003e. \u003cb\u003e213\u003c/b\u003e, 158\u0026ndash;166 (2010).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLinder Ewert, J. W. K. Free-living Amoebae Protecting Legionella in Water: The Tip of an Iceberg? \u003cem\u003eScand. J. Infect. Dis.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e, 383\u0026ndash;385 (1999).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eScheid, P. Relevance of free-living amoebae as hosts for phylogenetically diverse microorganisms. \u003cem\u003eParasitol. Res.\u003c/em\u003e \u003cb\u003e113\u003c/b\u003e, 2407\u0026ndash;2414 (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLetchumanan, V., Chan, K. G. \u0026amp; Lee, L. H. Vibrio parahaemolyticus: a review on the pathogenesis, prevalence, and advance molecular identification techniques. \u003cem\u003eFront Microbiol\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, (2014).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePang, Z., Raudonis, R., Glick, B. R., Lin, T. J. \u0026amp; Cheng, Z. Antibiotic resistance in Pseudomonas aeruginosa: mechanisms and alternative therapeutic strategies. \u003cem\u003eBiotechnol. Adv.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 177\u0026ndash;192 (2019).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMaycock, N. J. R. \u0026amp; Jayaswal, R. Update on Acanthamoeba Keratitis: Diagnosis, Treatment, and Outcomes. \u003cem\u003eCornea\u003c/em\u003e 35, (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJain, M., Olsen, H. E., Paten, B. \u0026amp; Akeson, M. The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community. \u003cem\u003eGenome Biol.\u003c/em\u003e \u003cb\u003e17\u003c/b\u003e, 239 (2016).\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiddiqui, R. \u0026amp; Khan, N. A. Biology and pathogenesis of Acanthamoeba. \u003cem\u003eParasit. Vectors\u003c/em\u003e. \u003cb\u003e5\u003c/b\u003e, 6 (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"ocular microbiology, acanthamoeba, endosymbionts, metagenomics","lastPublishedDoi":"10.21203/rs.3.rs-7554315/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7554315/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eAcanthamoeba\u003c/em\u003e, a free-living amoeba found in recreational waters, is both a pathogen and a host for microbial endosymbionts implicated in human infections, particularly in the eye. This study used metagenomic sequencing to characterize known endosymbionts associated with \u003cem\u003eAcanthamoeba\u003c/em\u003e infection in recreational water sites across Miami. Over a three-week sampling period, 51 unique microbial species were identified, with the highest diversity observed at sites with high human activity. \u003cem\u003eVibrio parahaemolyticus\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e - known ocular pathogens and \u003cem\u003eAcanthamoeba\u003c/em\u003e endosymbionts - were among the most abundant. Overall, 51% of species were associated with eye infections and 43% were linked to both keratitis and endophthalmitis. As well, 53% of the endosymbionts were associated with skin infections and 47% with respiratory infections. Sites with greater microbial diversity also demonstrated higher numbers of disease-associated species. The frequent detection of \u003cem\u003eAcanthamoeba\u003c/em\u003e endosymbionts in popular aquatic settings raises public health concerns, especially for contact lens users who may be at increased risk of ocular infections. Metagenomic surveillance of recreational waters may help inform prevention strategies and reduce the burden of vision-threatening and systemic infections.\u003c/p\u003e","manuscriptTitle":"Metagenomic study of acanthamoeba endosymbionts in recreational waters: role in ocular and systemic infections","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-03 15:27:28","doi":"10.21203/rs.3.rs-7554315/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9be29145-0427-4f9e-a02b-35bce11debf7","owner":[],"postedDate":"December 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":58919989,"name":"Health sciences/Diseases"},{"id":58919990,"name":"Biological sciences/Microbiology"}],"tags":[],"updatedAt":"2026-03-23T09:27:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-03 15:27:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7554315","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7554315","identity":"rs-7554315","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}