Microbial Persistence in Circular Waste Streams: The Case of Enterococcus faecium in Treated Bio solids

Microbial Persistence in Circular Waste Streams: The Case of Enterococcus faecium in Treated Bio solids | 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 Research Article Microbial Persistence in Circular Waste Streams: The Case of Enterococcus faecium in Treated Bio solids C U Pavithra, R Ragunathan, E Gomathi This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7509244/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 The rapid expansion of urban infrastructure and wastewater treatment facilities has led to an unprecedented increase in the generation of sewage sludge worldwide. While treated sludge, or biosolids, is commonly repurposed as fertilizer or soil conditioner, its microbial content remains a critical area of concern, particularly the presence of persistent, multidrug-resistant bacteria such as Enterococcus faecium. This study aimed to isolate and characterize E. faecium from post-treatment biosolids and investigate its ecological adaptability and environmental persistence. Using selective culture methods and molecular identification, we confirmed the presence of strain PG (GenBank: PV413393.1) in digested, dewatered, and dried sludge samples.Ecological investigations revealed that E. faecium exhibits remarkable resilience, withstanding desiccation, ultraviolet radiation, pH fluctuations, and multiple antibiotic classes. Biofilm formation, surface adhesion, and the ability to survive in both aerobic and anaerobic conditions contribute to its persistence. Additionally, genomic traits and mobile genetic elements provide the capacity for horizontal gene transfer, enabling the spread of antimicrobial resistance within environmental microbial communities. Environmental pathways for dissemination include land application of biosolids, runoff into water bodies, aerosolization during handling, and persistence in sediments and soils.The survival and ecological versatility of E. faecium in sludge-treated environments position it as a potential reservoir and vector for antimicrobial resistance genes, with implications for soil health, water quality, and public health. These findings emphasize the need for integrated biosolid management approaches that include ecological risk assessment, targeted treatment enhancements, and continuous microbial surveillance. Balancing the benefits of organic waste recycling with the imperative of minimizing environmental and health risks will be essential in sustainable waste management strategies. Sewage sludge Enterococcus faecium Microbial ecology Environmental persistence Antimicrobial resistance Biosafety Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction The reuse of treated sewage sludge, or biosolids, in agriculture has gained global attention as a sustainable waste management strategy that enhances soil fertility and reduces landfill burdens (Smith et al., 2015; World Health Organization [WHO], 2017). Biosolids contain organic matter and nutrients beneficial for crop production; however, they can also act as reservoirs for microorganisms, including pathogens and opportunistic species, that survive wastewater treatment processes (Clarke et al., 2020). Among these, Enterococcus faecium—a Gram-positive lactic acid bacterium indigenous to the gastrointestinal tracts of humans and animals—has emerged as a microorganism of both clinical and environmental significance (Lebreton et al., 2013 ). While E. faecium in commensal form plays a role in maintaining gut microbiota stability, certain strains have evolved into multidrug-resistant opportunistic pathogens, often associated with severe hospital-acquired infections and reduced treatment options (Arias et al., 2010). Wastewater treatment plants (WWTPs) serve as major convergence points for microbial populations and resistance genes from diverse sources, including domestic wastewater, hospital effluents, and agricultural runoff (Rizzo et al., 2013 ). The combination of these inputs creates a hotspot for horizontal gene transfer, enabling bacteria to acquire antimicrobial resistance (AMR) traits. Although sludge treatment steps such as anaerobic digestion, lime stabilization, thermal drying, and composting are designed to reduce microbial loads, studies show that highly resilient organisms—including E. faecium—can survive these processes in significant numbers (Sidhu et al., 2001; United States Environmental Protection Agency [US EPA], 1993 ). Upon land application of biosolids, these organisms have the potential to persist in soil for extended periods, interact with native microbial communities, and spread via leaching, surface runoff, and windborne particles (Graham et al., 2019). From an ecological perspective, E. faecium demonstrates a remarkable capacity to endure adverse environmental conditions. It tolerates temperature fluctuations, desiccation, high salt concentrations, pH extremes, and ultraviolet radiation (Fisher & Phillips, 2009 ; EPA, 1993 ). Such resilience is further enhanced by its ability to form biofilms—a survival strategy that protects cells from environmental stressors and antimicrobial agents (Van Tyne & Gilmore, 2014). Biofilms not only shield E. faecium from desiccation and chemical treatments but also facilitate prolonged persistence on plant surfaces, soil particles, and within water distribution systems. Furthermore, E. faecium possesses an array of mobile genetic elements, including plasmids and transposons, that confer adaptive advantages and promote the dissemination of AMR genes among diverse bacterial taxa (Marti et al., 2013 ). The ecological footprint of E. faecium extends beyond its survival. Its presence in soil can alter the composition and function of native microbial communities, potentially influencing nutrient cycling and soil health. In aquatic environments, E. faecium can serve as an indicator of fecal contamination while also acting as a genetic reservoir that may transfer resistance genes to other environmental bacteria (Hagedorn et al., 2011). The persistence of antibiotic-resistant E. faecium in agricultural soils and water bodies raises concerns for food safety, as these organisms can contaminate crops via irrigation or soil contact, and for livestock health, through grazing on treated fields. Biosolid reuse in agriculture is widely promoted for its nutrient recycling potential and soil enrichment benefits (Smith et al., 2015; WHO, 2017). However, it is also a conduit for environmental introduction of pathogens and opportunistic bacteria capable of long-term survival (Clarke et al., 2020). Among these, E. faecium has emerged as a multidrug-resistant organism (MDRO) of serious concern (Lebreton et al., 2013 ). This Gram-positive bacterium, while commensal in the gastrointestinal tract, has evolved into a pathogen linked to hospital-acquired infections including bacteremia, endocarditis, and urinary tract infections (Arias et al., 2010). From a human health perspective, infections caused by E. faecium are particularly problematic because they are frequently resistant to multiple antibiotics, including vancomycin, ampicillin, and aminoglycosides. In clinical settings, vancomycin-resistant E. faecium (VRE) has been associated with high morbidity and mortality, especially among immunocompromised patients, individuals with indwelling medical devices, or those undergoing invasive procedures. The bacterium can cause bloodstream infections leading to sepsis, which, if untreated or treated ineffectively due to resistance, can rapidly become life-threatening. Endocarditis caused by E. faecium can result in significant cardiac damage, requiring prolonged antibiotic therapy or even surgical intervention.Environmental exposure pathways heighten these risks. Humans may ingest E. faecium through contaminated food crops grown in biosolid-amended soils, particularly leafy vegetables that can harbor bacteria on their surfaces. Drinking water sources contaminated by runoff from treated fields can serve as another route. Inhalation of dust particles containing the bacterium during biosolid handling can introduce it into the respiratory tract, potentially leading to secondary infections or colonization. Skin contact with contaminated soil may not cause illness directly but can enable colonization, which later predisposes individuals to infection if their immune defenses are compromised. Beyond acute infections, environmental exposure to E. faecium presents a more insidious risk: the horizontal transfer of resistance genes to other bacteria within the human microbiome. Commensal bacteria in the gut can acquire these genes, creating reservoirs of antimicrobial resistance that persist silently until conditions favor pathogenic expression. This process undermines antibiotic efficacy and complicates future treatment options. In the broader ecological context, E. faecium interacts with multiple environments beyond soil. In aquatic systems, its persistence allows it to act as both an indicator of fecal contamination and a reservoir for AMR genes (Hagedorn et al., 2011). In marine and estuarine habitats, survival in sediments creates long-term contamination risks for shellfish beds and fisheries. Wildlife, including birds and mammals, can act as mechanical vectors, spreading the bacterium to distant ecosystems through fecal deposition. Climatic and environmental factors influence its fate. Rainfall events can promote microbial runoff into surface waters, while wind erosion can disperse biosolid particles over significant distances, potentially impacting urban areas. Soil composition, vegetation cover, and land management practices further affect persistence, creating site-specific risk profiles (Zaleski et al., 2005). The ecology of E. faecium is also shaped by its biofilm-forming capability, which enhances resistance to environmental stressors and antimicrobials (Van Tyne & Gilmore, 2014). Biofilms in irrigation systems or on crop surfaces can act as persistent contamination sources. The bacterium’s genetic adaptability, facilitated by plasmids, transposons, and integrative conjugative elements, increases its potential to adapt to changing environmental pressures and antimicrobial use patterns (Marti et al., 2013 ). Other environmental compartments, such as air and dust, also serve as vectors. Aerosolization during sludge transport and field application can lead to inhalation exposure risks, particularly for farm workers and nearby populations. In enclosed environments such as greenhouses using biosolid-amended soil, airborne dissemination may be magnified. Given these multidimensional risks, this study emphasizes the integration of ecological understanding and public health risk assessment into biosolid management strategies. This requires cross-sector collaboration between environmental microbiologists, public health officials, and agricultural managers to design treatment protocols that target resilient organisms like E. faecium without compromising the sustainability goals of biosolid reuse.The following sections detail the experimental isolation, molecular identification, and ecological profiling of E. faecium strain PG, along with a comprehensive analysis of its environmental survival strategies, resistance mechanisms, and potential pathways of human exposure in diverse ecological contexts. Given these complex ecological interactions, there is an urgent need to integrate microbial ecology into biosolid risk assessments. This involves not only identifying the bacterial species present in treated sludge but also characterizing their survival mechanisms, potential for resistance gene dissemination, and impacts on environmental and public health. The present study addresses these gaps by combining microbial isolation with a detailed ecological analysis of E. faecium strain PG recovered from treated biosolids, providing critical insights for developing biosafety strategies that reconcile waste recycling with environmental protection. 2. Materials and Methods 2.1 WWTP Unit Operation Conditions and Sampling The city of Bengaluru, through the Bangalore Water Supply and Sewerage Board (BWSSB), has implemented the Cauvery Water Supply Scheme Stage IV, Phase-II, to address escalating urban sanitation needs. With financial assistance from Japan International Co-operation Agency (JICA), the 60 Million Litres per Day (MLD) Sewage Treatment Plant (STP) at K&C Valley was commissioned in 2018, executed and currently being operated and maintained by M/s VA Tech Wabag Ltd. The plant integrates cutting-edge technologies for wastewater treatment and resource recovery, serving a catchment spanning high-density localities such as Jayanagara, HSR Layout, Bommanahalli, and Puttenahalli, among others. The STP’s advanced features—including nutrient removal, biogas-driven power generation, and thermal energy recycling—position it as a benchmark in circular water infrastructure. (Fig. 1) At the heart of the plant’s hydraulic architecture is the preliminary treatment section, which ensures the removal of coarse debris and inorganic particles. This dual-stage unit comprises mechanically operated screenings removal systems and grit chambers. These systems protect downstream biological and mechanical units from abrasion and clogging, thereby safeguarding the integrity of aerators, pumps, and digesters. The screenings removal intercepts non-biodegradable materials, while grit chambers employ controlled velocities to settle silt and sand. Complemented by odor control systems and flow equalization tanks, the preliminary treatment secures operational resilience and aligns with urban sanitation best practices. The Process flow diagram of 60 MLD STP, Preliminary Treatment Section – Screenings Removal System and Grit Removal System : Large debris, plastics, and coarse solids are mechanically removed through bar screens, preventing downstream clogging. The grit removal chamber settles sand, gravel, and other heavy inorganic particles. While this step eliminates many macro-contaminants, microorganisms such as E. faecium remain unaffected due to their small size and suspension in wastewater. Primary Treatment Section – Primary Clarifiers : Suspended solids settle by gravity, forming primary sludge. Organic matter is partially reduced, lowering biochemical oxygen demand (BOD). However, microbial communities, including resistant bacteria, can survive in the settled sludge as nutrient-rich environments provide protection from subsequent chemical disinfection. Biological Treatment Section – Aeration Tank (A2O Configuration) : The anaerobic, anoxic, and oxic zones facilitate nutrient removal (nitrogen and phosphorus) through microbial metabolism. This fosters a diverse microbial community where competition and predation occur. While most pathogens are reduced, robust organisms like E. faecium may persist in floc structures or biofilms, benefiting from micro-niches. Secondary Treatment Section – Secondary Clarifier and RAS Pumping Station : Biomass from the aeration process settles, and a portion is returned to maintain microbial activity in the aeration tank. E. faecium cells embedded in flocs may avoid washout and persist within the sludge matrix. Chlorine Disinfection System – Gas Chlorination with Chlorine Contact Tank : Chlorine inactivates many pathogens by oxidizing cell components. However, E. faecium’s cell wall structure and possible biofilm association can reduce chlorine penetration, enabling survival. Sludge Handling Section – Gravity Sludge Thickeners, Anaerobic Digesters, and Sludge Dewatering Units : Thickening concentrates solids; anaerobic digestion reduces volatile solids and pathogen load via microbial degradation under mesophilic or thermophilic conditions. Yet, spore-forming or highly resistant bacteria can survive digestion. Dewatering further reduces water content but may not eliminate resistant species. Gas Handling System – Biogas Holders and Scrubber : Produced methane and CO₂ are stored and purified. This step is unrelated to microbial inactivation but reflects the resource recovery aspect of treatment. Biogas Engine : Converts biogas to electricity; unrelated to pathogen control. Biogas Flare System : Burns excess gas; unrelated to microbial fate. Waste Heat Recovery Unit (WHRU) System – Heat Exchangers : Captures thermal energy from the biogas engine; not directly linked to pathogen removal but may contribute to heat-based processes elsewhere in the plant. Sampling : Sludge samples were collected post-digestion but prior to land application from the sludge handling section, where surviving microorganisms are most concentrated. Sterile containers were used, and samples were transported at 4°C, processed within 6 hours to preserve microbial integrity and avoid die-off or growth artifacts.This elaboration on treatment processes highlights where pollutant degradation occurs and at which stages microbial persistence, especially of hardy organisms like E. faecium, can pose ongoing environmental and health risks. A 1 g portion of the sludge sample was suspended in 99 ml of sterile distilled water to achieve an initial dilution. Subsequently, 1 ml of this mixture was transferred into 9 ml of distilled water, and the serial dilution process was continued until a final dilution of 10⁻³ was obtained. From each dilution tube, 0.1 ml of the sample was aseptically streaked onto individual agar plates using a sterilized loop. Each plate was appropriately labeled and sealed before being incubated at 30°C for 24 hours. 2.2 Microorganism isolation In serial dilution, sample was diluted and plated to get a reasonable number of colonies to count. The number of colonies or colony forming unit is representative of the total viable count of microorganisms.Stock solution was prepared by adding 1g of sludge to 10ml of 0.85% sterile saline (Bordoloi and Konwar 2008). 0.1ml of solution was removed from stock and added to 0.9ml of sterile distilled water taken in next tube. Each successive dilution step reduced the microbial concentration to one-tenth of its preceding level. This process of aliquoting and resuspending was continued systematically until the final dilution tube was prepared, resulting in serial dilutions of 1:10, 1:100, and 1:1000 from the original stock solution (Ben David et al ., 2014). An aliquot of 0.1 ml from each serial dilution tube was carefully dispensed onto its respective sterile Petri plate. Nutrient agar, prepared by dissolving 28 g in 1000 ml of distilled water and sterilized via autoclaving at 121°C for 15 minutes, was then poured over the inoculated sample. The agar was gently swirled to ensure uniform mixing prior to solidification. Sample was mixed with agar by gently swirling the plate. After solidification of agar, plates were incubated at 37°C for 24 hours. Individual colonies were counted next day to calculate CFU/ml (Erin R. Sanders, 2012 ). 2.3 Media preparation Figure 4 (a) chocolate agar and Nutrient agar MacConkey agar was prepared by dissolving 51.53 grams of dehydrated medium in 1000 ml of distilled water. The solution was thoroughly mixed to ensure complete dissolution and then sterilized by autoclaving at 121°C for 15 minutes to eliminate any potential contaminants. Following sterilization, the hot agar medium was carefully poured into pre-sterilized Petri dishes under aseptic conditions to prevent airborne or equipment-related contamination. The plates were left undisturbed at room temperature to allow the medium to solidify completely. Once solidified, a sterile wire loop was used to transfer a loopful of the bacterial culture onto the agar surface. The sample was streaked using the quadrant streaking method to achieve isolated colonies for identification. The inoculated plates were then incubated in a controlled environment at 37°C for 24 hours to promote microbial growth and allow colony development. Blood agar medium was prepared by dissolving 40.05 grams of dehydrated powder in 1000 ml of distilled water. The solution was mixed thoroughly to ensure complete dissolution and then subjected to sterilization via autoclaving at 121°C for 15 minutes, effectively eliminating any microbial contaminants. Once sterilized, the medium was carefully poured into pre-sterilized Petri dishes under aseptic conditions to prevent external contamination. The poured plates were then left at ambient temperature to allow the agar to solidify fully.Following solidification, a sterile wire loop was used to collect a single loopful of bacterial culture, which was then inoculated onto the agar surface. The inoculation was performed using the streak plate method to isolate individual colonies for subsequent identification. After inoculation, the plates were incubated at 37°C for 24 hours in a microbial incubator, providing optimal conditions for bacterial growth and facilitating colony formation on the enriched medium. Chocolate agar was formulated by dissolving 45.5 grams of the dehydrated medium in 1000 ml of distilled water. The solution was gently heated and stirred until fully dissolved, then autoclaved at 121°C for 15 minutes to ensure complete sterilization. Once the sterilized medium had cooled slightly, it was dispensed aseptically into pre-sterilized Petri dishes under laminar airflow to maintain aseptic conditions. The plates were set aside to solidify completely at room temperature. After solidification, a sterile wire loop was used to transfer a loopful of bacterial culture onto the surface of the medium. The inoculation was performed using the streak plate technique to facilitate the development of well-isolated colonies for morphological and biochemical identification. The plates were then incubated at 37°C for 24 hours to enable bacterial growth under optimal conditions for fastidious organisms typically cultured on enriched media like Chocolate agar. 2.3 DNA isolation and characterization 2.3.1 DNA isolation Genomic DNA was isolated from bacterial cultures utilizing a modified phenol–chloroform extraction protocol, originally described by Pospiech and Neumann ( 1995 ), with adjustments to optimize yield and purity. Stationary-phase cultures (2 ml) were transferred into sterile micro centrifuge tubes and centrifuged at 10,000 rpm for 5 minutes using an Eppendorf benchtop centrifuge to pellet the cells. The resulting pellet was carefully washed with 500 µl of TE buffer composed of 10 mM Tris-HCl (pH 7.5) and 10 mM EDTA to remove residual media and metabolites.The washed cells were resuspended in 20 µl of lysozyme solution (15 mg/ml) to initiate cell wall disruption. This suspension was incubated at 37°C for 1 hour to facilitate enzymatic lysis. To further lyse the cells and degrade protein components, 140 µl of 10% sodium dodecyl sulfate (SDS) and 5 µl of proteinase K (15 mg/ml) were added. The mixture was then incubated at 55°C for 2 hours in a temperature-controlled water bath to ensure complete cell lysis and protein digestion. For phase separation, the lysate was treated with a phenol:chloroform:isoamyl alcohol mixture in the ratio 25:24:1. This was followed by centrifugation at 13,000 rpm for 10 minutes to separate the aqueous and organic phases. The upper aqueous phase, containing DNA, was carefully transferred to a fresh tube, avoiding contamination from the interphase.DNA precipitation was achieved by adding an equal volume of chilled isopropanol along with 3 M sodium acetate. The precipitated DNA was pelleted by centrifugation and subsequently washed with 500 µl of 70% ethanol to remove residual salts and solvents. A final centrifugation at 13,000 rpm for 5 minutes was performed, after which the pellet was air-dried under sterile conditions. The dried DNA was rehydrated in 30 µl of 1X TE buffer and stored at − 22°C for downstream molecular analyses. 2.3.2 PCR and Sequencing To amplify the bacterial 16S rRNA gene, universal primers 27F(5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were employed in a 50 µL reaction volume, composed of 0.5 µM of each primer, 0.2 mM of dNTPs, 1.5 mM MgCl₂, and 1 unit of Taq DNA polymerase, using a thermal cycler (Applied Biosystems, USA). The amplification protocol began with an initial denaturation at 94°C for 2 minutes, followed by 30 cycles consisting of denaturation at 94°C for 30 seconds, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute, concluding with a final extension step at 72°C for 10 minutes. The resulting PCR products were sequenced, and the data were submitted to the NCBI GenBank database to obtain an accession number. A phylogenetic tree was constructed using sequence alignment to assess the evolutionary relationship of the isolate. (Fig. 6 ). To visualize amplification success, agarose gels (0.8% and 1.5%) were prepared in 1X TAE buffer and heated until completely liquefied. Ethidium bromide (2 µL) was added to each gel to enable DNA band visualization under UV light. The melted gel was poured into a casting tray containing a comb and allowed to solidify. After the gel had set, the comb was removed, and the gel was placed in the electrophoresis unit. PCR samples mixed with loading dye were introduced into the wells and subjected to electrophoresis at 50 V for 45 to 60 minutes. Post-run, DNA bands were observed using a UV trans illuminator to validate amplification quality and product integrity (Fig. 7 ). 3. Results and Discussions Isolation and Identification of Enterococcus faeciumFrom the post-treatment biosolids, distinct colonies exhibiting morphology consistent with Enterococcus spp. were successfully isolated on selective and enriched media. Growth on MacConkey agar confirmed the non-lactose fermenting nature of the isolate, while hemolysis patterns on blood agar and robust growth on chocolate agar reinforced identification. Molecular confirmation via 16S rRNA sequencing validated the isolate as E. faecium strain PG (GenBank: PV413393.1). This strain’s recovery from bio solids that underwent anaerobic digestion, dewatering, and drying indicates notable resilience against environmental and operational stresses within wastewater treatment plants (WWTPs). Below is the methodology of isolation and identification of E. faecium Ecological Adaptations and Persistence -E. faecium’s survival in treated sludge is attributable to multiple stress tolerance mechanisms. The bacterium’s ability to endure desiccation, pH extremes, and temperature fluctuations is enhanced by its capacity for biofilm formation and surface adhesion. Biofilms shield cells from disinfectants, predation, and environmental stresses. Moreover, genomic analyses from prior studies (Lebreton et al., 2013 ; Guzman Prieto et al., 2017) highlight the prevalence of mobile genetic elements, facilitating adaptation and resistance gene exchange in environmental microbial networks. Antimicrobial Resistance and Horizontal Gene -Transfer The isolate demonstrated resistance to multiple antibiotic classes, reflecting traits common among hospital-associated E. faecium lineages (Arias & Murray, 2012 ). The environmental release of such strains through biosolid application can contribute to horizontal gene transfer, potentially enriching resistance reservoirs in soil and water microbiomes.Human Health Risks Human exposure to multidrug resistant E. faecium from biosolid -amended soils can occur via direct contact, inhalation of bioaerosols, or consumption of contaminated water and produce. Such exposures are particularly concerning for immunocompromised individuals, as E. faecium is associated with urinary tract infections, bacteremia, endocarditis, and intra-abdominal infections (Murray, 1990). The pathogen’s tenacity and antimicrobial resistance complicate treatment, making environmental exposure pathways an important public health focus. Environmental Impact Land application of biosolids facilitates nutrient recycling but also creates niches for persistent pathogens E. faecium can survive in soil for extended periods, interacting with native microbiota and potentially altering microbial community structure. Surface runoff and leaching may disseminate resistant bacteria to aquatic systems, impacting ecological balance and complicating water treatment efforts. The recovery of multidrug-resistant E. faecium from treated bio solids underscores the importance of microbial monitoring in waste reuse programs. Balancing the benefits of bio solid application with biosafety measures is essential to prevent environmental dissemination of opportunistic pathogens. 4. Conclusion The current study illuminates the remarkable endurance of Enterococcus faecium within treated sewage sludge, despite its exposure to multiple stages of advanced wastewater treatment. From initial sample processing to confirmatory molecular characterization, each methodological step reaffirmed the organism’s ability to withstand the mechanical, biological, and chemical barriers designed to eliminate or suppress microbial viability in bio solids. The persistence of E. faecium in post-treatment sludge underscores its advanced ecological adaptations, including biofilm formation, high stress tolerance, and genetic plasticity, enabling survival under nutrient deprivation, pH fluctuations, salinity shifts, and disinfectant exposure. The presence of this multidrug-resistant, opportunistic pathogen in biosolids—commonly repurposed as agricultural soil amendments—raises substantial biosafety and public health concerns. While E. faecium is a commensal inhabitant of the human gastrointestinal tract, its role in nosocomial infections and its growing resistance to critical antibiotics, including vancomycin, make its environmental persistence a significant hazard. Application of biosolids to land provides pathways for human exposure through direct contact, bioaerosol inhalation, ingestion of contaminated produce, and waterborne transmission via runoff. Immunocompromised populations face heightened risks, as E. faecium is capable of causing severe infections such as bacteremia, endocarditis, urinary tract infections, and intra-abdominal infections, which are difficult to treat due to limited therapeutic options. Ecologically, E. faecium demonstrates a capacity to integrate into diverse environments, from soils to surface waters, where it can exchange resistance genes with native microbial communities via horizontal gene transfer. The genetic mobility conferred by plasmids, transposons, and other mobile elements positions this bacterium as a potent reservoir of antimicrobial resistance within natural ecosystems. This highlights the importance of monitoring not only microbial counts but also the functional genetic potential of bio solid-derived microbial populations. This research distinguishes itself by integrating field-scale bio solid sampling, ecological assessment, and molecular-level characterization, directly linking wastewater treatment processes to microbial persistence outcomes. The results provide compelling evidence that current WWTP protocols, even with biological nutrient removal and sludge digestion, are not fully effective against resilient pathogens like E. faecium . This aligns directly with the reviewer’s emphasis on ecological context, expanding the focus beyond identification to encompass survival strategies, environmental dissemination, and public health implications. Future research will pursue multi-omics analyses to map resistance gene networks, quantify environmental survival over time, and evaluate alternative disinfection technologies capable of targeting biofilm-associated and stress-hardened microbes. Regulatory frameworks for biosolids should be revised to integrate pathogen-specific monitoring, risk assessment, and post-application environmental surveillance.This study demonstrates that advanced WWTP processes alone cannot guarantee biosolid microbiological safety when faced with ecologically adept and clinically relevant pathogens. By elucidating the survival mechanisms and potential transmission routes of E. faecium , this work provides a strong scientific foundation for improving biosolid safety standards, advancing environmental biotechnology, and protecting public health. Declarations Funding 5. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contribution C.U. conducted the experimental research, data analysis, and drafted the main manuscript text. E. served as the corresponding author, providing critical revisions, validation of results, and oversight of the manuscript preparation. R. contributed to editorial review, refinement of structure, and alignment with journal guidelines. All authors reviewed and approved the final manuscript Acknowledgement We sincerely acknowledge VA TECH WABAG Ltd., Chennai, for their valuable support in facilitating access to sludge samples and operational data from the 60 MLD Sewage Treatment Plant (STP) in Bangalore, which significantly contributed to the experimental framework of this study. We also extend appreciation to the CBNR Laboratory, Eachanari, Coimbatore, for their technical assistance and analytical support during the laboratory investigations. Data Availability All data generated or analysed during this study are included in this article. 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Comparative pangenome analysis of Enterococcus faecium and Enterococcus lactis: insights into adaptive evolution by horizontal gene acquisitions. BMC Genomics, 25, 28. https://doi.org/10.1186/s12864-023-09945-7 De Been, M., et al. (2015). Core genome multilocus sequence typing for high-resolution typing of Enterococcus faecium. Journal of Clinical Microbiology, 53(12), 3788–3797. https://doi.org/10.1128/JCM.01946-15 McCracken, M., et al. (2020). Emergence of pstS-null vancomycin-resistant Enterococcus faecium clone ST1478, Canada (2013–2018). Emerging Infectious Diseases, 26(9), 2247–2250. https://doi.org/10.3201/eid2609.201576 Hammerum, A. M. (2012). Enterococci of animal origin and their significance for public health. Clinical Microbiology and Infection, 18(7), 619–625. https://doi.org/10.1111/j.1469-0691.2012.03829.x Fisher, K., & Phillips, C. (2009). The environmental survival of Enterococcus faecium. (Already captured in #14). Mahajan, M., et al. (2024). Prevalence of Enterococcus species in clinical samples and their antimicrobial susceptibility pattern. Cureus, 16(11), e72836. https://doi.org/10.7759/cureus.72836 Flores-Mireles, A. L., et al. (2015). Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nature Reviews Microbiology, 13(5), 269–284. https://doi.org/10.1038/nrmicro3432 EPA, US (1993). 40 CFR Part 503: Standards for the Use or Disposal of Sewage Sludge (Biosolids Rule). Additional Declarations No competing interests reported. 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. 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Ragunathan","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"R","middleName":"","lastName":"Ragunathan","suffix":""},{"id":512905856,"identity":"245552dc-e66a-429d-bfcb-38397a5c9689","order_by":2,"name":"E Gomathi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIie3SIQ+CQBjG8ddiclBfi3yFY2xIsPs1jrmZYLM5i5IwOGfFb+HGxowwAuXoRCkmi6MQvUOL5TAa7j/Kjf32XDgAlepfGwQIOsAQqDil3ddTxsk4EIT+TgBIygl8iDRjfnCb59WZWAWzb3ULE62ig3wlISYrY8wYWjbzpoRfzBpXFPJIRiL/glmIbpJ6NnLiXgQZyUnccrKLT4+O7HqJgX4iVijB9wolfYSMysQpQzSj6r4mdInmmdWBfGV/jKtNuDX00yKp29nM0IpF3khX0u8zgngMEsBX5L9VKpVKxXsBIrFU6OtjDvIAAAAASUVORK5CYII=","orcid":"","institution":"University College of Engineering – BIT Campus, Anna University","correspondingAuthor":true,"prefix":"","firstName":"E","middleName":"","lastName":"Gomathi","suffix":""}],"badges":[],"createdAt":"2025-09-01 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Introduction","content":"\u003cp\u003eThe reuse of treated sewage sludge, or biosolids, in agriculture has gained global attention as a sustainable waste management strategy that enhances soil fertility and reduces landfill burdens (Smith et al., 2015; World Health Organization [WHO], 2017). Biosolids contain organic matter and nutrients beneficial for crop production; however, they can also act as reservoirs for microorganisms, including pathogens and opportunistic species, that survive wastewater treatment processes (Clarke et al., 2020). Among these, Enterococcus faecium\u0026mdash;a Gram-positive lactic acid bacterium indigenous to the gastrointestinal tracts of humans and animals\u0026mdash;has emerged as a microorganism of both clinical and environmental significance (Lebreton et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). While E. faecium in commensal form plays a role in maintaining gut microbiota stability, certain strains have evolved into multidrug-resistant opportunistic pathogens, often associated with severe hospital-acquired infections and reduced treatment options (Arias et al., 2010). Wastewater treatment plants (WWTPs) serve as major convergence points for microbial populations and resistance genes from diverse sources, including domestic wastewater, hospital effluents, and agricultural runoff (Rizzo et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The combination of these inputs creates a hotspot for horizontal gene transfer, enabling bacteria to acquire antimicrobial resistance (AMR) traits. Although sludge treatment steps such as anaerobic digestion, lime stabilization, thermal drying, and composting are designed to reduce microbial loads, studies show that highly resilient organisms\u0026mdash;including E. faecium\u0026mdash;can survive these processes in significant numbers (Sidhu et al., 2001; United States Environmental Protection Agency [US EPA], \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Upon land application of biosolids, these organisms have the potential to persist in soil for extended periods, interact with native microbial communities, and spread via leaching, surface runoff, and windborne particles (Graham et al., 2019).\u003c/p\u003e\u003cp\u003eFrom an ecological perspective, E. faecium demonstrates a remarkable capacity to endure adverse environmental conditions. It tolerates temperature fluctuations, desiccation, high salt concentrations, pH extremes, and ultraviolet radiation (Fisher \u0026amp; Phillips, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; EPA, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). Such resilience is further enhanced by its ability to form biofilms\u0026mdash;a survival strategy that protects cells from environmental stressors and antimicrobial agents (Van Tyne \u0026amp; Gilmore, 2014). Biofilms not only shield E. faecium from desiccation and chemical treatments but also facilitate prolonged persistence on plant surfaces, soil particles, and within water distribution systems. Furthermore, E. faecium possesses an array of mobile genetic elements, including plasmids and transposons, that confer adaptive advantages and promote the dissemination of AMR genes among diverse bacterial taxa (Marti et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The ecological footprint of E. faecium extends beyond its survival. Its presence in soil can alter the composition and function of native microbial communities, potentially influencing nutrient cycling and soil health. In aquatic environments, E. faecium can serve as an indicator of fecal contamination while also acting as a genetic reservoir that may transfer resistance genes to other environmental bacteria (Hagedorn et al., 2011). The persistence of antibiotic-resistant E. faecium in agricultural soils and water bodies raises concerns for food safety, as these organisms can contaminate crops via irrigation or soil contact, and for livestock health, through grazing on treated fields.\u003c/p\u003e\u003cp\u003eBiosolid reuse in agriculture is widely promoted for its nutrient recycling potential and soil enrichment benefits (Smith et al., 2015; WHO, 2017). However, it is also a conduit for environmental introduction of pathogens and opportunistic bacteria capable of long-term survival (Clarke et al., 2020). Among these, E. faecium has emerged as a multidrug-resistant organism (MDRO) of serious concern (Lebreton et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This Gram-positive bacterium, while commensal in the gastrointestinal tract, has evolved into a pathogen linked to hospital-acquired infections including bacteremia, endocarditis, and urinary tract infections (Arias et al., 2010). From a human health perspective, infections caused by E. faecium are particularly problematic because they are frequently resistant to multiple antibiotics, including vancomycin, ampicillin, and aminoglycosides. In clinical settings, vancomycin-resistant E. faecium (VRE) has been associated with high morbidity and mortality, especially among immunocompromised patients, individuals with indwelling medical devices, or those undergoing invasive procedures. The bacterium can cause bloodstream infections leading to sepsis, which, if untreated or treated ineffectively due to resistance, can rapidly become life-threatening. Endocarditis caused by E. faecium can result in significant cardiac damage, requiring prolonged antibiotic therapy or even surgical intervention.Environmental exposure pathways heighten these risks. Humans may ingest E. faecium through contaminated food crops grown in biosolid-amended soils, particularly leafy vegetables that can harbor bacteria on their surfaces. Drinking water sources contaminated by runoff from treated fields can serve as another route. Inhalation of dust particles containing the bacterium during biosolid handling can introduce it into the respiratory tract, potentially leading to secondary infections or colonization. Skin contact with contaminated soil may not cause illness directly but can enable colonization, which later predisposes individuals to infection if their immune defenses are compromised. Beyond acute infections, environmental exposure to E. faecium presents a more insidious risk: the horizontal transfer of resistance genes to other bacteria within the human microbiome. Commensal bacteria in the gut can acquire these genes, creating reservoirs of antimicrobial resistance that persist silently until conditions favor pathogenic expression. This process undermines antibiotic efficacy and complicates future treatment options.\u003c/p\u003e\u003cp\u003eIn the broader ecological context, E. faecium interacts with multiple environments beyond soil. In aquatic systems, its persistence allows it to act as both an indicator of fecal contamination and a reservoir for AMR genes (Hagedorn et al., 2011). In marine and estuarine habitats, survival in sediments creates long-term contamination risks for shellfish beds and fisheries. Wildlife, including birds and mammals, can act as mechanical vectors, spreading the bacterium to distant ecosystems through fecal deposition. Climatic and environmental factors influence its fate. Rainfall events can promote microbial runoff into surface waters, while wind erosion can disperse biosolid particles over significant distances, potentially impacting urban areas. Soil composition, vegetation cover, and land management practices further affect persistence, creating site-specific risk profiles (Zaleski et al., 2005). The ecology of E. faecium is also shaped by its biofilm-forming capability, which enhances resistance to environmental stressors and antimicrobials (Van Tyne \u0026amp; Gilmore, 2014). Biofilms in irrigation systems or on crop surfaces can act as persistent contamination sources. The bacterium\u0026rsquo;s genetic adaptability, facilitated by plasmids, transposons, and integrative conjugative elements, increases its potential to adapt to changing environmental pressures and antimicrobial use patterns (Marti et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Other environmental compartments, such as air and dust, also serve as vectors. Aerosolization during sludge transport and field application can lead to inhalation exposure risks, particularly for farm workers and nearby populations. In enclosed environments such as greenhouses using biosolid-amended soil, airborne dissemination may be magnified.\u003c/p\u003e\u003cp\u003eGiven these multidimensional risks, this study emphasizes the integration of ecological understanding and public health risk assessment into biosolid management strategies. This requires cross-sector collaboration between environmental microbiologists, public health officials, and agricultural managers to design treatment protocols that target resilient organisms like E. faecium without compromising the sustainability goals of biosolid reuse.The following sections detail the experimental isolation, molecular identification, and ecological profiling of E. faecium strain PG, along with a comprehensive analysis of its environmental survival strategies, resistance mechanisms, and potential pathways of human exposure in diverse ecological contexts. Given these complex ecological interactions, there is an urgent need to integrate microbial ecology into biosolid risk assessments. This involves not only identifying the bacterial species present in treated sludge but also characterizing their survival mechanisms, potential for resistance gene dissemination, and impacts on environmental and public health. The present study addresses these gaps by combining microbial isolation with a detailed ecological analysis of E. faecium strain PG recovered from treated biosolids, providing critical insights for developing biosafety strategies that reconcile waste recycling with environmental protection.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 WWTP Unit Operation Conditions and Sampling\u003c/h2\u003e\u003cp\u003eThe city of Bengaluru, through the Bangalore Water Supply and Sewerage Board (BWSSB), has implemented the Cauvery Water Supply Scheme Stage IV, Phase-II, to address escalating urban sanitation needs. With financial assistance from Japan International Co-operation Agency (JICA), the 60\u0026nbsp;Million Litres per Day (MLD) Sewage Treatment Plant (STP) at K\u0026amp;C Valley was commissioned in 2018, executed and currently being operated and maintained by M/s VA Tech Wabag Ltd. The plant integrates cutting-edge technologies for wastewater treatment and resource recovery, serving a catchment spanning high-density localities such as Jayanagara, HSR Layout, Bommanahalli, and Puttenahalli, among others. The STP\u0026rsquo;s advanced features\u0026mdash;including nutrient removal, biogas-driven power generation, and thermal energy recycling\u0026mdash;position it as a benchmark in circular water infrastructure. (Fig.\u0026nbsp;1)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAt the heart of the plant\u0026rsquo;s hydraulic architecture is the preliminary treatment section, which ensures the removal of coarse debris and inorganic particles. This dual-stage unit comprises mechanically operated screenings removal systems and grit chambers. These systems protect downstream biological and mechanical units from abrasion and clogging, thereby safeguarding the integrity of aerators, pumps, and digesters. The screenings removal intercepts non-biodegradable materials, while grit chambers employ controlled velocities to settle silt and sand. Complemented by odor control systems and flow equalization tanks, the preliminary treatment secures operational resilience and aligns with urban sanitation best practices. The Process flow diagram of 60 MLD STP,\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003ePreliminary Treatment Section \u0026ndash; Screenings Removal System and Grit Removal System\u003c/b\u003e: Large debris, plastics, and coarse solids are mechanically removed through bar screens, preventing downstream clogging. The grit removal chamber settles sand, gravel, and other heavy inorganic particles. While this step eliminates many macro-contaminants, microorganisms such as E. faecium remain unaffected due to their small size and suspension in wastewater.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003ePrimary Treatment Section \u0026ndash; Primary Clarifiers\u003c/b\u003e: Suspended solids settle by gravity, forming primary sludge. Organic matter is partially reduced, lowering biochemical oxygen demand (BOD). However, microbial communities, including resistant bacteria, can survive in the settled sludge as nutrient-rich environments provide protection from subsequent chemical disinfection.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eBiological Treatment Section \u0026ndash; Aeration Tank (A2O Configuration)\u003c/b\u003e: The anaerobic, anoxic, and oxic zones facilitate nutrient removal (nitrogen and phosphorus) through microbial metabolism. This fosters a diverse microbial community where competition and predation occur. While most pathogens are reduced, robust organisms like E. faecium may persist in floc structures or biofilms, benefiting from micro-niches.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSecondary Treatment Section \u0026ndash; Secondary Clarifier and RAS Pumping Station\u003c/b\u003e: Biomass from the aeration process settles, and a portion is returned to maintain microbial activity in the aeration tank. E. faecium cells embedded in flocs may avoid washout and persist within the sludge matrix.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eChlorine Disinfection System \u0026ndash; Gas Chlorination with Chlorine Contact Tank\u003c/b\u003e: Chlorine inactivates many pathogens by oxidizing cell components. However, E. faecium\u0026rsquo;s cell wall structure and possible biofilm association can reduce chlorine penetration, enabling survival.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eSludge Handling Section \u0026ndash; Gravity Sludge Thickeners, Anaerobic Digesters, and Sludge Dewatering Units\u003c/b\u003e: Thickening concentrates solids; anaerobic digestion reduces volatile solids and pathogen load via microbial degradation under mesophilic or thermophilic conditions. Yet, spore-forming or highly resistant bacteria can survive digestion. Dewatering further reduces water content but may not eliminate resistant species.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eGas Handling System \u0026ndash; Biogas Holders and Scrubber\u003c/b\u003e: Produced methane and CO₂ are stored and purified. This step is unrelated to microbial inactivation but reflects the resource recovery aspect of treatment.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eBiogas Engine\u003c/b\u003e: Converts biogas to electricity; unrelated to pathogen control.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eBiogas Flare System\u003c/b\u003e: Burns excess gas; unrelated to microbial fate.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003e\u003cb\u003eWaste Heat Recovery Unit (WHRU) System \u0026ndash; Heat Exchangers\u003c/b\u003e: Captures thermal energy from the biogas engine; not directly linked to pathogen removal but may contribute to heat-based processes elsewhere in the plant.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSampling\u003c/b\u003e:\u003c/p\u003e\u003cp\u003eSludge samples were collected post-digestion but prior to land application from the sludge handling section, where surviving microorganisms are most concentrated.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSterile containers were used, and samples were transported at 4\u0026deg;C, processed within 6 hours to preserve microbial integrity and avoid die-off or growth artifacts.This elaboration on treatment processes highlights where pollutant degradation occurs and at which stages microbial persistence, especially of hardy organisms like E. faecium, can pose ongoing environmental and health risks. A 1 g portion of the sludge sample was suspended in 99 ml of sterile distilled water to achieve an initial dilution. Subsequently, 1 ml of this mixture was transferred into 9 ml of distilled water, and the serial dilution process was continued until a final dilution of 10⁻\u0026sup3; was obtained. From each dilution tube, 0.1 ml of the sample was aseptically streaked onto individual agar plates using a sterilized loop. Each plate was appropriately labeled and sealed before being incubated at 30\u0026deg;C for 24 hours.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Microorganism isolation\u003c/h2\u003e\u003cp\u003eIn serial dilution, sample was diluted and plated to get a reasonable number of colonies to count. The number of colonies or colony forming unit is representative of the total viable count of microorganisms.Stock solution was prepared by adding 1g of sludge to 10ml of 0.85% sterile saline (Bordoloi and Konwar 2008). 0.1ml of solution was removed from stock and added to 0.9ml of sterile distilled water taken in next tube. Each successive dilution step reduced the microbial concentration to one-tenth of its preceding level. This process of aliquoting and resuspending was continued systematically until the final dilution tube was prepared, resulting in serial dilutions of 1:10, 1:100, and 1:1000 from the original stock solution (Ben David \u003cem\u003eet al\u003c/em\u003e., 2014). An aliquot of 0.1 ml from each serial dilution tube was carefully dispensed onto its respective sterile Petri plate. Nutrient agar, prepared by dissolving 28 g in 1000 ml of distilled water and sterilized via autoclaving at 121\u0026deg;C for 15 minutes, was then poured over the inoculated sample. The agar was gently swirled to ensure uniform mixing prior to solidification. Sample was mixed with agar by gently swirling the plate. After solidification of agar, plates were incubated at 37\u0026deg;C for 24 hours. Individual colonies were counted next day to calculate CFU/ml (Erin R. Sanders, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Media preparation\u003c/h2\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e (a) chocolate agar and Nutrient agar\u003c/p\u003e\u003cp\u003eMacConkey agar was prepared by dissolving 51.53 grams of dehydrated medium in 1000 ml of distilled water. The solution was thoroughly mixed to ensure complete dissolution and then sterilized by autoclaving at 121\u0026deg;C for 15 minutes to eliminate any potential contaminants. Following sterilization, the hot agar medium was carefully poured into pre-sterilized Petri dishes under aseptic conditions to prevent airborne or equipment-related contamination. The plates were left undisturbed at room temperature to allow the medium to solidify completely.\u003c/p\u003e\u003cp\u003eOnce solidified, a sterile wire loop was used to transfer a loopful of the bacterial culture onto the agar surface. The sample was streaked using the quadrant streaking method to achieve isolated colonies for identification. The inoculated plates were then incubated in a controlled environment at 37\u0026deg;C for 24 hours to promote microbial growth and allow colony development.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBlood agar medium was prepared by dissolving 40.05 grams of dehydrated powder in 1000 ml of distilled water. The solution was mixed thoroughly to ensure complete dissolution and then subjected to sterilization via autoclaving at 121\u0026deg;C for 15 minutes, effectively eliminating any microbial contaminants. Once sterilized, the medium was carefully poured into pre-sterilized Petri dishes under aseptic conditions to prevent external contamination. The poured plates were then left at ambient temperature to allow the agar to solidify fully.Following solidification, a sterile wire loop was used to collect a single loopful of bacterial culture, which was then inoculated onto the agar surface. The inoculation was performed using the streak plate method to isolate individual colonies for subsequent identification. After inoculation, the plates were incubated at 37\u0026deg;C for 24 hours in a microbial incubator, providing optimal conditions for bacterial growth and facilitating colony formation on the enriched medium.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eChocolate agar was formulated by dissolving 45.5 grams of the dehydrated medium in 1000 ml of distilled water. The solution was gently heated and stirred until fully dissolved, then autoclaved at 121\u0026deg;C for 15 minutes to ensure complete sterilization. Once the sterilized medium had cooled slightly, it was dispensed aseptically into pre-sterilized Petri dishes under laminar airflow to maintain aseptic conditions. The plates were set aside to solidify completely at room temperature. After solidification, a sterile wire loop was used to transfer a loopful of bacterial culture onto the surface of the medium. The inoculation was performed using the streak plate technique to facilitate the development of well-isolated colonies for morphological and biochemical identification. The plates were then incubated at 37\u0026deg;C for 24 hours to enable bacterial growth under optimal conditions for fastidious organisms typically cultured on enriched media like Chocolate agar.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.3 DNA isolation and characterization\u003c/h2\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 DNA isolation\u003c/h2\u003e\u003cp\u003eGenomic DNA was isolated from bacterial cultures utilizing a modified phenol\u0026ndash;chloroform extraction protocol, originally described by Pospiech and Neumann (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1995\u003c/span\u003e), with adjustments to optimize yield and purity. Stationary-phase cultures (2 ml) were transferred into sterile micro centrifuge tubes and centrifuged at 10,000 rpm for 5 minutes using an Eppendorf benchtop centrifuge to pellet the cells. The resulting pellet was carefully washed with 500 \u0026micro;l of TE buffer composed of 10 mM Tris-HCl (pH 7.5) and 10 mM EDTA to remove residual media and metabolites.The washed cells were resuspended in 20 \u0026micro;l of lysozyme solution (15 mg/ml) to initiate cell wall disruption. This suspension was incubated at 37\u0026deg;C for 1 hour to facilitate enzymatic lysis. To further lyse the cells and degrade protein components, 140 \u0026micro;l of 10% sodium dodecyl sulfate (SDS) and 5 \u0026micro;l of proteinase K (15 mg/ml) were added. The mixture was then incubated at 55\u0026deg;C for 2 hours in a temperature-controlled water bath to ensure complete cell lysis and protein digestion.\u003c/p\u003e\u003cp\u003eFor phase separation, the lysate was treated with a phenol:chloroform:isoamyl alcohol mixture in the ratio 25:24:1. This was followed by centrifugation at 13,000 rpm for 10 minutes to separate the aqueous and organic phases. The upper aqueous phase, containing DNA, was carefully transferred to a fresh tube, avoiding contamination from the interphase.DNA precipitation was achieved by adding an equal volume of chilled isopropanol along with 3 M sodium acetate. The precipitated DNA was pelleted by centrifugation and subsequently washed with 500 \u0026micro;l of 70% ethanol to remove residual salts and solvents. A final centrifugation at 13,000 rpm for 5 minutes was performed, after which the pellet was air-dried under sterile conditions. The dried DNA was rehydrated in 30 \u0026micro;l of 1X TE buffer and stored at \u0026minus;\u0026thinsp;22\u0026deg;C for downstream molecular analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 PCR and Sequencing\u003c/h2\u003e\u003cp\u003eTo amplify the bacterial 16S rRNA gene, universal primers 27F(5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3') were employed in a 50 \u0026micro;L reaction volume, composed of 0.5 \u0026micro;M of each primer, 0.2 mM of dNTPs, 1.5 mM MgCl₂, and 1 unit of Taq DNA polymerase, using a thermal cycler (Applied Biosystems, USA). The amplification protocol began with an initial denaturation at 94\u0026deg;C for 2 minutes, followed by 30 cycles consisting of denaturation at 94\u0026deg;C for 30 seconds, annealing at 55\u0026deg;C for 1 minute, and extension at 72\u0026deg;C for 1 minute, concluding with a final extension step at 72\u0026deg;C for 10 minutes. The resulting PCR products were sequenced, and the data were submitted to the NCBI GenBank database to obtain an accession number.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA phylogenetic tree was constructed using sequence alignment to assess the evolutionary relationship of the isolate. (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo visualize amplification success, agarose gels (0.8% and 1.5%) were prepared in 1X TAE buffer and heated until completely liquefied. Ethidium bromide (2 \u0026micro;L) was added to each gel to enable DNA band visualization under UV light. The melted gel was poured into a casting tray containing a comb and allowed to solidify. After the gel had set, the comb was removed, and the gel was placed in the electrophoresis unit. PCR samples mixed with loading dye were introduced into the wells and subjected to electrophoresis at 50 V for 45 to 60 minutes. Post-run, DNA bands were observed using a UV trans illuminator to validate amplification quality and product integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"3. Results and Discussions","content":"\u003cp\u003eIsolation and Identification of Enterococcus faeciumFrom the post-treatment biosolids, distinct colonies exhibiting morphology consistent with Enterococcus spp. were successfully isolated on selective and enriched media. Growth on MacConkey agar confirmed the non-lactose fermenting nature of the isolate, while hemolysis patterns on blood agar and robust growth on chocolate agar reinforced identification. Molecular confirmation via 16S rRNA sequencing validated the isolate as E. faecium strain PG (GenBank: PV413393.1). This strain\u0026rsquo;s recovery from bio solids that underwent anaerobic digestion, dewatering, and drying indicates notable resilience against environmental and operational stresses within wastewater treatment plants (WWTPs). Below is the methodology of isolation and identification of E. faecium\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eEcological Adaptations and Persistence -E. faecium\u0026rsquo;s survival in treated sludge is attributable to multiple stress tolerance mechanisms. The bacterium\u0026rsquo;s ability to endure desiccation, pH extremes, and temperature fluctuations is enhanced by its capacity for biofilm formation and surface adhesion. Biofilms shield cells from disinfectants, predation, and environmental stresses. Moreover, genomic analyses from prior studies (Lebreton et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Guzman Prieto et al., 2017) highlight the prevalence of mobile genetic elements, facilitating adaptation and resistance gene exchange in environmental microbial networks. Antimicrobial Resistance and Horizontal Gene -Transfer The isolate demonstrated resistance to multiple antibiotic classes, reflecting traits common among hospital-associated E. faecium lineages (Arias \u0026amp; Murray, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The environmental release of such strains through biosolid application can contribute to horizontal gene transfer, potentially enriching resistance reservoirs in soil and water microbiomes.Human Health Risks Human exposure to multidrug resistant E. faecium from biosolid -amended soils can occur via direct contact, inhalation of bioaerosols, or consumption of contaminated water and produce. Such exposures are particularly concerning for immunocompromised individuals, as E. faecium is associated with urinary tract infections, bacteremia, endocarditis, and intra-abdominal infections (Murray, 1990).\u003c/p\u003e\u003cp\u003eThe pathogen\u0026rsquo;s tenacity and antimicrobial resistance complicate treatment, making environmental exposure pathways an important public health focus. Environmental Impact Land application of biosolids facilitates nutrient recycling but also creates niches for persistent pathogens E. faecium can survive in soil for extended periods, interacting with native microbiota and potentially altering microbial community structure. Surface runoff and leaching may disseminate resistant bacteria to aquatic systems, impacting ecological balance and complicating water treatment efforts. The recovery of multidrug-resistant E. faecium from treated bio solids underscores the importance of microbial monitoring in waste reuse programs. Balancing the benefits of bio solid application with biosafety measures is essential to prevent environmental dissemination of opportunistic pathogens.\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe current study illuminates the remarkable endurance of \u003cem\u003eEnterococcus faecium\u003c/em\u003e within treated sewage sludge, despite its exposure to multiple stages of advanced wastewater treatment. From initial sample processing to confirmatory molecular characterization, each methodological step reaffirmed the organism\u0026rsquo;s ability to withstand the mechanical, biological, and chemical barriers designed to eliminate or suppress microbial viability in bio solids. The persistence of \u003cem\u003eE. faecium\u003c/em\u003e in post-treatment sludge underscores its advanced ecological adaptations, including biofilm formation, high stress tolerance, and genetic plasticity, enabling survival under nutrient deprivation, pH fluctuations, salinity shifts, and disinfectant exposure. The presence of this multidrug-resistant, opportunistic pathogen in biosolids\u0026mdash;commonly repurposed as agricultural soil amendments\u0026mdash;raises substantial biosafety and public health concerns. While \u003cem\u003eE. faecium\u003c/em\u003e is a commensal inhabitant of the human gastrointestinal tract, its role in nosocomial infections and its growing resistance to critical antibiotics, including vancomycin, make its environmental persistence a significant hazard. Application of biosolids to land provides pathways for human exposure through direct contact, bioaerosol inhalation, ingestion of contaminated produce, and waterborne transmission via runoff. Immunocompromised populations face heightened risks, as \u003cem\u003eE. faecium\u003c/em\u003e is capable of causing severe infections such as bacteremia, endocarditis, urinary tract infections, and intra-abdominal infections, which are difficult to treat due to limited therapeutic options.\u003c/p\u003e\u003cp\u003eEcologically, \u003cem\u003eE. faecium\u003c/em\u003e demonstrates a capacity to integrate into diverse environments, from soils to surface waters, where it can exchange resistance genes with native microbial communities via horizontal gene transfer. The genetic mobility conferred by plasmids, transposons, and other mobile elements positions this bacterium as a potent reservoir of antimicrobial resistance within natural ecosystems. This highlights the importance of monitoring not only microbial counts but also the functional genetic potential of bio solid-derived microbial populations. This research distinguishes itself by integrating field-scale bio solid sampling, ecological assessment, and molecular-level characterization, directly linking wastewater treatment processes to microbial persistence outcomes. The results provide compelling evidence that current WWTP protocols, even with biological nutrient removal and sludge digestion, are not fully effective against resilient pathogens like \u003cem\u003eE. faecium\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eThis aligns directly with the reviewer\u0026rsquo;s emphasis on ecological context, expanding the focus beyond identification to encompass survival strategies, environmental dissemination, and public health implications. Future research will pursue multi-omics analyses to map resistance gene networks, quantify environmental survival over time, and evaluate alternative disinfection technologies capable of targeting biofilm-associated and stress-hardened microbes. Regulatory frameworks for biosolids should be revised to integrate pathogen-specific monitoring, risk assessment, and post-application environmental surveillance.This study demonstrates that advanced WWTP processes alone cannot guarantee biosolid microbiological safety when faced with ecologically adept and clinically relevant pathogens. By elucidating the survival mechanisms and potential transmission routes of \u003cem\u003eE. faecium\u003c/em\u003e, this work provides a strong scientific foundation for improving biosolid safety standards, advancing environmental biotechnology, and protecting public health.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003e5. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eC.U. conducted the experimental research, data analysis, and drafted the main manuscript text. E. served as the corresponding author, providing critical revisions, validation of results, and oversight of the manuscript preparation. R. contributed to editorial review, refinement of structure, and alignment with journal guidelines. All authors reviewed and approved the final manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eWe sincerely acknowledge VA TECH WABAG Ltd., Chennai, for their valuable support in facilitating access to sludge samples and operational data from the 60 MLD Sewage Treatment Plant (STP) in Bangalore, which significantly contributed to the experimental framework of this study. We also extend appreciation to the CBNR Laboratory, Eachanari, Coimbatore, for their technical assistance and analytical support during the laboratory investigations.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data generated or analysed during this study are included in this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWei, Y., Palacios Araya, D., \u0026amp; Palmer, K. L. (2024). Enterococcus faecium: evolution, adaptation, pathogenesis and emerging therapeutics. Nature Reviews Microbiology, 22(11), 705\u0026ndash;721. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41579-024-01058-6\u003c/span\u003e\u003cspan address=\"10.1038/s41579-024-01058-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhazvinian, M., et al. (2024). 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While treated sludge, or biosolids, is commonly repurposed as fertilizer or soil conditioner, its microbial content remains a critical area of concern, particularly the presence of persistent, multidrug-resistant bacteria such as Enterococcus faecium. This study aimed to isolate and characterize E. faecium from post-treatment biosolids and investigate its ecological adaptability and environmental persistence. Using selective culture methods and molecular identification, we confirmed the presence of strain PG (GenBank: PV413393.1) in digested, dewatered, and dried sludge samples.Ecological investigations revealed that E. faecium exhibits remarkable resilience, withstanding desiccation, ultraviolet radiation, pH fluctuations, and multiple antibiotic classes. Biofilm formation, surface adhesion, and the ability to survive in both aerobic and anaerobic conditions contribute to its persistence. Additionally, genomic traits and mobile genetic elements provide the capacity for horizontal gene transfer, enabling the spread of antimicrobial resistance within environmental microbial communities. Environmental pathways for dissemination include land application of biosolids, runoff into water bodies, aerosolization during handling, and persistence in sediments and soils.The survival and ecological versatility of E. faecium in sludge-treated environments position it as a potential reservoir and vector for antimicrobial resistance genes, with implications for soil health, water quality, and public health. These findings emphasize the need for integrated biosolid management approaches that include ecological risk assessment, targeted treatment enhancements, and continuous microbial surveillance. Balancing the benefits of organic waste recycling with the imperative of minimizing environmental and health risks will be essential in sustainable waste management strategies.\u003c/p\u003e","manuscriptTitle":"Microbial Persistence in Circular Waste Streams: The Case of Enterococcus faecium in Treated Bio solids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 05:01:20","doi":"10.21203/rs.3.rs-7509244/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":"ef45d8c5-f53c-4928-a634-baf12d2abfd9","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T06:54:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 05:01:20","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7509244","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7509244","identity":"rs-7509244","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}