Characterization of a NovelTequatrovirusPhage from Pristine Stretch of The Ganges River, India, in Reducing Bacterial Load from Sewage Water

26 Effective treatment of wastewater (WW) and its reuse is necessary to meet certain sustainable 27 development goals and a circular economy. Escherichia coli is one of the primary contaminants 28 in the WW, and its extra-intestinal occurrence poses a considerable threat under one health. 29 This study is the first report of a novel broad-spectrum phage (ф ERS-1) isolated from a pristine 30 stretch of the Ganges River in the biocontrol of E. coli , resistant to 3 rd-and 4 th generation 31 cephalosporins and aztreonam. This is the first report of a phage from the Tequatrovirus genus 32 to infect P. aeruginosa. The ф ERS-1 could reduce the abundance of E. coli cells by 8.22 log 10 33 CFU/mL ≤ 24 hrs. Additionally, /i3 ERS-1 disrupted the biofilm of E. coli with a reduction of 34 3.88 log10 CFU/mL. Further, /i3 ERS-1 could inhibit biofilm by multiple strains of E. coli and 35 multiple genera (E. coli, S. boydii, and P. aeruginosa). The phage /i3 ERS-1 reduced bacterial 36 c o un ts i n r a w W W by 2 l og10 CFU/mL and 4 log 10 CFU/mL reduction in coliform-enriched 37 WW in ≤ 24 hours. Overall, this study suggests that /i3 ERS-1 could be used as an effective 38 alternative to be combined with other treatments for improving the quality of WW disposal and 39 environmental health by reducing the bacterial load. 40 41 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 3 42 Graphical Abstract 43 44

Phages, Ganges, Biofilm, Wastewater, Coliform, Green approach 45 1. Introduction 46 The treatment and recycling of wastewater (WW) for non-potable purposes has been 47 recognized progressively as a sustainable alternative as it is a cost-effective option to tackle the 48 existing water scarcity crisis. Reusing domestic WW allows sustainable use of water resources 49 for agriculture or environmental benefits, contributing to the circular economy (Hernández-50 Crespo et al., 2022). Considering the load of inorganic and organic discharges in the sewage, 51 the conventional indicators include estimating the carbon pollutants and nitrogen and 52 phosphorus content. However, in recent times, biological indicators have been at the centre of 53 attention for sewage treatment and reuse (Al-Gheethi et al., 2018). 54 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 4 The raw (untreated) WW comprises various pathogenic bacteria, which include the 55 members of Enterobacter sp., Staphylococcus aureus, Klebsiella sp ., Acinetobacter sp., 56 Escherichia coli, Enterococcus sp., Proteus sp., Salmonella sp., Shigella sp., Pseudomonas 57 aeruginosa, and Citrobacter sp. Such microbial contamination in water has a detrimental effect 58 on the public health (Soliman et al., 2023). The key bacterial pathogens in sewage that cause 59 environmental and health problems are E. coli, Salmonella, Shigella, and Pseudomonas. The 60 contamination of these microbes in the environment occurs through intestinal and extra-61 intestinal routes (Jang et al., 2017). Therefore, biological indicators should be more focused on 62 reflecting the connotation of sewage recycling better and providing an answer to the current 63 water-environmental sanitation practices. The current WW treatment includes a combination of 64 physical, chemical, and biological methods for eliminating microbial contamination. (Qian et 65 al., 2022). However, the rise of antibiotic resistance in bacteria has considerably challenged the 66 treatment of microbial pollution in WW. Therefore, to tackle the existing antibiotic resistance 67 crisis, there is a need to safeguard human and animal health by shielding environmental health 68 through the ‘one health’ approach (Garvey, 2020). 69 E. coli members are an essential indicator in evaluating the extent of environmental 70 pollution. Additionally, E. coli has been identified as a key enteric pathogen responsible for 71 diarrhoeal disease due to its transmission through contaminated food, water, soil, surfaces, and 72 hands (Navab-Daneshmand et al., 2018). The fate of E. coli strains as commensals or pathogens 73 (expressing virulence factors) depends upon a complex balance between the host's status and 74 expression of the virulence determinants. Certain signature characteristics of pathogenic strains 75 of E. coli include adhesion, biofilm formation, toxin production, and evasion of host defense 76 mechanisms. The environmental transmission of E. coli and its pathotypes include animal 77 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 5 wastes, manure, WW, and sewage sludge exiled from WW treatment plants (Osi ń ska et al., 78 2023). Besides, the presence of E. coli in treated WW remains a considerable public health 79 concern due to the high expense of removal via traditional techniques employed in sewage 80 treatment plants. Therefore, to address the issue of AMR and control microbial contamination, 81 bacteriophages as green biocontrol agents have re-emerged (Soliman et al., 2023). 82 Recent studies have highlighted the extended ability of phages to control bacterial 83 populations in various fields of agriculture, aquaculture, biomedicine, the food industry, and 84 wastewater treatment. However, there are limited studies detailing the use of cocktail of phages 85 in their natural or engineered form to treat the WW for the reduction of bacterial load and the 86 elimination of waterborne pathogenic bacteria (Beheshti Maal et al., 2015; Bhargava et al., 87 2023; Grami et al., 2022; Jassim et al., 2016; Periasamy and Sundaram, 2013; Withey et al., 88 2005). Most of the phages are host-specific and facilitate the killing of their host through lysis, 89 followed by the release of the phage progeny. Moreover, phages, being abundant in nature, 90 make them ideal candidates for their use in a variety of applications spanning various domains 91 under one health canopy (Samson et al., 2024). However, a major limitation of the use of 92 phages in environmental settings is their narrow host range. Isolation of phages that could lyse 93 multiple hosts effectively is challenging. However, this constraint is overcome by combining 94 various monovalent phages as a cocktail or engineering naturally occurring phages to extend 95 their host spectra. Combining different phages may often lead to antagonistic outcomes and 96 cause a bacteriostatic effect instead of bactericidal ones (Zhou et al., 2022). 97 The Ganges River is India's national river and is well known for its unique properties of 98 ‘self-cleansing (non-putrefying) and special healing’ since the ancient past (Khairnar, 2016). 99 The waters of the River Ganges have a rich history of demonstrating antibacterial properties, 100 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 6 first studied by Ernst Hankin against Vibrio cholerae in 1896 (Hankin, 1896). However, a 101 detailed insight into these antibacterial aspects was provided by Félix d'Hérelle, who 102 discovered and termed the antagonist microbe of V. cholerae as ‘bacteriophage’ (D’Herelle, 103 1917). 104 It is well-known that despite tremendous anthropogenic activities and their related pollution 105 load, the Ganges can rejuvenate itself rapidly (Paul, 2017; Reddy and Dubey, 2019; Samson et 106 al., 2019; Zhang et al., 2018). This fact has also been witnessed recently during COVID-19 107 lockdown times with remarkably clean water even at the most polluted sites (Dutta et al., 108 2020). However, there are limited studies with this riverine system wherein novel virulent 109 phages were isolated from its waters against MDR strains of K. pneumoniae (Sundaramoorthy 110 et al., 2021) and P. aeruginosa (Rathor et al., 2022) . Our recent study on the sediments of the 111 river Ganges along its 1500 km has identified the repertoire of bacteriophages and their 112 associated host-phage functions against putative human, plant, and putrefying pathogens 113 (Samson et al., 2023). Therefore, this unique aquatic ecosystem provides an opportunity to 114 bioprospect its untapped and unique phage diversity for its potential applications. Given this, 115 the present study was initiated to explore the untapped phage diversity from the pristine stretch 116 of the river Ganges to isolate novel phages and explore their potential for use as biocontrol 117 agents against E. coli, facilitating improved environmental health. 118 2. Materials and Methods 119 2.1. Bacterial strain and antimicrobial susceptibility profile 120 Escherichia coli (ATCC 8739) was the bacterial host used in this study. The genomic 121 identity of the isolate was confirmed using the MinION Mk1C Nanopore sequencer (Oxford 122 Nanopore Technologies). The resistance profile of the isolate was ascertained with the VITEK 123 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 7 2 system (bioMérieux) using two different sets of antimicrobial susceptibility testing (AST) 124 cards, AST-N235 and AST-N281, having a defined set of parameters as recommended by the 125 Clinical and Laboratory Standards Institute (CLSI) (Supporting Information, E. 126 coli_AST_281). 127 2.2. Isolation, enrichment, and propagation of Escherichia coli phage ERS-1 128 Water-saturated sediment samples of the Ganges River from the Harshil (31°02'18.0"N 129 78°44'22.7" E) location were used as the source of phage isolation against the primary host E. 130 coli (ATCC 8739). A different approach was used for the isolation of bacteriophages. The 131 sediment samples (n=3, 50 gm) were vortexed at maximum speed for 30 minutes at room 132 temperature. The sediment was allowed to settle briefly while the sediment-laden water (~40 133 mL) was equilibrated with 10 mL of SM buffer for 1 hour in an incubator at 37 /i3 with a speed 134 of 180 rpm. The resulting mixture was removed and centrifuged (Eppendorf centrifuge, 5804 135 R) at 6000×g for 10 mins. The supernatant (~40 mL) was then passed through a combination of 136 0.45μ m 0.22 μ m of Polyether sulfone (PES) membrane-based syringe filters (Hi-Media), 137 respectively, and 10 mL of filtrate from each set of the syringe filters was used for enrichment. 138 A total of 5mL of double-strength broth of soybean-casein digest broth (SCDB) 139 (MH011-500G, Hi-Media) and 5/i3 mL of exponential phase culture of E. coli (host) was added 140 to 20 mL of the filtrate obtained from the previous step. The enrichment flasks were incubated 141 at 37°C for 24 h with shaking (180rpm). The presence of lytic phages in the samples was 142 confirmed by observing clear zones (plaques) against the bacterial lawn through the qualitative 143 spot assay. The quantitative enumeration of phages in the enriched lysate was done using the 144 soft agar overlay method. A single, well-isolated plaque was picked and suspended in SM 145 (Sodium-Magnesium) buffer (NaCl 5.8g/L, MgSO 4.7H2O 2g/L, Tris-HCl (pH7.4) 50mL, 146 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 8 autoclaved distilled water 950mL). The phage titer of the released phages from the single 147 plaque was determined in triplicates for over four times by making serial dilutions and infecting 148 the log-phase culture of E. coli, followed by double-layer agar plating and incubation at 37 °C 149 for 16-18 h. Purification and concentration of /i3 ERS-1 was carried out with Amicon® Ultra-150 15 centrifugal filter with Ultracel100 kDa membrane (Merck Millipore) (Sun et al., 2014). The 151 multiplicity of infection (MOI) (Kasman et al., 2002) and Poisson distribution for predicting 152 the probable number of infected cells in a population (P 0) (Abedon, 2023) were calculated 153 using the following formula: 154 155 156 A high titer phage stock of the purified phage lysate containing 1017 PFU/mL was made 157 using replicates of a total of 30 confluent lysis plates using replicates of a total of 30 confluent 158 lysis plates flooded with 7mL of SM buffer and incubated at 15 /i3 with shaking (100 rpm) for 4 159 hours followed by ultracentrifugation at 14000×g for 30 mins at 4/i3 and subsequent filtration of 160 the lysate with 0.2 μ m PES syringe filters (Bonilla and Barr, 2018). Enumeration of the high 161 titer phage stock was done using the double-layer agar overlay method. The purified high-titer 162 phage stock was stored at 4°C. 163 2.3. Characterization of Escherichia coli phage (/i1) ERS-1 164 2.3.1 Morphological features 165 The morphology of /i3 ERS-1 was visualized using Jeol JEM-F2100 high resolution-166 transmission electron microscope (HR-TEM) at 200 kV and imaged with a Xarosa emsis 167 camera coupled to the microscope. To obtain a detailed (3D) view of the phage morphology, 168 /g4666 /g1790 /g2777 /g4667 /g3404/g2778/g3398/g1805 /g2879/g1787/g1789/g1783 /g1787/g1789/g1783 /g3404 /g1788/g1821/g1813/g1802/g1805/g1818 /g1815/g1806 /g1816/g1812/g1801/g1817/g1821/g1805 /g1806/g1815/g1818/g1813/g1809/g1814/g1807 /g1821/g1814/g1809/g1820/g1819 /g4666/g1790/g1780/g1795/g4667//g1813/g1786/g3402 /g1788/g1821/g1813/g1802/g1805/g1818 /g1815/g1806 /g1777/g1815/g1812/g1815/g1814/g1825 /g1806/g1815/g1818/g1813/g1809/g1814/g1807 /g1821/g1814/g1809/g1820/g1819 /g4666/g1777/g1780/g1795/g4667//g1813/g1786 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 9 HR-TEM was used in STEM (Scanning Transmission Electron Microscopy) mode, enabling 169 scanning images at nanometer (nm) resolution. Briefly, 1 mL of the high titer stock of /i3 ERS-1 170 was centrifuged at 12,000 ×g for 30 mins, and the pellet was resuspended with 10 µL of SM 171 buffer. A total of 4 µL of the sample was placed on a lacey formvar/carbon, 200 mesh, copper 172 grid followed by staining with VitroEase™ Methylamine Tungstate Negative Stain (Thermo 173 Fisher Scientific), following the manufacturer's protocol. Morphological classification of phage 174 was done following the guidelines recommended by the International Committee on Taxonomy 175 of Viruses (ICTV) (Turner et al., 2023). 176 2.3.2 Phage adsorption rate and one-step-growth curve 177 To understand how fast phage virions can adsorb a target bacterial host cell, an 178 adsorption experiment was performed as described by (Heineman and Bull, 2007) with certain 179 variations. The host cell culture at the exponential phase was mixed with the fresh phage lysate 180 at an MOI of 0.1 and incubated at 37°C, 100 for 30 mins. After 5 minutes, an aliquot was 181 drawn and centrifuged to pellet the adsorbed fraction of the phage each time. At the same time, 182 the supernatant was filtered using a 0.2 μ m PES syringe filter to obtain the fraction of 183 unadsorbed phages. The fractions were plated using the agar overlay method to get total phage 184 (N total) and free phage (N free) densities, respectively. The assay was carried out in three 185 replicates, and the adsorption curve as a function of the percentage of adsorbed and unadsorbed 186 phages versus time was plotted using GraphPad Prism v9.5.1. The adsorption rate ( α ) for each 187 time point was calculated as: 188 189 One one-step growth curve assay for /i3 ERS-1 was carried out as described by (Jagdale 190 et al., 2019) with few modifications. Briefly, fresh phage lysate of ERS-1 at a MOI of 0.1 was 191 /g4666 /g2745 /g4667 /g3404/g1812 /g1814 /g4666 /g1788/g1820/g1815/g1820/g1801/g1812 /g1788/g1806/g1818/g1805/g1805 /g4667//g1801/g1804/g1819/g1815/g1818/g1816/g1820/g1809/g1815/g1814 /g1820/g1809/g1813/g1805 /g4666/g1820/g4667 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 10 added to the E. coli cells (108 cells/mL) and allowed to adsorb for 20 mins at 37°C, 100 /i3 rpm. 192 The adsorbed phages were centrifuged at 10,000 ×g for 5 min, and subsequently, the bacteria-193 phage complex (pellet) was resuspended in 2 /i3 mL of SCDB and incubated at 37°C, 100 /i3 rpm 194 for 100 mins. A 100 µL aliquot was collected every 10 /i3 minutes, followed by plaque assay to 195 determine the phage count (PFU/mL) at each time interval. The assay was carried out in 196 triplicates. 197 2.3.3 Evaluation of pH and temperature stability 198 The stability of /i3 ERS-1 under different acidic and basic conditions was assessed as 199 described by (Oliveira et al., 2020). Briefly, the pH of the SM buffer was adjusted to 3, 5, 7, 9, 200 and 11, followed by adding 100 μ L of the phage suspension (10 17 PFU/mL) to 900 μ L of SM 201 buffer with respective pH. The samples were incubated for four hours at 37°C, 100 rpm in a dry 202 bath, followed by plaque assay. For determining the thermal stability, suspensions of 100 μ L of 203 the phage (1017 PFU/mL) in 900 μ L of SM buffer were made and incubated at a diverse set of 204 temperatures (4, 15, 25, 37, 45, 55, and 65°C) for four hours at 100 rpm followed by measuring 205 the phage titer with plaque assay. The assays for pH and thermal stabi lity of /i3 ERS-1 were 206 carried out at two independent times in triplicates. GraphPad Prism v9.5.1 was used to compute 207 the statistical significance of the results using repeated measures (RM) one-way analysis of 208 variance (ANOVA). Add itionally, multiple comparisons with a false discovery rate (FDR 209 correction) and a p-value of 0.05 were used to compare the mean between the two groups. 210 2.3.4 Evaluation of the Lytic Spectrum 211 The host range of /i3 ERS-1 was evaluated using in-house bacterial host cultures of 212 Escherichia coli O157:H7 (ATCC 43888), Shigella boydii (ATCC 9207, 8700) , Pseudomonas 213 aeruginosa (ATCC 9027), Salmonella enterica (ATCC 12011, 13314), Staphylococcus aureus 214 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 11 (ATCC 6538), Listeria monocytogenes (ATCC 19112) , Enterococcus faecalis (ATCC 19443) 215 Yersinia enterocolitica (ATCC 27729). Details of the susceptibility profile of each host strain 216 with phage ERS-1 have been tabulated (Table S1). 217 2.3.5 DNA extraction, Genome Sequencing, and Analysis of /i1 ERS-1 218 A high-titer phage stock was used to isolate DNA from phage ERS-1. The DNA was 219 extracted using PureLink Viral RNA/DNA Mini Kit (Thermo Fisher Scientific), following the 220 manufacturer's protocol. A 16S rRNA gene amplification was done to ascertain that the 221 extracted DNA was devoid of host DNA. The PCR products were examined on 0.8% agarose 222 gel electrophoresis along with controls and standard molecular weight markers. The gel was 223 visualized using the BioEra Gel Documentation System (Fig S1). 224 The phage DNA concentration was quantified on a Qubit 4 Fluorometer (Invitrogen) 225 using a dsDNA HS (High Sensitivity) assay kit fluorometer (Invitrogen). Library preparation of 226 the samples for whole genome sequencing was carried out with Ligation Sequencing Kit (SQK-227 LSK114) and Native Barcoding Kit (SQK-NBD 114.24) as per the manufacturer's instructions 228 with certain modifications (Text S1). The library was loaded onto flow cell R10.4.1(Oxford 229 Nanopore Technologies), and the sequencing run was carried out for ~56 hours using the 230 MinION Mk1C sequencing platform. 231 The reads were processed from the sequencing data for quality control and adapter 232 trimming using FastQC (Galaxy Version 0.25.1+Galaxy0) and Porechop (Galaxy Version 233 0.2.4+galaxy0). The genome assembly was performed using Flye, a de novo genome assembler 234 v 2.9.1. Validation of sequence homology for the generated assembly with known phage 235 sequences was done with NCBI nucleotide BLAST(BLASTN). Further, the phage genome 236 annotation of the putative proteins was predicted with Rapid Annotation using the Subsystem 237 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 12 Technology (RAST) annotation server ( https://rast.nmpdr.org). The GenBank file generated 238 from RAST was used in Proksee for an in-depth analysis and visualization of the phage 239 genome (Grant et al., 2023). 240 Additionally, an open reading frame (ORF) finder from NCBI 241 (https://www.ncbi.nlm.nih.gov/orffinder/) was used to identify the protein-coding sequences in 242 the phage genome. Further, VipTree was used to understand genome-wide similarities between 243 /i3 ERS-1 and other reference viral genomes (Nishimura et al., 2017). Whole genome Average 244 nucleotide identity (ANI) to ascertain genetic relatedness between ERS-1 and other closely 245 related phages was computed with OAT v0.93.1 (Orthologous Average Identity Tool) software 246 (Lee et al., 2016). PhaBOX, a comprehensive web tool for phage identification, taxonomic 247 classification, and prediction of phage lifestyle and its host, was used for an integrated phage 248 analysis. Furthermore, intergenomic comparisons were made to ascertain relatedness between 249 the phages using Virus Intergenomic Distance Calculator (VIRIDIC) (Moraru et al., 2020). 250 2.4. Antibiofilm Potentials of /i1ERS-1 251 2.4.1 Quantification and Visualization of Biofilm Inhibition Spectrum of /i1 ERS-1 252 A mixed culture biofilm inhibition assay was performed to understand /i3 ERS-1's effect 253 in inhibiting broad-spectrum biofilm. In this experiment, the biofilm from mixed cultures was 254 formed in a 35 mm treated tissue culture dish (Hi-Media) that supports cell adherence as 255 described previously with certain modifications (Duarte et al., 2021). The assay was divided 256 into four sets, each having five replicates of control and treated groups as follows: 257 Set I (Single species biofilm of Gram-negative bacterium- E. coli) 258 Set II (Multi-strains biofilm of Gram-negative bacteria- E. coli) (ATCC 8739, 25922, 43888) 259 Set III (Multi-genera biofilm of Gram-negative bacteria- E. coli, S. boydii, P. aeruginosa) 260 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 13 Set IV (Single species biofilm of Gram-positive bacterium- S. aureus) 261 The overnight grown cultures of the above bacteria were diluted in SCDB (1×10 8 CFU/mL) 262 and inoculated in a tissue culture dish. For each set of the treated group, 0.1 MOI of /i3 ERS-1 263 was immediately added, while the control group was devoid of the phage treatment. Three 264 replicates of each set were incubated for 24 h at 37 /i3 . Subsequently, the biofilms were washed 265 with 1X-phosphate-buffered saline (PBS), pH 7.4 (Hi Media) to remove the planktonic cells, 266 followed by quantifying three of the replicates crystal violet (CV) assay as described by 267 (Matysik and Kline, 2019). Briefly, the biofilms were stained with 0.1% CV for 15 mins and 268 air-dried. The dye was solubilized with acetic acid 33% (v/v), and 200 µL aliquots were 269 transferred from each replicate into a 96-well microtiter plate (Tarsons) followed by measuring 270 the absorbance at 595 nm with BioTek Synergy H1 microplate reader (Agilent Technologies). 271 The microscopic examination of the biofilm was done under 100× using an OLYMPUS optical 272 microscope U-CMAD3 T7 coupled with Lumenera Infinity 1 camera. GraphPad Prism v9.5.1 273 was used to compute the statistical significance of the results using RM-one-way ANOVA with 274 multiple comparisons (FDR correction) and a p-value of 0.05. 275 2.4.2 Time-dependent evaluation of cell viability of E. coli biofilm 276 In-vitro efficacy of /i3 ERS-1 was assessed against E. coli ATCC 8739 in biofilm 277 control through inhibition and disruption assays using Film Tracer™ LIVE/DEAD® Biofilm 278 Viability kit (Invitrogen). The experiment was carried out as described by (Mulani et al., 2022) 279 and divided into two sets as follows: 280 Set I: Biofilm Inhibition ( /i3 ERS-1 treatment at 0 hr. followed by imaging the replicates at 6, 281 12, and 24 hrs.). 282 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 14 Set II: Biofilm Disruption (/i3 ERS-1 treatment after 24 hrs. on pre-formed biofilm and imaging 283 the replicates at 6, 12, and 24 hrs.). 284 For biofilm inhibition, the overnight grown host culture ( E. coli) cells were diluted in SCDB 285 (1×108) and inoculated in a tissue culture dish, followed by the addition of /i3 ERS-1 at 0.1 286 MOI. CFU was determined after each time point of 6, 12, and 24 hours. 287 For biofilm disruption, the overnight grown host culture ( E. coli) cells were diluted in SCDB 288 (1×108), inoculated in a tissue culture dish, and allowed to form biofilm for 24 hours. The 289 planktonic cells were washed, and /i3 ERS-1 was added to the pre-formed biofilm at 0.1 MOI. 290 Untreated cells of E. coli served as culture control. CFU was determined after each time point 291 of 6, 12, and 24 hours. Statistical analysis of the results was performed in GraphPad Prism 292 v9.5.1 using RM-one-way ANOVA with multiple comparisons and a p-value of 0.05. 293 For each time point, three replicates were used. The cells were washed with 1X-PBS (Hi 294 Media) and stained with a mixture of SYTO ® 9 and propidium iodide (PI) stains from Film 295 Tracer™ LIVE/DEAD® Biofilm Viability kit following manufacturers protocol. The stained 296 dishes were visualized with an inverted confocal laser scanning microscope (CLSM) (Leica 297 Stellaris 5, DMi8) using a 20× objective. The fluorescence from live and dead bacteria was 298 visualized using excitation wavelengths of 488 nm (SYTO ® 9) and 588 nm (PI), respectively. 299 Additionally, to understand the effect of phage treatment on the biofilm morphology, the 300 control and 24 h sample of disruption were imaged using Field emission scanning electron 301 microscopy (FESEM, Nova Nano SEM 450). 302 2.5. Bacteriophage-based biocontrol for reduction of bacterial counts from wastewater 303 2.5.1. Sample collection and details of the sampling site 304 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 15 Phytorid sewage treatment plant (STP) (18˚ 32.8246 '/i3 N, 73˚ 48.8338 '/i3 E) situated at 305 CSIR- National Chemical Laboratory, India, with a capacity of 0.15 million liters per day 306 (MLD) was chosen for sampling of untreated wastewater (Fig. S2). A total of 1 L (n=3) of 307 untreated wastewater samples were collected in a sterile bottle by grab-sampling method. The 308 samples were brought immediately to the lab and processed for experimental setup. 309 2.5.2. Evaluation of bacterial biocontrol by /i1 ERS-1 in untreated wastewater 310 The raw sewage (untreated wastewater) samples from Phytorid STP were divided into test 311 and control groups. For the test group (100 mL aliquot of the raw sewage was challenged with 312 1 mL of high titer phage stock of /i3 ERS-1(1017 PFU/mL), while the control group was devoid 313 of any treatment. The experiment was carried out in triplicates, and the flasks from each group 314 were incubated at 37˚C for 24 hours, followed by recording the results as CFU/mL, while the 315 reduction in the bacterial counts was calculated in the form of log reduction and percent 316 reduction as described previously (Bashir et al., 2022). 317 2.5.3. Time kill assay for evaluation of bacterial biocontrol by /i1 ERS-1 at lab-scale 318 A time-kill assay was performed to harness the broad-spectrum potentials of /i3 ERS-1 in 319 improving the reusability of wastewater and associated environmental health. The experimental 320 setup comprised of two sets as follows: 321 Set I: Unenriched group (Test: 100 mL raw sewage +1 mL /i3 ERS-1), (Control: 100 mL raw 322 sewage). 323 Set II: Coliform enriched group (Test: 100 mL raw sewage +10 mL Mc Conkey broth + 1 mL 324 /i3 ERS-1), (Control: 100 mL raw sewage + 10 mL Mc Conkey broth). 325 The experiment was performed in three replicates, and aliquots from each set were drawn at 0, 326 6, 12, and 24 hours, followed by serial dilutions of the aliquots and plating onto selective media 327 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 16 of Brilliant Green Bile Lactose Broth agar (BGLB-A) (Hi Media), and Eosin-methylene blue 328 agar (EMB-A). For each time point, the bacterial count was determined as CFU/mL, and the 329 efficacy of /i3 ERS-1 in reducing the bacterial counts was calculated through log reduction and 330 (%) reduction as described by (Bashir et al., 2022). Additionally, at each time point, live-dead 331 cell staining of the aliquots was carried out as described above and visualized under 20× 332

using CLSM. Statistical analysis of the results was performed in GraphPad Prism 333 v9.5.1 using two-way ANOVA with multiple comparisons (FDR correction) and a p-value of 334 0.05 for statistical significance. 335 3. Results and Discussion 336 3.1 Isolation, identification, and growth parameters of /i1 ERS-1 337 Amongst the 15 isolated phages, Escherichia phage (/i3 ) ERS-1 showed a broad-spectrum 338 lytic activity against E. coli, P. aeruginosa, S. boydii, Y. enterocolitica and partial lysis with 339 cloudy plaques for S. aureus, and therefore, was selected for detailed characterization in this 340 study. The host range of /i3 ERS-1 has been tabulated (Table S1). 341 The antimicrobial resistant profile of E. coli (ATCC 8739) assessed through VITEK-2 342 suggested its resistance toward third and fourth-generation cephalosporins (3GC, 4GC) and 343 beta-lactam class of antibiotics, namely ceftazidime, cefepime, and Aztreonam (Supporting 344 information_ E. coli_AST281) . The World Health Organization (WHO) declared 3GCs and 345 4GCs as antimicrobials of critical importance for human and animal health in 2019. However, 346 due to an increase in the prevalence of plasmid-encoded β -lactamases in E. coli, members of 347 this group have become resistant to these crucial antimicrobials (Kang et al., 2022). 348 The genome annotation of E. coli (ATCC 8739) showed the presence of β -lactamase 349 enzymes (EC 3.5.6.2) responsible for multidrug resistance toward extended-spectrum beta-350 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 17 lactam antibiotics such as penicillins, cephalosporins, and aztreonam. Additionally, type 1 351 fimbriae are among the essential virulence factors in pathogenic E. coli strains and are known 352 to promote their survival by antibiotic evasion. Besides, this strain was also found to possess 353 resistance mechanisms (enzymes and efflux pumps) for multiple antibiotics (Table S2) . 354 Therefore, the isolation of phage for improved management of E. coli and control of resistance 355 transfer for 3, 4 GCs, and multidrugs highlights the importance of this study. 356 The plaque morphology of /i3 ERS-1 with this host comprised clear plaques encircled in a 357 translucent halo (Fig 1A). This type of plaque morphology could be attributed to the 358 occurrence of polysaccharide depolymerases in either the structural proteins or tail fibers of /i3 359 ERS-1. These enzymes are non-lytic and are known to cleave the extracellular polysaccharides 360 of bacteria, reducing their virulence (Rice et al., 2021). This preliminary observation suggested 361 that /i3 ERS-1 could have potentials antibiofilm activity. 362 A detailed morphological characterization of the purified high titer stock of /i3 ERS-1 was 363 carried out with HR-TEM and STEM, which revealed typical "T4- like" features of myovirus. 364 The morphological architecture of /i3 ERS-1 comprised of a prolate head (length, 114 ± 5 nm; 365 width, 66/i3 ±/i3 3 nm) showing an icosahedral symmetry, a collar with whiskers, a contractile tail 366 (length, 90/i3 ±/i3 10 nm; width, 15 /i3 ±/i3 2 nm), a small baseplate with short spikes, and six long 367 terminal (tail) fibers (Fig 1B) . Based on the above morphological features, /i3 ERS-1 was 368 classified into the class Caudoviricetes and family Straboviridae, as suggested by the recent 369 taxonomic update of ICTV (Turner et al., 2023). 370 The titer of /i3 ERS-1 was determined as 4.8×10 17 PFU/mL. An MOI of five or greater 371 indicates the host cell being infected by more than one phage particle, thereby portraying the 372 virulent ability of phage to set up a productive infection within the susceptible host (Abedon, 373 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 18 2023). The MOI for /i3 ERS-1 was calculated as 8.8, which could achieve a bacterial clearance 374 of 99.98%, with a probability of infected cells being P 0=0.998. This indicates the virulent 375 nature of /i3 ERS-1 and its capability of mounting a productive infection in E. coli (ATCC 376 8739) cells. 377 The binding of the phage to specific receptors on the bacterial cell surface initiates 378 bacteriophage infection of the susceptible host. This event is termed phage adsorption, an 379 essential parameter in deciding the application of a bacteriophage (Ge et al., 2020). The 380 percentage of adsorbed fraction of /i3 ERS-1 particles was 50% up to 5 mins, which increased 381 to >90% by 20 mins. Additionally, the percentage of free phages in the unadsorbed fraction 382 decreased rapidly at 5 mins (~70%), and only ~10 % of free phages were observed from 20-30 383 mins (Fig 1C). Details of the adsorption rate have been summarised (Table S3). 384 The fundamental nature of replication of /i3 ERS-1 in E. coli (ATCC 8739) was determined 385 with one step growth experiment. This enables us to understand the duration of the different 386 phases in the lifecycle and the burst size (yield of the viral cycle) (Adams and Wassermann, 387 1956). Phage ERS-1 showed a latent period of 20 mins and a burst size of 45 (±5) phage 388 particles per infected cell of E. coli (host) (Fig 1D). 389 Various external factors are known to affect phage persistence. However, according to 390 Ackermann, tailed phages tend to be most stable under adverse external factors. The 391 temperature is a crucial factor in determining the survival and infection cycle of the phage. This 392 factor is also essential for phages' short-term and long-term storage to retain their activity 393 (Joń czyk et al., 2011). The temperature stability of /i3 ERS-1 was determined from 4 /i3 to 65/i3 . 394 Interestingly, it was observed that cold conditions of 4 and 15 /i3 were found to be optimal 395 temperatures as the phage titer increased from 10 17 PFU/mL to 10 20 PFU/mL. This could be 396 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 19 because the phage was isolated from the Ganges River's upper stretch (Harshil location), which 397 has a relatively cold temperature. An increase in the temperature from 37/i3 to 45/i3 resulted in a 398 significant loss in the phage titer (ANOVA, p<0.001). Additionally, a significant decline was 399 observed in the phage titer at 55/i3 and 65/i3 (ANOVA, p<0.001) (Fig 1E). 400 Another essential factor that limits the stability and activity of phages is the pH of the 401 environment(Joń czyk et al., 2011). The pH stability of /i3 ERS-1 was determined from pH 3 to 402 11. From the phage titration, pH 7 was the optimum pH for sustaining phage stability. On either 403 side of the pH scale, it was observed that the phage titer reduced from 10 17 PFU/mL to 10 10 404 PFU/mL (pH 3) and 10 12 PFU/mL (pH 5, pH 9), respectively (Fig 1F). However, a phage titer 405 of 109 PFU/mL is considered a high titer (Bonilla and Barr, 2018). This suggests that /i3 ERS-1 406 continues to have an infectious nature for E. coli even at a diverse pH range. Additionally, in 407 the environment, especially in wastewater, most microbes thrive at pH 6-9; therefore, most of 408 the biological treatment of wastewater occurs at this pH range (Bouchaala et al., 2021). The 409 ability of /i3 ERS-1 to mount infection to its target host at various ranges of temperature and pH 410 substantiates its candidacy for use in wastewater treatment. 411 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 20 412 Figure 1: Morphology and growth parameters of /i1 ERS-1. (A) Plaque morphology. The 413 blue arrows point to the zoomed view of plaques, showing a clear center surrounded by a 414 translucent halo. (B) HR-TEM micrographs of /i3 ERS-1 at 80000 ×, and scalebar of 50 nm. 415 The inset is STEM images of /i3 ERS-1 in extended and contracted states. (C) Graphical 416 representation of the percentage of adsorbed and unadsorbed phage particles of /i3 ERS-1 as a 417 function of time. (D) One-step growth curve of /i3 ERS-1 with the phases of its lifecycle 418 marked (pink). (E) Illustration of the effect of temperature on the stability of /i3 ERS-1. (F) 419 Effect of various pH ranges on the stability of /i3 ERS-1. Data in the graph panels from C-F 420 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 21 represent means of the experimental replicates, standard deviations (SD), and statistical 421 significance of the data (*, **, ***, ****) using RM- one-way ANOVA. The acronym 'ns' 422 implies no significant difference between the test groups. 423 3.2 Genome analysis, annotation, and proteome of /i1 ERS-1 424 The genome length of /i3 ERS-1 consists of 175,242 /i3 bp and 60.9% of G+C content. 425 Analysis of ORFs revealed the presence of 796 ORFs, mainly comprising hypothetical proteins 426 followed by phage proteins involved in defining phage structure, DNA replication, repair, 427 packing, and lysis of the host. The details and position of various proteins in the genome of /i3 428 ERS-1 have been illustrated in Fig 2A . Phage lysins are considered highly efficient and 429 evolved molecules that can digest the peptidoglycan in the cell wall of bacteria, thereby 430 facilitating the release of viral progeny (Fischetti, 2005). From genome annotation using RAST 431 and Prokka v1314.6 tool, it was evident that lysin was the key enzyme responsible for the host 432 lysis by /i3 ERS-1. 433 Further, from the orthoANI comparison of the genome of /i3 ERS-1 and closely related 434 Tequatrovirus members, it was observed that /i3 ERS-1 has an ANI of 94.2% with Escherichia 435 phage LH2, indicating the close relatedness of its genome (Fig 2B) . Additionally, the 436 intergenomic similarities analysis with VIRDIC tool revealed only 19% similarity of this phage 437 with its closest relatives, indicating distant relatedness of the genome of phage ERS-1 (Fig 2C). 438 However, VIRIDIC is not able to capture similarity relationships between the related viruses 439 which have regions of similarity of less than 65%, a limitation inherent to BLASTN. Therefore, 440 protein-based analysis is recommended to clarify the phylogenetic relationships. Analysis of 441 amino-acid sequences in the whole genome of /i3 ERS-1 with all the available reference phages, 442 and closely related phages was performed using VipTree. A total of 3036 reference sequences 443 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 22 of the phage genomes were used to construct the phylogenetic tree (Fig 2D) . The preliminary 444 observation of the circular phylogenetic tree of /i3 ERS-1 was consistent with morphological 445 features of 'T4 like myovirus'. Further, depending on the S G scores of tblastx, a subset of 21 446 closely related phages and three distantly related phages were chosen to construct a rectangular 447 phylogenetic tree (Fig 2E). The genome of /i3 ERS-1 clustered with some Escherichia phages, 448 followed by Shigella phages, all belonging to Tequatrovirus, a genus of the Straboviridae 449 family, according to ICTV. As per ICTV, the isolated phage(s) are classified as a member of 450 the same genus if their identities of nucleotide sequences are >70% and a distinct species if 451 ANI is ≤ 95% (Bin Jang et al., 2019). The above nucleotide and amino-acid-based analysis of /i3 452 ERS-1 suggested that /i3 ERS-1 could be classified as a new member within the Straboviridae 453 family and Tequatrovirus genus. The genome of this phage has been submitted to NCBI and 454 has been assigned accession number PP337211. 455 The members of Tequatrovirus are known to infect Escherichia, Enterobacteria, Shigella, 456 Salmonella, Yersinia, Aeromonas, Burkholderia, Stenotrophomonas, Prochlorococcus, 457 Synechococcus, Citrobacter and Staphylococcus 458 (https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=10663). However, it is 459 noteworthy that there are no reports of Tequatrovirus infecting P. aeruginosa in the NCBI 460 database. Hence, to our knowledge, this could be the first report detailing a complete genome 461 of a Tequatrovirus member capable of infecting P. aeruginosa ATCC 9027. 462 463 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 23 464 Figure 2: Genome details and amino-acid-based comparative genomic and phylogenetic 465 analysis of /i1ERS-1. (A) Linear genome map of /i1 ERS-1 created with Proksee viewer. The 466 outer blue arrows are the RAST annotated protein-coding genes on the positive and negative 467 strands of the DNA. The inner orange skew displays the total GC content, followed by GC 468 skew information in the positive (green) and negative (purple) strands. (B) Heatmap of 469 OrthoANI between /i1 ERS-1 and closely related Tequatroviruses, computed with OAT 470 software. The color scale (top right) contains values defining the similarity percentage between 471 the genomes. The numerical values of intergenomic genetic distance (from OAT software) are 472 marked on the branches. (C) Heatmap showing alignment indicators (left half) and 473 intergenomic similarity values (right-half) generated through VIRDIC. (D) A circular 474 phylogenetic tree generated by VipTree for all 3036 reference genomes of phages related to /i1 475 ERS-1. The genome distance matrix of the phage under consideration and its relatives are 476 calculated by BIONJ (an algorithm based on the distance for phylogeny reconstruction 477 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 24 (Gascuel, 1997). The midpoint of the tree marks the root. The outer-colored rings (host taxa) 478 and the inner-colored rings (viral families). (E) Based on the SG scores, a rectangular 479 phylogenetic tree of /i3 ERS-1 with its 21 closely related and 3 distantly related phage genomes. 480 The red stars represent Escherichia phage /i3 ERS-1. 481 3.3 Anti-Biofilm Potential of /i1 ERS-1. 482 A. Single, Multi-strain, Mixed-Genera Biofilm Inhibition Spectra 483 Several reports have suggested the occurrence of single species biofilm and mixed or multi-484 species/ mixed biofilms in nature. The most frequently encountered bacteria in formation of 485 biofilm in environment, medicine, food industry, agriculture and animal husbandry include 486 members of Escherichia, Shigella, Salmonella, Pseudomonas, Acinetobacter, Klebsiella, 487 Enterococcus, Streptococcus, and Staphylococcus (Burmølle et al., 2014; Denissen et al., 2022; 488 Mouiche et al., 2019; Sharma et al., 2023; Toushik et al., 2022) . Given this, we evaluated the 489 potentials of /i3 ERS-1 for inhibition of biofilm by single species, multiple strains, and multiple 490 genera, using the information of its host range obtained through spot assay. Results of the CV 491 assay indicated that /i3 ERS-1 could inhibit and reduce 96.17% of the biofilm mass produced by 492 E. coli (ATCC 8739) (Fig 3A, E). 493 Interestingly, it was observed that the addition of /i3 ERS-1 to multiple strains of E. coli 494 (ATCC 8739, 25922, 43888) (Fig 3B, E) and multiple genera (P. aeruginosa (ATCC 9027), S. 495 boydii (ATCC 9207), and E. coli (ATCC 8739) (Fig 3 C, E) inhibited the formation of biofilm 496 by 56.2% and 54% respectively. This is an important observation, as bacterial biofilms are 497 more resistant to external factors and treatment by antimicrobial and chemical agents than their 498 planktonic counterparts. The ability of /i3 ERS-1 to render the cells in their planktonic form 499 could be attributed to the presence of polysaccharide depolymerases in either free or bound 500 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 25 state, which is defined by the semi-transparent halo around phage plaques as evident in (Fig 501 1A). The other plausible mechanism behind the broad-spectrum antibacterial and antibiofilm 502 nature of /i3 ERS-1 could be the production of certain quorum-quenching enzymes or lipases. 503 Quorum-quenching enzymes in phages are responsible for inhibiting quorum sensing by mixed 504 species/genera biofilms composed of P. aeruginosa and E. coli (Liu et al., 2022; Pei and 505 Lamas-Samanamud, 2014). On the other hand, lipases disperse biofilms by hydrolyzing lipids 506 in the bacterial cell membrane (Azeredo et al., 2021). However, there is limited information 507 about these enzymes. Therefore, to have a holistic understanding of the mechanism of bacterial 508 lysis and broad host specificity, an in-depth study of phage-derived proteins of /i3 ERS-1 is 509 necessary. 510 511 512 Figure 3: Effects of /i1 ERS-1 in biofilm inhibition by single species, multi-strains, and 513 multiple genera of bacteria with CV assay. Figure 3A-D are CV-stained microscopic images 514 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 26 of biofilm, observed under an oil immersion lens (100×) after the control and treated groups 515 were incubated for 24 hours. (A) Single species biofilm of E. coli culture control (left), E. coli 516 treated with /i3 ERS-1 (right). (B) Multiple strains of E. coli mixed to form biofilm-culture 517 control (left), multi-strain group treated with /i3 ERS-1(right). (C) Biofilm formation by 518 multiple genera – culture control (left), multiple genera group treated with /i3 ERS-1. (D) 519 Biofilm formation by S. aureus- culture control (left), and /i3 ERS-1 treated (right). (E) 520 Quantification of biofilm formation with CV assay, followed by absorbance at 590 nm. 521 B. Time-dependent evaluation of E. coli biofilm inhibition and disruption 522 E. coli is a prominent fecal indicator in the waterways. The biofilm formation ability of E. 523 coli on the surface of water treatment pipes and filters poses a significant challenge for treating 524 and preventing this bacteria during disinfection and recycling practices of water and wastewater 525 (Qian et al., 2022; Steven et al., 2022). From the genome annotation study, the biofilm 526 composition of the E. coli host in this study was linear homopolymer poly-beta-1,6-N-acetyl-D-527 glucosamine (beta-1,6-GlcNAc; PGA) (Table S2). The pgaABCD operon's gene products are 528 necessary for forming and maintaining biofilm structural stability in several enteric pathogenic 529 E. coli (Itoh et al., 2008) . Therefore, in this study, we evaluated the potentials of /i3 ERS-1 in 530 the prevention (inhibition) and dispersal (disruption) of biofilm by E. coli ATCC 8739 in a 531 time-dependent manner. 532 The CLSM analysis of /i3 ERS-1 treated E. coli showed red fluorescence at 6, 12, and 24 533 hours, indicating the dead biomass of E. coli cells (Fig 4 B-D), as compared to the control 534 (devoid of /i3 ERS-1 treatment) (Fig 4A). The presence of live biomass was ascertained through 535 CFU counts of viable cells. Results indicated a significant inhibitory effect on biofilm 536 formation at 6 hrs., which continued up to 24 hours (ANOVA, p=<0.0001) (Fig 4D). Previous 537 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 27 studies have demonstrated the antibiofilm effect of Tequatrovirus against E. coli and 538 Salmonella, with bacterial reduction from 2-5 logs within 6-24 hours (Liao et al., 2022; Zhou et 539 al., 2022). However, in our study, for the first time we report the potential of /i3 ERS-1 in 540 reducing the abundance of E. coli cells from 6.14 log 10 CFU/mL at 6 hours to 8.22 log 10 541 CFU/mL at 24 hours, with a ~100% reduction in the total viable counts. This data suggests that, 542 /i3 ERS-1 poses an inhibitory effect that could be used for biological control of surface 543 colonization by E. coli cells either through inhibition of their initial attachment or impediment 544 of bacterial establishment on various surfaces. 545 546 Figure 4: Inhibitory effect of /i1 ERS-1 on biofilm formation by E. coli. Samples were 547 observed under a Leica Stellaris 5, DMi8 microscope with a 20× objective. The red 548 fluorescence indicates dead E. coli cells, while the green fluorescence indicates viable cells at 549 different time intervals. (A) CLSM micrograph of E. coli cells with no treatment, capable of 550 forming biofilm after 24 hours. (B) CLSM micrograph of /i3 ERS-1 treated group imaged after 551 6 hours. (C) CLSM micrograph of /i3 ERS-1 treated group imaged after 12 hours. (D) CLSM 552 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 28 micrograph of /i3 ERS-1 treated group imaged after 24 hours. (E) Quantification of the viable 553 cells as CFU/mL of the control (non-treated) and /i3 ERS-1 treated sets. 554 In the natural environments, most of the biofilms are in their mature state, and therefore, to 555 detach the biofilm, disruption of EPS polymers followed by planktonic cell dispersal is 556 essential (Shrestha et al., 2022). Capsular polysaccharides as major virulence factors have been 557 reported for A. baumannii (Wang et al., 2020), K. pneumoniae (García et al., 2019), and E. coli 558 (Guo et al., 2017) in biofilm formation. Thus, the antibiofilm effect of /i3 ERS-1 in 559 disintegrating matured or pre-formed biofilms was evaluated with CLSM and counts of viable 560 cells using CFU/mL. 561 It was observed that the number of dead/ non-viable cells increased in the phage-treated 562 group for 24 hours (Fig 5A-D). This indicates a gradual disruption of E. coli biofilm by /i3 563 ERS-1. The total viable counts at each time point revealed that /i3 ERS-1 was capable of 564 disrupting the pre-formed biofilm layer of E. coli with a reduction of 2.4 log 10 CFU/mL at 6 565 hours, followed by 2.70 log10 CFU/mL at 12 hours, and 3.88 log 10 CFU/mL at 24 hours. These 566 values indicate that /i3 ERS-1 can disrupt biofilm with a percent reduction of viable cells up to 567 99% with 6 hours of treatment. After 24 hours, the percent reduction of E. coli cells observed in 568 the biofilm was 99.98% (ANOVA, p=0.0022). (Fig 5 K). 569 Additionally, the FESEM imaging of the control and /i3 ERS-1 treated biofilm after 24 hours 570 confirmed disruption of the pre-formed biofilms. The cells of the control set showed a well-571 defined, rod-shaped morphology embedded in a matrix of EPS (Fig 5 E-G), while the /i3 ERS-1 572 (24 hrs.) treated set showed a distorted morphology within the biofilm (Fig 5 H-J) . The 573 planktonic bacteria within biofilms are highly resistant to the action of antibiotics, disinfectants, 574 and disruption by physical or chemical ways (Kovacs et al., 2023). However, the experimental 575 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 29 evidence in this study suggests the efficacy of /i3 ERS-1 in biofilm disruption and its 576 bacteriolytic effects against the planktonic cells, demonstrating its potential applications. 577 578 Figure 5: Effect of /i1 ERS-1 on pre-formed/ matured biofilm of E. coli (ATCC 8739). The 579 control and treatment sets visualized through CLSM with 20 × objective at specified time 580 intervals are represented in the panels as (A) CLSM micrograph of culture control with pre-581 formed biofilm of E. coli cells devoid of any treatment. (B-D) CLSM micrograph of /i3 ERS-1 582 treated pre-formed biofilm imaged after 6 hours, 12 hours (C), and 24 hours (D). (E-G) FE-583 SEM images of control biofilm at different magnifications 16000× (E), 30000× (F) , and 584 60000× (G), showing intact rods immersed in the EPS matrix of the biofilm. (H-J) FE-SEM 585 images of /i3 ERS-1 treated biofilm at different magnifications 16000× (H), 30000× (I) , and 586 60000× (J), showing disrupted EPS matrix and distorted morphology of compromised cells in 587 the biofilm. (K) Quantification of the viable cells in disrupted biofilm as CFU/mL of the 588 control (non-treated) and /i3 ERS-1 treated sets. 589 3.4 Bacteriophage-based reduction of bacterial counts from wastewater 590 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 30 The disinfection of the WW to make it safe is crucial to protecting the health of the 591 environment, humans, and animals (Regulation EU 2020/741). Failure to do so could be 592 responsible for different diseases because of various pathogens. According to this regulation, 593 the reclaimed water is monitored based on various pathogen indicators. For bacteria: E. coli; 594 for pathogenic viruses: coliphages; and protozoa: the spores of Clostridium perfringens or 595 sulphate-reducing bacteria (Hernández-Crespo et al., 2022; Kadlec and Wallace, 2008). 596 Therefore, considering the broad-spectrum host range, bacteriolytic, and antibiofilm potentials 597 of /i3 ERS-1, we focused its application on reducing the bacterial load from untreated 598 wastewater/ raw sewage. 599 The initial screening results showed a significant reduction of the bacterial load of 2.27 600 log10 CFU/mL in the /i3 ERS-1 treated group of the raw sewage after 24 hours (p<0.001) (Fig 6 601 A, B). Further, a time-kill assay was performed to evaluate the magnitude of reduction in the 602 viable coliform load in control and /i3 ERS-1-treated groups. The rationale behind plating the 603 raw sewage and coliform enriched (in McConkey broth) on BGLB agar and EMB agar was 604 differentiation and specific enumeration of the coliform bacterial load. Results after 24 -48 605 hours of incubation revealed a significantly lower number of viable bacteria in the /i3 ERS-1 606 treated group in both raw sewage (ANOVA, p<0.01) and coliform enriched group (ANOVA, 607 p<0.001). Interestingly, in the set of /i3 ERS-1 treated raw sewage, after 24 hours, a 2- 2.4 log10 608 reduction was observed compared to the control group. Notably, in the coliform enriched set of 609 raw sewage, 4.2 log10 CFU/mL reduction was observed in the /i3 ERS-1 treated group (Fig 6 C-610 E). Additionally, the CLSM imaging of aliquots also revealed an increase in the dead cells 611 (red) over time in the /i3 ERS-1 treated group of unenriched and coliform-enriched sewage (Fig 612 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 31 6 F) compared to the control group. This indicates that /i3 ERS-1 could be used as a green 613 approach to reduce the coliform counts. 614 There are few reports describing the use of phages in wastewater treatment to reduce the 615 bacterial load and the use of phage cocktail to eliminate waterborne pathogenic bacteria (Grami 616 et al., 2022; Jassim et al., 2016; Periasamy and Sundaram, 2013). However, to the best of our 617 knowledge, this is the first study to highlight the use of single polyvalent, non-engineered 618 (naturally occurring) phage /i3 ERS-1 to reduce the coliform load in wastewater. Also, this is 619 the very first kind of study detailing an in-depth characterization, antibacterial, and antibiofilm 620 potentials, together with lab-scale evaluation of reduction in coliform counts in raw sewage by 621 a novel phage /i3 ERS-1 isolated from the untapped location of the Ganges River. However, a 622 detailed understanding of the optimized phage dose and its effect on physicochemical and 623 microbiological parameters before and after /i3 ERS-1 treatment would be necessary for its on-624 site application. Additionally, to overcome resistance by bacteria in the long run, combining 625 various phages with /i3 ERS-1 or using phage-derived enzymes from /i3 ERS-1 would benefit its 626 application under one health canopy, promoting human, animal, and environmental health. 627 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 32 628 Figure 6: Evaluation of /i1 ERS-1 in reduction of coliform counts. (A) Graphical 629 representation of quantification of the viable cells in raw sewage as CFU/mL of the control 630 (non-treated) and /i3 ERS-1 treated sets. (B) Log reduction and percentage reduction values 631 indicate /i3 ERS-1 efficacy in reducing bacterial load in raw sewage after 24 hours. (C, D) 632 Graphical representation of quantification of the viable cells in raw and coliform enriched 633 sewage as CFU/mL of the control (non-treated) and /i3 ERS-1 treated sets on BGLB-agar (C), 634 and EMB agar (D), as a function of time. (E) Log reduction and percentage reduction values 635 indicate the time-dependent efficacy of /i3 ERS-1 in reducing bacterial load in raw and 636 coliform-enriched (McConkey broth) sewage. (F) CLSM micrographs showing live (green) and 637 dead (red) bacterial cells in the raw (unenriched) sewage and coliform-enriched (McConkey) 638 sewage at different time intervals. 639 4. Conclusion 640 This study demonstrates the isolation and characterization of a novel phage /i3 ERS-1 from 641 an untapped location of the Ganges River. The strength of our research lies in the novelty of 642 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 33 phage and its bacteriolytic and antibiofilm potentials in reducing the bacterial load of 643 environmentally relevant waterborne pathogens in their planktonic cells and biofilm form. The 644 concept of controlling bacteria using phages and their enzymes without the aid of chemical or 645 UV disinfection is a challenging one. However, the data also shows the efficacy of the newly 646 isolated, high titer /i3 ERS-1 in controlling the bacterial (coliform) load in the sewage water and 647 possibly improving the water quality. Our approach of using polyvalent phage as a green 648 alternative for bacterial biocontrol of sewage water highlights the importance of the potential 649 applications of such broad-spectrum bacteriophages to improve the quality of the effluent and 650 disposal of sludge in the environment. This approach can significantly impact the delivery of 651 sustainable development goals 6: clean water and sanitation, 3: good health and well-being, and 652 11 sustainable cities and communities. Moreover, using lytic phages to improve wastewater 653 quality would substantially reduce the load on other treatment methods, thereby contributing to 654 low energy consumption levels, reduced use of harmful chemicals, and improved access to 655 water reuse with reduced biological contamination, promoting a circular economy. 656 5. Credit authorship contribution statement 657 RS: Conceptualization, visualization, experimentation, sampling, writing- review & editing, 658 and Data analysis. KK: Project monitoring, Sample collection, editing, MSD: 659 Conceptualization, Supervision, review, and editing. 660 6. Declaration of Interest: The authors declare no conflict of interest. 661 7. Acknowledgments: 662 Authors are thankful to the National Mission for Clean Ganga (NMCG), Government of India, 663 New Delhi, India, for the project (GKC-01/2016-17, 212, NMCG- Research), Directors of 664 CSIR-NCL, and CSIR-NEERI for infrastructure and support. RS acknowledges Mr. Manan 665 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted November 2, 2024. ; https://doi.org/10.1101/2024.11.01.621489doi: bioRxiv preprint 34 Shah for his help in sampling. RS is grateful to Mr. Tushar Kolhe and Mr. Chetan from Central 666 Analytical Facility, CSIR-NCL, for their help in HR-TEM and FE-SEM. RS is thankful to 667 HRDG-CSIR and NMCG, New Delhi, for fellowship and AcSIR, New Delhi, for the academic 668 support. The manuscript has been checked for plagiarism using iThenticate software with an 669 institutional license. 670 8. References 671 Abedon, S.T., 2023. Automating Predictive Phage Therapy Pharmacology. Antibiotics. 672 https://doi.org/10.3390/antibiotics12091423 673 Adams, M.H., Wassermann, F.E., 1956. Frequency distribution of phage release in the one-step 674 growth experiment. 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