Characterization of Novel Straboviridae Phages Enhancing Antibiotic Efficacy and Biofilm Inhibition Against MDR Escherichia coli

Characterization of Novel Straboviridae Phages Enhancing Antibiotic Efficacy and Biofilm Inhibition Against MDR Escherichia coli | 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 Characterization of Novel Straboviridae Phages Enhancing Antibiotic Efficacy and Biofilm Inhibition Against MDR Escherichia coli Md Shamsuzzaman, Shukho kim, Yoon-Jung Choi, Jungmin Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6102499/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 Multidrug-resistant Escherichia coli (MDR- E. coli ) is a significant public health concern due to its resistance to multiple antibiotics, which complicates infection treatment. Phages are gaining attention for their specific lytic activity against pathogenic bacteria. This study aimed to isolate and characterize lytic bacteriophages designated as EC.W1-1 and EC.W15-3, targeting different sequence types (STs) of multidrug-resistant E. coli . The isolated phages, EC.W1-1 and EC.W15-3, belonged to the Straboviridae family and the genus Tequatrovirus . Phages remained stable at pH 2–10 for 4h and below 80°C for 1h. They exhibited in vitro bacterial lytic activity at various MOIs (10–0.001). The one-step growth curve of phages showed a short latent period of about 10–15 min and a moderate burst size of 64–83 (pfu/cell). Phages genome size ranged from 37,736–123,792bp, with G + C content of 35.6–37.2%. No virulence or drug-resistance genes were detected, enhancing their safety profile. Most predicted coding sequences (CDSs) in the phages analyzed were associated with various putative functions like tail protein, holin, lysis protein, head protein, structural proteins, and DNA replication, transcription, and repair proteins. Furthermore, the study explored the combined effects of phages and antibiotics, showing effective inhibition of ESBL-producing and carbapenem-resistant E. coli (CREC) with sub-lethal antibiotic doses. In addition, phages suspensions can eliminate biofilm formed against different STs of MDR E. coli . Therefore, we concluded that EC.W1-1 and EC.W15-3 have potential therapeutic properties, providing an alternative to antibiotic for treating various pathogenic MDR E. coli sequence types. Virology General Microbiology Multidrug-Resistant Escherichia coli phage therapy genome analysis phage-antibiotic synergy biofilm Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Escherichia coli has become a primary concern in healthcare settings due to its multidrug resistance, causing many infections [ 1 ]. This pathogen is a major cause of various infections in hospitalized patients, such as urinary tract infections (UTIs) [ 2 ], nosocomial pneumonia, cholangitis, peritonitis, cellulitis, cholecystitis, infectious arthritis, osteomyelitis, diarrhea, bloodstream infections, and neonatal meningitis [ 3 ][ 4 ]. The rise in MDR E. coli infections and limited antibiotic efficacy present significant challenges in antimicrobial resistance and disease spread in human and veterinary medicine [ 5 ]. E. coli is typically sensitive to many antibiotics but can acquire resistance genes via horizontal gene transfer [ 6 ]. E. coli that produce β -lactamases are a significant and widespread source of antimicrobial resistance [ 7 ]. For example, bla TEM-1, commonly found in E. coli from animals, produces narrow-spectrum β -lactamases that inactivate penicillin and aminopenicillins [ 6 ][ 8 ]. Recently, genes responsible for extended-spectrum β -lactamases (ESBLs) and AmpC β -lactamases have been identified in E. coli [ 9 ]. The emergence of resistance mechanisms in E. coli involves various mechanisms beyond β -lactamases, including, 16S rRNA methylases, carbapenem resistance, plasmid-mediated quinolone resistance, and mcr genes. [ 10 ][ 11 ]. Moreover, mobile genetic elements like transposons are crucial in disseminating resistance genes among bacterial populations, contributing to the global challenge of MDR E. coli infections [ 12 ]. Understanding these genetic mechanisms is essential for developing effective treatment strategies and combating the spread of antimicrobial resistance in E. coli . However, there is limited information on specific antimicrobial therapies for infections caused by various E. coli clones. This situation highlights the urgent need for innovative therapeutic approaches and the development of new antimicrobial agents to combat these infections effectively [ 13 ]. Phages have emerged as a promising alternative to combat MDR bacterial infections. Phage cocktails, combining multiple phages, have shown efficacy in targeting MDR pathogens like Escherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae [ 14 ][ 15 ]. Phage therapy, an age-old approach, declined with the advent of antibiotics but has regained popularity due to concerns about antibiotic resistance [ 16 ] [ 17 ]. Clinical successes in the United States, Georgia, Poland, and Russia highlight its potential as an alternative to antibiotics [ 18 ][ 19 ]. Phages specificity against target bacteria enhances their efficacy compared to broad-spectrum antibiotics [ 20 ]. Their natural origin and narrow therapeutic window make them a safe substitute for antibiotics [ 21 ]. However, the emergence of phage-resistant mutants poses a significant challenge. Researchers have explored the development of phage cocktails and phage-antibiotic combinations to address bacterial resistance. Studies have shown that the use of phage cocktails has been found to significantly reduce the growth of bacteria and inhibit the formation of phage-resistant mutants [ 22 ]. Furthermore, combining phages that target different host receptors in a cocktail has been shown to reduce the emergence of phage resistance during biocontrol applications, highlighting the potential of this approach to maintain the effectiveness of phage therapy [ 23 ]. To ensure the safety and efficacy of these cocktails, ongoing efforts focus on isolating new phages and conducting comprehensive in vitro and in vivo experiments for characterization [ 24 ][ 25 ]. In this study, we successfully isolated two E. coli phages from hospital sewage water in Daegu, South Korea, demonstrating their efficacy against different STs types of MDR E.coli . This study identified effective phages against ESBL-producing E.coli and CREC. These findings have important implications for the scientific community, providing insights into the development of novel strategies for combating MDR E. coli . Overall, this study will help advance researchers’ understanding of phage therapy and present the development of new treatment options for antibiotic-resistant infections. 2. Materials and Methods 2.1. E. coli isolates used in this study In this research, 60 ESBL-producing and CREC isolates were collected from the Pathogen Culture Collection at Kyungpook National University Hospital. Standard strains E. coli ATCC25922 was purchased form the American Type Culture Collection for this study. Initially, the bacteria were grown on 5% sheep blood agar plates at 37°C for 24 h, followed by culture in BHI media. For future experiments, they were preserved at − 70°C in 50% glycerol stocks. 2.2. Phage isolation, purification, and preparation Phage isolation, purification, and preparation were performed using modified liquid and agar overlay methods [ 26 ][ 15 ]. Initially, host bacteria were grown in BHI medium, while sewage samples from Kyungpook National University Hospital were processed by centrifugation at 12,000×g for 10 min to remove debris. The filtered sewage was used to infect E. coli cultures at their early exponential growth phase. After incubating overnight at 30°C with shaking, the mixture was kept at 4°C for 48 h. The supernatants were then centrifuged and filtered. Phage titers were measured using the double-layer method. A single plaque was isolated, resuspended in SM buffer, and purified by vortexing, centrifugation, and filtration. This purified phage suspension was then mixed with E. coli and incubated at 30°C with shaking at 150 rpm for 16 h. Further purification involved repeated treatments with 10% chloroform, gradually increasing the volume each time. Finally, the phage solution was mixed with 15% glycerol and stored at − 70°C for future use. 2.3. Determination of lytic activity of phages against 60 MDR E. coli A total of 60 E. coli isolates, representing 14 different ST types, were evaluated for their susceptibility to phage lytic activity using spot test assays, following the method outlined by Kim Shukho (2018)[ 13 ]. To prepare the bacterial lawn, 10 ml of 0.75% soft agar with 100 µl of bacterial culture (OD-0.5 nm ) was poured onto BHA agar plate. Once the overlay solidified, a 15 µl aliquot of the phage solution, with a concentration of approximately 10 11–13 PFU/ml, was directly spotted on the bacterial lawn. The plates were dried and incubated overnight at 37°C. The bacteria's susceptibility was determined by clear lytic zones on the plates. 2.4. Measurement of adsorption rate and burst size of two novel E. coli phages The phage adsorption rate and burst size of phages were determined by the method of Marzia Rahman (2011)[ 27 ]. For adsorption rate, E. coli is infected with phages at a low MOI (0.0001) and incubated at room temperature. Phage samples were taken at intervals (0, 1, 3, 5, 10, 12, 15, 20, and 25 min) to measure unabsorbed phage titers via plaque assays. Burst size was determined through a single growth curve by infecting E. coli at the same MOI, allowing adsorption for 30 min at 4°C, and sampling every 5 min for phage counts. This method is repeated three times independently. 2.5. Temperature and pH stability assays The thermal and pH stability of phages were tested by exposing them to different temperatures and pH levels. For thermal stability, phages in SM buffer were exposed to temperatures from − 20°C to 80°C. For pH stability, phages were incubated in buffers with pH ranging from 2.0 to 10 for 18 h at room temperature. Phage survival was assessed by comparing the percentage of surviving phages to those stored in SM buffer at pH 7.4 and 4°C. Survival rates were calculated using a double-layer plaque assay and counting PFUs. 2.6. In vitro bactericidal activity under different MOIs The experiments involved culturing phages with E. coli in BHI medium at various MOIs (10, 1, 0.1, 0.01, and 0.001) and incubating at 37°C with gentle shaking. Absorbance at 600 nm was measured using a UV-vis spectrophotometer (Molecular Devices, LLC, San Jose, CA, USA) in a 96-well plate to assess optical density. These measurements were taken every hour during the first 10 h and at 24 h. A bacterial culture without phages served as the positive control, while BHI medium alone was the negative control. Each sample was tested in triplicate [ 28 ]. 2.7. Genome sequencing, annotation, and genome analysis Phage DNA extraction was performed using the phenol-chloroform procedure described by Džiuginta Jakočiūnė (2018) )[ 29 ]. For next-generation sequencing (NGS), phage genomic DNA sequencing was performed on an Illumina Miseq platform in San Diego, CA, USA. Sequencing reads were assembled using the Celemics pipeline in South Korea ( https://btseq.celemics.com/ ). Genome sequence similarities were analyzed through alignment using BLASTn ( https://blast.ncbi.nlm.nih.gov/Blast.cgi ). The RAST server ( https://rast.nmpdr.org/rast.cgi ) was utilized to predict open reading frames (ORFs), with verification via the NCBI database. Gene function maps were created using a lab-developed custom program and enhanced with Geneious Prime 2023.2 ( https://www.geneious.com/ ). tRNAscan-SE v.2.0 ( http://lowelab.ucsc.edu/cgi-bin/tRNAscan-SE2.cgi ) was employed to predict tRNA, while ResFinder ( https://cge.cbs.dtu.dk/services/ResFinder/ ) and VirulenceFinder ( https://cge.cbs.dtu.dk/services/VirulenceFinder/ ) identified drug resistance and virulence genes. Phylogenetic trees, based on the phage proteome and circular tree, were developed using VIPTree ( https://www.genome.jp/viptree/ ). The VICTOR tool ( https://ggdc.dsmz.de/submit_victor_job.php ) constructed a complete genome phylogenetic tree based on whole-genome sequences of isolated phages. Full genome sequences of isolated phages were aligned with others using the BLASTn tool in the NCBI database. Genome sequence similarities were visualized with the Mauve algorithm (v2.3.1) ( https://darlinglab.org/mauve/mauve.html ), comparing isolated phages to their nearest E. coli phage relatives. 2.8. Combination effect of phage and antibiotic targeting ESBL-producing and CREC E. coli isolates. Phage antibiotic synergy testing was carried out in LB medium following the method by Carmen Gu Liu [ 30 ]. ESBL-producing and CREC isolates were cultured in BHI for 6 h. After centrifugation and washing, the cell pellets were resuspended in BHI medium and adjusted to a concentration of approximately 1 × 10 9 CFU/ml (OD600nm = 1). Then, 100 µl of this suspension was added to each well of a microtiter plate, which contained a checkerboard of phage and antibiotic concentrations (50 µl of each antimicrobial). The optical density at 600 nm (OD600nm) was measured every hour over a 24-h period at 37°C, using continuous shaking in a Hercuvan Lab Systems thermo shaker incubator (Hercuvan Lab Systems, London). 2.9. Biofilm formation assay To start the biofilm experiment, E. coli isolates were cultured overnight in LB medium at 37°C with shaking. Subsequently, 200 µl of a bacterial cell suspension (10 7 CFU/ml) was added to each well of a 96-well plate and incubated at 37°C for 24 h. Then, 100 µl of the culture medium for the sample wells was replaced with 100 µl of LB-containing phages (10 9 PFU/ml), while control wells received plain LB medium. The plate was incubated again at 37°C for an additional 24 h. After 48 h of biofilm growth, the liquid medium was carefully removed by aspiration, and the wells were washed twice with 1× PBS. The biofilms were stained with 200 µl of 0.1% crystal violet for 20 min at 37°C, followed by three washes with 1× PBS to remove excess stain. 200 µl of 95% ethanol was added and incubated to solubilize the crystal violet for 10 to 15 min. For biofilm reduction tests using serum, we used 20% serum in LB media instead of regular media. Finally, a plate reader quantified biofilm formation by measuring absorbance at 570 nm [ 31 ]. 3. Results 3.1. Bacteriophage isolation, host spectrum analysis and determination of antibiotic resistance profile of 60 MDR- E. coli This study isolated phages EC.W1-1 and EC.W15-3 from hospital sewage samples using E. coli ATCC25922 as the host. Spot test results showed that both phages exhibited high lytic activity against highly virulent MDR E. coli . Phage EC.W1-1 lysed 51.6% (31/60) of the isolates, including 57.6% (15/26) of ESBL-producing and 43.7% (21/48) of CREC isolates. Phage EC.W15-3 lysed 33.3% (20/60) of the isolates, including 30.7% (8/26) of ESBL-producing and 31.2% (15/48) of CREC isolates. The susceptibility of the two novel bacteriophages against various ST types of MDR E. coli was tested on 14 different ST types. Phage EC.W1-1 was 100% effective against ST648, ST2311, ST405, ST13003, ST167, and ST73, while phage EC.W15-3 was 100% effective against ST73, ST13003, and ST167. Against ST131, phage EC.W1-1 showed 88.8% (16/18) susceptibility, and phage EC.W15-3 showed 66.6% (12/18) susceptibility. However, against ST410, the effectiveness dropped to 19.2% (5/26) for phage EC.W1-1 and 15.3% (4/26) for phage EC.W15-3 (Table 1 ) and (Table S.1) According to the antibiotic resistance analysis of 60 clinically isolated E. coli isolates, all were multidrug-resistant. Among these, 80.4% were identified as CREC, and 43.33% of the isolates possessed ESBL, as previously determined by the Microbiology Laboratory at Kyungpook National University [ 15 ]. Table 1 Host range of phages EC.W1-1 and EC.W15-3 against 14 different ST types of 60 ESBL-producing and CREC E.coli. ST type E. coli isolates ESBL CREC Φ E.C-W1-1 Φ E.C-W15-3 IMI MEM 648 KBN10P04004 + - + S S 648 KBN10P03442 + - + S I 648 KBN10P01217 + - N/A R S 648 KBN10P03385 + + + S S 2311 KBN10P06803 + - + R R 405 KBN10P03987 + - + R S 607 KBN10P06211 - - + R R 13003 KBN10P06225 + + - R R 1139 KBN10P00235 - - N/A R R 1487 KBN10P00237 - - N/A R R 10 KBN10P00710 - - N/A R S 7962 KBN10P03386 - - + S S 7962 KBN10P05623 - - + R I 131 KBN10P03440 - - + S I 131 KBN10P03979 - + + S S 131 KBN10P07282 + + + S S 131 KBN10P07288 + + + S I 131 KBN10P01569 + + R R 131 KBN10P03452 + + + R R 131 KBN10P03005 + + - R I 131 KBN10P00128 + + N/A R R 131 KBN10P00238 + + N/A R R 131 KBN10P02048 + - N/A R R 131 KBN10P00547 + - + S S 131 KBN10P05638 + - N/A I S 131 KBN10P06781 + + + S S 131 KBN10P06658 - + + I R 131 KBN10P02511 + + + R I 131 KBN10P05702 + + - I R 131 KBN10P05883 + + - R S 131 KBN10P01583 + - + S S 410 KBN10P05295 - - + R R 410 KBN10P06393 - - - R R 410 KBN10P06382 - + - R R 410 KBN10P06490 - - - R R 410 KBN10P06021 - - - R R 410 KBN10P06501 - - - R R 410 KBN10P06636 - - - R R 410 KBN10P06786 + + - R R 410 KBN10P06210 - - + S R 410 KBN10P06390 - - - R R 410 KBN10P06244 - - - R R 410 KBN10P05957 - - - R R 410 KBN10P04869 - - - R R 410 KBN10P06204 - - - R R 410 KBN10P06388 - - - R R 410 KBN10P07290 - - I S 410 KBN10P05685 + + N/A R R 410 KBN10P05617 - - N/A R R 410 KBN10P03459 + - + S S 410 KBN10P03733 - - + S R 410 KBN10P03950 - - + S R 410 KBN10P06241 + - + S R 410 KBN10P06779 - - + R R 410 KBN10P01261 - - N/A R S 410 KBN10P07377 + - - R I 410 KBN10P06718 - + - I S 206 KBN10P00137 - - N/A R R 167 KBN10P00139 + + N/A R S Phage susceptibility is represented by symbols: S (Susceptible), R (Resistant), I (Intermediate), + (ESBL Present)/ Susceptible, - (ESBL Absent)/ Resistant, and N/A (Not Available) 3.2. Biological and morphological characterization The study examined the lytic activity, growth patterns, and stability of phages EC.W1-1 and EC.W15-3 under varying temperatures and pH levels. Both phages showed significant lytic activity against E. coli ATCC25922 at 37°C over 24 h at MOIs of 10 and 0.001 (Fig. 1 ). Phages suppressed E. coli growth within the first 10 h at all MOIs, evident from reduced OD600nm values. After 10 h, OD600nm values gradually rose, peaking at 24 h, with bacterial counts exceeding 10 6 CFU/ml. A notable difference between MOIs 10 and 0.001 and the positive control indicated the phages ongoing impact. Combining EC.W1-1 and EC.W15-3 showed no synergistic effect against E. coli ATCC25922. Additionally, when phages EC.W1-1 and EC.W15-3 were tested against E. coli KBN7288, it was observed that the MOIs of 10 and 0.1 facilitated enhanced lytic activity. (Figure S.1). The phage adsorption assay showed that over 90% of the phages could adsorb to E. coli ATCC25922 within the first 10–12 min. Among the tested phages, EC.W15-3 exhibited the fastest adsorption, with 71% attaching within 5 min, compared to 56% for EC.W1-1(Fig. 2 ). The one-stop growth curve analysis revealed that the phages have a latent period of approximately 10–15 min, followed by a rapid release of virus particles. The final titer ranged from 10 7.5 to 10 8 PFU/mL after a burst phase lasting 35–45 min, with an average burst size of 64–83 phage particles per cell (PFUs/cell), indicating efficient replication and production of new phage particles (Fig. 2 ). The phages demonstrated thermal stability between 20°C and 70°C for 60 min. However, their titers significantly decreased at 70°C and were completely inactivated at 80°C (Figure S.2). They also showed stability across pH levels from 2 to 10 within 4 h, with optimal stability observed at pH 6 to 8 (Figure S.3). After 18 h of incubation at 37°C on a double-layer agar plate, the phages formed clear plaques measuring about 1–2 mm in diameter. Transmission electron microscopy (TEM) images showed that phages EC.W1-1 and EC.W15-3 possessed an icosahedral head with a diameter of 105 ± 5 to 115 ± 5 nm and a contractile tail approximately 100 ± 2 to 110 ± 2 nm in length [ 15 ]. 3.3. Phylogenetic analysis of phages The phylogenetic relationship of the new phages was determined by analyzing their whole genome sequences against the closest known E. coli phages. The analysis revealed that EC.W1-1 and EC.W15-3 exhibited the highest DNA sequence similarities to the Tequatrovirus genus in the Straboviridae family, and phages Escherichia phage YUEEL01 (complete genome) and Escherichia phage D5505 (complete genome). Escherichia phage teqhad (complete genome) and Escherichia phage PE37 (complete genome) (Figure S.4). The query coverage and percentage identity were recorded as 98/98.27%,97.92%, 96.45% and 96.37% respectively for these isolates (Table S.2). 3.4. Genomic feature of two novel E. coli phage The comprehensive analysis of the whole-genome sequences for bacteriophages EC.W1-1 and EC.W15-3 reveals they each possess circular double-stranded DNA. The genome sizes are 37,736 bp for EC.W1-1 and 123,792 bp for EC.W15-3. The G + C content of these phages is measured at 35.61% for EC.W1-1 and 37.27% for EC.W15-3. These genome sequences have been cataloged in the NCBI GenBank under accession numbers PP170089 and PQ030847, respectively. For EC.W1-1, the genome annotation identified 93 protein-coding genes, while EC.W15-3 has 202 such genes. These genes are entirely located on the positive strand. The genomes contain various structural and assembly genes, including those necessary for terminase subunits, capsid construction, assembly processes, and tail fiber formation. In addition, both phages have genes responsible for DNA replication and transcription processes, such as DNA helicase, helicase loader, DNA polymerase, DNA topoisomerase, and RNA polymerase (Fig. 3 ). Using ResFinder, no antimicrobial resistance or virulence genes were detected. Nevertheless, the presence of particular genes like holin and several structural proteins indicates potential virulence for both phages. The absence of lysogeny-associated genes such as integrase, excisionase, or transposase signifies that the phages do not integrate into host genomes. When aligned using the MAUVE tool, over 95% of the genome sequences were conserved, showing close evolutionary relations with common ancestry. However, some small genetic variations were observed, indicating both conserved and unique regions, which point to potential functional differences and distinctive capabilities. 3.5. Assessment of phage-antibiotic synergy (PAS) effect on selected ESBL-producing and CREC isolates This study investigated synergistic interactions between phages and antibiotics focusing on three antibiotics colistin, meropenem, and tigecycline. The combination of antibiotics with phages EC.W1-1 and EC.W15-3 generally reduced the MICs, indicating an enhanced bactericidal effect (Table 2 ) and (Figure S.5) Colistin's efficacy improved significantly for E. coli KBN 7288 and E. coli KBN4004 by combining both phages, while E. coli KBN0128 showed no change of MIC. The median MIC value of colistin alone (n = 4) was 18 µg/mL and in combination with phage EC.W1-1, it was reduced to 4.5 µg/mL (13.5-fold reduction). The combination of colistin and EC.W15-3 resulted in a 9.5-fold reduction from 18 µg/mL to 8.5 µg/mL. Meropenem displayed substantial improvement, especially notable in E. coli KBN0128 and E. coli KBN4004 when combined with EC.W1-1, reducing the MIC from 128 µg/mL to 32 µg/mL and from 8 µg/mL to 2 µg/mL, respectively. The combination of phage EC.W15-3 and meropenem reduced the MIC for E. coli KBN7288 from 1 µg/mL to 0.25 µg/mL. Similarly, in tigecycline, only 33.3% of isolates (n = 4) had MIC values of ≤ 2 µg/mL. However, after combining phage EC.W1-1 and tigecycline, 75% of isolates showed MIC values of ≤ 2 µg/mL, indicating a synergistic effect. The combination of phage EC.W15-3 and tigecycline reduced the median MIC from 4 µg/mL to 1.5 µg/mL. Table 2 Reduction in MIC values due to phage antibiotics combination against different multidrug resistant sequence types. Name of Antibiotic Name of E. coli ST type MIC µg/ml Combination MIC of φ-EC.W1-1 and antibiotic(µg/ml) Combination MIC of φ-EC.W15-3 and antibiotic(µg/ml) FIC-Value Combination MIC φ-EC.W1-1 and antibiotic Combination MIC φ-EC.W15-3 and antibiotic Colistin KBN10P07288 ST131 32 8 16 0.25 0.5 KBN10P00128 ST131 256 256 256 1 1 KBN10P04004 ST648 4 1 1 0.25 0.25 KBN10P05617 ST410 2 1 1 0.5 0.5 Meropenem KBN10P07288 ST131 1 0.5 0.25 0.5 0.25 KBN10P00128 ST131 128 32 64 0.25 0.5 KBN10P05617 ST410 16 16 16 1 1 KBN10P04004 ST648 8 2 4 0.25 0.5 Tigecycline KBN10P07288 ST131 4 2 1 0.5 0.25 KBN10P00128 ST131 8 4 8 0.5 1 KBN10P05617 ST410 4 2 2 0.5 0.5 KBN10P04004 ST-648 2 1 1 0.5 0.5 3.6. Effect of phages on the biofilm biomass of E. coli isolates This research focused on the effects of bacteriophages on the development of biofilms in E. coli ATCC25922 and four distinct ESBL-producing and CREC isolates (Fig. 4 ). Applying phages EC.W1-1 and EC.W15-3 to cultures of multidrug-resistant E. coli resulted in a significant decrease in biofilm biomass compared to untreated samples. Specifically, phage EC.W1-1 reduced biofilm biomass by an average of 59.05% in E. coli ATCC25922, 54.03% in E. coli MG1655, 34.99% in E. coli KBN7288, 54.19% in E. coli KBN7282, and 23.86% in E. coli KBN4004. In contrast, phage EC.W15-3 led to an average biofilm reduction of 55.67% for E. coli ATCC25922, 48.61% for E. coli MG1655, 28.29% for E. coli KBN7288, 48.63% for E. coli KBN7282, and 36.69% for E. coli KBN4004. The reference strain used in this study was E. coli MG1655. Similarly, 20% serum to the medium significantly increased the biofilm formation. However, phages EC.W1-1 and EC.W15-3 demonstrated significant biofilm reduction against E. coli ATCC25922 and E.coli KBN7288, achieving 49.41% and 45.70% reduction, respectively, for E.coli ATCC 25922 and 27.92% and 19.08% reduction for E.coli KBN7288. 4. Discussion The rise of MDR E. coli infections poses a significant public health challenge, necessitating innovative therapeutic strategies to combat this growing threat [ 32 ]. Phage therapy, utilizing phages to target and destroy bacterial pathogens selectively, has emerged as a promising strategy to combat MDR infections. To ensure the efficacy and safety of phage therapy, selecting phages with a broad host range and devoid of toxin genes or lysogenic capabilities is crucial [ 33 ]. Recent studies have demonstrated the potential of phage therapy in treating MDR E. coli , highlighting its specificity and efficacy in combating ESBL-producing and CREC infections[ 15 ]. This study aimed to isolate and characterize two Straboviridae phages that target 14 distinct ST types of 60 E. coli . The E. coli isolates were found 100% MDR with 80.4% being CREC isolates. Additionally, 43.33% of the isolates carried extended β -lactamase [ 15 ]. Phages are very specific, targeting only one or a few strains of bacteria. This specificity makes it unlikely that harmless or useful bacteria will be killed when fighting an infection, unlike traditional antibiotics which have a more wide-ranging effect [ 34 ]. The lysis ability of phages 33.3–51.6% revealed phages have the potential to be a candidate for phage therapy [ 35 ]. Phage receptor binding proteins exhibit specificity in their interaction with bacterial cell receptors, influencing the bacterial host range of the phages. This specificity ensures that phages can infect only certain bacteria bearing receptors to which they can bind, thereby determining the phage's host range [ 36 ]. According to this study phage EC.W1-1 and EC.W15-3 recorded a latent period of 10–15 min accompanied by a 64–84 PFU/cell burst size. Phages with a short latent period and moderate burst size are crucial for effective therapeutic agents against E. coli infections. Previous studies have reported phages with varying burst sizes, such as 74–127 particles/cell [ 37 ], 93 particles/cell [ 38 ], and about 139 particles/cell [ 39 ]. Additionally, the latent periods observed in these studies ranged from 10–20 min [ 38 ], 20 min for 90% of to adsorb [ 39 ], and 15–20 min [ 37 ]. These characteristics indicate of the phage EC.W1-1 and EC.W15-3 lytic activity and ability to efficiently replicate within the host cells, making them promising candidates for combating MDR E. coli infections. Research indicates that temperatures such as 70°C can inactivate phages by denaturing their nucleic acids and proteins [ 40 ]. Extreme pH levels, between 2 and 11, can also inactivate phages, likely due to the dissociation of capsid proteins caused by the high concentrations of hydrogen and hydroxyl ions. Understanding these effects is crucial for optimizing phage therapy and ensuring effective treatments in clinical settings [ 41 ]. Specifically, EC.W1-1 and EC.W15-3 phages became inactive when incubated at pH 2–10, likely due to capsid protein dissociation from the high ion concentrations. Combination of phages and antibiotics has shown significant promise in reducing antibiotic doses and combating antibiotic resistance. In this study, phage combined with colistin, meropenem, and tigecycline showed better synergistic effects against different ST types of ESBL-producing and CREC isolates. Additionally, previous research has highlighted the synergistic effects of phage cocktails with antibiotics in lowering the minimum inhibitory concentration (MIC) values of antibiotics, display the potential of this approach in enhancing antibacterial activity and addressing antibiotic resistance [ 30 ]. Furthermore, phage combination with antibiotics has shown several potential advantages, including a broadened host range, reduced chances of emergence of phage-resistant bacteria, and more effective treatment of biofilm-producing uropathogens [ 42 ]. Phages have demonstrated effectiveness in targeting and eradicating bacterial biofilms, including those formed by E. coli , as indicated in various studies [ 43 ][ 44 ]. In this study, we investigated phage therapy against mature biofilms of different ST types of E. coli , which revealed a significant reduction in biofilm formation, highlighting the potential of phages in combating biofilm-related infections. Annotated phage genomes have identified proteins, such as glycoside hydrolase family proteins, that play crucial roles in degrading E. coli biofilms by targeting the polysaccharide matrix and breaking down the biofilm's structural integrity [ 45 ]. Moreover, the genomic analysis of Escherichia phages EC.W1-1 and EC.W15-3 has provided valuable insights into their genetic composition and evolutionary relationships, further enhancing our understanding of phage-mediated biofilm eradication mechanisms. Phylogenetic analysis revealed that phages EC.W1-1 and EC.W15-3 belong to the Straboviridae family and are closely related to other E. coli phages. These phages exhibit a strictly lytic nature due to the absence of temperate or lysogenic life cycle genes and the lack of virulence and antibiotic resistance genes, making them safe candidates for therapeutic applications. The absence of genes associated with lysogeny, virulence, or antibiotic resistance confirms their potential for targeted bacterial cell lysis without the risk of horizontal gene transfer or negative impacts on host cells, highlighting their suitability for phage therapy in combating E. coli infections. 5. Conclusion In conclusion, our comprehensive characterization of the phages EC.W1-1 and EC.W15-3 reveals their potential as effective agents against MDR E. coli . These lytic phages, identified within the Tequatrovirus genus and Straboviridae family, were confirmed through whole genome sequencing to lack virulence and antibiotic-resistance genes, making them safe candidates for therapeutic applications. Notably, they demonstrate significant lytic activity, with favourable attributes such as a short latent period, moderate burst size, and exceptional stability across diverse thermal and pH environments. These characteristics highlight their promise in phage therapy, especially for combating antibiotic-resistant strains. Future examinations should focus on validating the roles of hypothetical proteins within these phages and assessing their therapeutic efficacy in vivo , paving the way for their inclusion in strategies designed to tackle antibiotic-resistant infections. Declarations Acknowledgments This research was funded by a grant from the Korea Government National Research Foundation (Grant No.2022R1A2C2010683) and the Korea Disease Control and Prevention Agency (Grant No. 2022-ER2202-00). Conflicts of interest The authors declare no conflict of interest. Repositories: The complete genome sequences of phages EC. W1-1 and EC.W15-3 are available in GenBank with the NCBI (https://www.ncbi.nlm.nih.gov/) accession numbers PP170089 and PQ030847, respectively. References Kourtis AP, Sheriff EA, Weiner-Lastinger LM, Elmore K, Preston LE, Dudeck M, et al. Antibiotic Multidrug Resistance of Escherichia coli Causing Device- and Procedure-related Infections in the United States Reported to the National Healthcare Safety Network, 2013–2017. Clin Infect Dis 2021;73:e4552–e4559. Liu CM, Stegger M, Aziz M, Johnson TJ, Waits K, Nordstrom L, et al. Escherichia coli ST131-H22 as a foodborne uropathogen. MBio;9. Epub ahead of print 1 July 2018. DOI: 10.1128/MBIO.00470-18/SUPPL_FILE/MBO004184016SD2.XLSX. Jnani D, Ray SD. Escherichia coli Infection. Encycl Toxicol Fourth Ed Vol 1-9 2023;4:V4-357-V4-367. Kiliç SG, Öcal D, Tekeli A, Dolapçi İ. [Investigation of Virulence Factors, Phylogenetic Group Features, and the Presence of ST131 Clone in Escherichia coli Isolates, a Urinary Tract Infection Agent in Children]. Mikrobiyol Bul 2023;57 4:535–552. Taati Moghadam M, Mirzaei M, Fazel Tehrani Moghaddam M, Babakhani S, Yeganeh O, Asgharzadeh S, et al. The Challenge of Global Emergence of Novel Colistin-Resistant Escherichia coli ST131. https://home.liebertpub.com/mdr 2021;27:1513–1524. Poirel L, Madec J-Y, Lupo A, Schink A-K, Kieffer N, Nordmann P, et al. Antimicrobial Resistance in Escherichia coli . Microbiol Spectr;6. Epub ahead of print 27 July 2018. DOI: 10.1128/MICROBIOLSPEC.ARBA-0026-2017. Yan Y, Liu N, Tang Y. Recent developments in self-resistance gene directed natural product discovery. Epub ahead of print 2020. DOI: 10.1039/c9np00050j. Effendi MH, Hartadi EB, Witaningrum AM, Permatasari DA, Ugbo EN. Molecular identification of blaTEM gene of extended-spectrum beta-lactamase-producing Escherichia coli from healthy pigs in Malang district, East Java, Indonesia. J Adv Vet Anim Res 2022;9:447. Ewers C. Extended-Spectrum β -Lactamase and AmpC β -Lactamase-Producing Bacteria in Livestock Animals. Zoonoses Infect Affect Humans Anim 2023;547–578. Jariremombe RC, Jariremombe RC. Mechanisms of Antimicrobial Resistance of E. coli . Escherichia coli - Old New Insights. Epub ahead of print 17 August 2022. DOI: 10.5772/INTECHOPEN.101671. Amaro A, Leão C, Guerra V, Albuquerque T, Clemente L. Plasmid-Mediated Colistin Resistance Genes mcr-1 and mcr-4 in Multidrug-Resistant Escherichia coli Strains Isolated from a Healthy Pig in Portugal. https://home.liebertpub.com/mdr 2023;29:78–84. Ullah W, Ali S. Antimicrobial Resistance in Escherichia coli . Escherichia coli - Old New Insights. Epub ahead of print 1 March 2023. DOI: 10.5772/INTECHOPEN.101583. Kim S, Kim SH, Rahman M, Kim J. Characterization of a Salmonella Enteritidis bacteriophage showing broad lytic activity against Gram-negative enteric bacteria. J Microbiol 2018;56:917–925. Nawaz R, Husnain A, Ali M, Sajjad M, Ahad A, Shahid M, et al. Development of computationally-guided workflow for designing therapeutic phage cocktail: targeting multidrug-resistant (MDR) bacteria. Epub ahead of print 6 July 2023. DOI: 10.21203/RS.3.RS-3086398/V2. Shamsuzzamn M, Kim S, Choi Y-J, Kim B, Dahal RH, Shin M, et al. Therapeutic Phage Candidates for Targeting Prevalent Sequence Types of Carbapenem-Resistant Escherichia coli . https://home.liebertpub.com/fpd. Epub ahead of print 24 July 2024. DOI: 10.1089/FPD.2024.0023. Fabijan AP, Iredell J, Danis-Wlodarczyk K, Kebriaei R, Abedon ST. Translating phage therapy into the clinic: Recent accomplishments but continuing challenges. PLOS Biol 2023;21:e3002119. Hassan AY, Lin JT, Ricker N, Anany H. pharmaceuticals The Age of Phage: Friend or Foe in the New Dawn of Therapeutic and Biocontrol Applications? Epub ahead of print 2021. DOI: 10.3390/ph14030199. Cisek AA, Da ˛browska I, Karolina •, Gregorczyk P, Zbigniew •, Zewski W. Phage Therapy in Bacterial Infections Treatment: One Hundred Years After the Discovery of Bacteriophages. DOI: 10.1007/s00284-016-1166-x. Lin DM, Koskella B, Lin HC. Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World J Gastrointest Pharmacol Ther 2017;8:162. Hibstu Z, Belew H, Akelew Y, Mengist HM. Phage Therapy: A Different Approach to Fight Bacterial Infections. Biol Targets Ther 2022;16:173–186. Ling H, Lou X, Luo Q, He Z, Sun M, Sun J. Recent advances in bacteriophage-based therapeutics: Insight into the post-antibiotic era. Acta Pharm Sin B 2022;12:4348–4364. Yoo S, Lee K-M, Kim N, Vu TN, Abadie R, Yong D. Designing phage cocktails to combat the emergence of bacteriophage-resistant mutants in multidrug-resistant Klebsiella pneumoniae. Microbiol Spectr;12. Epub ahead of print 29 November 2023. DOI: 10.1128/SPECTRUM.01258-23. Martinez-Soto CE, McClelland M, Kropinski AM, Lin JT, Khursigara CM, Anany H. Multi-receptor phage cocktail against Salmonella enterica to circumvent phage resistance. microLife;5. Epub ahead of print 21 March 2024. DOI: 10.1093/FEMSML/UQAE003. Oromí-Bosch A, Antani JD, Turner PE. Developing Phage Therapy That Overcomes the Evolution of Bacterial Resistance. Annu Rev Virol 2023;10:503–524. Jones JD, Trippett C, Suleman M, Clokie MRJ, Clark JR. The Future of Clinical Phage Therapy in the United Kingdom. Viruses 2023;15:721–721. Hyman P. Phages for Phage Therapy: Isolation, Characterization, and Host Range Breadth. Pharm 2019, Vol 12, Page 35 2019;12:35. Rahman M, Kim S, Kim SM, Seol SY, Kim J. Characterization of induced Staphylococcus aureus bacteriophage SAP-26 and its anti-biofilm activity with rifampicin. Biofouling 2011;27:1087–1093. Tang Z, Tang N, Wang X, Ren H, Zhang C, Zou L, et al. Characterization of a lytic Escherichia coli phage CE1 and its potential use in therapy against avian pathogenic Escherichia coli infections. Front Microbiol 2023;14:1091442. Jakoči D, Un˙ E Id ¯, Moodley A. A Rapid Bacteriophage DNA Extraction Method. DOI: 10.3390/mps1030027. Liu CG, Green SI, Min L, Clark JR, Salazar KC, Terwilliger AL, et al. Phage-antibiotic synergy is driven by a unique combination of antibacterial mechanism of action and stoichiometry. MBio 2020;11:1–19. Gu Y, Xu Y, Xu J, Yu X, Huang X, Liu G, et al. Identification of novel bacteriophage vB_EcoP-EG1 with lytic activity against planktonic and biofilm forms of uropathogenic Escherichia coli. Appl Microbiol Biotechnol 2019;103:315–326. Aitken SL, Pierce VM, Pogue JM, Kline EG, Tverdek FP, Shields RK. The Growing Threat of NDM-Producing E. Coli With Penicillin-Binding Potein 3 Mutations in the United States—Is There a Potential Role for Durlobactam? Clin Infect Dis. Epub ahead of print 25 April 2024. DOI: 10.1093/CID/CIAE229. Azam AH, Sato K, Miyanaga K, Nakamura T, Ojima S, Kondo K, et al. Selective bacteriophages reduce the emergence of resistant bacteria in bacteriophage-antibiotic combination therapy. Microbiol Spectr;12. Epub ahead of print 4 June 2024. DOI: 10.1128/SPECTRUM.00427-23/SUPPL_FILE/SPECTRUM.00427-23-S0001.DOCX. Bhati T, Kumar S, Khandelwal S, Dhruw R. Bacteriophages: complementary therapy in antimicrobial resistant bacterial strains. Futur Trends Agric Eng Food Sci Vol 3 B 21 2024;96–106. Khorshidtalab M, Durukan İ, Tufekci EF, Nas SS, Abdurrahman MA, Kiliç AO. Isolation and Characterization of Lytic Bacteriophages from Wastewater with Phage Therapy Potentials Against Gram-Negative Bacteria. Eurasian J Med 2022;54:157–164. Krusche J, Beck C, Lehmann E, Gerlach D, Wolz C, Peschel A. Systematic classification of phage receptor-binding proteins predicts surface glycopolymer structure in Staphylococcus pathogens. bioRxiv 2024;2024.03.04.583386. Dhungana G, Nepal R, Houtak G, Bouras GS, Vreugde S, Malla R. Characterization and Preclinical In Silico Safety Assessment of Three Virulent Bacteriophages Targeting Carbapenem-Resistant Uropathogenic Escherichia coli. Epub ahead of print 6 November 2023. DOI: 10.20944/PREPRINTS202311.0329.V1. Abdulhussein AA, Abdulsattar BO. Identification and Characterization of a Bacteriophage with Lytic Activity against Multidrug Resistant E. coli. Maǧallaẗ ʻulūm al-mustanṣiriyyaẗ 2023;34:24–31. Khunti P, Chantakorn K, Tantibhadrasapa A, Htoo HH, Thiennimitr P, Nonejuie P, et al. A novel coli myophage and antibiotics synergistically inhibit the growth of the uropathogenic E. coli strain CFT073 in stoichiometric niches. Microbiol Spectr;11. Epub ahead of print 21 September 2023. DOI: 10.1128/SPECTRUM.00889-23. Phuong LNN, Anh LH, Huan PKN, Loc HT, Trang CTH, Mo TTH, et al. The effect of different media and temperature conditions for Salmonella bacteriophage preservation. Vet Integr Sci 2022;20:489–496. Ochirbat E, Zbonikowski R, Sulicka A, Bończak B, Bonarowska M, Łoś M, et al. Heteroaggregation of virions and microplastics reduces the number of active bacteriophages in aqueous environments. J Environ Qual 2023;52:665–677. Malik S, Nehra K, Rana JS. Bacteriophage cocktail and phage antibiotic synergism as promising alternatives to conventional antibiotics for the control of multi-drug-resistant uropathogenic Escherichia coli. Virus Res 2021;302:198496. Guo Z, Liu M, Zhang D. Potential of phage depolymerase for the treatment of bacterial biofilms. Virulence;14. Epub ahead of print 31 December 2023. DOI: 10.1080/21505594.2023.2273567. Meneses L, Brandão AC, Coenye T, Braga AC, Pires DP, Azeredo J. A systematic review of the use of bacteriophages for in vitro biofilm control. Eur J Clin Microbiol Infect Dis 2023;42:919–928. Wang D, Naqvi STA, Lei F, Zhang Z, Yu H, Ma LZ. Glycosyl hydrolase from Pseudomonas fluorescens inhibits the biofilm formation of Pseudomonads. Biofilm 2023;6:100155. Additional Declarations The authors declare no competing interests. Supplementary Files SupplimentaryPhage.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6102499","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":420566887,"identity":"beb5fded-3363-43b0-9a7e-5008516be06f","order_by":0,"name":"Md Shamsuzzaman","email":"","orcid":"https://orcid.org/0009-0009-1916-5888","institution":"Department of Biomedical Science, School of Medicine, Kyungpook National University, Daegu, Republic of Korea","correspondingAuthor":false,"prefix":"","firstName":"Md","middleName":"","lastName":"Shamsuzzaman","suffix":""},{"id":420567331,"identity":"f959eefc-5916-4ea5-b7e3-d3d14721f3e3","order_by":1,"name":"Shukho kim","email":"","orcid":"","institution":"Department of Microbiology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea","correspondingAuthor":false,"prefix":"","firstName":"Shukho","middleName":"","lastName":"kim","suffix":""},{"id":420567332,"identity":"1b15df77-fb1b-47ee-925c-cab1e0651b2b","order_by":2,"name":"Yoon-Jung Choi","email":"","orcid":"","institution":"Department of Microbiology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea","correspondingAuthor":false,"prefix":"","firstName":"Yoon-Jung","middleName":"","lastName":"Choi","suffix":""},{"id":420567333,"identity":"98bc2301-c0bf-4229-9166-08f91ee4360d","order_by":3,"name":"Jungmin Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAArUlEQVRIiWNgGAWjYDACZiBMYLAxgHITiNaSRooWkCYGhsMkaNFt5z1s8DDnvLG5RALjhx8MafkEtZgd5ktOSNx228xyRgKzZA9DjmUDYS08xgeAWmwMbiQwSDMwVBgQ0gHTcg6khfk30VqADjtgBtTCBrQlhzgtBonbko0Nzjxss+wxSCNCy/kzxpI/t9kZbjiefPjGj4pkwlqQAGMDAwNJGkbBKBgFo2AU4AQAI4k27nrQ8zsAAAAASUVORK5CYII=","orcid":"","institution":"Department of Microbiology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea","correspondingAuthor":true,"prefix":"","firstName":"Jungmin","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-02-25 07:36:11","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-6102499/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6102499/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":77225624,"identity":"52f89d15-493d-49c3-ac0d-a9c776e3871d","added_by":"auto","created_at":"2025-02-26 11:21:10","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":453230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e lytic activity of phages EC.W1-1 and EC.W15-3 against \u003cem\u003eE. coli\u003c/em\u003e ATCC25922. Killing curves show phage efficacy at various MOIs (10, 1, 0.1, 0.01, 0.001) over 24 h.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6102499/v1/244bf2b0e047d74e7b7c0e68.png"},{"id":77226649,"identity":"604915fd-fcde-45fb-9fff-b893b6ae6559","added_by":"auto","created_at":"2025-02-26 11:29:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":214974,"visible":true,"origin":"","legend":"\u003cp\u003eAdsorption rate and burst size of phages EC.W1-1 and EC.W15-3 to \u003cem\u003eE. coli\u003c/em\u003e ATCC25922. (A-B) Adsorption assay quantifies the percentage of free phages remaining after 20 min at MOI of 0.0001 and (C-D) one-step growth curve showing the latent period and burst size of four novel \u003cem\u003eE. coli\u003c/em\u003e bacteriophages in BHI medium at MOI of 0.0001. Values represent means ± standard deviations from the duplication of each treatment.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6102499/v1/ba00a469fd5f7d401a4127d7.png"},{"id":77225630,"identity":"ea1c49c6-5155-43cd-80bb-93290e4f2753","added_by":"auto","created_at":"2025-02-26 11:21:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1576918,"visible":true,"origin":"","legend":"\u003cp\u003eGenomic maps of phages EC.W1-1 and EC.W15-3. The outermost circle represents open reading frames (ORFs) encoded by the genome. Colors indicate different gene functions.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6102499/v1/fb3f84e312a9c82da83dc06c.png"},{"id":77225625,"identity":"ee24b242-6a69-4d9c-996a-5b7053a42223","added_by":"auto","created_at":"2025-02-26 11:21:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":888277,"visible":true,"origin":"","legend":"\u003cp\u003ePhage-mediated biofilm Reduction in LB Medium and Serum. (A-B) Phages EC.W1-1 and EC.W15-3 effectively reduced biofilm biomass in LB medium. (B-C) Importantly, biofilm reduction was also observed in the presence of 20% serum. Experiments were performed in duplicate (n=3 per condition).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6102499/v1/6d76bdf93b54a6cf42618cad.png"},{"id":77226882,"identity":"af21cf94-ec46-41c9-8c66-4cc580f83a60","added_by":"auto","created_at":"2025-02-26 11:37:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4473071,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6102499/v1/4736c06a-30cf-4f45-b940-39a191986ed1.pdf"},{"id":77225628,"identity":"06096db8-0799-4825-8496-869c5049aba7","added_by":"auto","created_at":"2025-02-26 11:21:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2028928,"visible":true,"origin":"","legend":"","description":"","filename":"SupplimentaryPhage.docx","url":"https://assets-eu.researchsquare.com/files/rs-6102499/v1/21baa05d3d0158fdffb32b36.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eCharacterization of Novel \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eStraboviridae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e Phages Enhancing Antibiotic Efficacy and Biofilm Inhibition Against MDR \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia coli\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003e \u003cem\u003eEscherichia coli\u003c/em\u003e has become a primary concern in healthcare settings due to its multidrug resistance, causing many infections [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This pathogen is a major cause of various infections in hospitalized patients, such as urinary tract infections (UTIs) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], nosocomial pneumonia, cholangitis, peritonitis, cellulitis, cholecystitis, infectious arthritis, osteomyelitis, diarrhea, bloodstream infections, and neonatal meningitis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e][\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The rise in MDR \u003cem\u003eE. coli\u003c/em\u003e infections and limited antibiotic efficacy present significant challenges in antimicrobial resistance and disease spread in human and veterinary medicine [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003eE. coli\u003c/em\u003e is typically sensitive to many antibiotics but can acquire resistance genes via horizontal gene transfer [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eE. coli\u003c/em\u003e that produce \u003cem\u003eβ\u003c/em\u003e-lactamases are a significant and widespread source of antimicrobial resistance [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For example, \u003cem\u003ebla\u003c/em\u003eTEM-1, commonly found in \u003cem\u003eE. coli\u003c/em\u003e from animals, produces narrow-spectrum \u003cem\u003eβ\u003c/em\u003e-lactamases that inactivate penicillin and aminopenicillins [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e][\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Recently, genes responsible for extended-spectrum \u003cem\u003eβ\u003c/em\u003e-lactamases (ESBLs) and AmpC \u003cem\u003eβ\u003c/em\u003e-lactamases have been identified in \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The emergence of resistance mechanisms in \u003cem\u003eE. coli\u003c/em\u003e involves various mechanisms beyond \u003cem\u003eβ\u003c/em\u003e-lactamases, including, 16S rRNA methylases, carbapenem resistance, plasmid-mediated quinolone resistance, and \u003cem\u003emcr\u003c/em\u003e genes. [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e][\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, mobile genetic elements like transposons are crucial in disseminating resistance genes among bacterial populations, contributing to the global challenge of MDR \u003cem\u003eE. coli\u003c/em\u003e infections [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnderstanding these genetic mechanisms is essential for developing effective treatment strategies and combating the spread of antimicrobial resistance in \u003cem\u003eE. coli\u003c/em\u003e. However, there is limited information on specific antimicrobial therapies for infections caused by various \u003cem\u003eE. coli\u003c/em\u003e clones. This situation highlights the urgent need for innovative therapeutic approaches and the development of new antimicrobial agents to combat these infections effectively [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Phages have emerged as a promising alternative to combat MDR bacterial infections. Phage cocktails, combining multiple phages, have shown efficacy in targeting MDR pathogens like \u003cem\u003eEscherichia coli, Acinetobacter baumannii, Pseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e][\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Phage therapy, an age-old approach, declined with the advent of antibiotics but has regained popularity due to concerns about antibiotic resistance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Clinical successes in the United States, Georgia, Poland, and Russia highlight its potential as an alternative to antibiotics [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e][\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Phages specificity against target bacteria enhances their efficacy compared to broad-spectrum antibiotics [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Their natural origin and narrow therapeutic window make them a safe substitute for antibiotics [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the emergence of phage-resistant mutants poses a significant challenge.\u003c/p\u003e \u003cp\u003eResearchers have explored the development of phage cocktails and phage-antibiotic combinations to address bacterial resistance. Studies have shown that the use of phage cocktails has been found to significantly reduce the growth of bacteria and inhibit the formation of phage-resistant mutants [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Furthermore, combining phages that target different host receptors in a cocktail has been shown to reduce the emergence of phage resistance during biocontrol applications, highlighting the potential of this approach to maintain the effectiveness of phage therapy [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. To ensure the safety and efficacy of these cocktails, ongoing efforts focus on isolating new phages and conducting comprehensive \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e experiments for characterization [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e][\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we successfully isolated two \u003cem\u003eE. coli\u003c/em\u003e phages from hospital sewage water in Daegu, South Korea, demonstrating their efficacy against different STs types of MDR \u003cem\u003eE.coli\u003c/em\u003e. This study identified effective phages against ESBL-producing \u003cem\u003eE.coli\u003c/em\u003e and CREC. These findings have important implications for the scientific community, providing insights into the development of novel strategies for combating MDR \u003cem\u003eE. coli\u003c/em\u003e. Overall, this study will help advance researchers\u0026rsquo; understanding of phage therapy and present the development of new treatment options for antibiotic-resistant infections.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. \u003cem\u003eE. coli\u003c/em\u003e isolates used in this study\u003c/h2\u003e \u003cp\u003eIn this research, 60 ESBL-producing and CREC isolates were collected from the Pathogen Culture Collection at Kyungpook National University Hospital. Standard strains \u003cem\u003eE. coli\u003c/em\u003e ATCC25922 was purchased form the American Type Culture Collection for this study. Initially, the bacteria were grown on 5% sheep blood agar plates at 37\u0026deg;C for 24 h, followed by culture in BHI media. For future experiments, they were preserved at \u0026minus;\u0026thinsp;70\u0026deg;C in 50% glycerol stocks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Phage isolation, purification, and preparation\u003c/h2\u003e \u003cp\u003ePhage isolation, purification, and preparation were performed using modified liquid and agar overlay methods [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e][\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Initially, host bacteria were grown in BHI medium, while sewage samples from Kyungpook National University Hospital were processed by centrifugation at 12,000\u0026times;g for 10 min to remove debris. The filtered sewage was used to infect \u003cem\u003eE. coli\u003c/em\u003e cultures at their early exponential growth phase. After incubating overnight at 30\u0026deg;C with shaking, the mixture was kept at 4\u0026deg;C for 48 h. The supernatants were then centrifuged and filtered. Phage titers were measured using the double-layer method. A single plaque was isolated, resuspended in SM buffer, and purified by vortexing, centrifugation, and filtration. This purified phage suspension was then mixed with \u003cem\u003eE. coli\u003c/em\u003e and incubated at 30\u0026deg;C with shaking at 150 rpm for 16 h. Further purification involved repeated treatments with 10% chloroform, gradually increasing the volume each time. Finally, the phage solution was mixed with 15% glycerol and stored at \u0026minus;\u0026thinsp;70\u0026deg;C for future use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Determination of lytic activity of phages against 60 MDR \u003cem\u003eE. coli\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eA total of 60 \u003cem\u003eE. coli\u003c/em\u003e isolates, representing 14 different ST types, were evaluated for their susceptibility to phage lytic activity using spot test assays, following the method outlined by Kim Shukho (2018)[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. To prepare the bacterial lawn, 10 ml of 0.75% soft agar with 100 \u0026micro;l of bacterial culture (OD-0.5\u003csub\u003enm\u003c/sub\u003e) was poured onto BHA agar plate. Once the overlay solidified, a 15 \u0026micro;l aliquot of the phage solution, with a concentration of approximately 10\u003csup\u003e11\u0026ndash;13\u003c/sup\u003e PFU/ml, was directly spotted on the bacterial lawn. The plates were dried and incubated overnight at 37\u0026deg;C. The bacteria's susceptibility was determined by clear lytic zones on the plates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Measurement of adsorption rate and burst size of two novel \u003cem\u003eE. coli\u003c/em\u003e phages\u003c/h2\u003e \u003cp\u003eThe phage adsorption rate and burst size of phages were determined by the method of Marzia Rahman (2011)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For adsorption rate, \u003cem\u003eE. coli\u003c/em\u003e is infected with phages at a low MOI (0.0001) and incubated at room temperature. Phage samples were taken at intervals (0, 1, 3, 5, 10, 12, 15, 20, and 25 min) to measure unabsorbed phage titers via plaque assays. Burst size was determined through a single growth curve by infecting \u003cem\u003eE. coli\u003c/em\u003e at the same MOI, allowing adsorption for 30 min at 4\u0026deg;C, and sampling every 5 min for phage counts. This method is repeated three times independently.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Temperature and pH stability assays\u003c/h2\u003e \u003cp\u003eThe thermal and pH stability of phages were tested by exposing them to different temperatures and pH levels. For thermal stability, phages in SM buffer were exposed to temperatures from \u0026minus;\u0026thinsp;20\u0026deg;C to 80\u0026deg;C. For pH stability, phages were incubated in buffers with pH ranging from 2.0 to 10 for 18 h at room temperature. Phage survival was assessed by comparing the percentage of surviving phages to those stored in SM buffer at pH 7.4 and 4\u0026deg;C. Survival rates were calculated using a double-layer plaque assay and counting PFUs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. \u003cem\u003eIn vitro\u003c/em\u003e bactericidal activity under different MOIs\u003c/h2\u003e \u003cp\u003eThe experiments involved culturing phages with \u003cem\u003eE. coli\u003c/em\u003e in BHI medium at various MOIs (10, 1, 0.1, 0.01, and 0.001) and incubating at 37\u0026deg;C with gentle shaking. Absorbance at 600 nm was measured using a UV-vis spectrophotometer (Molecular Devices, LLC, San Jose, CA, USA) in a 96-well plate to assess optical density. These measurements were taken every hour during the first 10 h and at 24 h. A bacterial culture without phages served as the positive control, while BHI medium alone was the negative control. Each sample was tested in triplicate [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Genome sequencing, annotation, and genome analysis\u003c/h2\u003e \u003cp\u003ePhage DNA extraction was performed using the phenol-chloroform procedure described by Džiuginta Jakočiūnė (2018) )[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For next-generation sequencing (NGS), phage genomic DNA sequencing was performed on an Illumina Miseq platform in San Diego, CA, USA. Sequencing reads were assembled using the Celemics pipeline in South Korea (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://btseq.celemics.com/\u003c/span\u003e\u003cspan address=\"https://btseq.celemics.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Genome sequence similarities were analyzed through alignment using BLASTn (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://blast.ncbi.nlm.nih.gov/Blast.cgi\u003c/span\u003e\u003cspan address=\"https://blast.ncbi.nlm.nih.gov/Blast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The RAST server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://rast.nmpdr.org/rast.cgi\u003c/span\u003e\u003cspan address=\"https://rast.nmpdr.org/rast.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was utilized to predict open reading frames (ORFs), with verification via the NCBI database. Gene function maps were created using a lab-developed custom program and enhanced with Geneious Prime 2023.2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.geneious.com/\u003c/span\u003e\u003cspan address=\"https://www.geneious.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). tRNAscan-SE v.2.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://lowelab.ucsc.edu/cgi-bin/tRNAscan-SE2.cgi\u003c/span\u003e\u003cspan address=\"http://lowelab.ucsc.edu/cgi-bin/tRNAscan-SE2.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was employed to predict tRNA, while ResFinder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.cbs.dtu.dk/services/ResFinder/\u003c/span\u003e\u003cspan address=\"https://cge.cbs.dtu.dk/services/ResFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and VirulenceFinder (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cge.cbs.dtu.dk/services/VirulenceFinder/\u003c/span\u003e\u003cspan address=\"https://cge.cbs.dtu.dk/services/VirulenceFinder/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) identified drug resistance and virulence genes. Phylogenetic trees, based on the phage proteome and circular tree, were developed using VIPTree (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.genome.jp/viptree/\u003c/span\u003e\u003cspan address=\"https://www.genome.jp/viptree/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The VICTOR tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ggdc.dsmz.de/submit_victor_job.php\u003c/span\u003e\u003cspan address=\"https://ggdc.dsmz.de/submit_victor_job.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) constructed a complete genome phylogenetic tree based on whole-genome sequences of isolated phages. Full genome sequences of isolated phages were aligned with others using the BLASTn tool in the NCBI database. Genome sequence similarities were visualized with the Mauve algorithm (v2.3.1) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://darlinglab.org/mauve/mauve.html\u003c/span\u003e\u003cspan address=\"https://darlinglab.org/mauve/mauve.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), comparing isolated phages to their nearest E. coli phage relatives.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Combination effect of phage and antibiotic targeting ESBL-producing and CREC \u003cem\u003eE. coli\u003c/em\u003e isolates.\u003c/h2\u003e \u003cp\u003ePhage antibiotic synergy testing was carried out in LB medium following the method by Carmen Gu Liu [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. ESBL-producing and CREC isolates were cultured in BHI for 6 h. After centrifugation and washing, the cell pellets were resuspended in BHI medium and adjusted to a concentration of approximately 1 \u0026times; 10\u003csup\u003e9\u003c/sup\u003e CFU/ml (OD600nm\u0026thinsp;=\u0026thinsp;1). Then, 100 \u0026micro;l of this suspension was added to each well of a microtiter plate, which contained a checkerboard of phage and antibiotic concentrations (50 \u0026micro;l of each antimicrobial). The optical density at 600 nm (OD600nm) was measured every hour over a 24-h period at 37\u0026deg;C, using continuous shaking in a Hercuvan Lab Systems thermo shaker incubator (Hercuvan Lab Systems, London).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Biofilm formation assay\u003c/h2\u003e \u003cp\u003eTo start the biofilm experiment, \u003cem\u003eE. coli\u003c/em\u003e isolates were cultured overnight in LB medium at 37\u0026deg;C with shaking. Subsequently, 200 \u0026micro;l of a bacterial cell suspension (10\u003csup\u003e7\u003c/sup\u003e CFU/ml) was added to each well of a 96-well plate and incubated at 37\u0026deg;C for 24 h. Then, 100 \u0026micro;l of the culture medium for the sample wells was replaced with 100 \u0026micro;l of LB-containing phages (10\u003csup\u003e9\u003c/sup\u003e PFU/ml), while control wells received plain LB medium. The plate was incubated again at 37\u0026deg;C for an additional 24 h. After 48 h of biofilm growth, the liquid medium was carefully removed by aspiration, and the wells were washed twice with 1\u0026times; PBS. The biofilms were stained with 200 \u0026micro;l of 0.1% crystal violet for 20 min at 37\u0026deg;C, followed by three washes with 1\u0026times; PBS to remove excess stain. 200 \u0026micro;l of 95% ethanol was added and incubated to solubilize the crystal violet for 10 to 15 min. For biofilm reduction tests using serum, we used 20% serum in LB media instead of regular media. Finally, a plate reader quantified biofilm formation by measuring absorbance at 570 nm [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Bacteriophage isolation, host spectrum analysis and determination of antibiotic resistance profile of 60 MDR-\u003cem\u003eE. coli\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eThis study isolated phages EC.W1-1 and EC.W15-3 from hospital sewage samples using \u003cem\u003eE. coli\u003c/em\u003e ATCC25922 as the host. Spot test results showed that both phages exhibited high lytic activity against highly virulent MDR \u003cem\u003eE. coli\u003c/em\u003e. Phage EC.W1-1 lysed 51.6% (31/60) of the isolates, including 57.6% (15/26) of ESBL-producing and 43.7% (21/48) of CREC isolates. Phage EC.W15-3 lysed 33.3% (20/60) of the isolates, including 30.7% (8/26) of ESBL-producing and 31.2% (15/48) of CREC isolates. The susceptibility of the two novel bacteriophages against various ST types of MDR \u003cem\u003eE. coli\u003c/em\u003e was tested on 14 different ST types. Phage EC.W1-1 was 100% effective against ST648, ST2311, ST405, ST13003, ST167, and ST73, while phage EC.W15-3 was 100% effective against ST73, ST13003, and ST167. Against ST131, phage EC.W1-1 showed 88.8% (16/18) susceptibility, and phage EC.W15-3 showed 66.6% (12/18) susceptibility. However, against ST410, the effectiveness dropped to 19.2% (5/26) for phage EC.W1-1 and 15.3% (4/26) for phage EC.W15-3 (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and (Table S.1) According to the antibiotic resistance analysis of 60 clinically isolated \u003cem\u003eE. coli\u003c/em\u003e isolates, all were multidrug-resistant. Among these, 80.4% were identified as CREC, and 43.33% of the isolates possessed ESBL, as previously determined by the Microbiology Laboratory at Kyungpook National University [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eHost range of phages EC.W1-1 and EC.W15-3 against 14 different ST types of 60 ESBL-producing and CREC \u003cem\u003eE.coli.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eST type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e isolates\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eESBL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"3\" nameend=\"c9\" namest=\"c7\"\u003e \u003cp\u003eCREC\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΦ E.C-W1-1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eΦ E.C-W15-3\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eIMI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eMEM\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P04004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P03442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P01217\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e 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align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e607\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06211\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e13003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P00235\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1487\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P00237\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e 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namest=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e7962\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P03386\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e 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align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P07282\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P07288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P01569\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P03452\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e 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colname=\"c9\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P00128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P00238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P02048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e 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align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P02511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P05702\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P05883\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P01583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P05295\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06393\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e 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align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06786\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06244\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e 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align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06388\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P07290\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P05685\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e 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colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P03733\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P03950\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06241\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06779\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e+\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P01261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P07377\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P06718\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e206\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P00137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P00139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e+\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c7\" namest=\"c5\"\u003e \u003cp\u003eN/A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eR\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"9\"\u003ePhage susceptibility is represented by symbols: S (Susceptible), R (Resistant), I (Intermediate), + (ESBL Present)/ Susceptible, - (ESBL Absent)/ Resistant, and N/A (Not Available)\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Biological and morphological characterization\u003c/h2\u003e \u003cp\u003eThe study examined the lytic activity, growth patterns, and stability of phages EC.W1-1 and EC.W15-3 under varying temperatures and pH levels. Both phages showed significant lytic activity against \u003cem\u003eE. coli\u003c/em\u003e ATCC25922 at 37\u0026deg;C over 24 h at MOIs of 10 and 0.001 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Phages suppressed \u003cem\u003eE. coli\u003c/em\u003e growth within the first 10 h at all MOIs, evident from reduced OD600nm values. After 10 h, OD600nm values gradually rose, peaking at 24 h, with bacterial counts exceeding 10\u003csup\u003e6\u003c/sup\u003e CFU/ml. A notable difference between MOIs 10 and 0.001 and the positive control indicated the phages ongoing impact. Combining EC.W1-1 and EC.W15-3 showed no synergistic effect against \u003cem\u003eE. coli\u003c/em\u003e ATCC25922. Additionally, when phages EC.W1-1 and EC.W15-3 were tested against \u003cem\u003eE. coli\u003c/em\u003e KBN7288, it was observed that the MOIs of 10 and 0.1 facilitated enhanced lytic activity. (Figure S.1).\u003c/p\u003e \u003cp\u003eThe phage adsorption assay showed that over 90% of the phages could adsorb to \u003cem\u003eE. coli\u003c/em\u003e ATCC25922 within the first 10\u0026ndash;12 min. Among the tested phages, EC.W15-3 exhibited the fastest adsorption, with 71% attaching within 5 min, compared to 56% for EC.W1-1(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The one-stop growth curve analysis revealed that the phages have a latent period of approximately 10\u0026ndash;15 min, followed by a rapid release of virus particles. The final titer ranged from 10\u003csup\u003e7.5\u003c/sup\u003e to 10\u003csup\u003e8\u003c/sup\u003e PFU/mL after a burst phase lasting 35\u0026ndash;45 min, with an average burst size of 64\u0026ndash;83 phage particles per cell (PFUs/cell), indicating efficient replication and production of new phage particles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The phages demonstrated thermal stability between 20\u0026deg;C and 70\u0026deg;C for 60 min. However, their titers significantly decreased at 70\u0026deg;C and were completely inactivated at 80\u0026deg;C (Figure S.2). They also showed stability across pH levels from 2 to 10 within 4 h, with optimal stability observed at pH 6 to 8 (Figure S.3). After 18 h of incubation at 37\u0026deg;C on a double-layer agar plate, the phages formed clear plaques measuring about 1\u0026ndash;2 mm in diameter. Transmission electron microscopy (TEM) images showed that phages EC.W1-1 and EC.W15-3 possessed an icosahedral head with a diameter of 105\u0026thinsp;\u0026plusmn;\u0026thinsp;5 to 115\u0026thinsp;\u0026plusmn;\u0026thinsp;5 nm and a contractile tail approximately 100\u0026thinsp;\u0026plusmn;\u0026thinsp;2 to 110\u0026thinsp;\u0026plusmn;\u0026thinsp;2 nm in length [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Phylogenetic analysis of phages\u003c/h2\u003e \u003cp\u003eThe phylogenetic relationship of the new phages was determined by analyzing their whole genome sequences against the closest known \u003cem\u003eE. coli\u003c/em\u003e phages. The analysis revealed that EC.W1-1 and EC.W15-3 exhibited the highest DNA sequence similarities to the \u003cem\u003eTequatrovirus\u003c/em\u003e genus in the \u003cem\u003eStraboviridae\u003c/em\u003e family, and phages Escherichia phage YUEEL01 (complete genome) and Escherichia phage D5505 (complete genome). Escherichia phage teqhad (complete genome) and Escherichia phage PE37 (complete genome) (Figure S.4). The query coverage and percentage identity were recorded as 98/98.27%,97.92%, 96.45% and 96.37% respectively for these isolates (Table S.2).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Genomic feature of two novel \u003cem\u003eE. coli\u003c/em\u003e phage\u003c/h2\u003e \u003cp\u003eThe comprehensive analysis of the whole-genome sequences for bacteriophages EC.W1-1 and EC.W15-3 reveals they each possess circular double-stranded DNA. The genome sizes are 37,736 bp for EC.W1-1 and 123,792 bp for EC.W15-3. The G\u0026thinsp;+\u0026thinsp;C content of these phages is measured at 35.61% for EC.W1-1 and 37.27% for EC.W15-3. These genome sequences have been cataloged in the NCBI GenBank under accession numbers PP170089 and PQ030847, respectively. For EC.W1-1, the genome annotation identified 93 protein-coding genes, while EC.W15-3 has 202 such genes. These genes are entirely located on the positive strand. The genomes contain various structural and assembly genes, including those necessary for terminase subunits, capsid construction, assembly processes, and tail fiber formation. In addition, both phages have genes responsible for DNA replication and transcription processes, such as DNA helicase, helicase loader, DNA polymerase, DNA topoisomerase, and RNA polymerase (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Using ResFinder, no antimicrobial resistance or virulence genes were detected. Nevertheless, the presence of particular genes like holin and several structural proteins indicates potential virulence for both phages. The absence of lysogeny-associated genes such as integrase, excisionase, or transposase signifies that the phages do not integrate into host genomes. When aligned using the MAUVE tool, over 95% of the genome sequences were conserved, showing close evolutionary relations with common ancestry. However, some small genetic variations were observed, indicating both conserved and unique regions, which point to potential functional differences and distinctive capabilities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.5. Assessment of phage-antibiotic synergy (PAS) effect on selected ESBL-producing and CREC isolates\u003c/h2\u003e \u003cp\u003eThis study investigated synergistic interactions between phages and antibiotics focusing on three antibiotics colistin, meropenem, and tigecycline. The combination of antibiotics with phages EC.W1-1 and EC.W15-3 generally reduced the MICs, indicating an enhanced bactericidal effect (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and (Figure S.5) Colistin's efficacy improved significantly for \u003cem\u003eE. coli\u003c/em\u003e KBN 7288 and \u003cem\u003eE. coli\u003c/em\u003e KBN4004 by combining both phages, while \u003cem\u003eE. coli\u003c/em\u003e KBN0128 showed no change of MIC. The median MIC value of colistin alone (n\u0026thinsp;=\u0026thinsp;4) was 18 \u0026micro;g/mL and in combination with phage EC.W1-1, it was reduced to 4.5 \u0026micro;g/mL (13.5-fold reduction). The combination of colistin and EC.W15-3 resulted in a 9.5-fold reduction from 18 \u0026micro;g/mL to 8.5 \u0026micro;g/mL. Meropenem displayed substantial improvement, especially notable in \u003cem\u003eE. coli\u003c/em\u003e KBN0128 and \u003cem\u003eE. coli\u003c/em\u003e KBN4004 when combined with EC.W1-1, reducing the MIC from 128 \u0026micro;g/mL to 32 \u0026micro;g/mL and from 8 \u0026micro;g/mL to 2 \u0026micro;g/mL, respectively. The combination of phage EC.W15-3 and meropenem reduced the MIC for \u003cem\u003eE. coli\u003c/em\u003e KBN7288 from 1 \u0026micro;g/mL to 0.25 \u0026micro;g/mL. Similarly, in tigecycline, only 33.3% of isolates (n\u0026thinsp;=\u0026thinsp;4) had MIC values of \u0026le;\u0026thinsp;2 \u0026micro;g/mL. However, after combining phage EC.W1-1 and tigecycline, 75% of isolates showed MIC values of \u0026le;\u0026thinsp;2 \u0026micro;g/mL, indicating a synergistic effect. The combination of phage EC.W15-3 and tigecycline reduced the median MIC from 4 \u0026micro;g/mL to 1.5 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eReduction in MIC values due to phage antibiotics combination against different multidrug resistant sequence types.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eName of Antibiotic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eName of \u003cem\u003eE. coli\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eST type\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMIC\u003c/p\u003e \u003cp\u003e\u0026micro;g/ml\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCombination MIC of φ-EC.W1-1 and antibiotic(\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCombination MIC of φ-EC.W15-3 and antibiotic(\u0026micro;g/ml)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eFIC-Value\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCombination MIC φ-EC.W1-1 and antibiotic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCombination MIC φ-EC.W15-3 and antibiotic\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eColistin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P07288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P00128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P04004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P05617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eMeropenem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P07288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P00128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P05617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P04004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eTigecycline\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P07288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P00128\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST131\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P05617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eKBN10P04004\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eST-648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Effect of phages on the biofilm biomass of \u003cem\u003eE. coli\u003c/em\u003e isolates\u003c/h2\u003e \u003cp\u003eThis research focused on the effects of bacteriophages on the development of biofilms in \u003cem\u003eE. coli\u003c/em\u003e ATCC25922 and four distinct ESBL-producing and CREC isolates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Applying phages EC.W1-1 and EC.W15-3 to cultures of multidrug-resistant \u003cem\u003eE. coli\u003c/em\u003e resulted in a significant decrease in biofilm biomass compared to untreated samples. Specifically, phage EC.W1-1 reduced biofilm biomass by an average of 59.05% in \u003cem\u003eE. coli\u003c/em\u003e ATCC25922, 54.03% in \u003cem\u003eE. coli\u003c/em\u003e MG1655, 34.99% in \u003cem\u003eE. coli\u003c/em\u003e KBN7288, 54.19% in \u003cem\u003eE. coli\u003c/em\u003e KBN7282, and 23.86% in \u003cem\u003eE. coli\u003c/em\u003e KBN4004. In contrast, phage EC.W15-3 led to an average biofilm reduction of 55.67% for \u003cem\u003eE. coli\u003c/em\u003e ATCC25922, 48.61% for \u003cem\u003eE. coli\u003c/em\u003e MG1655, 28.29% for \u003cem\u003eE. coli\u003c/em\u003e KBN7288, 48.63% for \u003cem\u003eE. coli\u003c/em\u003e KBN7282, and 36.69% for \u003cem\u003eE. coli\u003c/em\u003e KBN4004. The reference strain used in this study was \u003cem\u003eE. coli\u003c/em\u003e MG1655. Similarly, 20% serum to the medium significantly increased the biofilm formation. However, phages EC.W1-1 and EC.W15-3 demonstrated significant biofilm reduction against \u003cem\u003eE. coli\u003c/em\u003e ATCC25922 and \u003cem\u003eE.coli\u003c/em\u003e KBN7288, achieving 49.41% and 45.70% reduction, respectively, for \u003cem\u003eE.coli\u003c/em\u003e ATCC 25922 and 27.92% and 19.08% reduction for \u003cem\u003eE.coli\u003c/em\u003e KBN7288.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe rise of MDR \u003cem\u003eE. coli\u003c/em\u003e infections poses a significant public health challenge, necessitating innovative therapeutic strategies to combat this growing threat [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Phage therapy, utilizing phages to target and destroy bacterial pathogens selectively, has emerged as a promising strategy to combat MDR infections. To ensure the efficacy and safety of phage therapy, selecting phages with a broad host range and devoid of toxin genes or lysogenic capabilities is crucial [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Recent studies have demonstrated the potential of phage therapy in treating MDR \u003cem\u003eE. coli\u003c/em\u003e, highlighting its specificity and efficacy in combating ESBL-producing and CREC infections[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aimed to isolate and characterize two \u003cem\u003eStraboviridae\u003c/em\u003e phages that target 14 distinct ST types of 60 \u003cem\u003eE. coli\u003c/em\u003e. The \u003cem\u003eE. coli\u003c/em\u003e isolates were found 100% MDR with 80.4% being CREC isolates. Additionally, 43.33% of the isolates carried extended \u003cem\u003eβ\u003c/em\u003e -lactamase [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Phages are very specific, targeting only one or a few strains of bacteria. This specificity makes it unlikely that harmless or useful bacteria will be killed when fighting an infection, unlike traditional antibiotics which have a more wide-ranging effect [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The lysis ability of phages 33.3\u0026ndash;51.6% revealed phages have the potential to be a candidate for phage therapy [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Phage receptor binding proteins exhibit specificity in their interaction with bacterial cell receptors, influencing the bacterial host range of the phages. This specificity ensures that phages can infect only certain bacteria bearing receptors to which they can bind, thereby determining the phage's host range [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to this study phage EC.W1-1 and EC.W15-3 recorded a latent period of 10\u0026ndash;15 min accompanied by a 64\u0026ndash;84 PFU/cell burst size. Phages with a short latent period and moderate burst size are crucial for effective therapeutic agents against \u003cem\u003eE. coli\u003c/em\u003e infections. Previous studies have reported phages with varying burst sizes, such as 74\u0026ndash;127 particles/cell [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], 93 particles/cell [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], and about 139 particles/cell [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, the latent periods observed in these studies ranged from 10\u0026ndash;20 min [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], 20 min for 90% of to adsorb [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], and 15\u0026ndash;20 min [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. These characteristics indicate of the phage EC.W1-1 and EC.W15-3 lytic activity and ability to efficiently replicate within the host cells, making them promising candidates for combating MDR \u003cem\u003eE. coli\u003c/em\u003e infections.\u003c/p\u003e \u003cp\u003eResearch indicates that temperatures such as 70\u0026deg;C can inactivate phages by denaturing their nucleic acids and proteins [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Extreme pH levels, between 2 and 11, can also inactivate phages, likely due to the dissociation of capsid proteins caused by the high concentrations of hydrogen and hydroxyl ions. Understanding these effects is crucial for optimizing phage therapy and ensuring effective treatments in clinical settings [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Specifically, EC.W1-1 and EC.W15-3 phages became inactive when incubated at pH 2\u0026ndash;10, likely due to capsid protein dissociation from the high ion concentrations.\u003c/p\u003e \u003cp\u003eCombination of phages and antibiotics has shown significant promise in reducing antibiotic doses and combating antibiotic resistance. In this study, phage combined with colistin, meropenem, and tigecycline showed better synergistic effects against different ST types of ESBL-producing and CREC isolates. Additionally, previous research has highlighted the synergistic effects of phage cocktails with antibiotics in lowering the minimum inhibitory concentration (MIC) values of antibiotics, display the potential of this approach in enhancing antibacterial activity and addressing antibiotic resistance [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Furthermore, phage combination with antibiotics has shown several potential advantages, including a broadened host range, reduced chances of emergence of phage-resistant bacteria, and more effective treatment of biofilm-producing uropathogens [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhages have demonstrated effectiveness in targeting and eradicating bacterial biofilms, including those formed by \u003cem\u003eE. coli\u003c/em\u003e, as indicated in various studies [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e][\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In this study, we investigated phage therapy against mature biofilms of different ST types of \u003cem\u003eE. coli\u003c/em\u003e, which revealed a significant reduction in biofilm formation, highlighting the potential of phages in combating biofilm-related infections. Annotated phage genomes have identified proteins, such as glycoside hydrolase family proteins, that play crucial roles in degrading \u003cem\u003eE. coli\u003c/em\u003e biofilms by targeting the polysaccharide matrix and breaking down the biofilm's structural integrity [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Moreover, the genomic analysis of Escherichia phages EC.W1-1 and EC.W15-3 has provided valuable insights into their genetic composition and evolutionary relationships, further enhancing our understanding of phage-mediated biofilm eradication mechanisms.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis revealed that phages EC.W1-1 and EC.W15-3 belong to the \u003cem\u003eStraboviridae\u003c/em\u003e family and are closely related to other \u003cem\u003eE. coli\u003c/em\u003e phages. These phages exhibit a strictly lytic nature due to the absence of temperate or lysogenic life cycle genes and the lack of virulence and antibiotic resistance genes, making them safe candidates for therapeutic applications. The absence of genes associated with lysogeny, virulence, or antibiotic resistance confirms their potential for targeted bacterial cell lysis without the risk of horizontal gene transfer or negative impacts on host cells, highlighting their suitability for phage therapy in combating \u003cem\u003eE. coli\u003c/em\u003e infections.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, our comprehensive characterization of the phages EC.W1-1 and EC.W15-3 reveals their potential as effective agents against MDR \u003cem\u003eE. coli\u003c/em\u003e. These lytic phages, identified within the \u003cem\u003eTequatrovirus\u003c/em\u003e genus and \u003cem\u003eStraboviridae\u003c/em\u003e family, were confirmed through whole genome sequencing to lack virulence and antibiotic-resistance genes, making them safe candidates for therapeutic applications. Notably, they demonstrate significant lytic activity, with favourable attributes such as a short latent period, moderate burst size, and exceptional stability across diverse thermal and pH environments. These characteristics highlight their promise in phage therapy, especially for combating antibiotic-resistant strains. Future examinations should focus on validating the roles of hypothetical proteins within these phages and assessing their therapeutic efficacy \u003cem\u003ein vivo\u003c/em\u003e, paving the way for their inclusion in strategies designed to tackle antibiotic-resistant infections.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by a grant from the Korea Government National Research Foundation (Grant No.2022R1A2C2010683) and the Korea Disease Control and Prevention Agency (Grant No. 2022-ER2202-00).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRepositories:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete genome sequences of phages EC. W1-1 and EC.W15-3 are available in GenBank with the NCBI (https://www.ncbi.nlm.nih.gov/) accession numbers PP170089 and PQ030847, respectively.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKourtis AP, Sheriff EA, Weiner-Lastinger LM, Elmore K, Preston LE, Dudeck M, et al. Antibiotic Multidrug Resistance of \u003cem\u003eEscherichia coli\u003c/em\u003e Causing Device- and Procedure-related Infections in the United States Reported to the National Healthcare Safety Network, 2013\u0026ndash;2017. Clin Infect Dis 2021;73:e4552\u0026ndash;e4559.\u003c/li\u003e\n\u003cli\u003eLiu CM, Stegger M, Aziz M, Johnson TJ, Waits K, Nordstrom L, et al. \u003cem\u003eEscherichia coli \u003c/em\u003eST131-H22 as a foodborne uropathogen. MBio;9. Epub ahead of print 1 July 2018. DOI: 10.1128/MBIO.00470-18/SUPPL_FILE/MBO004184016SD2.XLSX.\u003c/li\u003e\n\u003cli\u003eJnani D, Ray SD. \u003cem\u003eEscherichia coli\u003c/em\u003e Infection. 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A systematic review of the use of bacteriophages for in vitro biofilm control. Eur J Clin Microbiol Infect Dis 2023;42:919\u0026ndash;928.\u003c/li\u003e\n\u003cli\u003eWang D, Naqvi STA, Lei F, Zhang Z, Yu H, Ma LZ. Glycosyl hydrolase from \u003cem\u003ePseudomonas\u003c/em\u003e fluorescens inhibits the biofilm formation of Pseudomonads. Biofilm 2023;6:100155.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Department of Microbiology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Multidrug-Resistant Escherichia coli, phage therapy, genome analysis, phage-antibiotic synergy, biofilm","lastPublishedDoi":"10.21203/rs.3.rs-6102499/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6102499/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultidrug-resistant \u003cem\u003eEscherichia coli\u003c/em\u003e (MDR-\u003cem\u003eE. coli\u003c/em\u003e) is a significant public health concern due to its resistance to multiple antibiotics, which complicates infection treatment. Phages are gaining attention for their specific lytic activity against pathogenic bacteria. This study aimed to isolate and characterize lytic bacteriophages designated as EC.W1-1 and EC.W15-3, targeting different sequence types (STs) of multidrug-resistant \u003cem\u003eE. coli\u003c/em\u003e. The isolated phages, EC.W1-1 and EC.W15-3, belonged to the \u003cem\u003eStraboviridae\u003c/em\u003e family and the genus \u003cem\u003eTequatrovirus\u003c/em\u003e. Phages remained stable at pH 2\u0026ndash;10 for 4h and below 80\u0026deg;C for 1h. They exhibited \u003cem\u003ein vitro\u003c/em\u003e bacterial lytic activity at various MOIs (10\u0026ndash;0.001). The one-step growth curve of phages showed a short latent period of about 10\u0026ndash;15 min and a moderate burst size of 64\u0026ndash;83 (pfu/cell). Phages genome size ranged from 37,736\u0026ndash;123,792bp, with G\u0026thinsp;+\u0026thinsp;C content of 35.6\u0026ndash;37.2%. No virulence or drug-resistance genes were detected, enhancing their safety profile. Most predicted coding sequences (CDSs) in the phages analyzed were associated with various putative functions like tail protein, holin, lysis protein, head protein, structural proteins, and DNA replication, transcription, and repair proteins. Furthermore, the study explored the combined effects of phages and antibiotics, showing effective inhibition of ESBL-producing and carbapenem-resistant \u003cem\u003eE. coli\u003c/em\u003e (CREC) with sub-lethal antibiotic doses. In addition, phages suspensions can eliminate biofilm formed against different STs of MDR \u003cem\u003eE. coli\u003c/em\u003e. Therefore, we concluded that EC.W1-1 and EC.W15-3 have potential therapeutic properties, providing an alternative to antibiotic for treating various pathogenic MDR \u003cem\u003eE. coli\u003c/em\u003e sequence types.\u003c/p\u003e","manuscriptTitle":"Characterization of Novel Straboviridae Phages Enhancing Antibiotic Efficacy and Biofilm Inhibition Against MDR Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-02-26 11:21:05","doi":"10.21203/rs.3.rs-6102499/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":"60bd164a-416b-4e45-b0dc-0be0dab60e43","owner":[],"postedDate":"February 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":44831167,"name":"Virology"},{"id":44831168,"name":"General Microbiology"}],"tags":[],"updatedAt":"2025-02-26T11:21:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-02-26 11:21:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6102499","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6102499","identity":"rs-6102499","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}