Amoxicillin resistance in uropathogenic Escherichia coli: extracellular proteomic Insights into transporter and multidrug resistance transporter proteins

Amoxicillin resistance in uropathogenic Escherichia coli: extracellular proteomic Insights into transporter and multidrug resistance transporter proteins | 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 Amoxicillin resistance in uropathogenic Escherichia coli: extracellular proteomic Insights into transporter and multidrug resistance transporter proteins Neha Srivastava, Meenu Singh, Sheetal Verma, Abhinav Kumar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7634107/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 Antibiotic resistance in uropathogenic Escherichia coli (UPEC) is a major obstacle to the treatment of urinary tract infections (UTIs). Previous research has focused on antibiotic susceptibility, antibiotic resistance genes, and the underlying intracellular mechanisms therein. This study aims to go beyond existing research by examining the extracellular proteins specifically produced by UPEC in response to amoxicillin (AMX) resistance with the goal of identifying potential therapeutic targets. We selected intrinsically antibiotic-sensitive UPEC strains from previous studies and developed AMX-resistant strains through prolonged AMX exposure. Using nano-based LC-ESI orbitrap mass spectrometry, we analyzed the extracellular proteome of the developed AMX-resistant UPEC and the wild-type control. Proteomics analysis revealed a total of 516 common extracellular proteins in the untreated and AMX-treated developed resistant UPEC strain. While 1,299 extracellular proteins were identified exclusively in the AMX-treated evolved resistant strain, which were categorized into 14 distinct groups based on Gene Ontology (GO) annotation. Among them, the transporter class was the second most abundant class, which included primary active transporters, secondary carrier transporters, and ion channels. Further analysis revealed 57 transporter proteins, including 4 multidrug-resistant transporter proteins, suggesting their potential role in AMX resistance adaptation in UPEC. Such a study can also contribute to creating new drugs or therapies aimed at controlling a wide range of infections, including UTIs, by targeting important identified molecules. Uropathogenic Escherichia coli amoxicillin resistance extracellular proteins proteome analysis mass spectrometry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Urinary tract infections (UTIs) are among the most common bacterial infections affecting millions of people worldwide each year (Ho et al. 2025 ). Uropathogenic Escherichia coli (UPEC) is the primary causative agent, which alone is responsible for up to 80–90% of community-acquired UTIs (Mohapatra et al. 2022 ; Carmona-Cartaya et al. 2022 ; Bhargava et al. 2022 ). UPEC's success as a pathogen is attributed to its versatile arsenal of virulence factors and its ability to adapt to diverse host and environmental conditions, including antibiotic stress (Zhou et al. 2023 ). The release of extracellular proteins/molecules seems to be a key factor for bacterial adaptability, which is earlier reported by several researchers. Incidentally, these proteins have a variety of functions which includes adhesion, invasion, immunological regulation, and nutrition acquisition (Vaca et al. 2020 ; Masselot–Joubert et al. 2025). Additionally, these proteins are reported to be a key factor for bacterium's survival under stress- conditions, such as antibiotic exposure, by contributing to biofilm formation, a popular resistance mechanism (George and Halami 2019 ; Flores-Mireles 2015). Secreted toxins (cytotoxic necrotizing factor 1, secreted autotransporter, etc.), secreted iron acquisition siderophores (enterobactin, aerobactin, yersiniabactin, etc.), secreted proteases (to break down host immunoglobulin A (IgA) and host-derived antimicrobial peptides like defensins and LL-37), and secreted urease (to hydrolyze urea into ammonia, raising pH and promoting the formation of biofilms) are a few examples of secreted virulence factors that work synergistically to facilitate UPEC survival, colonization, and persistence in the urinary tract (Subashchandrabose and Mobley 2015 ). The other two important concerns regarding UTIs are the ineffectiveness of prescribed medication and the recurrence of the disease (Cornelius et al. 2024 ). The probable reason could be natural resistance against prescribed medication or acquired resistance due to long-term, inappropriate exposure to antibiotics (Ahmed et al. 2024 ). The underlying principle behind antibiotic resistance is mutational adaptation and altered membrane permeability through porin down-regulation (Viveiros et al. 2007 ), transformation, or alteration of gene expression, which can confer resistance to virtually all antibiotics currently available in clinical practice (Duval and Lister 2013 ; Nachin et al. 2005 ; Guo and Gross 2014 ). In UPECs several outer membrane porins—including OmpC, OmpF, TolC, OmpX, YddB, TosA and Lpp—have been identified as significant contributors to antimicrobial resistance (Rodrigues et al. 2022 ). Current research on UTIs has mainly focused on the pattern of antibiotic susceptibility (Rizvi et al. 2024 ), the presence of antibiotic-resistant genes (Kumar et al. 2021 ), and intracellular mechanisms of antibiotic resistance (Rozwadowski and Gawel 2022 ; Herrera-Espejo et al. 2025 ). However, this leaves a critical gap in our understanding of how extracellular proteins contribute to the survival of UPECs under antibiotic stress. In order to address antibiotic resistance, it is essential to examine extracellular proteomes expressed under conditions of antibiotic stress or established resistance. Recent advances in proteomic technologies, such as Mass Spectrometry (MS)/ LC-MS (Liquid Chromatography-Mass Spectrometry), have enabled high-throughput identification and characterization of extracellular proteins with precision (Sanders and Edwards 2020 ; Kim et al. 2020 ). These technologies present an opportunity to investigate aspects of the resistance mechanisms that have been understudied thus far. The primary objective of this study is to identify the important extracellular protein classes that are uniquely expressed under antibiotic resistance conditions by examining the extracellular proteome of UPEC. We selected amoxicillin (AMX), a commonly prescribed antibiotic for UTI treatment, as our model drug. Through an in vitro study, we compared extracellular protein profiles between AMX-resistant UPEC and wild type antibiotic-sensitive UPEC strains. This experiment was designed to replicate the natural development of antibiotic resistance that occurs through repeated AMX exposure to intrinsically sensitive UPEC populations, thereby identifying protein classes specifically associated with the resistance phenotype. Understanding the proteome analysis of these extracellular proteins associated with antibiotic resistance may reveal therapeutic targets and inform the development of treatment and diagnostic tools, and select vaccine candidates for combating UPEC-associated UTIs. Material and method Bacterial strains and Inclusion criteria of selection: The inclusion criteria for isolate selection were (i) susceptibility to all tested classes of antibiotics and (ii) existence of at least one virulence factor gene (Adamus-Białek et al. 2019 ). From our previous study, five clinical Escherichia coli ( E.coli ) isolates (14244, 16337, 17811, 9969, and 10385), which had shown susceptibility for all 21 tested antibiotics through the Kirby-Bauer agar disc diffusion method following the guidelines of the Clinical & Laboratory Standards Institute (CLSI, 2024), were selected for this study. These isolates were among the group of 110 confirmed E. coli isolates collected from the Microbiology Lab, King George's Medical University (KGMU), Lucknow, Uttar Pradesh, during Feb-July 2022. The isolates were obtained from urine samples of patients suffering from UTIs. The ethical approval was granted from the Institution Ethical Committee of KGMU, Lucknow (Ref. code: 112th ECM IIA/P7) (Srivastava et al. 2024 ). Detection of virulence-associated factor genes In order to satisfy the second inclusion criterion, a panel of five virulence factor genes has been further examined in the antibiotic-sensitive clinical isolates with colony polymerase chain reaction (PCR) using virulence factor gene-specific primers (Table 1 ). For the template, DNA was extracted by the simple boiling method (Queipo-Ortuño et al. 2008 ). For that, 2–3 colonies of each isolate were mixed in Nuclease-free Water (20 µl) separately and kept at 95°C for 10 minutes in a dry bath, followed by micro-centrifuging at 15,000 × g for 1 min. The supernatant obtained was used as the template for colony PCR (Firoozeh et al. 2014 ). PCR mixture (20 µl) was made containing 7 µl template DNA (1–10 ng/reaction), 0.5 pmol of primers, 10mM of each dNTP, 6U/reaction Taq polymerase (Sigma Aldrich, USA), 1X Taq buffer (Sigma Aldrich, USA), and nuclease-free water (Sigma Aldrich, USA). The thermocycler (DNA Engine, Bio-Rad, USA) was programmed for the PCR reaction as outlined in the Table. 2. Table 1 List of virulence factor gene-specific primers used to detect the presence of virulence factor genes in the selected intrinsically antibiotic-sensitive isolates. Virulence factor Target gene(s) Primer Name Primer Sequence (5′- 3′) Size of amplicon (bp) Reference Type 1 fimbriae fimH fimH-f 5′-AAC AGC GAT GAT TTC CAG TTT GTG TG-3′ 465 (Dadi et al. 2020 ; Usein et al. 2001 ; Tarchouna et al. 2013 ) fimH-r 5′-ATT GCG TAC CAG CAT TAG CAA TGT CC-3′ P fimbriae papC pap1 5′-GAC GGC TGT ACT GCA GGG TGT GGC G-3’ 328 pap2 5′-ATA TCC TTT CTG CAG GGA TGC AAT A-3’ S and FIC fimbriae Sfa/focDEh region sfa1 5′-CTC CGG AGA ACT GGG TGC ATC TTA C-3’ 410 sfa2 5′-CGG AGG AGT AAT TAC AAA CCT GGC A-3’ Afa adhesins afaC c afa-f 5′-CGG CTT TTC TGC TGA ACT GGC AGG C-3’ 672 afa-r 5′-CCGTCAGCCCCCACGGCAGACC-3’ Hemolysin hlyCA region hly s 5′-AGATTCTTGGGCATGTATCCT-3’ 556 hly as 5′-TTGCTTTGCAGACTGTAGTGT-3’ Table 2 Colony PCR reaction to detect the presence of virulence factor genes in the selected intrinsically antibiotic-sensitive isolates Primer fimH papC Sfa/focDE h afaC c hlyCA Parameters Temp Time Temp Time Temp Time Temp Time Temp Time Initial Denaturation 95 ̊C 3 min 94–95 ̊C 3–5 min 94–95 ̊C 3–5 min 95 ̊C 3 min 95 ̊C 3 min Denaturation 95 ̊C 30 sec 94–95 ̊C 30 sec 94–95 ̊C 30 sec 95 ̊C 30 sec 95 ̊C 30 sec Annealing 58 ̊C 1 min 60–70 ̊C 1 min 58–61 ̊C 1 min 68 ̊C 1 min 68 ̊C 1 min Extension 72 ̊C 30 sec 72 ̊C 30 sec 72 ̊C 30 sec 72 ̊C 30 sec 72 ̊C 30 sec Final Extension 72 ̊C 5 min 72 ̊C 5–10 min 72 ̊C 5–10 min 72 ̊C 5 min 72 ̊C 5 min The rows shaded in grey represent 30 cycles of the same program Experimental design The acquisition of antibiotic resistance in an innate antibiotic-sensitive UPEC is dependent on the continuous repetitive exposure of antibiotics. In this study, under in vitro conditions, antibiotic-sensitive and virulent E.coli isolates were continuously exposed to sub-MIC of AMX for a prolonged period of 15 days. Because of the solubility of AMX in broth, the antibiotic stress-related experiment was performed in liquid broth. The strategy for in vitro antibiotic stress experimentation is represented in Fig. 1 . The developed AMX-resistant strains obtained were further analyzed for their growth, antibiotic susceptibility pattern, and stability of the developed AMX resistance, with control strains. Extracellular proteins from wild-type and developed resistant strains were extracted, followed by their identification and characterization of proteins through in silico methods, as represented in Fig. 1 . Emergence of an antibiotic-sensitive UPEC strain into an AMX-resistant strain The screened antibiotic-sensitive UPEC strains have undergone an antibiotic stress-related study. First, the minimum inhibitory concentrations (MICs) and sub-MICs of AMX were determined for each UPEC in accordance with the serial dilution method (Wiegand et al. 2008 ). The MIC and sub-MIC values were obtained from three independent experiments. Bacterial suspensions in 0.9% NaCl were prepared from overnight cultures. The turbidity was equal to 0.5 in the McFarland standard (∼1 × 10 8 CFU/ml). The prepared suspensions were diluted to ∼1 × 10 6 CFU/ml in Mueller Hinton II (MHII) broth with varying concentrations of the AMX to the final volume of 3 ml and incubated at 220 rpm for 18 hours at 37°C. The purity of broth was verified via swabbing of 100 µl of sterile broth onto MHII agar plates. We chose the MIC as the starting point and the border for determining the sub-minimum inhibitory concentrations (sub-MIC) of AMX in accordance with the MIC definition. We believed that the sub-MIC was the maximum AMX concentration at which bacterial growth was detected. Consequently, for both strains, the optimal MIC and sub-MIC of AMX were found to be 10 ug/ml and 1 ug/ml, respectively. The UPECs were continuously exposed to sub-MIC concentrations of AMX for a prolonged period of 15 days. The 30 µl of overnight bacterial inoculum was transferred to the MH II broth supplemented with sub-MIC of AMX in a total volume of 3 ml, to the final culture concentration of ∼1 × 10 6 CFU/ml. After 16 h of incubation, the 30 µl of bacterial suspension was passed on to the next broth with the sub-MIC of the antibiotic. The passes were conducted according to this scheme for 15 days. The wild-type strains were passed on the same way without the antibiotic, and they were considered as a negative control for the experiment. AMX-exposed or developed AMX-resistant virulent test strains were designated as UPEC16337′ and UPEC14244′, while the wild-type antibiotic sensitive virulent strains were denoted without the prime symbol (UPEC16337 and UPEC14244). To evaluate the progression of antibiotic sensitivity into antibiotic resistance after every 3–4 day interval, they were plated on Mueller Hinton II (MH II) agar media (Himedia) supplemented with the > = MIC of the AMX according to CLSI guidelines (Adamus-Białek et al. 2019 ). Further, their antibiotic susceptibility pattern was also examined by the disc diffusion method as per CLSI guidelines (CLSI 2024). Growth curves A fresh inoculum of bacteria was obtained from an overnight-grown culture in the MH II broth under optimum conditions. The bacterial culture was diluted to obtain 0.125 ± 0.005 OD at 600 nm, which corresponds to approximately 103 CFU of bacteria. The developed resistant UPEC were grown in triplicate at 37°C in the Luria-Bertani (LB) broth (Himedia) with a sub-MIC of the AMX. The negative control (wild-type UPEC) and positive control ( E. coli ATCC 25,922) were cultured and incubated under the same optimal conditions. Bacterial growth at 600 nm was taken every 2 hours for a total of twenty hours of incubation. Readings were taken in three separate tests (Adamus-Białek et al. 2019 ). Stability of induced resistance The developed AMX-resistant strains were passaged under optimal conditions for 8 days in non-AMX-supplemented MH broth (Himedia) under optimum conditions. Following 8 days of passage, the stability of induced resistance for AMX was verified by the antibiotic disk diffusion method as per the CLSI guidelines (CLSI 2024). Extraction and quantification of extracellular protein The developed AMX-resistant and stable UPEC was cultured in MH II broth and incubated overnight under optimal conditions along with the respective wild-type strain. For extracellular protein extraction, overnight-grown culture was centrifuged at 15000 rpm for 10 minutes at 40°C. The obtained supernatant was filtered through a 0.22 um syringe filter (Angkawidjaja et al. 2006 ). The absorbance ratio A260/280 was used to verify the purity of the extracellular preparation. The obtained low value (0.1–0.5) assured the absence of nucleic acid contamination and indirectly indicated that there was no cell lysis during extraction (Raynal et al. 2014 ). LCMS analysis of extracellular proteins The extracted extracellular proteins from control and test were trypsinized (1:100 w/w) at 37°C overnight (Gundry et al. 2010 ). The trypsinized samples were desalted (AKTA, GE Healthcare) followed by lyophilization (Penguin Classic, Laboratory Freeze Dryer, VFL) for further usages and analysis. The samples were analyzed in an LC-MS Orbitrap fusion MS (Thermo Scientific). They were separated on a C-18 in-house column of 15 cm length, 1.5-micron particle size, and 150 cm internal diameter at 30°C. The Ultimate 3000 RSnano UHPLC system was used with a 0.500 µl/min flow rate. A mobile phase of 0.1% formic acid in 98% water (A) and 0.1% formic acid in 80% acetonitrile (B) was used. Peptides were eluted with a gradient of 2%-90% mobile phase B over 110 minutes. The gradient was as follows: 0–10 min, 2% B; 10–70 min, 25% B; 70–96 min, 25% to 38%; 90% B in 100 min; 100–105 min, 90%; returned to 2% B in 106 min. The total run time was 110 min. Peptides were analyzed in positive-ion mode of the nano-electrospray ionization source. The capillary voltage was adjusted at 2500 V with an ion transfer tube temperature of 275°C. Automated MS/MS data of peptides were acquired through an Orbitrap mass analyzer between 350 m/z and 2000 m/z above the 5.0e3 count threshold. The twenty most intense ions were selected for MS/MS acquisition using an ion trap mass analyzer. The ion charge states were + 2 to + 8. The HCD fragmentation energy was adjusted to 30%. Five hundred nanograms of digested protein samples were loaded onto the LC/MS system for the run. Protein identification Maxquant (Max Planck Institute, Germany, https://www.maxquant.org ) was used to process the recorded MS/MS data. UniPortKB provided the E. coli database for download. By comparing the recorded MS/MS data with the in-silico MS/MS data, peptides and proteins were identified. The initial and primary search in the matching process was conducted using a 20 ppm mass tolerance. Comparative analysis of extracellular proteomes in evolved AMX resistant and wild type AMX sensitive UPEC. The obtained result of LCMS was analyzed for common extracellular proteins in control as well as test. The uncommon extracellular proteins exclusively expressed under AMX stress were also segregated for further analysis. These exclusively expressed extracellular proteins were analyzed with Panther, the gene list software ( http://pantherdb.org ). The software is used to categorize proteins into different protein classes. The obtained results for major classes were further analyzed to identify the proteins that are contributing to antibiotic resistance. They were further categorized on the basis of molecular function also (Kocak and Özkul 2021 ). Statistical analysis The data were presented as the average of three separate assays, with each assay being carried out in triplicate. The acquired data are presented in the form of mean ± standard deviation. Statistical Package for Social Sciences (SPSS v26) was used to analyze data. Result Isolate screening: antibiotic susceptibility and virulence gene profiling Five clinical isolates (14244, 16337,17811, 9969, and 10385) that exhibited complete susceptibility to all 21 tested antibiotics were evaluated for virulence factor genes to meet the dual inclusion criteria of antibiotic sensitivity and presence of at least one virulence determinant (Adamus-Białek et al. 2019 ). PCR analysis of virulence factor genes revealed that isolate 16337 harbored both Type 1 fimbriae ( fimH ) as well as Afa adhesins ( afaC c ) genes (Fig. 2 a), while isolate 14244 carried only fimH (Fig. 2 b). Based on these virulence profiles, both isolates 16337 and 14244 were selected for further analyses (Table 2 ). Table 2 Profile of the virulence factor genes in five clinical E.coli isolates. Virulence factor Target gene(s) 14244 16377 17811 9969 10385 Type 1 fimbriae fimH √ √ X X X P fimbriae papC X X X X X S and FIC fimbriae Sfa/focDEh region X X X X X Afa adhesins afaC c X √ X X X Hemolysin hlyCA region X X X X X √ - presence of virulence factor genes, X – absence of virulence factor genes in clinical E. coli isolates. Antibiotic resistance acquisition in sensitive UPEC wild type strains The antibiotic-sensitive UPEC (14244 and 16337) were exposed to sub-MIC concentrations of AMX for 15 days to evaluate their potential for resistance emergence. UPEC16337 developed AMX resistance faster than UPEC14244. The antibiotic-sensitive UPEC14244 required 13 days to acquire AMX resistance (designated as UPEC14244'), producing 150 colonies when plated on media supplemented with AMX (Fig. 3 a). In contrast, UPEC16337 acquired AMX resistance in five producing 200 colonies under identical conditions (Fig. 3 b). Both the resistant strains showed growth capability on media supplemented with ≥ MIC of AMX. (Table 3 ). None of the UPEC control cultures passaged without AMX exposure showed spontaneous resistance development to AMX. This confirmed that resistance emergence was induced by antibiotic pressure. Table 3 Evaluation of antibiotic sensitive UPECs (UPEC14244 & UPEC16337) for their emergence as resistant strains UPEC Conc. of antibiotics (AMX) Duration of antibiotic exposure/Stress in days No. of colonies grown on media supplemented with > = MIC UPEC14244 MIC (10 µg/ml) 1st day X 3rd day X 5rd day X 9th day X 11th day X 13th day √ (150 colonies) 15th day √ (150 colonies) >MIC (15 µg/ml) 15th day √ (30–40 colonies) UPEC16337 MIC (10 µg/ml) 1st day X 3rd day X 5rd day √ (200 Colonies) 9th day √ (200 Colonies) 11th day √ (200 Colonies) 13th day √ (200 Colonies) 15th day √ (200 Colonies) >MIC (15 µg/ml) 15th day √ (100 Colonies) √ - presence of colonies, X – absence of colonies Growth curve The growth curve of developed resistant strains (UPEC16337’ and UPEC14244’) was analyzed during the incubation with sub-MIC of AMX. Both the resistant derivatives showed growth initiation and kinetic profiles comparable to the positive control, with only minor variations between strains. The wild type strains were unable to grow under the treatment with sub-MIC of AMX, confirming the acquired resistance phenotype. The growth curve of the developed resistant strains in comparison to positive and negative control are presented in Fig. 4 . Stability of induced resistance in emerged AMX resistant strains The developed resistant strains, UPEC14244' and UPEC16337', were successfully passaged in AMX-free broth media for 8 consecutive days along with respective wild type strains. The passaged cultures were subsequently evaluated for the retention of resistance by plating on media supplemented with MIC and > MIC of AMX. Both resistant strains maintained stabile resistance at MIC dosage of AMX. However, only UPEC16337' strain retained resistance at concentrations exceeding MIC. This result verified that UPEC16337' had greater stability of induced resistance than UPEC14244' (Table 4 ). Disc diffusion antibiotic susceptibility testing further revealed that both the resistant derivatives had acquired cross-resistance to ampicillin (AMP), suggesting a broader β-lactam resistance profile. Table 4 Determination of the antibiotic stability of UPECs at sub-MIC and ≥ MIC of AMX S.No. Wild type/Emerged resistant strain Passages (Days) Resistance stability at Induced resistance to sub-MIC MIC >MIC 1 UPEC14244 (control) 8 NA NA 2 UPEC14244’ 8 Yes Yes NO AMP and AMC 4 UPEC16337 (control) 8 NA NA 5 UPEC16337’ 8 Yes Yes Yes AMP and AMC Nano-LC based proteomics analysis (LC-ESI-Orbitrap) LC-ESI Orbitrap analysis of extracellular protein samples revealed distinct proteomic profiles for the control and the resistant strain. A total of 516 common extracellular proteins were identified in both the untreated control (UPEC16337) and the AMX-treated resistant test strain (UPEC16337’). Notably, 1299 extracellular proteins were exclusively detected in resistant strain (UPEC16337’), indicating substantial proteomic remodeling following resistance development. Gene ontology analysis of exclusively expressed extracellular proteins from developed AMX resistant UPEC strain (UPEC16337’) The 1299 proteins exclusively expressed in the AMX-resistant strain (UPEC16337’) were classified into 14 distinct protein classes using Gene Ontology (GO) analysis (Fig. 5 a). Metabolite interconversion enzymes constituted the predominant class (49%), followed by transporters (16.4%). The metabolite interconversion enzyme class included oxidoreductase (38.20%), transferase (22.90%), hydrolase (18.80%), lyase (12.40%), ligase (5.30%) and isomerase (2.40%). The transporter class included primary active transporters (45.60%), secondary carrier transporters (22.80%) and ion channels (1.80%). Further analysis of transporter class, identified the presence of total 57 proteins which included ATP-Binding Cassette (ABC) Transporters (21), primary active transporters (5), secondary carrier transporters (8), amino acid transporter (5), transporters (17) and ion channels (1) (supplementary file 1). LC-MS/MS identification parameters for these extracellular proteins are also presented in supplementary file 1. Molecular function classification revealed that the majority of proteins were involved in catalytic activity (53.4%), followed by binding functions (19.8%) and transporter activity (12.9%) (Fig. 5 b). Discussion The emergence of antibiotic resistance to commonly used antibiotics remains a significant concern for medical professionals treating UTIs. Current research has primarily focused on causative agents, antibiotic susceptibility patterns, genetic factors, and intracellular mechanisms of antibiotic resistance (Subashchandrabose and Mobley 2015 ; Viveiros et al. 2007 ; Duval and Lister 2013 ). While intracellular proteins are crucial for overall cell function and regulation, extracellular factors serve as the frontline in antibiotic resistance, along with host-pathogen interactions, virulence, and survival strategies (El-Halfawy et al. 2017 ; Zhou et al. 2015 ). However, these extracellular components receive less attention than intracellular proteins in UTI research. Bacterial outer membrane proteins can be shed or released into the extracellular environment through various mechanisms and play significant roles in bacterial physiology, pathogenesis, and immune interactions (Han et al. 2003 ; Nandakumar et al. 2006 ). Hence, the exclusive presence of specific extracellular proteins under stress conditions provides valuable insights into UPEC survival strategies, making their study especially relevant for understanding and combating bacterial resistance mechanisms. This study investigated how AMX exposure altered the extracellular proteome of UPEC, focusing on specific proteins exclusively found in AMX-resistant strains, but absent in antibiotic-sensitive wild type UPEC. We have selected AMX as our model antibiotic because it is a commonly prescribed beta-lactam antibiotic, FDA-approved for treating UTIs brought on by beta-lactamase-negative E. coli or Enterococcus faecalis , Proteus mirabilis , in primary care settings (Akhavan et al. 2025 ; Mortazavi-Tabatabaei et al. 2019 ). However, over the past decade, the effectiveness of AMX against UTIs has declined substantially due to rising antibiotic resistance, particularly among UPECs (Mareș et al. 2024 ; Jancel and Dudas 2002 ), limiting its recommendation as first-line empirical therapy for UTIs in many regions. In our experimental model, continuous in vitro sub-MIC AMX exposure converted intrinsically antibiotic-sensitive UPECs into AMX-resistant strains capable of surviving at concentrations exceeding the MIC. This transformation demonstrates a gradual increase in their tolerance following sustained exposure to antibiotics. This resistance persisted even after the withdrawal of antibiotic for short periods; this is consistent with previous findings (Adamus-Białek et al. 2019 ). However, since only one test strain (UPEC16337’) maintained stable resistance even after discontinuation of antibiotic exposure, these observations cannot be generalized across every strain. Nevertheless, both phenomena likely contribute to treatment ineffectiveness and UTI recurrence in clinical settings. Comparative proteomics revealed a total of 1299 extracellular proteins exclusively present in AMX-resistant UPEC compared to wild-type-sensitive strains. GO analysis was performed to functionally categorize extracellular proteins, thereby revealing the systematic impact of AMX on extracellular protein expression in UPEC. The proteins clustered into 14 different classes with metabolite interconversion enzymes and transporters representing the most abundant categories. Their predominance suggests these major classes may play a critical role in the survival mechanisms of UPEC under AMX stress conditions. Metabolite interconversion enzymes (PC00262) facilitate the conversion of small molecules, catalyzing specific biochemical reactions (Mi et al. 2021 ), which are necessary for cellular viability under antibiotic stress conditions. The abundance of this protein class was expected, given its role in metabolic processes necessary for bacterial survival. This category includes hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases, which participate in a variety of metabolic and regulatory processes that enable bacterial adaptation to environmental challenges. Aminopeptidases (e.g., PepA in E. coli ) (Gonzales and Robert-Baudouy 1996 ) and Cyclic Di-GMP and Phosphodiesterases (e.g., PdeA in Pseudomonas aeruginosa) (Ha and O'Toole 2015 ) are some examples demonstrating this. These extracellular enzymes contribute significantly to antibiotic resistance, through mechanisms, particularly those involving enzymatic modification, deactivation, or degradation of antibiotics outside the bacterial cell. For example, extracellular β-lactamases, hydrolyze the β-lactam ring of amoxicillin before it reaches the intracellular target (Bush and Bradford 2016 ). Transporters constituted the second most abundant extracellular protein class, with 57 proteins found, which include ATP-Binding Cassette (ABC) Transporters (21), primary active transporters [5], secondary carrier transporters [8], amino acid transporters [5], transporters [17], and ion channels [1]. Our finding aligns with established resistance mechanisms where membrane permeability alterations through outer membrane proteins (OMPs) (Zhou et al. 2023 ) and efflux pump overproduction (Gaurav et al. 2023 ) represent major strategies used by microbes for antibiotic resistance. The role of transporters in controlling the influx and efflux of antimicrobial agents, thereby affecting bacterial susceptibility, is well documented (Dalbanjan et al. 2024 ). Our results reiterate previous studies reporting porins, outer membrane proteins, and transport protein, under stress conditions (Zhao et al. 2025 ; Lee et al. 2020 ; Ying et al. 2008 ). ABC transporters (Orelle et al. 2019 ; Akhtar and Turner 2022 ; Shea et al. 2024 ), and Major Facilitator Superfamily (MFS) (Dalbanjan et al. 2024 ; Du et al. 2014 ; Drew et al. 2021 ) both major transporters known for antibiotic/multidrug resistance in E.coli were identified in our dataset. Given antibiotic stress conditions, we focused particularly on transporter proteins associated with drug resistance. In-silico analysis revealed the presence of four multidrug transporter proteins in the extracellular sample: multidrug resistance-like ATP-binding protein MdlA, multidrug export protein EmrA, multidrug export protein AcrE and probable multidrug resistance protein EmrK. Previous studies have documented their roles in the exportation of antibiotics and elimination of toxic compounds, thereby conferring resistance (Pasqua et al. 2021 ; Kumawat et al. 2023 ; Lin et al. 2017 ). Their exclusive presence in the extracellular environment signifies their contribution to AMX resistance in UPEC. Classification on the basis of molecular function revealed that the majority of extracellular proteins were involved in cellular and metabolic activities, followed by transport and binding functions, which is consistent with the mechanisms underlying AMX resistance acquisition as discussed. Conclusion The exclusive presence of transporter proteins as a major class of proteins, encompassing specific multidrug transporter proteins, provides strong evidence for their significance in AMX resistance and identifies them as potential therapeutic targets for combating antibiotic resistance in UTIs. Despite precautionary measures to minimize cell lysis during the extraction of extracellular proteins, potential cytosolic protein contamination cannot be completely excluded due to disruption or aging, and such proteins could not be removed from the extracellular proteome. Furthermore, these findings require further investigation at the molecular level to elucidate the specific role of exclusively expressed extracellular proteins in UPEC survival and persistence under AMX stress conditions. Similar studies should also be performed on other UPEC isolates, to sketch a universal conclusion regarding the significance of the extracellular proteins in UPEC survival under antibiotic stress. Declarations Acknowledgement Authors acknowledge the technical assistance provided by Ms. Priyam Srivastava from Microbiology laboratory at King George Medical University in Lucknow. Authors are also grateful to the assistance offered by Mr. Hari Babu, laboratory attendant, Biotechnology laboratory, IILM University, Greater Noida, during the study. Funding: The authors declare that no funds or grants received for this research work. Conflict of Interest: Authors declare no financial interest. Ethical approval: NA Author contributions Neha Srivastava : Conceptualization; investigation; conduction of research work, writing and editing. Sheetal Verma: Supervision, investigation and validation. Meenu Singh: Supervision, review and editing. Abhinav Kumar: Conceptualization; designing, supervision; review and editing. Data Availability Statement (Required): NA References Adamus-Białek W, Wawszczak M, Arabski M, Majchrzak M, Gulba M, Jarych D, et al. (2019) Ciprofloxacin, amoxicillin, and aminoglycosides stimulate genetic and phenotypic changes in uropathogenic Escherichia coli strains. Virulence 10:260-76. https://doi.org/10.1080/21505594.2019.1596507]. Ahmed SK, Hussein S, Qurbani K, Ibrahim RH, Fareeq A, Mahmood KA, Mohamed MG (2024) Antimicrobial resistance: Impacts, challenges, and future prospects. J. Med. Surg. Public Health 2:100081. https://doi.org/10.1016/j.glmedi.2024.100081 Akhavan, B. J., Khanna, N. R., & Vijhani, P. (2025) Amoxicillin, StatPearls [Internet]. StatPearls Publishing. Amoxicillin - StatPearls - NCBI Bookshelf Akhtar AA, Turner DP (2022) The role of bacterial ATP-binding cassette (ABC) transporters in pathogenesis and virulence: Therapeutic and vaccine potential. Microb Pathog 171:105734. https://doi.org/10.1016/j.micpath.2022.105734 Angkawidjaja C, Kuwahara K, Omori K, Koga Y, Takano K, Kanaya S (2006) Extracellular secretion of Escherichia coli alkaline phosphatase with a C-terminal tag by type I secretion system: purification and biochemical characterization. Protein Eng Des Sel 19:337-43. https://doi.org/10.1093/protein/gzl017 Bhargava K, Nath G, Bhargava A, Kumari R, Aseri GK, Jain N (2022) Bacterial profile and antibiotic susceptibility pattern of uropathogens causing urinary tract infection in the eastern part of Northern India. Front. Microbiol 13:965053. https://doi.org/10.3389/fmicb.2022.965053 Bush K, Bradford PA (2016) β-Lactams and β-lactamase inhibitors: an overview. Cold Spring Harb Perspect Med 6:a025247. 10.1101/cshperspect.a025247 Carmona-Cartaya Y, Hidalgo-Benito M, Borges-Mateus LM, Pereda-Novales N, González-Molina MK, Quiñones-Pérez D (2022) Community-acquired uropathogenic Escherichia coli, antimicrobial susceptibility, and extended-spectrum beta-lactamase detection. MEDICC review 24:20-5. https://doi.org/10.37757/MR2022.V24.N2.2 Clinical and Laboratory Standards Institute (2024) Performance Standards for Antimicrobial Susceptibility Testing. 34th ed. CLSI supplement M100. https://clsi.org/standards/products/microbiology/documents/m100 / Cornelius SA, Basu U, Zimmern PE, De Nisco NJ (2024) Overcoming challenges in the management of recurrent urinary tract infections. Expert Rev Anti-infect Ther 22:1157-69. https://doi.org/10.1080/14787210.2024.2412628 Dadi BR, Abebe T, Zhang L, Mihret A, Abebe W, Amogne W (2020) Distribution of virulence genes and phylogenetics of uropathogenic Escherichia coli among urinary tract infection patients in Addis Ababa, Ethiopia. BMC Infect Dis 20:1-2. https://doi.org/10.1186/s12879-020-4844-z Dalbanjan NP, Kadapure AJ, SK PK (2024) A comprehensive review on latent role of stress proteins in antibiotic resistance. The Microbe 4:100151. https://doi.org/10.1016/j.microb.2024.100151 Drew D, North RA, Nagarathinam K, Tanabe M (2021) Structures and general transport mechanisms by the major facilitator superfamily (MFS). Chem. Rev 121:5289-335. https://doi.org/10.1021/acs.chemrev.0c00983 Du D, Wang Z, James NR, Voss JE, Klimont E, Ohene-Agyei T, Venter H, Chiu W, Luisi BF (2014) Structure of the AcrAB–TolC multidrug efflux pump. Nature 509:512-5. https://doi.org/10.1038/nature13205 Duval V, Lister IM (2013) MarA, SoxS and Rob of Escherichia coli–Global regulators of multidrug resistance, virulence and stress response. Int J Biotechnol Wellness Ind. 2:101-124. 10.6000/1927-3037.2013.02.03.2 El-Halfawy OM, Klett J, Ingram RJ, Loutet SA, Murphy ME, Martín-Santamaría S, Valvano MA (2017) Antibiotic capture by bacterial lipocalins uncovers an extracellular mechanism of intrinsic antibiotic resistance. MBio 8:10-128. https://doi.org/10.1128/mbio.00225-17 Firoozeh F, Saffari M, Neamati F, Zibaei M (2014) Detection of virulence genes in Escherichia coli isolated from patients with cystitis and pyelonephritis. Int. J. Infect. Dis. 29:219-22. https://doi.org/10.1016/j.ijid.2014.03.1393 Flores-Mireles AL, Walker JN, Caparon M, Hultgren SJ (2015) Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 13:269-84. https://doi.org/10.1038/nrmicro3432 Gaurav A, Bakht P, Saini M, Pandey S, Pathania R (2023) Role of bacterial efflux pumps in antibiotic resistance, virulence, and strategies to discover novel efflux pump inhibitors. Microbiology 169:001333. 10.1099/mic.0.001333. George J, Halami PM (2019) Presence of extracellular DNA & protein in biofilm formation by gentamicin-resistant Lactobacillus plantarum. IJMR 149:257-62. https://doi.org/10.4103/ijmr.IJMR_2022_17 Gonzales T, Robert-Baudouy J (1996) Bacterial aminopeptidases: properties and functions. FEMS Microbiol Rev 18:319-44. https://doi.org/10.1111/j.1574-6976.1996.tb00247.x Gundry RL, White MY, Murray CI, Kane LA, Fu Q, Stanley BA, Van Eyk JE (2010) Preparation of proteins and peptides for mass spectrometry analysis in a bottom‐up proteomics workflow. Curr. Protoc. Mol. Biol 90:10-25. https://doi.org/10.1002/0471142727.mb1025s88 Guo MS, Gross CA (2014) Stress-induced remodeling of the bacterial proteome. Curr Biol 24:R424-34. 10.1016/j.cub.2014.03.023. Ha DG, O'Toole GA (2015) c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review. Microbiol Spectr 3:10-128. https://doi.org/10.1128/microbiolspec.mb-0003-2014 Han L, Enfors SO, Häggström L (2003) Escherichia coli high-cell-density culture: carbon mass balances and release of outer membrane components. Bioprocess Biosyst Eng 25:205-12. https://doi.org/10.1007/s00449-002-0300-2 Herrera-Espejo S, Rubio A, Ceballos-Romero L, Pachón J, Cordero E, Pérez-Pulido AJ, Pachón-Ibáñez ME (2025). Detection of Possible Resistance Mechanisms in Uropathogenic Escherichia coli Strains Isolated from Kidney Transplant Recipients Based on Whole Genome Sequencing. Biomolecules 15:260. https://doi.org/10.3390/biom15020260 Ho CS, Wong CTH, Aung TT, Lakshminarayanan R, Mehta JS, Rauz S, McNally A, Kintses B, Peacock SJ, de la Fuente-Nunez C, Hancock REW, Ting DSJ (2025) Antimicrobial resistance: a concise update. Lancet Microbe 6:100947. 10.1016/j.lanmic.2024.07.010. Jancel T, Dudas V. (2002) Management of uncomplicated urinary tract infections. West J Med 176:51. https://doi.org/10.1136/ewjm.176.1.51 Kim SW, Seo JS, Park SB, Lee AR, Lee JS, Jung JW, Chun JH, Lazarte JM, Kim J, Kim JH, Song JW (2020) Significant increase in the secretion of extracellular vesicles and antibiotics resistance from methicillin-resistant Staphylococcus aureus induced by ampicillin stress. Sci Rep .10:21066. https://doi.org/10.1038/s41598-020-78121-8 Kocak E, Özkul C (2021) Comparative proteomic analysis of Escherichia coli under ofloxacin stress. Turk J Pharm Sci 18:133-39. 10.4274/tjps.galenos.2020.47704 Kumar N, Chatterjee K, Deka S, Shankar R, Kalita D (2021) Increased isolation of extended-spectrum beta-lactamase-producing Escherichia coli from community-onset urinary tract infection cases in Uttarakhand, India. Cureus 13:e13837. 10.7759/cureus.13837 Kumawat M, Nabi B, Daswani M, Viquar I, Pal N, Sharma P, Tiwari S, Sarma DK, Shubham S, Kumar M (2023) Role of bacterial efflux pump proteins in antibiotic resistance across microbial species. Microb Pathog 181:106182. https://doi.org/10.1016/j.micpath.2023.106182 Lee SY, Yun SH, Lee H, Yi YS, Park EC, Kim W, Kim HY, Lee JC, Kim GH, Kim SI (2020) Analysis of the extracellular proteome of colistin-resistant Korean acinetobacter baumannii strains. ACS omega 5:5713-20. https://doi.org/10.1021/acsomega.9b03723 Lin MF, Lin YY, Lan CY (2017) Contribution of EmrAB efflux pumps to colistin resistance in Acinetobacter baumannii. J Microbiol 5:130-6. https://doi.org/10.1007/s12275-017-6408-5 Mareș C, Petca RC, Popescu RI, Petca A, Mulțescu R, Bulai CA, Ene CV, Geavlete PA, Geavlete BF, Jinga V (2024) Update on urinary tract infection antibiotic resistance—a retrospective study in females in conjunction with clinical data. Life 14:106. https://doi.org/10.3390/life14010106 Masselot--Joubert L, Di Renzo MA (2025) ATP-Binding Cassette (ABC) Transporters and Antibiotic Resistance: Specialized Systems for Capsular Polysaccharide Export in Gram-Negative Pathogens. Polysaccharides 6:38. https://doi.org/10.3390/polysaccharides6020038 Mi H, Ebert D, Muruganujan A, Mills C, Albou LP, Mushayamaha T, Thomas PD (2021) PANTHER version 16: a revised family classification, tree-based classification tool, enhancer regions and extensive API. Nucleic Acids Res 49:D394-403. https://doi.org/10.1093/nar/gkaa1106 Mohapatra S, Panigrahy R, Tak V, JV S, KC S, Chaudhuri S, Pundir S, Kocher D, Gautam H, Sood S, Das BK (2022) Prevalence and resistance pattern of uropathogens from community settings of different regions: an experience from India. Access Microbiol 4:000321. https://doi.org/10.1099/acmi.0.000321 Mortazavi-Tabatabaei SA, Ghaderkhani J, Nazari A, Sayehmiri K, Sayehmiri F, Pakzad I (2019) Pattern of antibacterial resistance in urinary tract infections: A systematic review and meta-analysis. Int J Prev Med 10:169. 10.4103/ijpvm.IJPVM_419_17 Nachin L, Nannmark U, Nyström T (2005) Differential roles of the universal stress proteins of Escherichia coli in oxidative stress resistance, adhesion, and motility. J Bacteriol. 187:6265-72. https://doi.org/10.1128/jb.187.18.6265-6272.2005 Nandakumar MP, Cheung A, Marten MR (2006) Proteomic Analysis of Extracellular Proteins from Escherichia coli W3110. J. Proteome Res 5:1155-61.https://doi.org/10.1021/pr050401j Orelle C, Mathieu K, Jault JM (2019). Multidrug ABC transporters in bacteria. Res. Microbiol 170:381-91. https://doi.org10.1016/j.resmic.2019.06.001 Pasqua M, Bonaccorsi di Patti MC, Fanelli G, Utsumi R, Eguchi Y, Trirocco R, Prosseda G, Grossi M, Colonna B (2021) Host-bacterial pathogen communication: the wily role of the multidrug efflux pumps of the MFS family. Front Mol Biosci 8:723274. https://doi.org/10.3389/fmolb.2021.723274 Queipo-Ortuño MI, De Dios Colmenero J, Macias M, Bravo MJ, Morata P (2008) Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis. Clin. Vaccine Immunol. 15:293-6. https://doi.org/10.1128/CVI.00270-07 Raynal B, Lenormand P, Baron B, Hoos S, England P (2014) Quality assessment and optimization of purified protein samples: why and how?. Microb Cell Fact 13:180. https://doi.org/10.1186/s12934-014-0180-6 Rizvi M, Malhotra S, Agarwal J, Siddiqui AH, Devi S, Poojary A, Thakuria B, Princess I, Sami H, Gupta A, Sultan A (2024) Regional variations in antimicrobial susceptibility of community-acquired uropathogenic Escherichia coli in India: Findings of a multicentric study highlighting the importance of local antibiograms. IJID regions 11:100370. https://doi.org/10.1016/j.ijregi.2024.100370 Rodrigues IC, Rodrigues SC, Duarte FV, Costa PM, Costa PM (2022) The role of outer membrane proteins in UPEC antimicrobial resistance: A systematic review. Membranes 12:981. https://doi.org/10.3390/membranes12100981 Rozwadowski M, Gawel D (2022) Molecular factors and mechanisms driving multidrug resistance in uropathogenic Escherichia coli—an update. Genes 13:1397. https://doi.org/10.3390/genes13081397 Sanders KL, Edwards JL (2020) Nano-liquid chromatography-mass spectrometry and recent applications in omics investigations. Anal. Methods 12:4404-17. https://doi.org/10.1039/D0AY01194K Shea AE, Forsyth VS, Stocki JA, Mitchell TJ, Frick-Cheng AE, Smith SN, Hardy SL, Mobley HL (2024) Emerging roles for ABC transporters as virulence factors in uropathogenic Escherichia coli. Proc. Acad. Natl. Sci. 121:e2310693121. https://doi.org/10.1073/pnas.2310693121 Srivastava N, Verma S, Singh M, Kumar A (2024) A short-term study on statistical numeration of multidrug resistant Escherichia coli isolates among the patients with urinary tract infection. IJEB 62:119-125. https://doi.org/10.56042/ijeb.v62i02.2549 Subashchandrabose S, Mobley HLT. Virulence and Fitness Determinants of Uropathogenic Escherichia coli (2015) Microbiol Spectr. 3:10.1128. 10.1128/microbiolspec.UTI-0015-2012 Tarchouna M, Ferjani A, Ben-Selma W, Boukadida J (2013) Distribution of uropathogenic virulence genes in Escherichia coli isolated from patients with urinary tract infection. Int J Infect Dis 17:e450-3. https://doi.org/10.1016/j.ijid.2013.01.025 Usein CR, Damian M, Tatu‐Chitoiu D, Capusa C, Fagaras R, Tudorache D, et al (2001) Prevalence of virulence genes in Escherichia coli strains isolated from Romanian adult urinary tract infection cases. J Cell Mol Med 5:303-10. https://doi.org/10.1111/j.1582-4934.2001.tb00164.x Vaca DJ, Thibau A, Schütz M, Kraiczy P, Happonen L, Malmström J, Kempf VA (2020) Interaction with the host: the role of fibronectin and extracellular matrix proteins in the adhesion of Gram-negative bacteria. Med Microbiol Immunol 209:277-99. https://doi.org/10.1007/s00430-019-00644-3 Viveiros M, Dupont M, Rodrigues L, Couto I, Davin-Regli A, Martins M, Pages JM, Amaral L (2007) Antibiotic stress, genetic response and altered permeability of E. coli . PloS one 2:e365. https://doi.org/10.1371/journal.pone.0000365 Wiegand I, Hilpert K, Hancock RE (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc . 3:163-75. https://doi.org/10.1038/nprot.2007.521 Ying N, Zheng Z, Xu H, Tian B, Hua Y (2008) Extracellular Proteome Changes of Deinococcus radiodurans Under γ-Irradiation Stress Conditions. Protein and Peptide Letters 15:595-9. https://doi.org/10.2174/092986608784966985 Zhao Q, Wu Y, Sun J, Zhang J, Li X, Pang X, Gu S (2025) The ABC Transport Protein PotC Plays a Crucial Role in Antibiotic Resistance in Escherichia coli. Appl Biochem Microbiol 61: 58–67. https://doi.org/10.1134/S0003683824603706 Zhou G, Shi QS, Huang XM, Xie XB (2015) The three bacterial lines of defense against antimicrobial agents. Int. J. Mol. Sci 16:21711-33. https://doi.org/10.3390/ijms160921711 Zhou G, Wang Q, Wang Y, Wen X, Peng H, Peng R, Shi Q, Xie X, Li L (2023) Outer Membrane Porins Contribute to Antimicrobial Resistance in Gram-Negative Bacteria. Microorganisms 11:1690. 10.3390/microorganisms11071690 Zhou Y, Zhou Z, Zheng L, Gong Z, Li Y, Jin Y, Huang Y, Chi M (2023) Urinary tract infections caused by uropathogenic Escherichia coli: mechanisms of infection and treatment options. Int J Mol Sci 24:10537.https://doi.org/10.3390/ijms241310537 Additional Declarations No competing interests reported. Supplementary Files Suplementaryfile1.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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7634107","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516168815,"identity":"2f1c7651-fe1a-4f2a-9ace-bb6a78e66232","order_by":0,"name":"Neha Srivastava","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAElEQVRIiWNgGAWjYFACxgY46/GPCiDFzNyAUzGSFgMQi9mY4QyIQhiCD4C1sEkztqHaixXIzz7cJvHjzx95g+MHmI0L59VG87cDtfyo2Ibb+HOJbZK9bQaGG84kMD6eue147ozDjA2MPWdu49bCw9hswNtgwDizIYHZgHfbsdwGoBZmxjbcWuR7GJsN//wxsJ/Z/4BNgnfOsdz5hLQwnGFsfMzDZpDYL5HAJs3bUJO7gZAWA5AW2Tbj5H6Jh82GM44dyN0I1HIQn1/ke9gfHHzzR862jT/54IMPNXW5884fPvjgRwUehyEAODoOg5kHiFEPA3WkKB4Fo2AUjIIRAgC4Aluv6cgoogAAAABJRU5ErkJggg==","orcid":"","institution":"Dr. A. P. J. Abdul Kalam Technical University","correspondingAuthor":true,"prefix":"","firstName":"Neha","middleName":"","lastName":"Srivastava","suffix":""},{"id":516168816,"identity":"fb2f882e-3014-48d7-97b8-8bbedb09cbcf","order_by":1,"name":"Meenu Singh","email":"","orcid":"","institution":"IILM Academy of Higher Learning","correspondingAuthor":false,"prefix":"","firstName":"Meenu","middleName":"","lastName":"Singh","suffix":""},{"id":516168817,"identity":"b472ae65-af1d-41ed-971f-7624366d4f81","order_by":2,"name":"Sheetal Verma","email":"","orcid":"","institution":"King Georges’ Medical University","correspondingAuthor":false,"prefix":"","firstName":"Sheetal","middleName":"","lastName":"Verma","suffix":""},{"id":516168818,"identity":"35cc1ebc-37f3-46ec-a02a-8f30e9e31fd1","order_by":3,"name":"Abhinav Kumar","email":"","orcid":"","institution":"IILM Academy of Higher Learning","correspondingAuthor":false,"prefix":"","firstName":"Abhinav","middleName":"","lastName":"Kumar","suffix":""}],"badges":[],"createdAt":"2025-09-16 23:08:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7634107/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7634107/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91587032,"identity":"e2037752-cf46-426a-a389-f62a0ea80aab","added_by":"auto","created_at":"2025-09-18 06:06:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117789,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentation of the experimental design of the research work\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7634107/v1/a7a4fdbffbff68c84c32f329.jpg"},{"id":91589510,"identity":"8b681aa9-92be-45fb-bbf3-7025331da25f","added_by":"auto","created_at":"2025-09-18 06:22:22","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":90479,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Detection of virulence factor genes in the selected \u003cem\u003eE .coli\u003c/em\u003e isolate (16337) by PCR: Figure represents the presence of fimH and afa virulence factor associated genes in the E. coli isolate (16337). M (Marker) - 1 kb DNA ladder (Fermentas, lane1); Fim H (lane2); pap (lane3); sfa (lane 4); afa (lane 5) and hyl (lane6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Detection of virulence factor genes in the selected \u003cem\u003eE. coli\u003c/em\u003e isolate (14244) by PCR: Figure represents the presence of fimH virulence factor associated gene in the \u003cem\u003eE. coli\u003c/em\u003e isolate (14244). M (Marker) - 1 kb DNA ladder (lane1); fimH (lane2); pap (lane3); sfa (lane 4); afa (lane 5) and hyl (lane6).\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7634107/v1/26b817b8b069e0bbe083479e.jpg"},{"id":91587038,"identity":"7a3ec7c9-25f9-4a0c-998f-04df881ef3d4","added_by":"auto","created_at":"2025-09-18 06:06:22","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":151421,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Plating of AMX-induced stressed cells of UPEC14244’ on HM agar supplemented with \u0026gt;=MIC\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb\u003c/strong\u003e Plating of AMX-induced stressed cells of UPEC16337’ on HM agar supplemented with \u0026gt;=MIC of AMX\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7634107/v1/145cc29bcd33ed0020298a85.jpg"},{"id":91588529,"identity":"4046a299-ffb6-41a3-b338-fc480fac6143","added_by":"auto","created_at":"2025-09-18 06:14:22","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":161395,"visible":true,"origin":"","legend":"\u003cp\u003eThe growth curves of wild type UPEC14244, UPEC16337 (negative controls), \u003cem\u003eE.coli\u003c/em\u003e \u0026nbsp;ATCC 25922 (positive control), and developed resistant UPEC14244A’ and UPEC16337’ (test) strains\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7634107/v1/4df31701af123274713ea99b.jpg"},{"id":91587037,"identity":"84274e1e-ab5a-46b2-8daf-0443c379dc83","added_by":"auto","created_at":"2025-09-18 06:06:22","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74255,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eCharacterization of extracellular proteins of evolved AMX-resistant UPEC16337’ into different protein classes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb \u003c/strong\u003eCharacterization of exclusively expressed extracellular proteins of developed AMX-resistant UPEC (16337’A15), on the basis of molecular function.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7634107/v1/b3b9cc65a7f7186e2bbb4393.jpg"},{"id":96087094,"identity":"0025dc33-ef3f-4718-b454-3fb8f8d8b1bb","added_by":"auto","created_at":"2025-11-17 12:38:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1922323,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7634107/v1/eaf4c896-1c52-4568-a371-6f8769ceb2fe.pdf"},{"id":91587033,"identity":"f10a5e16-85b2-4347-a83f-5fbe97ad7320","added_by":"auto","created_at":"2025-09-18 06:06:22","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":35548,"visible":true,"origin":"","legend":"","description":"","filename":"Suplementaryfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-7634107/v1/6ac97536db11c8c54c7ac5f0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Amoxicillin resistance in uropathogenic Escherichia coli: extracellular proteomic Insights into transporter and multidrug resistance transporter proteins","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUrinary tract infections (UTIs) are among the most common bacterial infections affecting millions of people worldwide each year (Ho et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Uropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (UPEC) is the primary causative agent, which alone is responsible for up to 80\u0026ndash;90% of community-acquired UTIs (Mohapatra et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Carmona-Cartaya et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Bhargava et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). UPEC's success as a pathogen is attributed to its versatile arsenal of virulence factors and its ability to adapt to diverse host and environmental conditions, including antibiotic stress (Zhou et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The release of extracellular proteins/molecules seems to be a key factor for bacterial adaptability, which is earlier reported by several researchers. Incidentally, these proteins have a variety of functions which includes adhesion, invasion, immunological regulation, and nutrition acquisition (Vaca et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Masselot\u0026ndash;Joubert et al. 2025). Additionally, these proteins are reported to be a key factor for bacterium's survival under stress- conditions, such as antibiotic exposure, by contributing to biofilm formation, a popular resistance mechanism (George and Halami \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Flores-Mireles 2015). Secreted toxins (cytotoxic necrotizing factor 1, secreted autotransporter, etc.), secreted iron acquisition siderophores (enterobactin, aerobactin, yersiniabactin, etc.), secreted proteases (to break down host immunoglobulin A (IgA) and host-derived antimicrobial peptides like defensins and LL-37), and secreted urease (to hydrolyze urea into ammonia, raising pH and promoting the formation of biofilms) are a few examples of secreted virulence factors that work synergistically to facilitate UPEC survival, colonization, and persistence in the urinary tract (Subashchandrabose and Mobley \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe other two important concerns regarding UTIs are the ineffectiveness of prescribed medication and the recurrence of the disease (Cornelius et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The probable reason could be natural resistance against prescribed medication or acquired resistance due to long-term, inappropriate exposure to antibiotics (Ahmed et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The underlying principle behind antibiotic resistance is mutational adaptation and altered membrane permeability through porin down-regulation (Viveiros et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), transformation, or alteration of gene expression, which can confer resistance to virtually all antibiotics currently available in clinical practice (Duval and Lister \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Nachin et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Guo and Gross \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In UPECs several outer membrane porins\u0026mdash;including OmpC, OmpF, TolC, OmpX, YddB, TosA and Lpp\u0026mdash;have been identified as significant contributors to antimicrobial resistance (Rodrigues et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eCurrent research on UTIs has mainly focused on the pattern of antibiotic susceptibility (Rizvi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), the presence of antibiotic-resistant genes (Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and intracellular mechanisms of antibiotic resistance (Rozwadowski and Gawel \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Herrera-Espejo et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). However, this leaves a critical gap in our understanding of how extracellular proteins contribute to the survival of UPECs under antibiotic stress. In order to address antibiotic resistance, it is essential to examine extracellular proteomes expressed under conditions of antibiotic stress or established resistance.\u003c/p\u003e\u003cp\u003eRecent advances in proteomic technologies, such as Mass Spectrometry (MS)/ LC-MS (Liquid Chromatography-Mass Spectrometry), have enabled high-throughput identification and characterization of extracellular proteins with precision (Sanders and Edwards \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These technologies present an opportunity to investigate aspects of the resistance mechanisms that have been understudied thus far. The primary objective of this study is to identify the important extracellular protein classes that are uniquely expressed under antibiotic resistance conditions by examining the extracellular proteome of UPEC. We selected amoxicillin (AMX), a commonly prescribed antibiotic for UTI treatment, as our model drug. Through an \u003cem\u003ein vitro\u003c/em\u003e study, we compared extracellular protein profiles between AMX-resistant UPEC and wild type antibiotic-sensitive UPEC strains. This experiment was designed to replicate the natural development of antibiotic resistance that occurs through repeated AMX exposure to intrinsically sensitive UPEC populations, thereby identifying protein classes specifically associated with the resistance phenotype.\u003c/p\u003e\u003cp\u003eUnderstanding the proteome analysis of these extracellular proteins associated with antibiotic resistance may reveal therapeutic targets and inform the development of treatment and diagnostic tools, and select vaccine candidates for combating UPEC-associated UTIs.\u003c/p\u003e"},{"header":"Material and method","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eBacterial strains and Inclusion criteria of selection:\u003c/h2\u003e\u003cp\u003eThe inclusion criteria for isolate selection were (i) susceptibility to all tested classes of antibiotics and (ii) existence of at least one virulence factor gene (Adamus-Białek et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). From our previous study, five clinical \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE.coli\u003c/em\u003e) isolates (14244, 16337, 17811, 9969, and 10385), which had shown susceptibility for all 21 tested antibiotics through the Kirby-Bauer agar disc diffusion method following the guidelines of the Clinical \u0026amp; Laboratory Standards Institute (CLSI, 2024), were selected for this study. These isolates were among the group of 110 confirmed \u003cem\u003eE. coli\u003c/em\u003e isolates collected from the Microbiology Lab, King George's Medical University (KGMU), Lucknow, Uttar Pradesh, during Feb-July 2022. The isolates were obtained from urine samples of patients suffering from UTIs. The ethical approval was granted from the Institution Ethical Committee of KGMU, Lucknow (Ref. code: 112th ECM IIA/P7) (Srivastava et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDetection of virulence-associated factor genes\u003c/h3\u003e\n\u003cp\u003eIn order to satisfy the second inclusion criterion, a panel of five virulence factor genes has been further examined in the antibiotic-sensitive clinical isolates with colony polymerase chain reaction (PCR) using virulence factor gene-specific primers (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For the template, DNA was extracted by the simple boiling method (Queipo-Ortuño et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). For that, 2–3 colonies of each isolate were mixed in Nuclease-free Water (20 µl) separately and kept at 95°C for 10 minutes in a dry bath, followed by micro-centrifuging at 15,000 × \u003cem\u003eg\u003c/em\u003e for 1 min. The supernatant obtained was used as the template for colony PCR (Firoozeh et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). PCR mixture (20 µl) was made containing 7 µl template DNA (1–10 ng/reaction), 0.5 pmol of primers, 10mM of each dNTP, 6U/reaction Taq polymerase (Sigma Aldrich, USA), 1X Taq buffer (Sigma Aldrich, USA), and nuclease-free water (Sigma Aldrich, USA). The thermocycler (DNA Engine, Bio-Rad, USA) was programmed for the PCR reaction as outlined in the Table. 2.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\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\u003eList of virulence factor gene-specific primers used to detect the presence of virulence factor genes in the selected intrinsically antibiotic-sensitive isolates.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVirulence factor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTarget gene(s)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003ePrimer Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePrimer Sequence (5′- 3′)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSize of amplicon (bp)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eReference\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eType 1 fimbriae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003efimH\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003efimH-f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5′-AAC AGC GAT GAT TTC CAG TTT GTG TG-3′\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e465\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\" morerows=\"9\" rowspan=\"10\"\u003e\u003cp\u003e(Dadi et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Usein et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Tarchouna et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003efimH-r\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5′-ATT GCG TAC CAG CAT TAG CAA TGT CC-3′\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eP fimbriae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003epapC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003epap1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5′-GAC GGC TGT ACT GCA GGG TGT GGC G-3’\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e328\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003epap2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5′-ATA TCC TTT CTG CAG GGA TGC AAT A-3’\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eS and FIC fimbriae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eSfa/focDEh region\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003esfa1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5′-CTC CGG AGA ACT GGG TGC ATC TTA C-3’\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e410\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003esfa2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5′-CGG AGG AGT AAT TAC AAA CCT GGC A-3’\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eAfa adhesins\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003eafaC\u003c/em\u003e \u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eafa-f\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5′-CGG CTT TTC TGC TGA ACT GGC AGG C-3’\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e672\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eafa-r\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5′-CCGTCAGCCCCCACGGCAGACC-3’\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eHemolysin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e\u003cem\u003ehlyCA region\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ehly s\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5′-AGATTCTTGGGCATGTATCCT-3’\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e556\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003ehly as\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e5′-TTGCTTTGCAGACTGTAGTGT-3’\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv class=\"gridtable\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\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\u003e Colony PCR reaction to detect the presence of virulence factor genes in the selected intrinsically antibiotic-sensitive isolates\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"11\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimer\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e\u003cp\u003e\u003cem\u003efimH\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e\u003cp\u003e\u003cem\u003epapC\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u003cp\u003e\u003cem\u003eSfa/focDE h\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u003cp\u003e\u003cem\u003eafaC\u003c/em\u003e \u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c11\" namest=\"c10\"\u003e\u003cp\u003e\u003cem\u003ehlyCA\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eParameters\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTemp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTemp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eTemp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e\u003cp\u003eTemp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c9\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c10\"\u003e\u003cp\u003eTemp\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c11\"\u003e\u003cp\u003eTime\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eInitial Denaturation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e95 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e94–95 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3–5 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e94–95 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3–5 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e95 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e3 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e95 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e3 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDenaturation\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e95 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30 sec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e94–95 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30 sec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e94–95 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e30 sec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e95 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e30 sec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e95 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e30 sec\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAnnealing\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e58 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e60–70 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e58–61 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e1 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e68 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e1 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e68 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e1 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtension\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e72 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e30 sec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e30 sec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e72 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e30 sec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e72 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e30 sec\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e72 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e30 sec\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFinal Extension\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e72 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e72 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5–10 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e72 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5–10 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e72 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e\u003cp\u003e5 min\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e\u003cp\u003e72 ̊C\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c11\"\u003e\u003cp\u003e5 min\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"11\" nameend=\"c11\" namest=\"c1\"\u003e\u003cp\u003eThe rows shaded in grey represent 30 cycles of the same program\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eExperimental design\u003c/h3\u003e\n\u003cp\u003eThe acquisition of antibiotic resistance in an innate antibiotic-sensitive UPEC is dependent on the continuous repetitive exposure of antibiotics. In this study, under \u003cem\u003ein vitro\u003c/em\u003e conditions, antibiotic-sensitive and virulent \u003cem\u003eE.coli\u003c/em\u003e isolates were continuously exposed to sub-MIC of AMX for a prolonged period of 15 days. Because of the solubility of AMX in broth, the antibiotic stress-related experiment was performed in liquid broth. The strategy for \u003cem\u003ein vitro\u003c/em\u003e antibiotic stress experimentation is represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe developed AMX-resistant strains obtained were further analyzed for their growth, antibiotic susceptibility pattern, and stability of the developed AMX resistance, with control strains. Extracellular proteins from wild-type and developed resistant strains were extracted, followed by their identification and characterization of proteins through \u003cem\u003ein silico\u003c/em\u003e methods, as represented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n\u003ch3\u003eEmergence of an antibiotic-sensitive UPEC strain into an AMX-resistant strain\u003c/h3\u003e\n\u003cp\u003eThe screened antibiotic-sensitive UPEC strains have undergone an antibiotic stress-related study. First, the minimum inhibitory concentrations (MICs) and sub-MICs of AMX were determined for each UPEC in accordance with the serial dilution method (Wiegand et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The MIC and sub-MIC values were obtained from three independent experiments. Bacterial suspensions in 0.9% NaCl were prepared from overnight cultures. The turbidity was equal to 0.5 in the McFarland standard (∼1 × 10\u003csup\u003e8\u003c/sup\u003e CFU/ml). The prepared suspensions were diluted to ∼1 × 10\u003csup\u003e6\u003c/sup\u003e CFU/ml in Mueller Hinton II (MHII) broth with varying concentrations of the AMX to the final volume of 3 ml and incubated at 220 rpm for 18 hours at 37°C. The purity of broth was verified via swabbing of 100 µl of sterile broth onto MHII agar plates. We chose the MIC as the starting point and the border for determining the sub-minimum inhibitory concentrations (sub-MIC) of AMX in accordance with the MIC definition. We believed that the sub-MIC was the maximum AMX concentration at which bacterial growth was detected. Consequently, for both strains, the optimal MIC and sub-MIC of AMX were found to be 10 ug/ml and 1 ug/ml, respectively.\u003c/p\u003e\u003cp\u003eThe UPECs were continuously exposed to sub-MIC concentrations of AMX for a prolonged period of 15 days. The 30 µl of overnight bacterial inoculum was transferred to the MH II broth supplemented with sub-MIC of AMX in a total volume of 3 ml, to the final culture concentration of ∼1 × 10\u003csup\u003e6\u003c/sup\u003e CFU/ml. After 16 h of incubation, the 30 µl of bacterial suspension was passed on to the next broth with the sub-MIC of the antibiotic. The passes were conducted according to this scheme for 15 days. The wild-type strains were passed on the same way without the antibiotic, and they were considered as a negative control for the experiment. AMX-exposed or developed AMX-resistant virulent test strains were designated as UPEC16337′ and UPEC14244′, while the wild-type antibiotic sensitive virulent strains were denoted without the prime symbol (UPEC16337 and UPEC14244).\u003c/p\u003e\u003cp\u003eTo evaluate the progression of antibiotic sensitivity into antibiotic resistance after every 3–4 day interval, they were plated on Mueller Hinton II (MH II) agar media (Himedia) supplemented with the \u0026gt; = MIC of the AMX according to CLSI guidelines (Adamus-Białek et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Further, their antibiotic susceptibility pattern was also examined by the disc diffusion method as per CLSI guidelines (CLSI 2024).\u003c/p\u003e\n\u003ch3\u003eGrowth curves\u003c/h3\u003e\n\u003cp\u003eA fresh inoculum of bacteria was obtained from an overnight-grown culture in the MH II broth under optimum conditions. The bacterial culture was diluted to obtain 0.125 ± 0.005 OD at 600 nm, which corresponds to approximately 103 CFU of bacteria. The developed resistant UPEC were grown in triplicate at 37°C in the Luria-Bertani (LB) broth (Himedia) with a sub-MIC of the AMX. The negative control (wild-type UPEC) and positive control (\u003cem\u003eE. coli\u003c/em\u003e ATCC 25,922) were cultured and incubated under the same optimal conditions. Bacterial growth at 600 nm was taken every 2 hours for a total of twenty hours of incubation. Readings were taken in three separate tests (Adamus-Białek et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStability of induced resistance\u003c/h2\u003e\u003cp\u003eThe developed AMX-resistant strains were passaged under optimal conditions for 8 days in non-AMX-supplemented MH broth (Himedia) under optimum conditions. Following 8 days of passage, the stability of induced resistance for AMX was verified by the antibiotic disk diffusion method as per the CLSI guidelines (CLSI 2024).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExtraction and quantification of extracellular protein\u003c/h3\u003e\n\u003cp\u003eThe developed AMX-resistant and stable UPEC was cultured in MH II broth and incubated overnight under optimal conditions along with the respective wild-type strain. For extracellular protein extraction, overnight-grown culture was centrifuged at 15000 rpm for 10 minutes at 40°C. The obtained supernatant was filtered through a 0.22 um syringe filter (Angkawidjaja et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). The absorbance ratio A260/280 was used to verify the purity of the extracellular preparation. The obtained low value (0.1–0.5) assured the absence of nucleic acid contamination and indirectly indicated that there was no cell lysis during extraction (Raynal et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eLCMS analysis of extracellular proteins\u003c/h3\u003e\n\u003cp\u003eThe extracted extracellular proteins from control and test were trypsinized (1:100 w/w) at 37°C overnight (Gundry et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The trypsinized samples were desalted (AKTA, GE Healthcare) followed by lyophilization (Penguin Classic, Laboratory Freeze Dryer, VFL) for further usages and analysis. The samples were analyzed in an LC-MS Orbitrap fusion MS (Thermo Scientific). They were separated on a C-18 in-house column of 15 cm length, 1.5-micron particle size, and 150 cm internal diameter at 30°C. The Ultimate 3000 RSnano UHPLC system was used with a 0.500 µl/min flow rate.\u003c/p\u003e\u003cp\u003eA mobile phase of 0.1% formic acid in 98% water (A) and 0.1% formic acid in 80% acetonitrile (B) was used. Peptides were eluted with a gradient of 2%-90% mobile phase B over 110 minutes. The gradient was as follows: 0–10 min, 2% B; 10–70 min, 25% B; 70–96 min, 25% to 38%; 90% B in 100 min; 100–105 min, 90%; returned to 2% B in 106 min. The total run time was 110 min. Peptides were analyzed in positive-ion mode of the nano-electrospray ionization source. The capillary voltage was adjusted at 2500 V with an ion transfer tube temperature of 275°C. Automated MS/MS data of peptides were acquired through an Orbitrap mass analyzer between 350 m/z and 2000 m/z above the 5.0e3 count threshold. The twenty most intense ions were selected for MS/MS acquisition using an ion trap mass analyzer. The ion charge states were + 2 to + 8. The HCD fragmentation energy was adjusted to 30%. Five hundred nanograms of digested protein samples were loaded onto the LC/MS system for the run.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eProtein identification\u003c/h2\u003e\u003cp\u003eMaxquant (Max Planck Institute, Germany, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.maxquant.org\u003c/span\u003e\u003cspan address=\"https://www.maxquant.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e)\u003c/span\u003e was used to process the recorded MS/MS data. UniPortKB provided the E. coli database for download. By comparing the recorded MS/MS data with the in-silico MS/MS data, peptides and proteins were identified. The initial and primary search in the matching process was conducted using a 20 ppm mass tolerance.\u003c/p\u003e\u003cp\u003e\u003cb\u003eComparative analysis of extracellular proteomes in evolved AMX resistant and wild type AMX sensitive UPEC.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe obtained result of LCMS was analyzed for common extracellular proteins in control as well as test. The uncommon extracellular proteins exclusively expressed under AMX stress were also segregated for further analysis.\u003c/p\u003e\u003cp\u003eThese exclusively expressed extracellular proteins were analyzed with Panther, the gene list software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pantherdb.org\u003c/span\u003e\u003cspan address=\"http://pantherdb.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e).\u003c/span\u003e The software is used to categorize proteins into different protein classes. The obtained results for major classes were further analyzed to identify the proteins that are contributing to antibiotic resistance. They were further categorized on the basis of molecular function also (Kocak and Özkul \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data were presented as the average of three separate assays, with each assay being carried out in triplicate. The acquired data are presented in the form of mean ± standard deviation. Statistical Package for Social Sciences (SPSS v26) was used to analyze data.\u003c/p\u003e\u003c/div\u003e"},{"header":"Result","content":"\u003ch2\u003eIsolate screening: antibiotic susceptibility and virulence gene profiling\u003c/h2\u003e\u003cp\u003eFive clinical isolates (14244, 16337,17811, 9969, and 10385) that exhibited complete susceptibility to all 21 tested antibiotics were evaluated for virulence factor genes to meet the dual inclusion criteria of antibiotic sensitivity and presence of at least one virulence determinant (Adamus-Białek et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). PCR analysis of virulence factor genes revealed that isolate 16337 harbored both Type 1 fimbriae (\u003cem\u003efimH\u003c/em\u003e) as well as Afa adhesins (\u003cem\u003eafaC\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e) genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), while isolate 14244 carried only \u003cem\u003efimH\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). Based on these virulence profiles, both isolates 16337 and 14244 were selected for further analyses (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cdiv class=\"gridtable\"\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\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eProfile of the virulence factor genes in five clinical E.coli isolates.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eVirulence factor\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTarget gene(s)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14244\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16377\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e17811\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e9969\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003e10385\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eType 1 fimbriae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003efimH\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e√\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP fimbriae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003epapC\u003c/em\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eS and FIC fimbriae\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eSfa/focDEh\u003c/em\u003e region\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAfa adhesins\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eafaC\u003c/em\u003e\u003csup\u003e\u003cem\u003ec\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHemolysin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003ehlyCA\u003c/em\u003e region\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"7\" nameend=\"c7\" namest=\"c1\"\u003e\u003cp\u003e√ - presence of virulence factor genes, X – absence of virulence factor genes in clinical \u003cem\u003eE. coli\u003c/em\u003e isolates.\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003eAntibiotic resistance acquisition in sensitive UPEC wild type strains\u003c/h2\u003e\u003cp\u003eThe antibiotic-sensitive UPEC (14244 and 16337) were exposed to sub-MIC concentrations of AMX for 15 days to evaluate their potential for resistance emergence. UPEC16337 developed AMX resistance faster than UPEC14244. The antibiotic-sensitive UPEC14244 required 13 days to acquire AMX resistance (designated as UPEC14244'), producing 150 colonies when plated on media supplemented with AMX (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In contrast, UPEC16337 acquired AMX resistance in five producing 200 colonies under identical conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Both the resistant strains showed growth capability on media supplemented with ≥ MIC of AMX. (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e3\u003c/span\u003e). None of the UPEC control cultures passaged without AMX exposure showed spontaneous resistance development to AMX. This confirmed that resistance emergence was induced by antibiotic pressure.\u003c/p\u003e\u003cdiv class=\"gridtable\"\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\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eEvaluation of antibiotic sensitive UPECs (UPEC14244 \u0026amp; UPEC16337) for their emergence as resistant strains\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUPEC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eConc. of antibiotics (AMX)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eDuration of antibiotic exposure/Stress in days\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eNo. of colonies grown on media supplemented with \u0026gt; = MIC\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eUPEC14244\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"6\" rowspan=\"7\"\u003e\u003cp\u003eMIC (10 µg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1st day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3rd day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5rd day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9th day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11th day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13th day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√ (150 colonies)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15th day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√ (150 colonies)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;MIC (15 µg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15th day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√ (30–40 colonies)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eUPEC16337\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"6\" rowspan=\"7\"\u003e\u003cp\u003eMIC (10 µg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1st day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3rd day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eX\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5rd day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√ (200 Colonies)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9th day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√ (200 Colonies)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11th day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√ (200 Colonies)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13th day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√ (200 Colonies)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15th day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√ (200 Colonies)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026gt;MIC (15 µg/ml)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e15th day\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e√ (100 Colonies)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e\u003cp\u003e√ - presence of colonies, X – absence of colonies\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003eGrowth curve\u003c/h2\u003e\u003cp\u003eThe growth curve of developed resistant strains (UPEC16337’ and UPEC14244’) was analyzed during the incubation with sub-MIC of AMX. Both the resistant derivatives showed growth initiation and kinetic profiles comparable to the positive control, with only minor variations between strains. The wild type strains were unable to grow under the treatment with sub-MIC of AMX, confirming the acquired resistance phenotype. The growth curve of the developed resistant strains in comparison to positive and negative control are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e\u003ch2\u003eStability of induced resistance in emerged AMX resistant strains\u003c/h2\u003e\u003cp\u003eThe developed resistant strains, UPEC14244' and UPEC16337', were successfully passaged in AMX-free broth media for 8 consecutive days along with respective wild type strains. The passaged cultures were subsequently evaluated for the retention of resistance by plating on media supplemented with MIC and \u0026gt; MIC of AMX. Both resistant strains maintained stabile resistance at MIC dosage of AMX. However, only UPEC16337' strain retained resistance at concentrations exceeding MIC. This result verified that UPEC16337' had greater stability of induced resistance than UPEC14244' (Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Disc diffusion antibiotic susceptibility testing further revealed that both the resistant derivatives had acquired cross-resistance to ampicillin (AMP), suggesting a broader β-lactam resistance profile.\u003c/p\u003e\u003cdiv class=\"gridtable\"\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\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDetermination of the antibiotic stability of UPECs at sub-MIC and ≥ MIC of AMX\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eS.No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eWild type/Emerged resistant strain\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003ePassages\u003c/p\u003e\u003cp\u003e(Days)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eResistance stability at\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eInduced resistance to\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003esub-MIC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMIC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026gt;MIC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUPEC14244 (control)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUPEC14244’\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eNO\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAMP and AMC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUPEC16337 (control)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c6\" namest=\"c4\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNA\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eUPEC16337’\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eYes\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eAMP and AMC\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003eNano-LC based proteomics analysis (LC-ESI-Orbitrap)\u003c/h2\u003e\u003cp\u003eLC-ESI Orbitrap analysis of extracellular protein samples revealed distinct proteomic profiles for the control and the resistant strain. A total of 516 common extracellular proteins were identified in both the untreated control (UPEC16337) and the AMX-treated resistant test strain (UPEC16337’). Notably, 1299 extracellular proteins were exclusively detected in resistant strain (UPEC16337’), indicating substantial proteomic remodeling following resistance development.\u003c/p\u003e\u003ch2\u003eGene ontology analysis of exclusively expressed extracellular proteins from developed AMX resistant UPEC strain (UPEC16337’)\u003c/h2\u003e\u003cp\u003eThe 1299 proteins exclusively expressed in the AMX-resistant strain (UPEC16337’) were classified into 14 distinct protein classes using Gene Ontology (GO) analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Metabolite interconversion enzymes constituted the predominant class (49%), followed by transporters (16.4%). The metabolite interconversion enzyme class included oxidoreductase (38.20%), transferase (22.90%), hydrolase (18.80%), lyase (12.40%), ligase (5.30%) and isomerase (2.40%). The transporter class included primary active transporters (45.60%), secondary carrier transporters (22.80%) and ion channels (1.80%).\u003c/p\u003e\u003cp\u003eFurther analysis of transporter class, identified the presence of total 57 proteins which included ATP-Binding Cassette (ABC) Transporters (21), primary active transporters (5), secondary carrier transporters (8), amino acid transporter (5), transporters (17) and ion channels (1) (supplementary file 1). LC-MS/MS identification parameters for these extracellular proteins are also presented in supplementary file 1.\u003c/p\u003e\u003cp\u003eMolecular function classification revealed that the majority of proteins were involved in catalytic activity (53.4%), followed by binding functions (19.8%) and transporter activity (12.9%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eb).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe emergence of antibiotic resistance to commonly used antibiotics remains a significant concern for medical professionals treating UTIs. Current research has primarily focused on causative agents, antibiotic susceptibility patterns, genetic factors, and intracellular mechanisms of antibiotic resistance (Subashchandrabose and Mobley \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Viveiros et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Duval and Lister \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). While intracellular proteins are crucial for overall cell function and regulation, extracellular factors serve as the frontline in antibiotic resistance, along with host-pathogen interactions, virulence, and survival strategies (El-Halfawy et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhou et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, these extracellular components receive less attention than intracellular proteins in UTI research. Bacterial outer membrane proteins can be shed or released into the extracellular environment through various mechanisms and play significant roles in bacterial physiology, pathogenesis, and immune interactions (Han et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Nandakumar et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Hence, the exclusive presence of specific extracellular proteins under stress conditions provides valuable insights into UPEC survival strategies, making their study especially relevant for understanding and combating bacterial resistance mechanisms.\u003c/p\u003e\u003cp\u003eThis study investigated how AMX exposure altered the extracellular proteome of UPEC, focusing on specific proteins exclusively found in AMX-resistant strains, but absent in antibiotic-sensitive wild type UPEC. We have selected AMX as our model antibiotic because it is a commonly prescribed beta-lactam antibiotic, FDA-approved for treating UTIs brought on by beta-lactamase-negative \u003cem\u003eE. coli\u003c/em\u003e or \u003cem\u003eEnterococcus faecalis\u003c/em\u003e, \u003cem\u003eProteus mirabilis\u003c/em\u003e, in primary care settings (Akhavan et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Mortazavi-Tabatabaei et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, over the past decade, the effectiveness of AMX against UTIs has declined substantially due to rising antibiotic resistance, particularly among UPECs (Mareș et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Jancel and Dudas \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), limiting its recommendation as first-line empirical therapy for UTIs in many regions.\u003c/p\u003e\u003cp\u003eIn our experimental model, continuous in vitro sub-MIC AMX exposure converted intrinsically antibiotic-sensitive UPECs into AMX-resistant strains capable of surviving at concentrations exceeding the MIC. This transformation demonstrates a gradual increase in their tolerance following sustained exposure to antibiotics. This resistance persisted even after the withdrawal of antibiotic for short periods; this is consistent with previous findings (Adamus-Białek et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, since only one test strain (UPEC16337\u0026rsquo;) maintained stable resistance even after discontinuation of antibiotic exposure, these observations cannot be generalized across every strain. Nevertheless, both phenomena likely contribute to treatment ineffectiveness and UTI recurrence in clinical settings.\u003c/p\u003e\u003cp\u003eComparative proteomics revealed a total of 1299 extracellular proteins exclusively present in AMX-resistant UPEC compared to wild-type-sensitive strains. GO analysis was performed to functionally categorize extracellular proteins, thereby revealing the systematic impact of AMX on extracellular protein expression in UPEC. The proteins clustered into 14 different classes with metabolite interconversion enzymes and transporters representing the most abundant categories. Their predominance suggests these major classes may play a critical role in the survival mechanisms of UPEC under AMX stress conditions.\u003c/p\u003e\u003cp\u003eMetabolite interconversion enzymes (PC00262) facilitate the conversion of small molecules, catalyzing specific biochemical reactions (Mi et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which are necessary for cellular viability under antibiotic stress conditions. The abundance of this protein class was expected, given its role in metabolic processes necessary for bacterial survival. This category includes hydrolases, isomerases, ligases, lyases, oxidoreductases, and transferases, which participate in a variety of metabolic and regulatory processes that enable bacterial adaptation to environmental challenges. Aminopeptidases (e.g., PepA in \u003cem\u003eE. coli\u003c/em\u003e) (Gonzales and Robert-Baudouy \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) and Cyclic Di-GMP and Phosphodiesterases (e.g., PdeA in Pseudomonas aeruginosa) (Ha and O'Toole \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) are some examples demonstrating this. These extracellular enzymes contribute significantly to antibiotic resistance, through mechanisms, particularly those involving enzymatic modification, deactivation, or degradation of antibiotics outside the bacterial cell. For example, extracellular β-lactamases, hydrolyze the β-lactam ring of amoxicillin before it reaches the intracellular target (Bush and Bradford \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTransporters constituted the second most abundant extracellular protein class, with 57 proteins found, which include ATP-Binding Cassette (ABC) Transporters (21), primary active transporters [5], secondary carrier transporters [8], amino acid transporters [5], transporters [17], and ion channels [1]. Our finding aligns with established resistance mechanisms where membrane permeability alterations through outer membrane proteins (OMPs) (Zhou et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and efflux pump overproduction (Gaurav et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) represent major strategies used by microbes for antibiotic resistance. The role of transporters in controlling the influx and efflux of antimicrobial agents, thereby affecting bacterial susceptibility, is well documented (Dalbanjan et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Our results reiterate previous studies reporting porins, outer membrane proteins, and transport protein, under stress conditions (Zhao et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Ying et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). ABC transporters (Orelle et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Akhtar and Turner \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Shea et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), and Major Facilitator Superfamily (MFS) (Dalbanjan et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Du et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Drew et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) both major transporters known for antibiotic/multidrug resistance in \u003cem\u003eE.coli\u003c/em\u003e were identified in our dataset.\u003c/p\u003e\u003cp\u003eGiven antibiotic stress conditions, we focused particularly on transporter proteins associated with drug resistance. In-silico analysis revealed the presence of four multidrug transporter proteins in the extracellular sample: multidrug resistance-like ATP-binding protein MdlA, multidrug export protein EmrA, multidrug export protein AcrE and probable multidrug resistance protein EmrK. Previous studies have documented their roles in the exportation of antibiotics and elimination of toxic compounds, thereby conferring resistance (Pasqua et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Kumawat et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lin et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Their exclusive presence in the extracellular environment signifies their contribution to AMX resistance in UPEC.\u003c/p\u003e\u003cp\u003eClassification on the basis of molecular function revealed that the majority of extracellular proteins were involved in cellular and metabolic activities, followed by transport and binding functions, which is consistent with the mechanisms underlying AMX resistance acquisition as discussed.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe exclusive presence of transporter proteins as a major class of proteins, encompassing specific multidrug transporter proteins, provides strong evidence for their significance in AMX resistance and identifies them as potential therapeutic targets for combating antibiotic resistance in UTIs. Despite precautionary measures to minimize cell lysis during the extraction of extracellular proteins, potential cytosolic protein contamination cannot be completely excluded due to disruption or aging, and such proteins could not be removed from the extracellular proteome.\u003c/p\u003e\u003cp\u003eFurthermore, these findings require further investigation at the molecular level to elucidate the specific role of exclusively expressed extracellular proteins in UPEC survival and persistence under AMX stress conditions. Similar studies should also be performed on other UPEC isolates, to sketch a universal conclusion regarding the significance of the extracellular proteins in UPEC survival under antibiotic stress.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors acknowledge the technical assistance provided by Ms. Priyam Srivastava from Microbiology laboratory at King George Medical University in Lucknow. Authors are also grateful to the assistance offered by Mr. Hari Babu, laboratory attendant, Biotechnology laboratory, IILM University, Greater Noida, during the study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e The authors declare that no funds or grants received for this research work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e Authors declare no financial interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval:\u003c/strong\u003e NA\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNeha Srivastava\u003c/strong\u003e: Conceptualization; investigation; conduction of research work, writing and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSheetal Verma:\u003c/strong\u003e Supervision, investigation and validation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeenu Singh:\u003c/strong\u003e Supervision, review and editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAbhinav Kumar:\u0026nbsp;\u003c/strong\u003eConceptualization; designing, supervision; review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e Statement (Required): NA\u003c/p\u003e"},{"header":"References","content":"\u003col class=\"decimal_type\"\u003e\n\u003cli\u003eAdamus-Białek W, Wawszczak M, Arabski M, Majchrzak M, Gulba M, Jarych D, et al. (2019) Ciprofloxacin, amoxicillin, and aminoglycosides stimulate genetic and phenotypic changes in uropathogenic Escherichia coli strains. Virulence 10:260-76. https://doi.org/10.1080/21505594.2019.1596507].\u003c/li\u003e\n\u003cli\u003eAhmed SK, Hussein S, Qurbani K, Ibrahim RH, Fareeq A, Mahmood KA, Mohamed MG (2024) Antimicrobial resistance: Impacts, challenges, and future prospects. J. Med. Surg. Public Health 2:100081. \u003cu\u003ehttps://doi.org/10.1016/j.glmedi.2024.100081\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eAkhavan, B. J., Khanna, N. R., \u0026amp; Vijhani, P. (2025) Amoxicillin, StatPearls [Internet]. StatPearls Publishing. Amoxicillin - StatPearls - NCBI Bookshelf\u003c/li\u003e\n\u003cli\u003eAkhtar AA, Turner DP (2022) The role of bacterial ATP-binding cassette (ABC) transporters in pathogenesis and virulence: Therapeutic and vaccine potential. Microb Pathog 171:105734. https://doi.org/10.1016/j.micpath.2022.105734\u003c/li\u003e\n\u003cli\u003eAngkawidjaja C, Kuwahara K, Omori K, Koga Y, Takano K, Kanaya S (2006) Extracellular secretion of Escherichia coli alkaline phosphatase with a C-terminal tag by type I secretion system: purification and biochemical characterization. Protein Eng Des Sel 19:337-43. https://doi.org/10.1093/protein/gzl017\u003c/li\u003e\n\u003cli\u003eBhargava K, Nath G, Bhargava A, Kumari R, Aseri GK, Jain N (2022) Bacterial profile and antibiotic susceptibility pattern of uropathogens causing urinary tract infection in the eastern part of Northern India. Front. Microbiol 13:965053. https://doi.org/10.3389/fmicb.2022.965053\u003c/li\u003e\n\u003cli\u003eBush K, Bradford PA (2016) \u0026beta;-Lactams and \u0026beta;-lactamase inhibitors: an overview. Cold Spring Harb Perspect Med 6:a025247. \u003cu\u003e10.1101/cshperspect.a025247\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eCarmona-Cartaya Y, Hidalgo-Benito M, Borges-Mateus LM, Pereda-Novales N, Gonz\u0026aacute;lez-Molina MK, Qui\u0026ntilde;ones-P\u0026eacute;rez D (2022) Community-acquired uropathogenic Escherichia coli, antimicrobial susceptibility, and extended-spectrum beta-lactamase detection. MEDICC review 24:20-5. https://doi.org/10.37757/MR2022.V24.N2.2 \u003c/li\u003e\n\u003cli\u003eClinical and Laboratory Standards Institute (2024) Performance Standards for Antimicrobial Susceptibility Testing. 34th ed. CLSI supplement M100. \u003cu\u003ehttps://clsi.org/standards/products/microbiology/documents/m100\u003c/u\u003e/\u003c/li\u003e\n\u003cli\u003eCornelius SA, Basu U, Zimmern PE, De Nisco NJ (2024) Overcoming challenges in the management of recurrent urinary tract infections. Expert Rev Anti-infect Ther 22:1157-69. \u003cu\u003ehttps://doi.org/10.1080/14787210.2024.2412628\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eDadi BR, Abebe T, Zhang L, Mihret A, Abebe W, Amogne W (2020) Distribution of virulence genes and phylogenetics of uropathogenic Escherichia coli among urinary tract infection patients in Addis Ababa, Ethiopia. BMC Infect Dis 20:1-2. https://doi.org/10.1186/s12879-020-4844-z\u003c/li\u003e\n\u003cli\u003eDalbanjan NP, Kadapure AJ, SK PK (2024) A comprehensive review on latent role of stress proteins in antibiotic resistance. The Microbe 4:100151. \u003cu\u003ehttps://doi.org/10.1016/j.microb.2024.100151\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eDrew D, North RA, Nagarathinam K, Tanabe M (2021) Structures and general transport mechanisms by the major facilitator superfamily (MFS). Chem. Rev 121:5289-335. \u003cu\u003ehttps://doi.org/10.1021/acs.chemrev.0c00983\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eDu D, Wang Z, James NR, Voss JE, Klimont E, Ohene-Agyei T, Venter H, Chiu W, Luisi BF (2014) Structure of the AcrAB\u0026ndash;TolC multidrug efflux pump. Nature 509:512-5. \u003cu\u003ehttps://doi.org/10.1038/nature13205\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eDuval V, Lister IM (2013) MarA, SoxS and Rob of Escherichia coli\u0026ndash;Global regulators of multidrug resistance, virulence and stress response. Int J Biotechnol Wellness Ind. 2:101-124. 10.6000/1927-3037.2013.02.03.2\u003c/li\u003e\n\u003cli\u003eEl-Halfawy OM, Klett J, Ingram RJ, Loutet SA, Murphy ME, Mart\u0026iacute;n-Santamar\u0026iacute;a S, Valvano MA (2017) Antibiotic capture by bacterial lipocalins uncovers an extracellular mechanism of intrinsic antibiotic resistance. MBio 8:10-128. https://doi.org/10.1128/mbio.00225-17\u003c/li\u003e\n\u003cli\u003eFiroozeh F, Saffari M, Neamati F, Zibaei M (2014) Detection of virulence genes in Escherichia coli isolated from patients with cystitis and pyelonephritis. Int. J. Infect. Dis. 29:219-22. https://doi.org/10.1016/j.ijid.2014.03.1393\u003c/li\u003e\n\u003cli\u003eFlores-Mireles AL, Walker JN, Caparon M, Hultgren SJ (2015) Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat Rev Microbiol 13:269-84. \u003cu\u003ehttps://doi.org/10.1038/nrmicro3432\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eGaurav A, Bakht P, Saini M, Pandey S, Pathania R (2023) Role of bacterial efflux pumps in antibiotic resistance, virulence, and strategies to discover novel efflux pump inhibitors. Microbiology 169:001333. \u003cu\u003e10.1099/mic.0.001333.\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eGeorge J, Halami PM (2019) Presence of extracellular DNA \u0026amp; protein in biofilm formation by gentamicin-resistant Lactobacillus plantarum. IJMR 149:257-62. \u003cu\u003ehttps://doi.org/10.4103/ijmr.IJMR_2022_17\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eGonzales T, Robert-Baudouy J (1996) Bacterial aminopeptidases: properties and functions. FEMS Microbiol Rev 18:319-44. https://doi.org/10.1111/j.1574-6976.1996.tb00247.x\u003c/li\u003e\n\u003cli\u003eGundry RL, White MY, Murray CI, Kane LA, Fu Q, Stanley BA, Van Eyk JE (2010) Preparation of proteins and peptides for mass spectrometry analysis in a bottom‐up proteomics workflow. Curr. Protoc. Mol. Biol 90:10-25. https://doi.org/10.1002/0471142727.mb1025s88\u003c/li\u003e\n\u003cli\u003eGuo MS, Gross CA (2014) Stress-induced remodeling of the bacterial proteome. Curr Biol 24:R424-34. 10.1016/j.cub.2014.03.023.\u003c/li\u003e\n\u003cli\u003eHa DG, O\u0026apos;Toole GA (2015) c-di-GMP and its effects on biofilm formation and dispersion: a Pseudomonas aeruginosa review. Microbiol Spectr 3:10-128. https://doi.org/10.1128/microbiolspec.mb-0003-2014\u003c/li\u003e\n\u003cli\u003eHan L, Enfors SO, H\u0026auml;ggstr\u0026ouml;m L (2003) \u003cem\u003eEscherichia coli\u003c/em\u003e high-cell-density culture: carbon mass balances and release of outer membrane components. \u003cem\u003eBioprocess Biosyst Eng \u003c/em\u003e 25:205-12. \u003cu\u003ehttps://doi.org/10.1007/s00449-002-0300-2\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eHerrera-Espejo S, Rubio A, Ceballos-Romero L, Pach\u0026oacute;n J, Cordero E, P\u0026eacute;rez-Pulido AJ, Pach\u0026oacute;n-Ib\u0026aacute;\u0026ntilde;ez ME (2025). Detection of Possible Resistance Mechanisms in Uropathogenic Escherichia coli Strains Isolated from Kidney Transplant Recipients Based on Whole Genome Sequencing. Biomolecules 15:260. https://doi.org/10.3390/biom15020260\u003c/li\u003e\n\u003cli\u003eHo CS, Wong CTH, Aung TT, Lakshminarayanan R, Mehta JS, Rauz S, McNally A, Kintses B, Peacock SJ, de la Fuente-Nunez C, Hancock REW, Ting DSJ (2025) Antimicrobial resistance: a concise update. Lancet Microbe 6:100947.\u003cu\u003e 10.1016/j.lanmic.2024.07.010. \u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eJancel T, Dudas V. (2002) Management of uncomplicated urinary tract infections. West J Med 176:51. https://doi.org/10.1136/ewjm.176.1.51\u003c/li\u003e\n\u003cli\u003eKim SW, Seo JS, Park SB, Lee AR, Lee JS, Jung JW, Chun JH, Lazarte JM, Kim J, Kim JH, Song JW (2020) Significant increase in the secretion of extracellular vesicles and antibiotics resistance from methicillin-resistant Staphylococcus aureus induced by ampicillin stress. \u003cem\u003eSci Rep\u003c/em\u003e.10:21066. https://doi.org/10.1038/s41598-020-78121-8\u003c/li\u003e\n\u003cli\u003eKocak E, \u0026Ouml;zkul C (2021) Comparative proteomic analysis of Escherichia coli under ofloxacin stress. Turk J Pharm Sci 18:133-39. 10.4274/tjps.galenos.2020.47704\u003c/li\u003e\n\u003cli\u003eKumar N, Chatterjee K, Deka S, Shankar R, Kalita D (2021) Increased isolation of extended-spectrum beta-lactamase-producing Escherichia coli from community-onset urinary tract infection cases in Uttarakhand, India. Cureus 13:e13837. \u003cu\u003e10.7759/cureus.13837\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eKumawat M, Nabi B, Daswani M, Viquar I, Pal N, Sharma P, Tiwari S, Sarma DK, Shubham S, Kumar M (2023) Role of bacterial efflux pump proteins in antibiotic resistance across microbial species. Microb Pathog 181:106182. https://doi.org/10.1016/j.micpath.2023.106182\u003c/li\u003e\n\u003cli\u003eLee SY, Yun SH, Lee H, Yi YS, Park EC, Kim W, Kim HY, Lee JC, Kim GH, Kim SI (2020) Analysis of the extracellular proteome of colistin-resistant Korean acinetobacter baumannii strains. ACS omega 5:5713-20. \u003cu\u003ehttps://doi.org/10.1021/acsomega.9b03723\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eLin MF, Lin YY, Lan CY (2017) Contribution of EmrAB efflux pumps to colistin resistance in Acinetobacter baumannii. J Microbiol 5:130-6. https://doi.org/10.1007/s12275-017-6408-5\u003c/li\u003e\n\u003cli\u003eMareș C, Petca RC, Popescu RI, Petca A, Mulțescu R, Bulai CA, Ene CV, Geavlete PA, Geavlete BF, Jinga V (2024) Update on urinary tract infection antibiotic resistance\u0026mdash;a retrospective study in females in conjunction with clinical data. Life 14:106. https://doi.org/10.3390/life14010106\u003c/li\u003e\n\u003cli\u003eMasselot--Joubert L, Di Renzo MA (2025) ATP-Binding Cassette (ABC) Transporters and Antibiotic Resistance: Specialized Systems for Capsular Polysaccharide Export in Gram-Negative Pathogens. Polysaccharides 6:38. https://doi.org/10.3390/polysaccharides6020038\u003c/li\u003e\n\u003cli\u003eMi H, Ebert D, Muruganujan A, Mills C, Albou LP, Mushayamaha T, Thomas PD (2021) PANTHER version 16: a revised family classification, tree-based classification tool, enhancer regions and extensive API. Nucleic Acids Res 49:D394-403. https://doi.org/10.1093/nar/gkaa1106\u003c/li\u003e\n\u003cli\u003eMohapatra S, Panigrahy R, Tak V, JV S, KC S, Chaudhuri S, Pundir S, Kocher D, Gautam H, Sood S, Das BK (2022) Prevalence and resistance pattern of uropathogens from community settings of different regions: an experience from India. Access Microbiol 4:000321. https://doi.org/10.1099/acmi.0.000321\u003c/li\u003e\n\u003cli\u003eMortazavi-Tabatabaei SA, Ghaderkhani J, Nazari A, Sayehmiri K, Sayehmiri F, Pakzad I (2019) Pattern of antibacterial resistance in urinary tract infections: A systematic review and meta-analysis. Int J Prev Med 10:169. 10.4103/ijpvm.IJPVM_419_17\u003c/li\u003e\n\u003cli\u003eNachin L, Nannmark U, Nyström T (2005) Differential roles of the universal stress proteins of \u003cem\u003eEscherichia coli\u003c/em\u003e in oxidative stress resistance, adhesion, and motility. J Bacteriol. 187:6265-72. https://doi.org/10.1128/jb.187.18.6265-6272.2005\u003c/li\u003e\n\u003cli\u003eNandakumar MP, Cheung A, Marten MR (2006) Proteomic Analysis of Extracellular Proteins from Escherichia coli W3110. J. Proteome Res 5:1155-61.https://doi.org/10.1021/pr050401j\u003c/li\u003e\n\u003cli\u003eOrelle C, Mathieu K, Jault JM (2019). Multidrug ABC transporters in bacteria. Res. Microbiol 170:381-91. https://doi.org10.1016/j.resmic.2019.06.001\u003c/li\u003e\n\u003cli\u003ePasqua M, Bonaccorsi di Patti MC, Fanelli G, Utsumi R, Eguchi Y, Trirocco R, Prosseda G, Grossi M, Colonna B (2021) Host-bacterial pathogen communication: the wily role of the multidrug efflux pumps of the MFS family. Front Mol Biosci 8:723274. https://doi.org/10.3389/fmolb.2021.723274\u003c/li\u003e\n\u003cli\u003eQueipo-Ortu\u0026ntilde;o MI, De Dios Colmenero J, Macias M, Bravo MJ, Morata P (2008) Preparation of bacterial DNA template by boiling and effect of immunoglobulin G as an inhibitor in real-time PCR for serum samples from patients with brucellosis. Clin. Vaccine Immunol. 15:293-6. https://doi.org/10.1128/CVI.00270-07\u003c/li\u003e\n\u003cli\u003eRaynal B, Lenormand P, Baron B, Hoos S, England P (2014) Quality assessment and optimization of purified protein samples: why and how?. Microb Cell Fact 13:180. https://doi.org/10.1186/s12934-014-0180-6\u003c/li\u003e\n\u003cli\u003eRizvi M, Malhotra S, Agarwal J, Siddiqui AH, Devi S, Poojary A, Thakuria B, Princess I, Sami H, Gupta A, Sultan A (2024) Regional variations in antimicrobial susceptibility of community-acquired uropathogenic Escherichia coli in India: Findings of a multicentric study highlighting the importance of local antibiograms. IJID regions 11:100370. https://doi.org/10.1016/j.ijregi.2024.100370\u003c/li\u003e\n\u003cli\u003eRodrigues IC, Rodrigues SC, Duarte FV, Costa PM, Costa PM (2022) The role of outer membrane proteins in UPEC antimicrobial resistance: A systematic review. Membranes 12:981. https://doi.org/10.3390/membranes12100981\u003c/li\u003e\n\u003cli\u003eRozwadowski M, Gawel D (2022) Molecular factors and mechanisms driving multidrug resistance in uropathogenic Escherichia coli\u0026mdash;an update. Genes 13:1397. https://doi.org/10.3390/genes13081397\u003c/li\u003e\n\u003cli\u003eSanders KL, Edwards JL (2020) Nano-liquid chromatography-mass spectrometry and recent applications in omics investigations. Anal. Methods 12:4404-17. https://doi.org/10.1039/D0AY01194K\u003c/li\u003e\n\u003cli\u003eShea AE, Forsyth VS, Stocki JA, Mitchell TJ, Frick-Cheng AE, Smith SN, Hardy SL, Mobley HL (2024) Emerging roles for ABC transporters as virulence factors in uropathogenic Escherichia coli. Proc. Acad. Natl. Sci. 121:e2310693121. https://doi.org/10.1073/pnas.2310693121\u003c/li\u003e\n\u003cli\u003eSrivastava N, Verma S, Singh M, Kumar A (2024) A short-term study on statistical numeration of multidrug resistant Escherichia coli isolates among the patients with urinary tract infection. IJEB 62:119-125. https://doi.org/10.56042/ijeb.v62i02.2549\u003c/li\u003e\n\u003cli\u003eSubashchandrabose S, Mobley HLT. Virulence and Fitness Determinants of Uropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (2015) Microbiol Spectr. 3:10.1128. \u003cu\u003e10.1128/microbiolspec.UTI-0015-2012\u003c/u\u003e \u003c/li\u003e\n\u003cli\u003eTarchouna M, Ferjani A, Ben-Selma W, Boukadida J (2013) Distribution of uropathogenic virulence genes in Escherichia coli isolated from patients with urinary tract infection. Int J Infect Dis 17:e450-3. https://doi.org/10.1016/j.ijid.2013.01.025\u003c/li\u003e\n\u003cli\u003eUsein CR, Damian M, Tatu‐Chitoiu D, Capusa C, Fagaras R, Tudorache D, \u003cem\u003eet al\u003c/em\u003e (2001) Prevalence of virulence genes in Escherichia coli strains isolated from Romanian adult urinary tract infection cases. J Cell Mol Med\u003cem\u003e \u003c/em\u003e5:303-10. https://doi.org/10.1111/j.1582-4934.2001.tb00164.x\u003c/li\u003e\n\u003cli\u003eVaca DJ, Thibau A, Sch\u0026uuml;tz M, Kraiczy P, Happonen L, Malmstr\u0026ouml;m J, Kempf VA (2020) Interaction with the host: the role of fibronectin and extracellular matrix proteins in the adhesion of Gram-negative bacteria. Med Microbiol Immunol\u003cem\u003e \u003c/em\u003e209:277-99. https://doi.org/10.1007/s00430-019-00644-3\u003c/li\u003e\n\u003cli\u003eViveiros M, Dupont M, Rodrigues L, Couto I, Davin-Regli A, Martins M, Pages JM, Amaral L (2007) Antibiotic stress, genetic response and altered permeability of \u003cem\u003eE. coli\u003c/em\u003e. PloS one 2:e365. https://doi.org/10.1371/journal.pone.0000365\u003c/li\u003e\n\u003cli\u003eWiegand I, Hilpert K, Hancock RE (2008) Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc\u003cem\u003e.\u003c/em\u003e 3:163-75. \u003cu\u003ehttps://doi.org/10.1038/nprot.2007.521\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eYing N, Zheng Z, Xu H, Tian B, Hua Y (2008) Extracellular Proteome Changes of Deinococcus radiodurans Under \u0026gamma;-Irradiation Stress Conditions. Protein and Peptide Letters 15:595-9. https://doi.org/10.2174/092986608784966985\u003c/li\u003e\n\u003cli\u003eZhao Q, Wu Y, Sun J, Zhang J, Li X, Pang X, Gu S (2025) The ABC Transport Protein PotC Plays a Crucial Role in Antibiotic Resistance in Escherichia coli. Appl Biochem Microbiol 61: 58\u0026ndash;67. \u003cu\u003ehttps://doi.org/10.1134/S0003683824603706\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eZhou G, Shi QS, Huang XM, Xie XB (2015) The three bacterial lines of defense against antimicrobial agents. Int. J. Mol. Sci 16:21711-33. https://doi.org/10.3390/ijms160921711\u003c/li\u003e\n\u003cli\u003eZhou G, Wang Q, Wang Y, Wen X, Peng H, Peng R, Shi Q, Xie X, Li L (2023) Outer Membrane Porins Contribute to Antimicrobial Resistance in Gram-Negative Bacteria. Microorganisms 11:1690. \u003cu\u003e10.3390/microorganisms11071690\u003c/u\u003e\u003c/li\u003e\n\u003cli\u003eZhou Y, Zhou Z, Zheng L, Gong Z, Li Y, Jin Y, Huang Y, Chi M (2023) Urinary tract infections caused by uropathogenic Escherichia coli: mechanisms of infection and treatment options. Int J Mol Sci 24:10537.https://doi.org/10.3390/ijms241310537\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Uropathogenic Escherichia coli, amoxicillin resistance, extracellular proteins, proteome analysis, mass spectrometry","lastPublishedDoi":"10.21203/rs.3.rs-7634107/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7634107/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAntibiotic resistance in uropathogenic \u003cem\u003eEscherichia coli\u003c/em\u003e (UPEC) is a major obstacle to the treatment of urinary tract infections (UTIs). Previous research has focused on antibiotic susceptibility, antibiotic resistance genes, and the underlying intracellular mechanisms therein. This study aims to go beyond existing research by examining the extracellular proteins specifically produced by UPEC in response to amoxicillin (AMX) resistance with the goal of identifying potential therapeutic targets. We selected intrinsically antibiotic-sensitive UPEC strains from previous studies and developed AMX-resistant strains through prolonged AMX exposure. Using nano-based LC-ESI orbitrap mass spectrometry, we analyzed the extracellular proteome of the developed AMX-resistant UPEC and the wild-type control. Proteomics analysis revealed a total of 516 common extracellular proteins in the untreated and AMX-treated developed resistant UPEC strain. While 1,299 extracellular proteins were identified exclusively in the AMX-treated evolved resistant strain, which were categorized into 14 distinct groups based on Gene Ontology (GO) annotation. Among them, the transporter class was the second most abundant class, which included primary active transporters, secondary carrier transporters, and ion channels. Further analysis revealed 57 transporter proteins, including 4 multidrug-resistant transporter proteins, suggesting their potential role in AMX resistance adaptation in UPEC. Such a study can also contribute to creating new drugs or therapies aimed at controlling a wide range of infections, including UTIs, by targeting important identified molecules.\u003c/p\u003e","manuscriptTitle":"Amoxicillin resistance in uropathogenic Escherichia coli: extracellular proteomic Insights into transporter and multidrug resistance transporter proteins","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 06:06:17","doi":"10.21203/rs.3.rs-7634107/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":"20f3fbc7-cc34-406c-86d0-9cb98874428a","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T12:38:16+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-18 06:06:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7634107","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7634107","identity":"rs-7634107","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}