Abstract
Background and objectives: Multidrug-resistant (MDR) Escherichia coli is a global concern. E. coli exhibits resistance through extended-spectrum beta-lactamase (ESBL) production, efflux pumps, and biofilm formation. Fosfomycin alone and in combination with amikacin and ciprofloxacin is effective against MDR E. coli. We conducted this study to evaluate the in vitro activity of fosfomycin, alone and in combination with amikacin and ciprofloxacin, against clinical MDR E. coli isolates, including ESBL and biofilm producers. We assessed the in vitro effects by calculating the minimum inhibitory concentration (MIC). The biofilm grades were evaluated using a spectrophotometer. We also assessed the drug interactions of fosfomycin with amikacin and ciprofloxacin using the fractional inhibitory concentration index (FICI) and Epsilometer test (E-test) cross methods.
Methods: This study was conducted from April 2023 to March 2025 at Kalinga Institute of Medical Sciences (KIMS), Bhubaneswar, India. We included MDR E. coli isolates collected from clinical specimens (i.e., urine, blood, cerebrospinal fluid, respiratory samples, pus, and tissues) of adult patients of both sexes. The bacteria in the specimens were cultivated using enriched and selective media, such as MacConkey agar and 5% sheep blood agar. Antibiotic susceptibility pattern of E. coli evaluated by VITEK AST card N-235 and N-405 (BioMérieux, Marcy-l'Étoile, France) for urinary and non-urinary isolates, respectively. The MDR strains were noted. The biofilm grades were assessed using a spectrophotometer by measuring optical density (OD). The higher OD values were suggestive of stronger biofilms by the MDR E. coli isolates. The drug interactions of fosfomycin with amikacin and ciprofloxacin were assessed through FICI and E-test cross methods. Lower FICI values indicated additive and synergistic interactions. We analyzed the data through R software (version 4.5.2) (R Foundation for Statistical Computing, Vienna, Austria).
Results: We analyzed 422 MDR E. coli isolates during the study period. The majority of the samples were urine samples (306, 72.5%). Most of the MDR E. coli isolates were ESBL producers (364, 86.3%). Biofilm production was seen in only 131 (31.0%) isolates. The median OD value of all 422 isolates was 0.073 (0.066-0.129). The majority of isolates were either biofilm non-producers or weak biofilm producers. The MIC90 values of fosfomycin, amikacin, and ciprofloxacin were 32, 8, and 0.5 micrograms/ml, respectively. The FICI values of the combinations of fosfomycin with amikacin and ciprofloxacin were 0.62 and 0.86 microgram/ml, respectively. These values supported the synergistic and additive interactions among the drugs. Drug combinations were more effective than monotherapy across all categories of biofilm producers.
Conclusion: The MDR E. coli isolates were more common in urine specimens. Most of them were ESBL producers. Around one-third of isolates were biofilm producers. The drug combinations were more effective than monotherapy with the three antimicrobials. We recommend prospective studies with larger sample sizes to confirm the clinical relevance of our findings.
Keywords: amikacin, biofilm layer, ciprofloxacin, drug-interaction, drug synergism, escherichia coli infection, extended-spectrum beta-lactamase (esbl), fosfomycin sensitivity, in vitro study, multidrug resistance (mdr)
Introduction
Escherichia coli is a facultative anaerobic, gram-negative (GN) bacillus of the Enterobacteriaceae family. It causes urinary tract infections (UTIs), intestinal and bloodstream infections, and surgical site infections [1-3]. Non-susceptibility to at least one antibiotic in three or more classes, as determined by in vitro susceptibility testing, is referred to as multidrug resistance [4]. β-lactamases, enzymes that hydrolyze β-lactam antibiotics and render them ineffective, are typically responsible for mediating β-lactam resistance [5]. Infections caused by multidrug-resistant (MDR) E. coli are responsible for millions of deaths worldwide [6,7]. MDR E. coli possess multiple resistance mechanisms (e.g., extended-spectrum beta-lactamase (ESBL) production, efflux pumps, and biofilm formation). Surveillance of ESBL-producing E. coli is crucial for directing empirical treatment and tracking resistance patterns [6-9].
Biofilm formation complicates the management of infectious conditions. Because the biofilms protect bacteria from antimicrobials and host defenses, thereby promoting persistence and recurrent infections. E. coli biofilms are particularly relevant in catheter-associated UTIs and healthcare settings, and their association with MDR phenotypes contributes to therapeutic challenges [8,10,11]. Approximately 67% of uropathogenic isolates of MDR E. coli strains produce biofilms. Biofilms are highly resistant to antibiotics owing to limited drug penetration and reduced metabolism [10-12]. The biofilm matrix, composed of extracellular DNA, proteins, and polysaccharides, protects bacteria from antimicrobials and host defenses, thereby promoting persistence and recurrent infections [12,13].
This highlights the need to explore the drugs with anti-biofilm effects. Antibacterial agents, such as fosfomycin, have a distinct mechanism of inhibiting MurA enzyme-mediated peptidoglycan synthesis, making them effective against MDR and ESBL-producing E. coli strains [14]. It has activity against biofilm-associated bacteria and can be used in combination with bactericidal antibiotics such as ciprofloxacin and amikacin [14,15]. Fosfomycin combined with amikacin or ciprofloxacin targets distinct bacterial pathways, thereby improving bacterial killing and biofilm penetration [14-16]. Their different targets increase the potential for synergistic interaction and reduce the likelihood of cross-resistance [15,16]. Evaluating drug interactions of these drugs using the fractional inhibitory concentration index (FICI) [17] and Epsilometer test (E-test) cross methods [18] helps identify clinically useful combinations for MDR E. coli isolates.
An in vitro study was conducted as a preliminary step to evaluate the antimicrobial efficacy and synergistic potential of fosfomycin combinations before clinical application. Such studies are essential for screening promising antibiotic combinations, determining MICs, and assessing synergy indices under controlled conditions. Given the rising prevalence of MDR E. coli in India and limited treatment options, this study was undertaken to evaluate the in vitro activity of fosfomycin, alone and in combination with amikacin and ciprofloxacin, against clinical MDR E. coli isolates, including ESBL producers and biofilm producers. We assessed the in vitro effects by calculating the minimum inhibitory concentration (MIC). The grade of biofilm production was evaluated by its optical density (OD) values. We gauged the drug interactions of fosfomycin with amikacin and ciprofloxacin through alluvial plots.
Materials and methods
Study design
This in vitro study was conducted in the Department of Microbiology at Kalinga Institute of Medical Sciences (KIMS), Bhubaneswar, from April 2023 to March 2025. Before conducting the study, we obtained ethical permission from the Institutional Ethics Committee (KIIT/KIMS/IEC/1199/2023 dated 27.02.2023).
Study criteria
Clinically significant (i.e., with confirmed infections), non-duplicate MDR E. coli isolates collected from various clinical specimens (urine, blood, pus, respiratory samples, cerebrospinal fluid, and tissues) of adult patients (admitted to the hospital during the study period) of both sexes were included. A single specimen was analyzed for each participant. Repeat isolates, colonizers, environmental samples, and isolates with incomplete clinical data were excluded.
Study procedure
The study was carried out on non-repeat clinically relevant isolates of MDR E. coli during the study period. Demographic details (age and sex) and specimen type were noted. The clinical samples were collected in sterile containers following aseptic measures and sent to the laboratory without delay. Urine samples were inoculated into Cysteine Lactose Electrolyte Deficient (CLED) media. The plates were then incubated at 37°C overnight. Blood samples were collected in BacT/Alert (BioMérieux, Marcy-l'Étoile, France) broth bottles. The bottles were incubated in a BacT/Alert 3D incubator (BioMérieux, Marcy-l'Étoile, France) for five days or until the bottles were flagged as positive. Then, plating was done on blood agar and MacConkey agar. Respiratory (sputum, endotracheal aspirate, and bronchoalveolar lavage fluid), pus, and tissue samples were also inoculated onto MacConkey agar and blood agar and incubated in a CO2 incubator for 24 to 48 hours.
The isolated colonies of E. coli were identified using the VITEK 2 automated system (BioMérieux, Marcy-l'Étoile, France) with the GN card. Antibiotic susceptibility pattern of E. coli evaluated by VITEK AST card N-405 for non-urinary isolates and N-235 for urinary isolates. MDR strain was defined as non-susceptibility to at least one agent in three or more antimicrobial categories. MDR E. coli were stored in 50% glycerol broth for testing ESBL production, biofilm formation, fosfomycin sensitivity, and the synergistic effects of fosfomycin combined with amikacin and ciprofloxacin. The tissue culture plate method for biofilm formation (Figure 1) was performed using 96-well microplates [19]. For quality control, E. coli (ATCC 25922) and K. pneumoniae (ATCC 700603) were used as negative and positive controls [20]. The MDR isolates were grown overnight in tryptic soy broth (TSB) with 1% glucose at 37°C. 200 μL of bacterial suspension was inoculated into sterile 96-well flat-bottom polystyrene microtiter plates. After 24 hours of incubation at 37°C, the wells were washed with phosphate-buffered saline (PBS, pH 7.2). Then they were fixed with methanol for 15 min and stained with 0.1% crystal violet for 15 min. The excess stain was washed off, wells were dried, and dye was solubilized with 33% glacial acetic acid. The optical density (OD₄₉₀) of stained adherent bacteria was measured using a microplate reader at 490 nm [21]. The classification of biofilm formation capacity is shown in Table 1. The optical density cut-off value (ODc) is defined as three times the standard deviation (SD) above the mean OD value of the negative control.
Table 1. Interpretation of biofilm formation capacity.
ODs: mean optical density of the sample. ODc: mean OD of the negative control plus three times the standard deviation (SD).
| Criteria | Biofilm formation capacity |
| ODs ≤ ODc | Not a biofilm producer |
| ODc ≤ ODs ≤ 2 × ODc | Weak biofilm producer |
| 2 × ODc ≤ ODs ≤ 4 × ODc | Moderate biofilm producer |
| 4 × ODc < ODs | Strong biofilm producer |
Figure 1. Biofilm formation in 96-well microplates.
NC: negative control, PC: positive control.
The detection of ESBL producers (Figure 2) was screened by reduced susceptibility to ceftazidime 30 μg and cefotaxime 30 μg. The production of ESBL was confirmed by the combined disk test (CDT). The CDT employs ceftazidime 30 μg versus ceftazidime 30 μg + clavulanate 10 μg. An increase of ≥5 mm in zone diameter with clavulanate was considered ESBL positive. For quality control, E. coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as negative and positive controls, respectively.
Figure 2. Detection of ESBL production by the combined disk method.
ESBL: extended-spectrum beta-lactamase, NC: negative control, PC: positive control.
The in vitro activity of fosfomycin against MDR E. coli was tested using the agar dilution method (Figure 3) as per Clinical and Laboratory Standards Institute (CLSI) guidelines [22]. Different concentrations of fosfomycin (i.e., 8, 16, 32, 64, and 128 µg/ml) were prepared. The bacterial isolates were spot-inoculated onto these drug-containing plates, and a separate growth-control plate was incubated at 37°C for 24 hours. After incubation, growth was assessed on the control plate to confirm the bacteria's viability. The lowest concentration of fosfomycin at which no visible bacterial growth occurred was recorded as the MIC for that isolate. The MIC90 value represents the MIC that inhibits the growth of 90% of the organisms.
Figure 3. MIC determination by agar dilution method.
MIC: minimum inhibitory concentration.
The effect of fosfomycin on biofilm disruption by MDR E. coli was tested in 96-well plates. After biofilm formation, the wells were washed to remove free-floating bacteria, then treated with fosfomycin at concentrations ranging from 0.25 µg/mL to 32 µg/mL. Following 24 hours of incubation at 37°C, the wells were washed, stained with crystal violet, and the optical density (OD₄₉₀) was measured to assess biofilm mass. Each test was done in triplicate for accuracy [21].
The MIC values of ciprofloxacin and amikacin for synergy testing were confirmed by the Epsilometer test (E-test) strip [18]. Inoculum of 0.5 McFarland was lawn cultured over a Mueller-Hinton agar (MHA) plate, and E-test strips of ciprofloxacin and amikacin were placed individually over them. The plates were incubated at 35-37°C in an incubator for 18 hours. The point at which the zone of inhibition meets the strip was determined to be the MIC. The in vitro activity of combination antibiotics was tested by placing E-test strips of the two antimicrobials on agar at a 90° angle, with the intersection at the respective MICs for the organism. Figures 4, 5 demonstrate the drug interactions of fosfomycin with amikacin and ciprofloxacin, respectively. The MHA agar plates were incubated at 35-37°C in an incubator for 18 hours, and the MICs of each antimicrobial combination were determined. The effects of the antimicrobial combinations were defined according to the FICI [17]. The formula for FICI is as follows:
FICI = (MIC drug A plus drug B/MIC drug A) + (MIC drug A plus drug B/MIC drug B).
Table 2 illustrates the interpretation of FICI values. Figures 4, 5 show the synergy testing of fosfomycin with amikacin and ciprofloxacin, respectively.
Table 2. Interpretation of FICI values.
FICI: fractional inhibitory concentration index.
| FICI value | Interpretation |
| ≤0.5 | Synergy |
| >0.5 to ≥1 | Additive |
| >1 to ≤4 | No interaction (indifference) |
| >4 | Antagonism |
Figure 4. Synergy testing of fosfomycin with amikacin.
Figure 5. Synergy testing of fosfomycin with ciprofloxacin.
Statistical analysis
A non-probability consecutive sampling was used to conduct this in vitro study. The Shapiro-Wilk test was used to verify that the data were normally distributed. The median and interquartile range (IQR) were used to describe the continuous data. For the categorical data, the frequency and proportion were shown. We used an upset plot to illustrate the distribution of the study population. The OD values of biofilms were depicted through a combination of half-eye, beeswarm, and jitter plots. The alluvial plots illustrated drug interactions between fosfomycin and amikacin or ciprofloxacin across various biofilm types and ESBL producers. R software version 4.5.2 (R Foundation for Statistical Computing, Vienna, Austria) was used to compute the data [23]. Statistical significance was defined as a p-value of 0.05 or less.
Results
In this study, we analyzed 422 clinical isolates of MDR E. coli. Table 3 shows the demographic and clinical traits of the study participants. 221 (52.4%) of 422 participants were males. The median age of the study population was 53.5 (38.5-71.0) years. The 306 (72.5%) urine samples accounted for the majority. There were 131 (31.0%) biofilm producers and 364 (86.3%) ESBL producers.
Table 3. Demographic and clinical traits of the study population.
The median (IQR) was used to depict the continuous variables. n (%) was used to display the category values. IQR: interquartile range, ESBL: extended-spectrum beta-lactamase.
| Parameters | Value |
| Total participants | 422 |
| Age (years) | 53.5 (38.5-71.0) |
| Elderly | 147 (34.8%) |
| Male | 221 (52.4%) |
| Urine samples | 306 (72.5%) |
| Biofilm producers | 131 (31.0%) |
| ESBL producers | 364 (86.3%) |
| Duration of hospitalization (days) | 12.5 (6.5-18.5) |
Figure 6 illustrates the distribution of the study population through an upset plot. The upset plot consists of two sets of bar plots (one horizontal and one vertical) and a matrix that demonstrates the intersections of the variables shown in the horizontal bar plot. The following data were shown in the horizontal bar plot: males, elderly participants, urine samples, ESBL-producing pathogens, and biofilm producers. Both bar plots were arranged in decreasing order. Urine samples from 73 (17.3%) young female and 55 (13.0%) young male subjects yielded ESBL-producing, biofilm-nonproducing MDR E. coli isolates.
Figure 6. Upset plot showing the distribution of the study population.
The distribution of the study population is illustrated through an upset plot. The horizontal bar plot (red) shows the number of participants across the five sets (i.e., male and elderly participants, urine samples, ESBL-producing pathogens, and biofilm producers). The matrix (blue color) shows various intersections through the highlighted dots and connecting lines. The vertical bar plot (green) shows the number of participants in each intersection. Both bar plots are arranged in decreasing order. ESBL: extended-spectrum beta-lactamase.
Figure 7 illustrates the OD values of biofilms produced by the E. coli isolates through half-eye, interval, and beeswarm plots. Figure 7a shows the median OD values of the entire study population. The median OD value was 0.073 (0.066-0.129) (male: 0.078 (0.069-0.129), female: 0.072 (0.066-0.135), p = 0.50). Figure 7b shows the median OD values of the study participants without ESBL-producing E. coli (n = 58). The median OD value of this subgroup was 0.076 (0.066-0.126) (male: 0.070 (0.069-0.080), female: 0.080 (0.066-0.186), p = 0.53). Figure 7c shows the median OD values of the study participants with ESBL-producing E. coli (n = 364). The median OD value of this subgroup was 0.073 (0.066-0.129) (male: 0.086 (0.069-0.129), female: 0.072 (0.065-0.123), p = 0.25). Figure 7d shows the median OD values of adult participants (n = 275). The median OD value of this subgroup was 0.073 (0.066-0.135) (male: 0.075 (0.067-0.133), female: 0.073 (0.067-0.132), p = 0.93). Figure 7e shows the median OD values of elderly participants (n = 147). The median OD value of this subgroup was 0.070 (0.066-0.123) (male: 0.082 (0.069-0.122), female: 0.070 (0.063-0.132), p = 0.34). The ODc value in our study was 0.04. Based on the criteria for OD values shown in Table 1, the majority of isolates were either biofilm non-producers or weak biofilm producers.
Figure 7. OD values of the biofilms produced by E. coli isolates.
The half-eye, interval, and beeswarm plots illustrate the OD values of biofilms produced by the E. coli isolates. Figures (a)-(e) represent the entire study population, participants without ESBL-producing E. coli, participants with ESBL-producing E. coli, adult participants, and elderly participants, respectively. OD: optical density, ESBL: extended-spectrum beta-lactamase.
Table 4 shows the MIC values of the three drugs (i.e., fosfomycin, amikacin, and ciprofloxacin) alone and the FICI values of the two combinations (i.e., fosfomycin plus amikacin and fosfomycin plus ciprofloxacin). The lower FICI values for combinations of two drugs (amikacin and ciprofloxacin) with fosfomycin indicate synergistic and additive interactions among the drugs.
Table 4. MIC90 and FICI values.
The MIC90 values were expressed in micrograms per milliliter. MIC: minimum inhibitory concentration, FICI: fractional inhibitory concentration index, ESBL: extended-spectrum beta-lactamase.
| Variables | MIC90 fosfomycin | MIC90 amikacin | MIC90 ciprofloxacin | FICI for fosfomycin plus amikacin | FICI for fosfomycin plus ciprofloxacin |
| Total (n = 422) | 32 | 8 | 0.5 | 0.62 | 0.86 |
| Male (n = 221) | 32 | 8 | 0.5 | 0.57 | 0.78 |
| Female (n = 201) | 32 | 8 | 0.5 | 0.68 | 0.95 |
| ESBL positive (n = 364) | 32 | 8 | 0.5 | 0.61 | 0.84 |
| ESBL negative (n = 58) | 32 | 8 | 0.5 | 0.68 | 0.99 |
| Biofilm producers (n = 131) | 32 | 8 | 0.5 | 0.56 | 0.79 |
| Biofilm non-producers (n = 291) | 32 | 8 | 0.5 | 0.65 | 0.89 |
| Urine samples (n = 306) | 32 | 8 | 0.5 | 0.57 | 0.83 |
| Non-urine samples (n = 116) | 64 | 16 | 1 | 0.75 | 0.94 |
The alluvial plots in Figures 8, 9 illustrate the association among fosfomycin-drug interactions (with amikacin and ciprofloxacin), biofilm grades, and the presence of ESBL-producing E. coli. There were 131 (31.0%) biofilm-producing and 364 (86.3%) ESBL-producing MDR E. coli. Figure 8 shows 158 (37.4%) cases of synergism and 113 (26.8%) cases of additive interactions between fosfomycin and amikacin. The remaining 151 (35.8%) interactions were insignificant. The incidence of synergism was higher than that of additive interactions across all three biofilm categories (i.e., strong, moderate, and weak). Figure 9 shows 133 (31.5%) cases of synergism and 96 (22.7%) cases of additive interactions between fosfomycin and ciprofloxacin. The remaining 193 (45.8%) interactions were insignificant. The incidence of synergism was higher than that of additive interactions across all three biofilm categories (i.e., strong, moderate, and weak). Combinations of fosfomycin with amikacin and ciprofloxacin were more effective (as evidenced by synergistic interactions) than monotherapy for all categories of biofilm producers.
Figure 8. Associations of biofilm and ESBL production with interaction of fosfomycin and amikacin.
The alluvial plot illustrates the association between biofilm and ESBL production in MDR E. coli, as well as the interactions between fosfomycin and amikacin. The number of participants is shown on the y-axis. The biofilm types (i.e., strong, moderate, weak, and no biofilm) are portrayed in different colors. The widths of the bands denote the proportions of participants with the relevant parameters. ESBL: extended-spectrum beta-lactamase, MDR: multidrug-resistant, AK: amikacin.
Figure 9. Associations of biofilm and ESBL production with interaction of fosfomycin and ciprofloxacin.
The alluvial plot illustrates the association between biofilm and ESBL production in MDR E. coli, as well as the interactions between fosfomycin and ciprofloxacin. The number of participants is shown on the y-axis. The biofilm types (i.e., strong, moderate, weak, and no biofilm) are portrayed in different colors. The widths of the bands denote the proportions of participants with the relevant parameters. ESBL: extended-spectrum beta-lactamase, MDR: multidrug-resistant, CIP: ciprofloxacin.
Discussion
In this in vitro study, we analyzed 422 MDR E. coli isolates (one isolate per participant). The majority of the samples were urine samples (306, 72.5%). Most of the MDR E. coli isolates were ESBL producers (364, 86.3%). Biofilm production was seen in only 131 (31.0%) isolates. The FICI values were lower for the following MDR E. coli isolates than for their counterparts: ESBL producers, biofilm producers, and isolates obtained from urine specimens or male participants. Lower FICI values suggested synergistic and additive interactions among the drugs. Our observations concurred with the findings of two recent studies [21,24,25].
Among the 422 samples analyzed, the urine samples from 73 (17.3%) young female and 55 (13.0%) young male subjects had the highest numbers of MDR E. coli isolates. An equilibrium between the urethra and bacteria can be maintained by the urethral mucosa and epithelial cells' ability to fight off harmful bacterial invasion. Highly pathogenic bacteria, such as uropathogenic E. coli (UPEC), disrupt this equilibrium and weaken the defense system. These reactions are frequently influenced by specific UPEC components, including polysaccharide capsules, flagella, fimbriae, outer-membrane proteins (OMPs), lipopolysaccharides (LPSs), and iron-acquisition receptors [1,8,9].
MDR E. coli exerts antimicrobial resistance through multiple mechanisms (e.g., ESBL production, biofilm formation, and efflux pumps). Biofilms not only shield pathogens from phagocytosis, antimicrobial peptides, and antibody opsonization but also prevent bacteria from being ejected by epithelial cells via ciliary action [6,7,26]. Numerous efflux pumps, which are responsible for eliminating a variety of chemicals from inside the cell, are frequently encoded in bacterial genomes. Efflux pumps can significantly affect a bacterial species' innate drug sensitivity and the emergence of clinically meaningful antibiotic resistance when overexpressed [7-9,26,27].
The FICI values and alluvial plots revealed that drug combinations were more effective than monotherapy with the three antimicrobials. Studies in animals have shown that fosfomycin prevents aminoglycoside-induced histamine release from mast cells, thereby providing protection against aminoglycoside-related nephrotoxicity [14,15,28]. Because of amikacin's concentration-dependent bactericidal activity, this combination will enable a higher, once-daily amikacin dosage if required [15,28].
Strengths and limitations
The calculations of OD value for assessing biofilm strength and FICI values for interpreting the drug interactions were the strengths of this study. Another plus was the graphical illustration of drug interactions through the alluvial plots. Our study had some limitations as well. First, clinical correlation could not be performed owing to the in vitro study design. Secondly, we analyzed neither non-MDR E. coli isolates nor any other MDR pathogens.
Conclusions
Urine specimens had more MDR E. coli isolates. They were mostly ESBL producers. Biofilms were produced by about one-third of the isolates. The medication combinations outperformed the three antimicrobials when used alone, as evidenced by the FICI values. Combinations of fosfomycin with amikacin and ciprofloxacin were more effective (as evidenced by synergistic interactions) than monotherapy for all categories of biofilm producers. To clinically correlate the results of our study, we advise conducting prospective studies with larger sample sizes.
Disclosures
Human subjects: Informed consent for treatment and open access publication was obtained or waived by all participants in this study. Institutional Ethics Committee Kalinga Institute of Medical Sciences (KIMS) issued approval KIIT/KIMS/IEC/1199/2023 dated 27.02.2023.
Animal subjects: All authors have confirmed that this study did not involve animal subjects or tissue.
Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following:
Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work.
Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work.
Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work.
Author Contributions
Concept and design: Jyoti Prakash Sahoo, Kumudini Panigrahi, Dipti Pattnaik
Acquisition, analysis, or interpretation of data: Jyoti Prakash Sahoo, Swadhin Choudhury, Kumudini Panigrahi, Subhra Snigdha Panda
Drafting of the manuscript: Jyoti Prakash Sahoo, Swadhin Choudhury, Kumudini Panigrahi, Subhra Snigdha Panda, Dipti Pattnaik
Critical review of the manuscript for important intellectual content: Jyoti Prakash Sahoo
Supervision: Jyoti Prakash Sahoo, Kumudini Panigrahi, Subhra Snigdha Panda, Dipti Pattnaik
References
- 1.Urinary tract infections caused by uropathogenic Escherichia coli: mechanisms of infection and treatment options. Zhou Y, Zhou Z, Zheng L, et al. Int J Mol Sci. 2023;24:10537. doi: 10.3390/ijms241310537. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.A nationwide population-based study of Escherichia coli bloodstream infections: incidence, antimicrobial resistance and mortality. Feldman SF, Temkin E, Wullfhart L, et al. Clin Microbiol Infect. 2022;28:879–870. doi: 10.1016/j.cmi.2021.12.009. [DOI] [PubMed] [Google Scholar]
- 3.Emergence and dissemination of antimicrobial resistance in Escherichia coli causing bloodstream infections in Norway in 2002-17: a nationwide, longitudinal, microbial population genomic study. Gladstone RA, McNally A, Pöntinen AK, et al. Lancet Microbe. 2021;2:0–41. doi: 10.1016/S2666-5247(21)00031-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Multidrug-resistant bacteria in surgical intensive care units: antibiotic susceptibility and β-lactamase characterization. Bandić Pavlović D, Pospišil M, Nađ M, et al. Pathogens. 2024;13:411. doi: 10.3390/pathogens13050411. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.The biogenesis of β-lactamase enzymes. Kaderabkova N, Bharathwaj M, Furniss RC, Gonzalez D, Palmer T, Mavridou DA. Microbiology (Reading) 2022;168:1217. doi: 10.1099/mic.0.001217. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Antibiotic resistance profiles and risk factors of multidrug-resistant Escherichia coli in a large tertiary care hospital in a low- and middle-income country. Mousa N, Aiesh BM, Jomaa R, et al. Sci Rep. 2025;15:26667. doi: 10.1038/s41598-025-12384-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Prevalence of multidrug-resistant diarrheagenic Escherichia coli in Asia: a systematic review and meta-analysis. Salleh MZ, Nik Zuraina NM, Hajissa K, Ilias MI, Deris ZZ. Antibiotics (Basel) 2022;11:1333. doi: 10.3390/antibiotics11101333. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.A multicenter analysis of trends in resistance in urinary Enterobacterales isolates from ambulatory patients in the United States: 2011-2020. Dunne MW, Aronin SI, Yu KC, Watts JA, Gupta V. BMC Infect Dis. 2022;22:194. doi: 10.1186/s12879-022-07167-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Antimicrobial resistance trends in urine Escherichia coli isolates from adult and adolescent females in the United States from 2011 to 2019: rising ESBL strains and impact on patient management. Kaye KS, Gupta V, Mulgirigama A, et al. Clin Infect Dis. 2021;73:1992–1999. doi: 10.1093/cid/ciab560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Catheter-associated urinary tract infections: understanding the interplay between bacterial biofilm and antimicrobial resistance. Tegegne DT, Abbott IJ, Poźniak B. Int J Mol Sci. 2025;26:9193. doi: 10.3390/ijms26189193. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Biofilm-associated Escherichia coli infections: pathogenesis, clinical implications, and treatment strategies. Thakur D, Kumar L. Crit Rev Microbiol. 2026;52:198–242. doi: 10.1080/1040841X.2025.2548490. [DOI] [PubMed] [Google Scholar]
- 12.Biofilms: formation, drug resistance and alternatives to conventional approaches. Mirghani R, Saba T, Khaliq H, et al. AIMS Microbiol. 2022;8:239–277. doi: 10.3934/microbiol.2022019. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Biofilm formation: mechanistic insights and therapeutic targets. Wang X, Liu M, Yu C, Li J, Zhou X. Mol Biomed. 2023;4:49. doi: 10.1186/s43556-023-00164-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Potential inhibitors targeting Escherichia coli UDP-N-acetylglucosamine enolpyruvyl transferase (MurA): an overview. Raina D, Kumar C, Kumar V, Khan IA, Saran S. Indian J Microbiol. 2022;62:11–22. doi: 10.1007/s12088-021-00988-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Assessment of in-vitro synergy of fosfomycin with meropenem, amikacin and tigecycline in whole genome sequenced extended and pan drug resistant Klebsiella pneumoniae: exploring a colistin sparing protocol. Al-Quraini M, Rizvi M, Al-Jabri Z, et al. Antibiotics (Basel) 2022;11:153. doi: 10.3390/antibiotics11020153. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Synergistic activity and molecular modelling of fosfomycin combinations with some antibiotics against multidrug resistant Helicobacter pylori. Abouwarda AM, Ismail TA, Abu El-Wafa WM, Faraag AH. World J Microbiol Biotechnol. 2022;38:102. doi: 10.1007/s11274-022-03289-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.The fractional inhibitory concentration (FIC) index as a measure of synergy. Hall MJ, Middleton RF, Westmacott D. J Antimicrob Chemother. 1983;11:427–433. doi: 10.1093/jac/11.5.427. [DOI] [PubMed] [Google Scholar]
- 18.Comparison of three different in vitro methods of detecting synergy: time-kill, checkerboard, and E test. White RL, Burgess DS, Manduru M, Bosso JA. Antimicrob Agents Chemother. 1996;40:1914–1918. doi: 10.1128/aac.40.8.1914. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Detection of biofilm formation among the clinical isolates of Staphylococci: an evaluation of three different screening methods. Mathur T, Singhal S, Khan S, Upadhyay DJ, Fatma T, Rattan A. Indian J Med Microbiol. 2006;24:25–29. doi: 10.4103/0255-0857.19890. [DOI] [PubMed] [Google Scholar]
- 20.Isolation and identification of bacteriophage against Escherichia coli ATCC 25922 and their biofilm inhibition studies. Sivakumar L, John Durai Kumar JV, Madhavan S, et al. Sci Rep. 2025;15:26964. doi: 10.1038/s41598-025-12366-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Efficacy of fosfomycin against planktonic and biofilm-associated MDR uropathogenic Escherichia coli clinical isolates. Dzib-Baak HE, Uc-Cachón AH, Dzul-Beh AJ, Rosado-Manzano RF, Gracida-Osorno C, Molina-Salinas GM. Trop Med Infect Dis. 2022;7:235. doi: 10.3390/tropicalmed7090235. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Clinical and Laboratory Standards Institute. Wayne, Pennsylvania: Clinical and Laboratory Standards Institute; [ Feb; 2026 ]. 2023. M100: Performance Standards for Antimicrobial Susceptibility Testing, 34th ed. [Google Scholar]
- 23.R: A language and environment for statistical computing, Vienna, Austria. [ Feb; 2026 ]. 2020. https://www.r-project.org/ https://www.r-project.org/
- 24.In vitro and in vivo effect of amikacin and imipenem combinations against multidrug-resistant E. coli. Farhan SM, El-Baky RM, Abdalla S, et al. Trop Med Infect Dis. 2022;7:281. doi: 10.3390/tropicalmed7100281. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.In vitro antibacterial activities of fosfomycin against Escherichia coli isolates from canine urinary tract infection. Jariyapamornkoon N, Nuanualsuwan S, Suanpairintr N. Animals (Basel) 2024;14:1916. doi: 10.3390/ani14131916. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 26.A review of the mechanisms that confer antibiotic resistance in pathotypes of E. coli. Nasrollahian S, Graham JP, Halaji M. Front Cell Infect Microbiol. 2024;14:1387497. doi: 10.3389/fcimb.2024.1387497. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Resistance mechanisms. Cag Y, Caskurlu H, Fan Y, Cao B, Vahaboglu H. Ann Transl Med. 2016;4:326. doi: 10.21037/atm.2016.09.14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.The revival of fosfomycin. Michalopoulos AS, Livaditis IG, Gougoutas V. Int J Infect Dis. 2011;15:0–9. doi: 10.1016/j.ijid.2011.07.007. [DOI] [PubMed] [Google Scholar]