Synergistic Effect of Polyhydroxybutyrate Nanoparticle on the Susceptibility of Escherichia coli to Cefotaxime In Vitro

1. Introduction

Resistance to antibiotics is considered a global challenge facing public health, responsible for increasing rates of morbidity, mortality, and healthcare costs worldwide [1]. It has been reported previously that Escherichia coli isolates cause various infections, including urinary tract infections (UTIs), bacteremia, wound infections, and neonatal sepsis [2].

Recently, E. coli has increasingly acquired resistance to different kinds of antibiotics, largely through the production of extended-spectrum β-lactamases (ESBLs), efflux pump overexpression, and porin alterations, which restrict antibiotic penetration into bacterial cells [3].

Cefotaxime (CTX) is a 3rd-generation cephalosporin. For many years, it has been considered a drug of choice for infections caused by various pathogenic bacteria because of its broad-spectrum activity and relative safety. In contrast, the rising outbreak of ESBL-producing E. coli has substantially compromised its clinical effectiveness, necessitating the development of new strategies to reactivate and restore this antibiotic's susceptibility [4].

Recently, nanotechnology has provided promising avenues to combat antibiotic resistance by enhancing antibiotic delivery, increasing antibiotic potency, and improving synergistic interactions between nanoparticles and conventional antibiotics [5]. Nanoparticles are considered an adjuvant capable of changing bacterial membrane permeability and enhancing antibiotic action [6].

Polyhydroxybutyrate (PHB) is a naturally occurring biopolyester synthesized by different bacteria and plants. It has attracted significant interest not only for this material's biodegradability and biocompatibility, but also for its antimicrobial properties, especially antibacterial effects, and its ability to interact with the cell membrane when PHB is formulated into nanoparticles [7]. Several studies have reported the biomedical applications of PHB, but limited data are available on their interactions with conventional antibiotics and their potential role in reducing pathogenic bacterial resistance to antibiotics [7,8].

No previous study has demonstrated the synergistic effect of PHB-NPs on the effectiveness of cefotaxime against E. coli isolated from wound infections. This project opens the door to new, safe strategies for restoring the efficacy of conventional antibiotics (cefotaxime) against multidrug-resistant E. coli isolated from infected wounds. Thus, the present study aims to assess the synergistic effect of PHB-NPs on the susceptibility of MDR E. coli to CTX.

2. Materials and Methods

2.1. Bacterial Isolates

In the current study, 83 urine samples were collected from the same number of patients. The patients attended two main hospitals in Baghdad, Iraq. Patients had stopped taking antibiotic therapy within 72 h prior to sample collection, and informed consent was obtained from all participants. The samples were inoculated onto various differential and selective culture media, including MacConkey agar. Pink colonies (lactose-fermenting colonies) were selected. Gram stain was performed, followed by biochemical tests. A VITEK DensiCheck instrument and fluorescence system (bioMérieux, Marcy-l'Étoile, France) (ID-GNB card) were used to finally identify the isolates as E. coli.

2.2. Kirby-Bauer Disc Diffusion Method

The Kirby-Bauer disk diffusion method described previously [9,10] was used to determine the antibiotic susceptibility of cefotaxime (CTX) against 10 E. coli isolates. Inhibition zone diameters, including the 6 mm disk diameter, were measured in millimeters. The Clinical and Laboratory Standards Institute (CLSI) M100 breakpoints for CTX were used to interpret the results and categorize isolates as susceptible (S), intermediate (I), or resistant (R).

2.3. PHB-NPs Preparation and Characterization

The PHB-NPs were prepared using a precipitation-solvent evaporation emulsification method. One hundred milligrams of PHB polymer (Sigma-Aldrich, USA) were dissolved in ten milliliters of chloroform (Fluke, UK) with mixing at 20°C. The organic polymer solution was added drop by drop to 100 mL of 0.5% polyvinyl alcohol (Sigma-Aldrich, USA) with ultrasonication (Sonics Vibra-cell, USA) at 40% amplitude for 5 min in an ice bath. The resulting emulsion was incubated overnight with gentle mixing at 20°C, producing stable aqueous PHB-NPs. The yielded PHB-NPs were harvested by centrifugation at 10,000×g for 20 min and washed three times with double-distilled water. The final product was stored at −20°C until use. Morphological characterization was performed using scanning electron microscopy (SEM; Hitachi High-Tech, Tokyo, Japan) after negative staining with 2% uranyl acetate. Fourier-transform infrared (FTIR) spectroscopy (Perkin-Elmer Spectrum 65; PerkinElmer, USA) was used to confirm polymer identity.

2.4. MTT Assay

In the current experiment, the cytotoxicity of PHB-NPs on MCF-7 human breast cancer cells was evaluated using the MTT assay, a colorimetric indicator of cell viability. The method was described in detail in previous studies [11-13]. Cell viability was expressed as a percentage relative to untreated control cells using the following formula:

The IC₅₀ was determined from the concentration-response curve.

2.5. Minimum Inhibitory Concentrations (MICs)

The MICs of CTX and PHB-NPs were determined by the microdilution method in 96-well U-shaped polystyrene microtiter plates (Thermo Scientific, USA) following standard procedures [9,10]. Serial two-fold dilutions of both agents (100 µL) were prepared in Mueller-Hinton broth (MHB, HiMedia, India). The standard inoculum of E. coli was prepared by washing overnight bacterial growth three times with phosphate-buffered saline (PBS, 151.5 mM, pH 7.2), then adjusting the optical density of the suspension to 0.1 at 600 nm. Five microliters of bacterial suspension was added to each well. The plates were incubated at 37°C for 24 h. Three controls were applied: growth control (MHB + bacteria), sterile control (MHB only), and antibiotic turbidity control. The experiments were repeated three times [9,10].

2.6. Biofilm Formation

Biofilm formation in the E. coli isolates was measured using the crystal violet microtiter plate assay, described in detail in several previous standard publications [9,10]. In this method, tryptic soy broth (TSB, HiMedia, India) supplemented with 0.25% glucose was used. Absorbance was measured at 590 nm (Bio-Rad, USA), with the absorbance of an empty well used as the blank. Sterility of the media and the effectiveness of bacterial growth controls were verified. The experiment was repeated three times. A cut-off value was calculated to define the ranges for the three categories of biofilm-producing bacteria (strong, moderate, and weak).

2.7. Synergistic Effect of PHB-NPs and CTX on MICs

The checkerboard microdilution method was used to evaluate the effectiveness of the interaction between PHB-NPs and CTX against the MDR E. coli isolate that produced a strong biofilm on polystyrene microtiter plates. In the U-shaped polystyrene microtiter plate, two-fold serial dilutions of CTX were prepared from 2000 µg/mL to 0.97 µg/mL across columns 1–12. Two-fold serial sub-MICs of PHB-NPs were prepared from 1/2 to 1/64 of the MIC. The agents were prepared in MHB (HiMedia, India). Five microliters of the standard inoculum of E. coli (prepared as described in Section 2.5) was added to each well and incubated at 37°C for 24 h. The fractional inhibitory concentration index (FICI) was calculated as:

FICI ≤0.5 was defined as synergistic, 0.5–1.0 as additive, 1.0–2.0 as indifferent, and >2.0 as antagonistic.

2.8. Effect of the Combination of CTX and PHB-NPs at Sub-MICs on Biofilm

The procedure used to evaluate the synergistic effect of the studied agents on E. coli (Section 2.7) was followed to check the effect of the combination of CTX and PHB-NPs at sub-MIC levels on the ability of the E. coli isolate to form biofilm on a flat-bottom polystyrene microtiter plate. In this experiment, TSB (HiMedia, India) was used instead of MHB. After incubation, the biofilm measurement protocol described in Section 2.6 was followed, with absorbance measured at 590 nm using a microplate reader (Bio-Rad, USA). The experiment was repeated three times [14-16].

2.9. Statistical Analysis

Microsoft Excel and IBM SPSS v.26 (IBM, USA) were used to analyze the current study's data. Data were expressed as mean ± SD. Student's t-test and one-way ANOVA with post-hoc Tukey's test were used for comparisons. A P-value of <0.05 was considered statistically significant.

3. Results

3.1. Bacterial Isolates and CTX Susceptibility

In the present study, 10 E. coli isolates were obtained from 83 urine samples collected from patients with UTIs. The incidence of UTI infection with E. coli was 12.04%. Species were identified using the VITEK® 2 system. The Kirby-Bauer disk diffusion method showed that inhibitory zone diameters of the 10 E. coli isolates ranged from 7.8 ± 0.9 mm (Ec9) to 32.1 ± 3.5 mm (Ec8). All reported inhibition zone diameters represent the total diameter, including the 6 mm disk diameter, consistent with CLSI measurement guidelines. MIC values of CTX ranged from 0.062 µg/mL to 256 µg/mL. Of the 10 isolates, 4 were susceptible (S), 1 intermediate (I), and 5 resistant (R) to CTX, indicating a high prevalence of CTX resistance in uropathogenic E. coli (Table 1). The microdilution method was also used to determine the MICs of PHB-NPs against the 10 E. coli isolates, which ranged from 0.12 µg/mL to 256 µg/mL.

3.2. Biofilm Formation and CTX Response

The study showed that six isolates were categorized as strong biofilm producers, three as moderate, and one as a weak biofilm producer. Susceptible isolates showed low to moderate biofilm production, while resistant isolates exhibited moderate to strong biofilm formation. Statistical analysis showed a negative correlation between biofilm formation and inhibition zone diameter (r = −0.83; P = 0.007), and a significant positive correlation between biofilm formation and CTX MICs (r = +0.801, P = 0.003). These data support that enhanced biofilm production is strongly associated with increased CTX resistance (Table 1).

3.3. PHB-NPs Characterization

Scanning electron microscopy revealed that the PHB nanoparticles exhibited a near-spherical morphology with aggregation. FTIR spectra showed the characteristic PHB carbonyl ester peak at 1700 cm⁻¹ and C-O-C stretching bands at 1278 and 1060 cm⁻¹. These findings confirmed the identity of the prepared PHB nanoparticles and the integrity of the polymer core.

3.4. MTT Cytotoxicity Assay

The results showed that PHB-NPs exhibited a concentration-dependent decrease in MCF-7 cell viability. The half-maximal inhibitory concentration (IC₅₀) was determined to be 52.4 µg/mL (Figure 2). The sub-MIC concentrations of PHB-NPs used in the synergistic assay were below the IC₅₀ value, indicating a potential therapeutic selectivity window and supporting the feasibility of the nanoparticle concentrations used in the in vitro antibacterial assays.

3.5. Synergistic Effect of PHB-NPs and CTX on MICs

Tables 2 and 3 demonstrate that the combination of PHB-NPs and CTX increased the antibacterial activity of CTX against E. coli (Ec9). The MIC of CTX alone was 250 µg/mL; however, when combined with PHB-NPs, bacterial growth inhibition was seen at a lower CTX concentration of 62.5 µg/mL when ½ and ¼ MICs of PHB-NPs were used. CTX MICs were reduced to 125 µg/mL when combined with 1/8, 1/16, and 1/32 sub-MICs of PHB-NPs. The combinations exhibited synergistic interactions based on FICI values. The strongest synergistic effect was observed with ¼ MIC PHB-NPs combined with CTX, which produced the lowest FICI value (0.5). In contrast, the combination containing ½ MIC PHB-NPs showed an additive effect (FICI = 0.75).

3.6. Effect of the Combination of PHB-NPs and CTX on Biofilm

Table 4 shows the effect of combining PHB-NPs and CTX at sub-MIC levels on biofilm formation by Ec9. The lowest biofilm formation, measured by OD₅₉₀, was observed in the untreated control (0.83 ± 0.21) and decreased to 0.13 ± 0.04 (P<0.05 vs. the first, second, and third controls) at the highest combination concentration (½ MIC PHB-NPs and ½ MIC CTX). At the lowest concentrations of the combined agents (1/32 MIC PHB-NPs and 1/32 MIC CTX), biofilm production was still significantly reduced compared with all controls (P<0.05). Thus, the current experiments report, for the first time, that the combination of CTX and PHB-NPs at sub-MIC levels reduced E. coli biofilm formation more effectively than either agent applied alone.

4. Discussion

The elevation of resistance in E. coli to third-generation cephalosporins represents one of the biggest challenges in infectious disease medicine and public health in general. The World Health Organization's 2024 priority pathogens list identifies ESBL-producing E. coli as a high-priority target for developing new treatments for infectious diseases [17]. The present study showed that a high percentage of uropathogenic E. coli isolates were resistant to cefotaxime (CTX), confirming the high outbreak of ESBL-producing E. coli documented in Iraqi clinical settings and reflecting global trends in beta-lactam resistance [18].

The study showed a negative correlation between biofilm formation in E. coli isolates and CTX inhibition zone diameter, and a positive correlation between the minimum inhibitory concentration of the same isolates and their biofilm formation. Thus, biofilm biomass restricts antibiotic diffusion, reduces pH and oxygen tension, and helps bacterial persistence, which promotes resistance gene expression — explaining the correlation between biofilm formation and resistance to antibiotics, especially to CTX [19,20].

The prepared PHB-NPs exhibited physicochemical characteristics, including nano-diameter, well suited for biomedical applications. The nanoscale size of the PHB-NPs facilitates their interaction with bacterial cell surfaces and biofilm matrix components, enhancing penetration into the biofilm matrix. The study also showed that the IC₅₀ of the prepared PHB-NPs against MCF-7 cells was 52.4 µg/mL, supporting the concept of using this material in therapeutic fields. Several previous studies have highlighted the use of PHB material at various sizes, including nanoscale, in therapeutic and antimicrobial applications [21].

Several mechanisms may underlie the potentiation of CTX action by PHB-NPs. The physical characteristics of PHB-NPs (i.e., nanoscale size and hydrophobicity) facilitate their adhesion to the bacterial outer membrane and disrupt the packing of LPS, which would increase membrane permeability to CTX, thus allowing CTX better access to its PBP targets. PHB-based NPs may also interfere with efflux pump function by changing membrane fluidity or altering the conformation of membrane proteins, decreasing the amount of CTX extruded from inside the cell by the efflux pump [22]. Within biofilms, PHB-NPs can penetrate and interact with the polysaccharides and proteins in the EPS matrix, thereby disrupting the structural integrity of the biofilm and lowering resistance to CTX diffusion. These processes are consistent with the known antibiofilm activity of NP systems based on other polymers against Gram-negative pathogens [23,24].

The outcomes of this study support that PHB nanoparticles display an additive or synergistic capacity with CTX against E. coli, based on combined FIC index values; these outcomes correlate with previous studies evaluating additive interactions between polymers and antibiotics [25]. Notably, although significant reductions in CTX MIC against Ec9 were observed at the maximum tested combination (compared to the baseline MIC of 256 µg/mL), Ec9 remained clinically resistant to CTX based on MIC testing, as all post-combination MIC values remained well above the clinical susceptibility breakpoint of 1 µg/mL established by CLSI. Thus, the authors do not claim that PHB-NPs restored clinical susceptibility to CTX, but rather that the NPs potentiated CTX's actions. Nonetheless, the FIC data support the need for further studies involving extended pharmacokinetic/pharmacodynamic modeling and in vivo validation before clinical translation can take place.

The combination of PHB-NPs and CTX displayed greater antibiofilm efficacy than either agent alone, based on the biofilm biomass produced by Ec9, with up to an 85% reduction relative to the untreated control at the maximum concentration tested. This finding is in agreement with previous studies showing that the combined activity of polymer NPs and antibiotics is greater than that of single antibiotics in penetrating and disrupting biofilms [26].

The current study has several limitations. First, the study focuses on the in vitro environment; future studies will use a mouse UTI model to evaluate combined PHB-NPs and CTX treatment in vivo. Second, the genetic basis of resistance was not investigated and will be addressed in subsequent investigations. Third, the synergistic effect of both agents, and the effect of their combination on biofilm formation, was tested on a single isolate (Ec9); future studies will address this by employing several isolates.

5. Conclusion

The present study's findings provide evidence that 50% of uropathogenic E. coli isolates are resistant to CTX and show a significant correlation between biofilm-forming ability and CTX resistance. PHB-NPs created by solvent evaporation with emulsification exhibited suitable physicochemical characteristics and low toxicity (IC₅₀: 52.4 µg/mL). Sub-MICs of CTX and PHB-NPs alone showed a concentration-dependent reduction in biofilm formation. This study is the first to show that PHB-NPs have a combined effect with CTX, both in restraining CTX-resistant E. coli and in inhibiting biofilm formation, supporting in vivo testing to further evaluate their use in conjunction with CTX as adjunctive antimicrobial therapy for treating drug-resistant E. coli and preventing biofilm-related persistence.

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