Escherichia coli DH5α, which possesses the RP4-7 plasmid containing chloramphenicol and ampicillin resistance genes, acted as the donor, while E. coli J53, resistant to sodium azide, acted as the recipient. Both isolates were procured from the laboratory for conjugation testing. Bronopol originated from Aladdin, and phosphate-buffered saline (PBS) was employed as the solvent.

Overnight cultures of J53 and DH5α were diluted in Luria-Bertani (LB) broth to a turbidity of 0.5 McFarland standard. Bronopol was administered to each bacterium to achieve final concentrations of 2 μg/L, 20 μg/L, and 200 μg/L. Samples devoid of bronopol served as controls. The mixes were incubated statically, and OD₆₀₀ was recorded hourly for a duration of 16 h. Each group was examined three times.

Conjugation experiments between donor and recipient bacteria were conducted under bronopol exposure using a modified protocol adapted from the previous study (Pallares-Vega et al., 2021). Bacteria were cultivated in LB broth at 37°C and harvested using centrifugation. A McFarland turbidity of 0.5 was then attained by resuspending the bacterial precipitate in PBS, resulting in a final bacterial density of 1.5 × 10⁸ CFU/mL, thereby forming the final conjugation system with a total volume of 2 mL. One milliliter of either donor or recipient bacteria was combined with varying concentrations of bronopol (2 μg/L and 20 μg/L), then incubated statically for 12 h at 37°C. Plates were prepared by adding 5 μL of conjugation mixture to LB agar supplemented with sodium azide (200 mg/L) and ampicillin (100 mg/L), then incubated under standard conditions. Transconjugant enumeration was performed by plating on LB agar medium containing 200 mg/L sodium azide to quantify recipient counts. CTF was calculated as the transconjugant-to-recipient ratio. Antimicrobial susceptibility tests verified representative colonies from conjugate crosses. All mating experiments were conducted in biological triplicate.

Intracellular ROS levels were measured with a Cellular ROS Assay Kit (Beyotime, Shanghai, China) per the manufacturer’s protocol. Donor and recipient cultures were grown overnight to an OD₆₀₀ of 0.5 and resuspended in PBS. Bacteria were incubated with 10 μM DCFH-DA at 37°C for 30 min in the dark. The unbound probe was removed via two PBS washes. After a 2-h incubation at 37°C, fluorescence intensity was recorded using Infinite M200 Microplate Reader at 488 nm excitation/525 nm emission. All experiments were performed in triplicate biological repeats. Membrane permeability was evaluated with 0.5 μM PI (propidium iodide) and 10 μM NPN (1-N-phenylnaphthylamine) (Beyotime). PI excitation/emission was 535 nm/615 nm, while NPN was 350 nm/420 nm. All tests were conducted in triplicate. According to the previous experimental protocol (Zhang et al., 2022), we analyzed bacterial membrane permeability using Confocal laser scanning microscopy (CLSM). Bacteria with a 0.5 McFarland turbidity were inoculated into PBS containing 2 μg/L bronopol or 20 μg/L bronopol and treated at 37°C for 12 h. The samples were then incubated at room temperature in PI (50 mg/L) for 20 min. Bright-field and fluorescent images were captured using CLSM (LSM800, Zeiss, Jena, Germany).

Following 12-h exposure to 20 μg/L bronopol, bacterial cell ultrastructure was analyzed via transmission electron microscopy (TEM). E. coli DH5α and J53 cultures were harvested by centrifugation at 5,000 × g for 6 min, washed twice with ice-cold PBS, and resuspended in PBS. Cells were fixed in 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) at 4°C overnight. Specimens were dehydrated through a graded ethanol series (50, 70, 90, 100% for 15 min each), infiltrated with Epon-Araldite epoxy resin, and polymerized at 60°C for 48 h. Ultrathin sections (70 nm) were prepared using an EM UC7 ultramicrotome (Leica, Germany), post-stained with 2% uranyl acetate and lead citrate, and imaged on a Tecnai T12 TEM (Thermo Fisher Scientific, USA) operated at 120 kV.

Bacterial cultures (1.5 × 10⁸ CFU/mL) were treated with 20 μg/L bronopol at 37°C for 12 h. Total RNA was extracted using the EASYspin Bacterial RNeasy Mini Kit (Aidlab, China) as per the manufacturer’s instructions. cDNA was synthesized via reverse transcription with the PrimeScript RT reagent Kit (TaKaRa, Japan). qRT-PCR was carried out on an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, USA) using SYBR Premix Ex Taq II (TaKaRa, Japan). Relative mRNA levels were normalized to the 16S rRNA using the 2^−ΔΔCt method. Primer sequences are in Supplementary Table S1. All experiments were done in triplicate.

GraphPad Prism version 8.2.1 was used to analyze the data. The mean ± standard deviation is used to display the data. Independent samples t-test was used to assess significant differences, and p < 0.05 was considered statistically significant.

Bronopol exhibited strain-specific minimum inhibitory concentrations (MICs), with values of 2 mg/L for DH5α and 4 mg/L for both J53 and DC8855 (Figure 1 and Table 1). After 24 h, all treatment groups with sub-inhibitory concentrations achieved growth levels comparable to the control group. However DH5α treated with 200 μg/L bronopol displayed a lower growth rate during the exponential phase (5–12 h post-treatment; Figure 1A). To ensure consistent bacterial growth, which is a critical requirement for comparing the changes in the conjugative transfer frequency, in the subsequent experiments, we selected the concentrations of 2 μg/L and 20 μg/L.

Sub-inhibitory concentrations of bronopol significantly enhanced the conjugative transfer frequency (CTF) of the RP4-7 plasmid in a dose-dependent manner (Figure 2A). At 20 μg/L, the CTF increased by 4.15-fold (p < 0.01) compared to the control, while a 1.76-fold increase was observed at 2 μg/L (p < 0.05). To ascertain whether bronopol could enhance the CTF of clinically relevant wide-host-range plasmids and to explore its implications for the spread of clinically significant carbapenem-resistant genes, we concurrently measured the CTF of the IncFII(K) plasmid harboring the bla_NDM-4 gene (Figure 2B). While bronopol induced a weaker CTF promotion for IncFII(K) plasmid (115,297 bp, >10 KB) compared to RP4-7 (60,002 bp), a 2.32-fold increase (p < 0.05) was detected for IncFII(K) at 20 μg/L. This indicates that bronopol at residual concentrations in the environment has a low capacity to increase the conjugative transfer frequency of large plasmids.

The cell membrane plays a pivotal role in the conjugation process. To delve into the biological mechanism of conjugative transfer, we investigated whether subinhibitory concentrations of bronopol could enhance cell membrane permeability by assessing inner and outer membrane permeability, conducting TEM, CLSM, and measuring ROS production. NPN staining revealed significant increases in outer membrane permeability, respectively, in both donor and recipient strains treated with 2 μg/L and 20 μg/L bronopol (Figure 3A). In addition, we evaluated the cell membrane permeability using PI staining through microplate reader assays and CLSM analysis. The results showed that after pre-incubation of cells with bronopol, the fluorescence intensity increased in a concentration-dependent manner due to PI uptake and DNA binding, indicating a gradual decline in cell membrane integrity (Figure 3B and Supplementary Figure S1). In contrast to the blank control group, the bronopol-treated donor and recipient strains did not display a significant increase in fluorescence associated with ROS accumulation (Figure 3C). Concurrently, TEM images vividly revealed distinct morphological alterations in the cells induced by bronopol (Figures 3D,E). Bronopol-treated cells exhibited shrunken, roughened surfaces with distinct cytoplasmic membrane detachment, whereas control cells retained smooth, intact membranes. Collectively, these data affirm that bronopol exposure enhances cell membrane permeability, potentially facilitating the colocalization-mediated transfer of antibiotic-resistance genes.

Plasmid conjugation is predominantly governed by three core systems: mating-pair formation, DNA transfer, and replication. To investigate bronopol’s impact on this mechanism, we analyzed transcriptional changes in genes encoding conjugation machinery components. Our results revealed significant divergences in gene expression patterns between treatment groups (Figure 4A). Following exposure to 20 μg/L bronopol during conjugation, mRNA expression levels of trfAp, kilA, and kilB were synergistically upregulated by 69.9, 37.5, and 49.9% compared to the control, respectively. Conversely, treatment with 2 μg/L bronopol induced no significant change in kilA expression, yet triggered marked increases in trfAp and kilB expression (83.5 and 27.0%, respectively). Furthermore, no significant effects of various treatment groups were detected on the global regulatory gene (korB). These findings suggest that bronopol promotes conjugative transfer channel formation and plasmid transmission through coordinate upregulation of global regulator genes (kilA/kilB) and the trfA promoter (trfAp).

Next, we investigated alterations in outer membrane porin and efflux pump gene expression at the bacterial cellular level. Among the tested genes, ompA expression did not differ significantly between bronopol-treated and control groups (Figure 4B). Notably, ompC transcription was significantly upregulated in cells exposed to both 2 μg/L and 20 μg/L bronopol. Additionally, examination of major efflux pump genes revealed a modest upregulation of acrA and acrB in bronopol-treated cells at sub-inhibitory concentrations. Collectively, these findings suggest that bronopol exposure induces coordinated increases in ompC membrane porins and efflux pumps.