Bile modulates phage–host interactions in multidrug-resistant Pseudomonas aeruginosa

Abstract

Biliary tract infections (BTIs) arise within a bile-rich environment that profoundly shapes microbial ecology and pathogen adaptation. Pseudomonas aeruginosa, a major opportunistic pathogen in nosocomial settings, exhibits remarkable physiological plasticity, that enable persistence in such challenging niches. However, the influence of bile on P. aeruginosa’s adaptive responses and phage–host interactions remains largely unexplored. Here, we demonstrate that ox-bile imposes concentration-dependent stress on P. aeruginosa strain ZS-PA-35, indicative of host-derived selective pressure. Notably, ox-bile enhances biofilm formation and promotes swarming and twitching motilities while concurrently suppressing swimming motility. Moreover, ox-bile modulates phage susceptibility, likely through altered receptor expression: exposure to ox-bile sensitizes P. aeruginosa to the type IV pili (T4P)-dependent phage phipa2, whereas susceptibility to the lipopolysaccharide (LPS)-targeting phage phipa10 remains unchanged. Genome-wide mutagenesis identified resistance-conferring mutations affecting T4P structures, LPS biosynthesis, and associated regulatory pathways. Among these, phage-resistant mutants ΔpilT and ΔgalU retained high fitness under ox-bile stress, accompanied by enhanced swarming and swimming motilities. Furthermore, in a lysogenic context, ox-bile markedly suppressed prophage accumulation in the T4P-dependent strain ZS-PA-05. These findings reveal that bile acts as a critical environmental cue shaping both adaptive physiology and phage susceptibility in P. aeruginosa, with broad implications for microbiome dynamics and the development of phage-based therapies targeting bile-impacted infections.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04379-5.

Introduction

The liver and biliary tract are critical interfaces within the gut–liver axis, where diverse microbial communities—including bacteria, archaea, fungi, and phages, dynamically interact with host physiology [1]. Among these, biliary tract infections (BTIs) present a significant clinical burden, particularly when caused by multidrug-resistant (MDR) pathogens such as Pseudomonas aeruginosa—a prominent member of the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa, and Enterobacter spp.), which collectively account for the majority of nosocomial infections worldwide [2]. P. aeruginosa exploit various adaptive mechanisms, including efflux pumps activation, outer membrane remodeling, and virulence modulation to evade antibiotics and host defenses [3, 4]. These adaptations enable immune evasion and confer resistance to bile, thereby enhancing bacterial survival in the hostile bile-rich environment, facilitating infection, and contributing to the high mortality rates in immunocompromised individuals [5].

The gallbladder secretes bile—a heterogeneous mixture of bile salts, phospholipids, cholesterol, and bilirubin—into the proximal section of the duodenum, where the concentration of bile components is estimated to range from 0.2 to 2% [6]. The primary role of bile is fat digestion and homeostasis, but it also exerts profound effects on microbial physiological behavior adaptation, including motility, biofilm formation, and gene expression in the gut microbiota by affecting the bacterial metabolism [7–9]. Indeed, recent studies have identified bile as a physiological gut signal that modulates biofilm formation in Shigella flexneri [10], Bacteroides thetaiotaomicron [11] and Vibrio cholerae [12], thereby influencing antimicrobial resistance and overall pathogenicity. Furthermore, bile has been shown to modulate bacterial surface structure, with subsequent effects on phage–host interactions in bile-rich environments and complicating therapeutic interventions [13].

Phage therapy has re-emerged as a promising alternative to antibiotics for treating MDR infections, leveraging phages’ ability to target specific bacterial receptors without disrupting commensal microbiota [14]. The phageome is an important modulator of bacterial community structure, controlling bacterial diversity and abundance [15]. Furthermore, phages require host-specific receptors to interact with bacteria, simultaneously influencing phage susceptibility and phage production. Many studies have explored phage therapy as a promising alternative or adjunct to antibiotics—either alone or in cocktails for treating the P. aeruginosa infections [14, 16]. A recent clinical trial has turned the spotlight on phage therapy, demonstrating its potential as an alternative or adjunct to antibiotics treatment for treating chronic P. aeruginosa infections in the biliary tract [17]. However, in this study, resistance rapidly emerged through mutations in lipopolysaccharide (LPS) biosynthesis. Subsequent treatment with the phage phiYY, specifically targeting O-antigen-deficient mutants, resulted in marked clinical improvement but did not achieve complete bacterial clearance [17]. This study highlights the complexities of phage resistance and underscores the necessity for further exploration of phage–host interactions in intricate environments, such as the biliary tract. In P. aeruginosa, LPS and type IV pili (T4P) serve as primary phage receptors [18]. These receptors also facilitate the host attachment, motility, and biofilm formation, which are essential for infection establishment and immune evasion. Despite these advantages, the presence of bile in the biliary tract further complicates host–pathogen interactions, potentially altering bacterial susceptibility to phages and modulating virulence gene expression. Therefore, we propose that bile may play a critical role in phage therapy, the interplay between bile, bacterial adaptation and associated phage efficacy remains largely unexplored.

In this study, we investigated how ox-bile exposure influences motility, biofilm formation, and prophage induction in clinical P. aeruginosa isolates, revealing distinct ox-bile-induced phenotypic adaptations. Given the potential role of these traits in early-stage phage adsorption, we assessed the infectivity and replication of two lytic phages, phipa2 and phipa10, under bile-enriched conditions that mimic the biliary environment. Our results show that ox-bile markedly modulates phage–host interactions, enhancing phage susceptibility—most notably for the T4P-dependent phage phipa2, while exerting comparatively modest effects on the LPS O-antigen-targeting phage phipa10. Gene-level analyses revealed that ox-bile exposure reshapes the expression of key genes involved in phage receptor pathways, including pilT, suggesting that P. aeruginosa ZS-PA-35 adapts to ox-bile stress through receptor-specific modulation to balance phage susceptibility and survival. Furthermore, we demonstrate that ox-bile-mediated regulation of T4P significantly influences prophage release in the lysogenic strain ZS-PA-05, highlighting a critical link between environmental stressors and phage dynamics. Together, these findings provide mechanistic insight into how ox-bile governs the interplay between bacterial physiology and phage interactions in the context of biliary tract infections (Fig. 1).

Fig. 1.

Impact of bile on P. aeruginosa adaptation and phage–host interactions This model illustrates how bile availability in the biliary tract shapes P. aeruginosa physiological adaptation and interactions with phages. During infections, pathogens such as P. aeruginosa compete with the resident microbiota while facing environmental stress and altered nutrient availability. Ox-bile directly modulates bacterial physiology by influencing key virulence factors, including type IV pili (T4P) and the LPS O-antigen, and promotes enhanced biofilm formation, motility, and genetic plasticity. These adaptations facilitate bacterial evasion of phage predation and modulate the induction of lytic and lysogenic cycles, collectively contributing to increased pathogenicity

Materials and methods

Bacterial strains, phages, and growth conditions

All bacterial strains and their corresponding phages used in this study are summarized in Table S1. The phages utilized include phipa2 and phipa10, which target P. aeruginosa ZS-PA-35. The parent strain ZS-PA-35 was originally isolated from Zhongshan Hospital, Shanghai, China [18]. Phage-resistant mutants ZS-PA-35-ΔpilT and ZS-PA-35-ΔgalU, derived from P. aeruginosa ZS-PA-35, were included; these mutants are no longer susceptible to phipa2 and phipa10, respectively. Additionally, P. aeruginosa ZS-PA-05 was used for prophage proliferation studies, and its isogenic mutant, ZS-PA-05-ΔpilT, was employed to validate T4P involvement in prophage activity [19]. A panel of six Pseudomonas aeruginosa clinical isolates was used, comprising T4P-specific strains ZS-PA-14, ZS-PA-20, and ZS-PA-27, and O-antigen–specific strains ZS-PA-07, ZS-PA-15, and ZS-PA-25, all obtained from Shanghai Public Health Clinical Center (SPHCC). All bacterial strains were preserved in 24% glycerol stocks and stored at − 80 °C. For experiments, strains were cultured overnight at 37 °C in Luria-Bertani (LB) broth (composed of 10 g/L Tryptone (Bacto, Gibco), 5 g/L Yeast Extract (Bacto, Gibco), 10 g/L NaCl) and/or on 1.5% agar (Bacto, Gibco) plates with or without ox-bile supplementation. To evaluate the effect of ox-bile on P. aeruginosa physiology, LB broth was supplemented with ox-bile (purified dehydrated ox-bile, Millipore-Sigma, USA) at final concentrations of 0.05, 0.1, 0.5, 2.5, and 5 g/L. Based on the dose–response observations, 0.5 g/L ox-bile was selected for subsequent experiments. For experimental procedures, overnight cultures were diluted to the required optical density (OD600). Bacterial growth was monitored by measuring OD600 at defined time points, and colony-forming unit (CFU) counts were used to determine the viable bacterial numbers.

Phage enumeration and titration

Overnight bacterial cultures were diluted 1:1000 in 10 mL of LB broth and grown to an optical density (OD600) of 0.3 at 37 °C with shaking. Phages phipa2 and phipa10 were propagated and titrated on their respective wild-type bacterial hosts using 0.5% (wt/vol) top agar for plaque assays. Plaque assays were performed at 37 °C for 6 h with serial dilutions, and PFU were quantified. To obtain high-titer phage lysates, plates with 30 and 200 plaques were selected. The top agar containing plaques was transferred into a stabilizing buffer composed of 10 mM MgSO₄ and 5 mM CaCl₂ (Sigma-Aldrich), supplemented with 1% chloroform to lyse any residual bacteria.The top agar containing plaques was transferred into a stabilizing buffer composed of 10 mM MgSO4 (Sigma-Aldrich), and 5 mM CaCl2 (Sigma-Aldrich), supplemented with 1% chloroform, to lyse any residual bacteria. After 4 h, the lysates were centrifuged at 12,000 × g for 10 min at 4 °C and the supernatant was transferred to sterile tubes. The resulting phage stocks were stored at 4 °C and subsequently tested for bacterial contamination using spot tests to ensure purity.

Motility and biofilm assays

To quantify the physiological adaptations of P. aeruginosa ZS-PA-35 strains, motility and biofilm formation assays were conducted. Biofilm formation was assessed using crystal violet staining in 10 mL polystyrene tubes. Overnight bacterial cultures were diluted to the OD600 of 0.4 and inoculated into 3 mL of LB broth or LB broth supplemented with 0.5% ox-bile. Cultures were incubated statically at 37 °C for 3 days to allow mature biofilm formation. Non-adherent cells were removed by washing the tubes three times with tap water, followed by staining with 4 mL of 0.4% crystal violet solution. Excess stain was removed by washing with tap water additional washes with tap water. Biofilms were quantified by dissolving the crystal violet stain in 4 mL of 75% ethanol using vortex mixing, and absorbance was measured at OD600. All experiments were performed in triplicate. Swarming motility was evaluated by spotting 2 µL aliquots of bacteria (OD600 = 1.0, diluted 100-fold) onto 0.5% LB agar or LB agar supplemented with 0.5% ox-bile. Plates were incubated horizontally at 37 °C for 16 h, and colony expansion was measured using ImageJ software. For swimming motility, a sterile toothpick was dipped into overnight cultures and used to vertically inoculate 0.5% LB agar or LB agar supplemented with 0.5% ox-bile. A 0.5% agar concentration. This agar concentration (0.5%) is commonly used to evaluate both swimming and swarming motilities. Plates were incubated horizontally at 37 °C for 16 h, and the diameter of bacterial spread was measured to quantify swimming motility. Twitching motility was assessed as previously described [20]. Briefly, 2 µL aliquots of overnight cultures were stabbed to the bottom center of petri dishes containing 1.5% LB agar or LB agar supplemented with 0.5% ox-bile. The higher (1.5%) agar concentration increases surface stiffness, facilitating T4P-dependent lateral movement. Plates were incubated horizontally at 37 °C for 7 days. After incubation, the agar was carefully removed, and the motility zones were stained with 0.4% crystal violet for 30 min at room temperature. Excess stain was removed by rinsing five times with tap water. The diameter of motility zones was measured using ImageJ software.

Phage–host interaction in LB and ox-bile broth

The inhibition assays were conducted to evaluate the interaction between phages phipa2 and phipa10 with P. aeruginosa ZS-PA-35. Overnight cultures of ZS-PA-35 were adjusted to an OD600 of 0.01 (approximately 8 × 10⁶ CFU/mL) and inoculated into 8 mL of LB broth with or without 0.5% ox-bile in 50 mL centrifuge tubes.inoculated into 8 mL of LB broth or LB broth supplemented with 0.5% ox-bile in 50 mL centrifuge tubes. Phages phipa2 and phipa10 were added at a multiplicity of infection (MOI) of 0.1. Tubes were incubated at 37 °C with shaking, and bacterial growth was monitored periodically by measuring OD600. To quantify the phage activity, 500 µL aliquots of each sample were treated with 1% chloroform, vortexed briefly and centrifuged at 12,000 × g for 3 min. The resulting supernatant was serially diluted and subjected to plaque assays using the double agar overlay method. Plaque counts were recorded under both LB and LB supplemented with ox-bile conditions to determine phage titers and inhibition efficiency. All assays were performed in triplicate to ensure reproducibility.

Determination of mutation rate in LB and ox-bile

P. aeruginosa ZS-PA-35 was cultured overnight in LB broth with or without 0.5% ox-bile, then diluted 1:1000 into 3 mL of fresh LB or LB supplemented with ox-bile and LB broth supplemented with 0.5% ox-bile, then diluted 1:1000 into 3 mL of fresh LB or ox-bile broth. Cultures were incubated with shaking at 37 °C until reaching an OD600 of 0.4. Subsequently, 1 mL of each bacterial suspension was mixed with phages phipa2 and phipa10 at a MOI of 4. An additional 1 mL of fresh LB or ox-bile broth was added to each mixture, followed by incubation at 37 °C for 60 min. After incubation, 1 mL and 100 µL aliquots were plated onto LB agar and ox-bile agar plates to enumerate colonies resistant to phipa2 or phipa10. Control samples (bacteria without phage treatment) were serially diluted and plated to determine total CFU of ZS-PA-35. Plates were incubated overnight at 37 °C, and colony counts were used to calculate the mutation rates. Mutation rates (µ) were determined using the formula µ = m/N, where m represents the mean numbers of resistant mutants per culture, and N is the total CFU. All experiments were performed in triplicate.

Comparative genomics analysis of phage-resistant mutants

Phage-resistant bacterial mutants of P. aeruginosa ZS-PA-35 were isolated on sodium citrate agar plates. The resistance phenotype of surviving colonies against phipa2 and phipa10 was confirmed via cross-streak assays and validated through three rounds of spot tests [18]. Verified resistant mutants were cultured, and glycerol stocks were prepared using sterile 24% glycerol (Macklin, China) and stored at − 80°C for future use.

For whole-genome sequencing, phipa2 and phipa10-resistant mutants (from both LB and ox-bile conditions) were cultured, and genomic DNA was extracted from cell pellets (5,000 × g, 10 min) using the Wizard^® Genomic DNA purification kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions, following the manufacturer’s instructions. DNA quality was assessed using a NanoDrop spectrophotometer. Samples were subjected to Illumina paired-end sequencing (~ 1 Gbp/sample) at Sangon Biotech (Shanghai, China).

Raw reads were quality-checked with FastQC and trimmed using Trimmomatic to remove adapters and low-quality bases. Sequences were aligned to the P. aeruginosa ZS-PA-35 reference genome using the Burrows-Wheeler Aligner (BWA), following GATK best practices. Duplicates were removed using MarkDuplicates (https://gatk.broadinstitute.org/). Single nucleotide polymorphisms (SNPs) and small insertions/deletions were identified, and read depth per gene was analyzed using BEDTools v2.28.0. Variant effects were annotated and categorized as high, moderate, low, or modifier using SnpEff v4.3T. To validate and visualize the results, annotated variants were aligned to the ZS-PA-35 reference genome using CLC Genomics Workbench. Mutations predicted to have high or moderate impact were manually verified and presented as annotated genome maps and summary tables. Mutations with high or moderate predicted impact were manually verified and presented as annotated genome maps and summary tables.

Characterization of mutant strains

To evaluate the impact of gene deletions (pilT and galU) on bacterial growth, previously constructed mutant strains were analyzed alongside wild-type controls [18]. Overnight cultures were diluted 1:1000 in LB broth, with or without 0.5% ox-bile supplementation, and incubated at 37 °C. Optical density (OD600) was measured at at 4-h intervals over a 24-h period 4 h intervals over a 24 h period. Linear growth rates and generation times were calculated from three independent biological replicates. Additionally, motility assays (swimming, swarming, and twitching) were performed for ΔpilT and ΔgalU mutants under both LB and ox-bile conditions, as previously described.

RNA extraction and cDNA synthesis

To examine the expression patterns of the galU and pilT genes in P. aeruginosa ZS-PA-35 under LB and ox-bile conditions, overnight cultures were diluted 1:1000 into fresh LB or LB supplemented with 0.5% ox-bile broth and incubated at 37 °C until reaching an OD600 of 0.3 incubated at 37 °C until the OD600 of 0.3. Cells were harvested into pre-chilled 2 mL microcentrifuge tubes and centrifuged at 12,000 × g for 3 min at 4 °C. Supernatants were discarded, and the bacterial pellets were immediately frozen at − 80 °C for subsequent RNA extraction.

Total RNA was extracted using the Trizol reagent (Invitrogen) following the manufacture'"s protocol. Briefly, bacterial pellets were resuspended, and 200 µL of chloroform was added, followed by vortexing and incubation at room temperature for 5 min. After centrifugation at 12,000 × g for 15 min, at 4 °C, the upper aqueous phase was transferred and mixed with 500 µL of isopropanol. RNA was precipitated by centrifugation, washed with 75% ethanol, and air-dried. The RNA pellet was then dissolved in 50 µL of diethyl pyrocarbonate (DEPC)-treated water. Genomic DNA contamination was removed using DNase I (1 U/µL; Thermo Scientific, USA) at 37 °C for 30 min, followed by inactivation with 50 mM EDTA. RNA concentration and purity were assessed using a NanoDrop spectrophotometer, and then all RNA samples were normalized to 500 ng/µL and stored at − 80 °C until use.

For cDNA synthesis, 500 ng of DNase-treated RNA was reverse transcribed using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) with random hexamer primers, following the manufacturer’s instructions. The reaction was performed under the following thermal cycling conditions: 25 °C for 5 min, 42 °C for 60 min, and 70 °C for 5 min. The resulting cDNA was serially diluted to obtain Ct values between 20 and 30, and stored at − 20 °C until further analysis.

RT-qPCR gene expression quantification

The relative expression levels of galU and pilT in P. aeruginosa ZS-PA-35 under LB and ox-bile conditions were determined by quantitative real-time PCR (qPCR) using the CFX96 Real-Time PCR Detection System (Bio-Rad, CA, USA) with SsoAdvanced SYBR Green supermix (Bio-Rad) and gene-specific primers listed in Table S2. Each qPCR reaction (25 µL) mixture contained 0.5 µL of each primer, 5 µL of cDNA, and 12.5 µL of SsoAdvanced SYBR Green Supermix. SYBR Green master mix. The thermal cycling program consisted of an initial denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 4 s and 55 °C for 30 s. Melting curve analysis was performed from 65 °C to 95 °C, with 0.5 °C increments every 5 s, which confirmed a single specific peak for each primer pair. Gene expression levels were normalized to rpoS, which served as the internal reference gene normalized to rpoS as the internal reference gene and calculated using the comparative threshold cycle (∆∆Ct) method [21]. All reactions were performed in triplicate with three independent biological replicates to ensure reproducibility.

Prophage induction assay

Overnight cultures of bacterial strains were diluted 1:1000 into 10 mL of LB broth and incubated at 37 °C until an OD600 of approximately 2.0 the OD600 reached approximately 2.0. The cultures were then centrifuged at 16,000 × g for 3 min at 4 °C, and the supernatants were collected and filtered through a 0.22 μm membrane filter to remove residual bacterial cells. To determine the phage titers, the filtered supernatant was serially diluted, and 200 µL of each appropriate dilution was mixed with 200 µL of fresh bacterial culture. The mixture was overlaid onto LB agar plates using the double agar overlay method. After the top layer solidified, plates were incubated overnight at 37 °C. Plaques were enumerated to calculate PFU and quantify phage abundance plaques were enumerated, and the number of PFUs was calculated to quantify phage abundance.

Statistical analysis

Statistical analyses and graph generation were performed using GraphPad Prism software (version 9.2, La Jolla, CA). All data are presented as the mean ± standard deviation (SD) from three biological replicates. For pairwise comparisons, statistical significance was assessed using an unpaired one-tailed t-test including analyses of swarming motility, twitching motility, biofilm formation, mutation rates, and RT-qPCR data. For comparisons involving multiple groups-such as growth curves (OD600) and phage abundance (PFUs) across different time points or treatment conditions-one-way ANOVA followed by Tukey’s multiple comparison test was used to assess intergroup differences. A P value < 0.05 was considered statistically significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; and ****, P ≤ 0.0001.

Results

Ox-bile exerts concentration-dependent inhibition on P. aeruginosa growth

To assess the growth impact of bile on P. aeruginosa strain ZS-PA-35, we conducted a synergistic synographic assay using a gradient of five ox-bile concentrations (0.05–5 g/L), encompassing over 80% of the physiological range encountered in the human gastrointestinal tract. This gradient was designed to mimic the bile-rich conditions faced by pathogens during biliary tract colonization. ZS-PA-35 was cultured in LB broth with or without ox-bile supplementation, and bacterial growth was monitored over a 10 h period by measuring optical density at 600 nm (OD600). During the initial 2 h, growth kinetics were similar across all conditions, with no statistically significant differences observed (P > 0.17), indicating that early-phase proliferation is largely unaffected by ox-bile. From 3 h onward, however, ox-bile exposure significantly inhibited bacterial growth in a dose-dependent manner. Specifically, concentrations of 0.1, 0.5, 2.5, and 5 g/L caused marked growth suppression compared to the LB control (p < 0.001), with significant differences emerging as early as 3 h (p < 0.0001). The inhibitory effect plateaued at concentrations ≥ 2.5 g/L, as no additional reduction was observed at 5 g/L relative to 2.5 g/L (p < 0.001). By contrast, the lowest tested concentration (0.05 g/L) had no measurable effect on growth. These results reveal a threshold-dependent inhibitory response, with ox-bile-mediated growth suppression emerging at concentrations ≥ 0.1 g/L and persisting throughout the 10 h exposure period (Fig. 2). This delayed yet sustained inhibition likely reflects membrane disruption or metabolic adaptation under bile-induced stress. For downstream assays, 5 g/L ox-bile was selected to ensure maximal physiological relevance and consistency with prior studies, facilitating cross-study comparability.

Fig. 2.

Synograph depicting the effect of ox-bile concentration on the growth of P. aeruginosa ZS-PA-35 Optical density at 600 nm (OD600) was measured hourly over a 10 h in LB broth supplemented with increasing concentrations of ox-bile (0.05–5 g/L), with LB alone serving as the control. Each cell in the synograph represents the OD600 value at the corresponding time point and ox-bile concentration, with darker shades indicating higher bacterial density

Ox-bile modulates biofilm formation and motility in P. aeruginosa

Biofilm formation is a hallmark of chronic infections, enabling microbial communities to adhere to surfaces, evade host defenses, and persist in hostile environments [22]. Motility, including flagella- and T4P-mediated movement, is closely linked to biofilm dynamics and is essential for surface colonization and biofilm dispersal. Motility-including flagella- and T4P-mediated movement-is closely linked to biofilm dynamics and is essential for surface colonization and biofilm dispersal [23]. Although bile is known to modulate bacterial physiology, its role in regulating these virulence-associated phenotypes in P. aeruginosa remains incompletely understood. To investigate this, we examined the effects of physiologically relevant ox-bile concentrations (5 g/L) on biofilm formation and motility in P. aeruginosa ZS-PA-35. Bile exposure induced distinct, pathway-specific responses. Biofilm biomass showed a modest but significant increase following ox-bile treatment, consistent with prior reports linking LPS O-antigen regulation to enhanced biofilm formation (P < 0.05; Fig. 3A). Similarly, ox-bile significantly promoted T4P-dependent surface motility: swarming motility was markedly enhanced (P < 0.01; Fig. 3B), and the twitching motility zone expanded significantly (P < 0.05; Fig. 3D), suggesting upregulation of pilus function and increased surface engagement under bile-rich conditions. In contrast, swimming motility, primarily mediated by flagella, was markedly reduced, showing a 31.4% decrease in migration radius compared with untreated controls. In contrast, swimming motility—primarily mediated by flagella- was substantially impaired, with a 31.4% reduction in migration radius compared to untreated controls (P < 0.001; Fig. 3C), indicating a negative effect on flagellar function or associated membrane components. These findings highlight the complex and modality-specific impact of ox-bile on P. aeruginosa motility and biofilm-related behaviors. The enhancement of T4P-dependent motility, coupled with repression of flagellar-driven swimming, suggests divergent regulatory or structural adaptations under bile-induced stress, with potential implications for host colonization and phage susceptibility.

Fig. 3.

Ox-bile enables diverse niche adaptations in P. aeruginosa ZS-PA-35 (A) Biofilm formation by ZS-PA-35 grown in LB supplemented with ox-bile compared to LB alone after 3 days of incubation. B–D Swimming, swarming, and twitching motilities assessed on agar plates containing LB or LB supplemented with ox-bile after 16 h of incubation. Data represent three independent experiments, with individual measurements shown as symbols. Biofilm biomass was quantified by optical density at 600 nm (OD600), and motility was measured as zone diameter (cm). Bars represent mean ± standard error of the mean (SEM). Statistical significance was determined by unpaired one-tailed t-test: P ≤ 0.05; *P ≤ 0.01; **P ≤ 0.001

Ox-bile modulates phage susceptibility and productivity in P. aeruginosaP. aeruginos

Given the observed bile-induced alterations in motility and biofilm formation in P. aeruginosa ZS-PA-35, we next investigated whether these phenotypic shifts affect phage–host dynamics. Many Pseudomonas phages rely on cell surface structures such as O-antigen and T4P for adsorption and infection. To dissect receptor-specific effects of bile, we employed two well-characterized lytic phages: phipa2, which targets T4P, and phipa10, which utilizes the O-antigen as its primary receptor [18, 24]. Consistent with prior results, ox-bile exposure significantly impaired the growth of ZS-PA-35 compared to LB alone (p < 0.01), confirming its stress-inducing effects (Fig. 4A). Under LB conditions, phipa2 exerted moderate antibacterial activity. However, in ox-bile-supplemented media, the inhibitory effect of phipa2 was dramatically potentiated (p < 0.0001; Fig. 4B), suggesting enhanced phage infectivity or increased bacterial susceptibility. Correspondingly, phage titers in ox-bile-treated cultures were significantly elevated between 2 and 8 h post-infection (Fig. 4C), indicating enhanced phage replication or release. These findings suggest that bile sensitizes the host to T4P-targeting phages and promotes more efficient phage amplification. In contrast, phipa10 maintained strong lytic activity under both LB and bile-rich conditions, with no significant differences in bacterial susceptibility or phage production (p > 0.05; Fig. 4D and E). This receptor-specific resilience indicates that ox-bile’s modulatory effects are phage-dependent and likely influenced by the nature of receptor engagement.

Fig. 4.

Regulation of phage–host interactions by ox-bile. Optical density (OD600) and phage abundance (plaque-forming units, PFUs) were monitored for P. aeruginosa ZS-PA-35 with or without infection by phipa2 or phipa10 under different conditions. A–E Cultures were incubated in LB or LB supplemented with 0.5% ox-bile at 37 °C with a multiplicity of infection (MOI) of 0.1 over 8 h. OD600 was measured every 2 h, and phage abundance was quantified by plaque assay. Data represent three biological replicates, with distinct symbols and colors indicating bacterial strains and corresponding phage abundance

To assess the generalizability of these observations, we evaluated phage susceptibility across six clinical P. aeruginosa isolates. In the T4P-specific strains ZS-PA-14, ZS-PA-20, and ZS-PA-27, bile significantly enhanced the antibacterial activity of phipa2, though the onset and magnitude varied. ZS-PA-14 and ZS-PA-20 exhibited delayed synergy, with significant effects emerging after 6 h and peaking at 8 h (p < 0.0001; Fig. S1A–B), whereas ZS-PA-27 showed immediate and sustained enhancement starting at 0 h (P = 0.0153) and continuing through 8 h (p < 0.0001; Fig. S1C). Conversely, in O-antigen–dependent strains (ZS-PA-07, ZS-PA-15, ZS-PA-25), the efficacy of phipa10 efficacy remained unchanged under bile exposure, with no additive or synergistic effects observed (p > 0.9 at all- time points; Fig. S1D–F). In some cases, ox-bile modestly suppressed bacterial growth, but co-treatment with phipa10 did not result in further inhibition. Collectively, these results demonstrate that ox-bile selectively modulates phage–host interactions in a receptor-dependent manner, enhancing infectivity and replication efficiency of T4P-targeting phages while having minimal impact on O-antigen-dependent systems. This underscores the importance of host surface architecture and environmental context-particularly in bile-rich niches such as the hepatobiliary tract—when designing and optimizing phage-based therapeutics.

Ox-bile slightly delays the emergence of phage resistance and modulates host fitness adaptations

To assess whether bile exposure alters the evolutionary dynamics of phage resistance, we evaluated mutation frequencies in P. aeruginosa ZS-PA-35 against two phages, phipa2 and phipa10, under standard and ox-bile-supplemented conditions. Mutation rates for both phages were marginally lower in the presence of ox-bile, though not statistically significant. Specifically, phipa2 exhibited a mean mutation rate of 3.72 × 10⁻⁵ in LB, which declined to 2.13 × 10⁻⁵ under ox-bile exposure (p > 0.05; Fig. 5A, C). Similarly, phipa10 mutation rates decreased from 4.63 × 10⁻⁵ to 4.14 × 10⁻⁵ with ox-bile exposure (p > 0.05; Fig. 5B, D). These findings indicate that while ox bile does not significantly suppress the spontaneous mutation rate, phage-specific trends, particularly with phipa2, suggest receptor-dependent modulation of resistance emergence. These findings indicate that while ox-bile does not significantly suppress the spontaneous mutation rate, phage-specific trends—particularly with phipa2- suggest receptor-dependent modulation of resistance emergence.

Fig. 5.

Phage resistance and host fitness in response to bile. A Mutation rate of P. aeruginosa ZS-PA-35 in LB and ox-bile media following infection with phipa2. B Mutation rate of P. aeruginosa ZS-PA-35 in LB and ox-bile media following infection with phipa10 (C–D) Representative plates showing resistant colonies corresponding to mutation rates for P. aeruginosa ZS-PA-35 against phipa2 and phipa10 in LB and ox-bile media. (E–F) Graphical representation of genome-wide T4P (phipa2)– and LPS (phipa10)-related mutations occurring in P. aeruginosa ZS-PA-35 strains with impaired phage adsorption. Colored dots represent different mutation types, and arrows indicate high-impact mutations. Data represent the averages of three biological replicates. Bars show the mean ± standard error of the mean (SEM), with statistical significance determined by unpaired one-tailed t-test (ns = not significant)

To characterize the phenotypic and genetic bases of resistance, we randomly isolated 40 surviving colonies per phage from both LB and ox-bile conditions. Resistance was confirmed by cross-streaking and spot assays. Four representative resistant phenotypes were selected for whole-genome sequencing, including small and large colony variants, pyomelanin-producing isolates, and phenotypically heterogeneous forms. Comparative genomic analysis revealed distinct mutational landscapes depending on the infecting phage. Phipa2-resistant mutants carried 11 unique mutations, including 5 predicted to have high functional impact and 6 with moderate impact (as classified by SnpEff). Breseq analysis identified disruptions in T4P biogenesis and chemotaxis pathways, including point mutations in cheB (chemotactic signaling), and indels in bcr/cflA (efflux transporter, which may influence membrane stability and pilus integrity). Notably, loss-of-function mutations in pilP, pilN, pilE, and pilV— genes essential for T4P assembly—were common and likely confer resistance by impairing phage receptor presentation (Fig. 5E). Additional mutations in a chemotaxis sensor and a putative lipoprotein suggest secondary adaptation that may limit twitching motility and biofilm formation, further reducing phage adsorption.

In contrast, phipa10-resistant mutants exhibited genetic alterations affecting lipopolysaccharide (LPS) biosynthesis and outer membrane architecture. Key variants were identified in genes encoding a multidrug transporter, a glycosyltransferase, and an amidotransferase—all of which are known to modulate LPS structure and antigenicity (Table S3). These mutations likely disrupt the structural integrity or accessibility of the O-antigen receptor required for phipa10 infection (Fig. 5F). Together, these findings demonstrate that bile exposure does not substantially alter the overall mutation rate, it appears to delay the emergence of resistant phenotypes, particularly against T4P-targeting phages. Moreover, resistance arises via distinct and functionally relevant mutational trajectories, highlighting receptor-specific evolutionary paths that shape phage–host dynamics under ox-bile-induced stress.

Ox-bile exposure compromises receptor-associated functions and alters fitness landscapes in P. aeruginosa

Phage receptors—surface-expressed organelles such as T4P and LPS—serve multifaceted roles in bacterial physiology, contributing to motility, membrane integrity, nutrient acquisition, and virulence regulation [25]. We hypothesized that bile, by targeting these surface structures, imposes receptor-associated fitness costs under host-mimicking conditions. To test this, we compared the growth dynamics of two previously characterized phage-resistant P. aeruginosa ZS-PA-35 mutants (ΔpilT, resistant to phage phipa2 and ΔgalU, resistant to phage phipa10) with the parental strain in LB and ox-bile-supplemented media over a 24 h period. Ox-bile significantly inhibited wild-type ZS-PA-35 growth, particularly during the mid-logarithmic and stationary phases (P < 0.0001; Fig. 6A). Both mutants exhibited exacerbated growth impairments in bile-rich environments, with ΔpilT showing heightened sensitivity throughout the assay (P < 0.0001) and ΔgalU demonstrating a pronounced reduction in stationary-phase growth (P < 0.0001). These results underscore the critical role of intact T4P retraction and LPS biosynthesis in mitigating bile-induced stress, highlighting receptor-associated vulnerabilities that compromise bacterial fitness in host-relevant conditions.

Fig. 6.

Growth kinetics and motility of bacterial strains under varying conditions. A Growth of P. aeruginosa wild-type (ZS-PA-35) and its mutants ΔpilT and ΔgalU in LB or LB supplemented with 0.5% ox-bile. B, C, E, F Swimming and swarming motility of ZS-PA-35, ΔpilT, and ΔgalU assessed on 0.5% agar plates containing LB or LB supplemented with ox-bile after 16 h of incubation. D, G Twitching motility of ZS-PA-35, ΔpilT, and ΔgalU assessed on 1.5% agar plates containing LB or LB supplemented with ox-bile after 3 days of incubation. Data represent the mean ± standard error of the mean (SEM) of three biological replicates, with color-coded lines and symbols indicating respective conditions. Optical density was quantified at OD600, while motility zone diameters were measured in centimeters (cm). Statistical significance was determined by unpaired one-tailed t-test: P ≤ 0.01; *P ≤ 0.001; **P ≤ 0.0001

Next, we assessed whether bile alters motility phenotypes linked to phage receptor function. The ΔpilT mutant exhibited a modest but statistically significant increase in swimming (P < 0.01) and swarming motility (P < 0.01) under ox-bile exposure (Fig. 6B–C), potentially reflecting compensatory upregulation of flagellar motility in response to defective T4P retraction. However, twitching motility was markedly suppressed (P < 0.001; Fig. 6D), consistent with impaired pili function under bile stress. In contrast, the ΔgalU mutant displayed a robust hypermotile phenotype, with significantly enhanced swimming, swarming, and twitching motility in the presence of ox-bile (P < 0.0001 for all; Fig. 6E–G). This behavior may result from altered surface hydrophilicity or ox-bile-induced overproduction of biosurfactants such as rhamnolipids or hydroxyalkanoyloxy alkanoic acids (HAAs), allowing motility compensation despite defective O-antigen synthesis. These data suggest that bile functions as a complex environmental cue that reshapes bacterial motility programs and drives adaptive plasticity under phage-receptor selective pressure.

Ox-bile modulates expression of phage receptor–associated genes

To assess how bile influences the transcriptional regulation of phage receptor-associated genes, P. aeruginosa ZS-PA-35 was cultured to logarithmic phase in LB broth with or without ox-bile supplementation. The expression levels of two key genes were quantified: pilT, which encodes a component of the T4P retraction machinery, and galU, which is involved in LPS core biosynthesis. Consistent with the increased susceptibility to the T4P-targeting phage phipa2, pilT expression was significantly upregulated in bile-exposed cells (P < 0.05; Fig. 7A), suggesting that bile modulates pilus dynamics and surface architecture. Likewise, galU expression was significantly elevated under ox-bile conditions (P < 0.01; Fig. 7B), indicating a potential role in outer membrane remodeling or stress mitigation. Together, these findings suggest that bile acts as a potent environmental cue, driving the expression of genes involved in motility, surface composition, and phage susceptibility, thereby shaping bacterial adaptation within host-associated, bile-rich niches.

Fig. 7.

Bile-regulated transcriptional alterations in phage receptor-related genes of ZS-PA-35. A Relative expression of pilT in P. aeruginosa ZS-PA-35 at OD600 = 0.3 in LB and LB supplemented with 0.5% ox-bile, normalized to rpoS. B Relative expression of galU in P. aeruginosa ZS-PA-35 under the same conditions, normalized to rpoS. Data represent the averages of three biological replicates. Bars indicate mean ± standard error of the mean (SEM), with statistical significance determined by unpaired one-tailed t-test: ns = not significant; P ≤ 0.05; *P ≤ 0.01

Ox-bile suppresses pilT-mediated prophage induction in lysogenic P. aeruginosa ZS-PA-05

Prophages are abundant and functionally active within the human gut microbiome, where their induction can dramatically reshape microbial communities, influence bacterial fitness, and drive ecological transitions [26]. Given the known role of environmental cues—particularly ox-bile—in modulating bacterial physiology and prophage dynamics, we investigated the effect of ox-bile on prophage induction in the lysogenic clinical isolate P. aeruginosa ZS-PA-05 and its isogenic pilT deletion mutant, which is deficient in T4P retraction and thereby prevents superinfection loss. During the first 2 h of growth, ZS-PA-05 exhibited comparable proliferation in both LB and ox-bile–supplemented LB media (P > 0.05), indicating that early-phase growth was not significantly impacted by ox-bile. However, by 4 h, cultures exposed to ox-bile showed significantly altered growth dynamics compared to LB control (P < 0.01), suggesting a transient, bile-induced stress response potentially linked to prophage activation (Fig. 8A). Notably, the ΔpilT ZS-PA-05 mutant displayed no significant growth differences between the two conditions at any time point, implicating T4P in mediating the bile response. By 6 h, the inhibitory effects of bile on growth had diminished in both strains.

Fig. 8.

Growth and prophage abundance of lysogenic strains in the presence of ox-bile. A Optical density at 600 nm (OD600) of P. aeruginosa ZS-PA-05 wild-type and ZS-PA-05 ΔpilT strains measured at 2 h intervals over 8 h in LB broth or LB supplemented with ox-bile. Corresponding phage particle abundance (PFUs/mL) was determined by plaque assay. Lines represent the mean of three biological replicates, with individual measurements shown as symbols

To directly evaluate prophage activity, we quantified phage titers in culture supernatants over time. The ΔpilT ZS-PA-05 mutant consistently showed slightly higher concentrations than the wild-type strain ZS-PA-05, potentially due to impaired T4P-mediated superinfection exclusion. At 2 h, phage induction increased significantly in both strains, and ox-bile further augmented this effect in the ΔpilT mutant (P < 0.0001). In contrast, in the wild-type strain, phage accumulation was significantly reduced under ox-bile exposure at 6 h (P < 0.01) and 8 h (P < 0.001) (Fig. 8B) relative to LB alone. These findings suggest that ox-bile suppresses extracellular prophage accumulation in wild-type ZS-PA-05—an effect not observed in the ΔpilT mutant. The data support a model wherein functional T4P in wild-type cells facilitates reinfection during prophage induction, generating a superinfection loop that ultimately diminishes extracellular phage titers. In contrast, the ΔpilT mutant—lacking pilus retraction—exhibits stable phage levels due to reduced reinfection efficiency. Collectively, these results indicate that bile constrains prophage dynamics in a T4P-dependent manner, highlighting a novel regulatory mechanism through which host-derived cues shape bacterial–phage interactions within the gut environment.

Discussion

MDR bacterial infections of the biliary tract represent a growing clinical challenge, often rendering conventional antibiotic therapies ineffective. Phage therapy has emerged as a promising alternative, due to its high specificity and potent bactericidal activity. However, its clinical efficacy is frequently undermined by the rapid emergence of phage resistance, primarily driven by mutations or modifications in bacterial surface receptors. Such resistance can arise swiftly during treatment, diminishing the therapeutic durability of single-phage interventions. Addressing this limitation requires continuous isolation and characterization of phages that target diverse and dynamic bacterial receptors to circumvent resistance development. Bacterial surface structures serve as essential interfaces for environmental sensing, virulence regulation, and phage adsorption [27, 28]. Their modulation represents a key adaptive strategy, enabling survival across fluctuating ecological niches [25]. In Gram-negative pathogens, receptor heterogeneity and regulatory plasticity have been widely recognized as central mechanisms of phage resistance [20, 29]. While factors such as growth phase and population density are known to influence receptor expression, the role of host-associated environmental signals, particularly bile, in modulating these responses remains poorly understood. Elucidating how bile shapes receptor landscapes and phage susceptibility is crucial for optimizing phage-based therapies against MDR infections in complex environments such as the biliary tract. In this study, we utilized two well-characterized phages, phipa2 and phipa10, to investigate their interactions with the clinical P. aeruginosa isolate ZS-PA-35 in both LB broth and LB supplemented with ox-bile. Additionally, we assessed bile’s impact on prophage induction in lysogenic P. aeruginosa strain (ZS-PA-05). Our findings demonstrate that ox-bile exerts multifaceted effects on bacterial physiology, including alterations in bacterial biofilm formation, motility, growth dynamics, and phage susceptibility. Specifically, ox-bile modulated the activity of the T4P-targeting phage phipa2, enhancing its infectivity and amplification, whereas the O-antigen–targeting phage phipa10 remained largely unaffected. These results suggest that ox-bile serves as a critical environmental cue, selectively influencing T4P mediated phage–host interactions and promoting bacterial phenotypic plasticity within bile-rich host niches. Understanding these interactions offers new insights for the rational design of phage therapies in hepatobiliary and other mucosal environments.

Most bacterial communities thrive within biofilms, structured surface-associated assemblies that enhance survival, persistence, and host colonization. The impact of bile on biofilm formation constitutes a potential survival strategy for enteric pathogens exposed to the harsh conditions of the gastrointestinal tract [30, 31]. Bile has been shown to play a dual role—promoting biofilm formation in certain pathogens such as Bifidobacterium spp, V. cholerae, C. jejuni, E. coli, and Salmonella spp., by enhancing extracellular matrix production and virulence gene expression [32–35], while inhibiting biofilm development in others by disrupting membrane integrity or repressing biofilm associated pathways. Our findings indicate that ox-bile modestly enhances biofilm formation in P. aeruginosa ZS-PA-35, a response accompanied by increased surface-associated motility. Notably, we observed significant expansion in both swimming and twitching motility zones—phenotypes that are closely tied to virulence, host tissue colonization, and dissemination. These findings align with previous reports suggesting that ox-bile can stimulate P. aeruginosa biofilm development by modulating virulence factors such as pyocyanin production and quorum sensing pathways [36, 37]. Collectively, these results support the notion that bile acts as a host-derived environmental cue, fine-tuning bacterial surface behaviors to promote niche adaptation and persistence.

Beyond biofilm regulation, ox-bile exerts strain-specific effects on bacterial motility by modulating motility-associated gene expression. For instance, in enterotoxigenic E. coli (ETEC) and Salmonella enterica serovar Typhimurium, bile has been shown to suppress motility by interfering pilus assembly, while in V. cholerae, bile enhances motility and virulence [38, 39]. Our study aligns with these observations, demonstrating that bile distinctly regulates motility in ZS-PA-35. Specifically, ox-bile significantly enhanced twitching motility—likely through modulation of the Type IV pilus (T4P) system via the Chp chemosensory system and cAMP-Vfr signaling cascade, which coordinate pilus assembly and surface engagement [40–43]. Additional regulation via the PilS–PilR two-component system and c-di-GMP–responsive PilB activity may further contribute to this enhanced T4P-mediated motility [44, 45]. This bile-induced motility shift may increase susceptibility to T4P-dependent phages like phipa2, emphasizing the interplay between environmental signals, surface structures, and phage–host dynamics in the biliary niche. Conversely, ox-bile significantly suppressed swimming motility, likely through downregulation of flagellar biosynthesis pathways. Prior studies have implicated the FleS/FleR two-component system and elevated c-di-GMP levels in repressing flagellar gene expression under stress conditions [46, 47]. This motility-to-sessility transition supports biofilm formation and surface colonization as a protective adaptation. It is consistent with previous findings linking bile-mediated repression of motility-associated genes (e.g., nirS, norC, nosZ) and virulence regulators (e.g., lecB) to enhanced survival in hostile environments [48–50]. Additionally, ox-bile enhanced swarming motility in ZS-PA-35, a behavior known to promote colonization and biofilm initiation.This response is in line with reports suggesting that bile exposure modulates chemotaxis and quorum sensing pathways, including suppression of the Type III secretion system (T3SS) [37, 51]. Bacteria isolated from bile-rich environments such as the gallbladder often display increased expression of flagellar and fimbrial proteins, supporting their role in surface colonization and persistence [52–54]. In clinical contexts, such as cystic fibrosis, bile reflux has been shown to alter P. aeruginosa physiology by promoting flagellar loss, reducing motility, and downregulating key virulence determinants, including T3SS, hydrogen cyanide biosynthesis (hcnABC), amidase (amiR), and phenazine biosynthesis genes (phzA-E) [55]. Taken together, our results underscore the nuanced regulatory effects of bile on P. aeruginosa motility, and surface behavior, demonstrating its critical role in shaping pathogen adaptation and virulence in host-associated environments.

Our study further demonstrates that ox-bile significantly modulates phage–bacterial interactions in P. aeruginosa, with direct implications for phage infectivity and efficacy. This modulation appears to be primarily mediated by changes in bacterial surface receptor availability and associated fitness costs, such as impaired growth in receptor-deficient mutants. These observations align with previous studies showing that bile alters phage susceptibility in diverse pathogens. For example, bile restricts phage ICP1 infectivity in V. cholerae by modifying O-antigen structure [56], and inhibits E. coli phage λ adsorption via bile-induced alterations of the Ag43 surface protein [57]. Similarly, bile activates virulence factors, such as T6SS1 in V. parahaemolyticus, indicating its dual role in both pathogenesis and phage resistance [58].

Pathogenic bacteria, such as E. coli, Salmonella spp, Shigella spp, Vibrio spp, utilize bile as both a stressor and a signaling molecule, adapting their surface structures to resist stress and regulate virulence gene expression for effective colonization of the GI tract [8]. In this context, bile-mediated remodeling of the bacterial envelope directly influences phage adsorption and infection dynamics, a crucial factor for the success of phage therapy [56, 59, 60].

Our results highlight a receptor-specific modulation of phage susceptibility by bile. Exposure to ox-bile significantly enhanced the infectivity and replication of the T4P-dependent phage phipa2 in P. aeruginosa ZS-PA-35, likely via upregulation of T4P expression and increased receptor accessibility. This observation is consistent with studies showing bile-induced enhancement of phage ICP2 adsorption to V. cholerae via receptor upregulation. In contrast, phage phipa10, which targets the LPS O-antigen, showed no change in infectivity. This stability suggests that the O-antigen may act as a protective barrier against bile-mediated disruption. Alternatively, bile treatment might alter the structural integrity of the O-antigen, thereby influencing the initial phage adsorption process. Another plausible explanation for the consistent infectivity and replication of phage phipa10 across LB and x-bile-supplemented media lies in the structural resilience and constitutive expression of LPS O-antigen components. These receptors appear less susceptible to ox-bile-induced membrane perturbations compared to T4P-associated structures. Supporting this, our qPCR analysis revealed higher expression of the galU gene (involved in LPS biosynthesis) relative to pilT, suggesting that bile exposure does not markedly affect phipa10 receptor availability or infection dynamics. These findings highlight the complex interplay between bile, phages, and bacterial surface structures in shaping infection dynamics [56].

Beyond receptor availability, bile may suppress phage infectivity through multiple bacterial defense mechanisms. For example, bile has been shown to hinder the PPO1 phage adsorption efficiency by altering the bacterial surface properties [24], and phase variation or DNA methylation in salmonella spp, may further restrict receptor availability [55]. Additionally, bile triggers the stress such as SOS response, which can activate antiphage defenses including CRISPR-Cas systems and restriction modification enzymes [34, 61]. The observed suppression of phage assembly in the presence of bile may represent an adaptive strategy by bacteria to evade phage predation under stress conditions. These findings are consistent with previous studies showing that bile exposure can induce bacterial resistance mechanisms and the production of virulence factors, thereby contributing to phage resistance [5, 61].

Strain-specific differences in receptor regulation and bile sensitivity further complicate this interplay, as seen in our clinical isolate panel, where phage susceptibility varied independently of growth inhibition. Bacteria utilize diverse strategies to impede phage adhesion through multilayered defense mechanisms, including receptor masking, the production of outer membrane vesicles (OMVs), activation of quorum sensing genes, and extracellular matrix reinforcement [1, 2, 17]. These multilayered defenses are particularly pronounced under stress conditions, which have been shown to markedly enhance phage resistance [62–65]. Future studies should investigate whether bile modulates these defense pathways directly. Mechanistically, the bile-induced modulation of motility and phage susceptibility in P. aeruginosa likely involves coordinated regulation via the Chp chemosensory system, the cAMP–Vfr axis, and c-di-GMP levels, which collectively promote T4P assembly while repressing flagellar synthesis. While our findings establish a functional link under controlled conditions, in vivo validation using bile-mimicking organoid systems or animal models is necessary to assess physiological relevance.

Prophage activation in the gastrointestinal tract influences bacterial fitness, pathogen virulence, microbial dynamics, and gut conditions. This process is triggered by environmental stimuli such as diet, antibiotics, metabolites, oxidative stress, and cell density [26]. Bile plays a potential role in shaping bacterial-prophage interactions, particularly by inducing prophage activation through DNA damage and disruption of bacterial surface structures, as observed in pathogens such as S. enterica [66]. Additionally, bile functions as a signaling molecule, regulating virulence and gene expression in E. coli, C. jejuni, and other enteric pathogens [44, 67]. Our findings demonstrate that bile regulates lysogenic bacteria and prophage dynamics, particularly under stress conditions in the gut like bile availability. These results highlight the dual role of T4P expression in modulating receptor accessibility and prophage–host interactions, with bile acting as a key regulatory factor. The ΔpilT strain showed enhanced prophage accumulation under bile, likely due to impaired T4P function preventing superinfection-induced reduction in phage titers. While these findings suggest a bile–T4P–prophage interaction axis, the precise molecular mechanism—whether it involves SOS pathway suppression, transcriptional inhibition, or impaired phage assembly—remains unresolved. Together, this study enhances our understanding of prophage dynamics in the gut and suggests potential implications for both health and dysbiosis, opening new avenues for phage therapy and microbiome research.

Conclusion

The emergence of MDR pathogens, notably P. aeruginosa, poses a critical global health challenge, particularly in healthcare settings where treatment options are limited. Phage therapy offers a promising alternative to antibiotics due to their host-specific precision. However, its efficacy is heavily influenced by host-associated environmental factors. The biliary tract, enriched in bile, imposes unique physiological stressors that modulate bacterial phenotypes and phage susceptibility. In this study, we investigated how ox-bile influences key traits of the MDR strain P. aeruginosa ZS-PA-35, including biofilm formation, motility, and phage resistance, and examined prophage induction dynamics in the lysogenic strain ZS-PA-05. Our findings reveal that bile acts as a potent environmental signal, reshaping surface structures, receptor availability, and resistance mechanisms in a receptor-dependent manner. Notably, bile enhanced the infectivity of T4P-targeting phages while exerting minimal impact on O-antigen-dependent phages, highlighting the complexity of bile-mediated modulation. By elucidating how bile shapes phage–bacteria interactions and prophage dynamics, this study advances our understanding of microbial adaptation in the biliary niche. These insights are critical for guiding the rational design and application of phage therapy against MDR pathogens in bile-rich environments such as the hepatobiliary tract.

Abbreviations

BWA

Burrows-wheeler aligner

BTIs

Biliary tract infections

CFU/mL

Colony-forming units per milliliter

CT

Comparative threshold cycle

ETEC

Enterotoxigenic Escherichia coli 

GATK

Genome analyzer toolkit

HAAs

Hydroxyalkanoyloxy alkanoic acids

LB

Luria Broth (Miller’s LB Broth)

LPS

Lipopolysaccharide

MDR

Multidrug-resistant

MOI

Multiplicity of infection

OMVs

Outer membrane vesicles

OD

Optical density

PFU/mL

Plaque-forming units per milliliter

SEM

Standard error of the mean

SNPs

Single nucleotide polymorphisms

T3SS

Type III secretion system

T4P

Type IV pili

µ (Mutation Rate)

Mutant per culture (m) divided by the total number of cells in the CFU (N), µ = m/N

ΔpilT/ΔgalU

Mutant genes of pilT and galU

ZS-PA-35

Wild-type pseudomonas aeruginosa strain

Authors’ contributions

Muhammad Saleem Iqbal Khan: Investigation, Methodology, Formal analysis, Data curation, Validation, Writing- original draft, review and editing. Ju Wu: Conceptualization, Methodology, Investigation, Visualization. Shuangshuang Hou: Investigation, Methodology, Formal analysis. Shenlin Ji: Methodology, Data curation. He Li: Methodology, Data curation. Yaoyuan Chang: Software, Data analysis, Review, and editing. Bingrui Sui: Methodology, Investigation. Demeng Tan: Resources, Study design, Conceptualization, Supervision, Project administration, Editing. Jiajun Yin: Funding acquisition, Supervision, Review & Editing, Project administration.

Funding

We sincerely thank the Dalian Deng Feng Program for supporting this work through the Key Medical Specialties Construction Grant provided by the People’s Government of Dalian Municipality (2021 − 243), and the Liaoning Provincial Department of Education University Fundamental Research Project (LJ242411258022). The funding bodies had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. 

Data availability

The genomic sequences of the bacterial strain and phages used in this study are available in the GenBank database under the following accession numbers: Pseudomonas aeruginosa ZS-PA-35 (GCA_020567355.1), phage phipa2 (OK539824.1), and phage phipa10 (OK539826.1). Additionally, a supplementary file containing the source data used to generate all main and supplementary figures is also provided in the supplemental file.

Declarations

Ethics approval and consent to participate

This study did not involve any experiments with human participants or animals conducted by the authors.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.