Chao An1,2,3,4
Ruan Chen5Baijian Wu5
Shanjian Chen1,2,3,4Shenghua Zou6Yulan Lin1,2,3,4
Bin Yang1,2,3,4*Chenshuo Luo1,2,3,4*- 1Department of Laboratory Medicine, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
- 2Department of Laboratory Medicine, National Regional Medical Center, Binhai Campus of the First Affiliated Hospital, Fujian Medical University, Fuzhou, China
- 3Fujian Key Laboratory of Laboratory Medicine, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
- 4Clinical Laboratory Diagnostics, The First Clinical College, Fujian Medical University, Fuzhou, China
- 5Department of Orthopaedic Surgery, The First Affiliated Hospital, Fujian Medical University, Fuzhou, China
- 6Department of Laboratory Medicine, Fuzhou Pulmonary Hospital of Fujian Province, Fuzhou, China
Background: Pseudomonas aeruginosa is the most prevalent pathogen responsible for persistent pulmonary infections. Iron concentrations in the human lung are known to vary considerably between health and disease states. We hypothesized that increased iron availability is a key driver of persistent infection establishment and sought to define the impact of iron on P. aeruginosa in the context of persistent lung infection.
Methods: Clinically isolated strains of P. aeruginosa from persistent lung infections and the reference strain PAO1 were collected. We examined bacterial growth rates, virulence determinants such as biofilm formation and pyocyanin production, and adhesion to lung epithelial cells under different iron conditions. Virulence was assessed using a Galleria mellonella model, and pathogenicity was evaluated in a mouse model.
Results: Compared to P. aeruginosa grown in iron-deficient medium, bacteria cultured in iron-rich medium exhibited significantly enhanced growth rates and biofilm formation, while virulence determinants were attenuated. The Galleria mellonella model also showed reduced virulence. Additionally, iron-rich conditions enhanced bacterial adhesion to lung epithelial cells. In the mouse model, weakened pathological damage and higher bacterial loads in the lungs were observed.
Conclusion: Our findings indicate that environmental iron facilitates the growth and biofilm formation of P. aeruginosa causing pulmonary infections, while attenuating its virulence. This iron-mediated adaptation may be associated with the persistence of P. aeruginosa pulmonary infections, and these findings merit further investigation.
Introduction
Pseudomonas aeruginosa (P. aeruginosa) is a common opportunistic pathogen in clinical settings, capable of causing wound infections, respiratory tract infections, urinary tract infections, and other conditions (Pont et al., 2025; Faure et al., 2018; Sorbets et al., 2019). Among these, persistent pulmonary infections caused by P. aeruginosa represent one of the most challenging clinical conditions to manage. A biofilm is a structured community of microbial cells enclosed in a self-produced polymeric matrix that adheres to a surface (Preda and Săndulescu, 2019). P. aeruginosa serves as a model organism for studying bacterial biofilms. The formation of biofilms enhances the ability of P. aeruginosa to adapt to external environments and is a major factor contributing to the recalcitrance of persistent lung infections to treatment (Jones and Wozniak, 2017).
The virulence of P. aeruginosa is mediated by an array of determinants, including siderophores, proteases, alkaline protease, flagella, pyocyanin, elastase, hemolysins, phospholipase C, exotoxin A, rhamnolipids, among others (Liao et al., 2022). These virulence factors have been demonstrated to play pivotal roles in the pathogenesis of infectious diseases caused by P. aeruginosa (Schaible et al., 2017). For example, exotoxin A targets eukaryotic elongation factor 2 (eEF-2), inhibiting host protein synthesis and inducing host cell death (Du et al., 2010). Pyocyanin causes infection by inducing oxidative stress in host cells and disrupting the host’s immune function (Lau et al., 2004). Extracellular invasive enzymes, including elastase, alkaline protease, and protease, degrade host connective tissue, leading to lung parenchymal damage and hemorrhage, and are associated with disseminated infections by P. aeruginosa (Sánchez-Jiménez et al., 2023).
P. aeruginosa possesses the ability to continuously sense and respond to environmental cues such as bicarbonate levels, osmotic stress, pH, and iron availability (Ruksakiet et al., 2021). Among these factors, iron stands out as one of the most critical elements. Previous studies have reported that iron plays an essential role in both biofilm formation and virulence expression of P. aeruginosa (Hamza et al., 2023; Banin et al., 2005; Minandri et al., 2016). However, host-bound iron remains largely inaccessible to most microorganisms, including P. aeruginosa. To overcome this limitation, the pathogen employs siderophore production and secretion to acquire iron necessary for growth and metabolism (Mayneris-Perxachs et al., 2022). These iron-chelating molecules not only facilitate iron acquisition and support infection establishment but also significantly influence bacterial survival within the host.
It has been demonstrated that the urogenital tract environment significantly influences the growth and virulence factor levels of P. aeruginosa and may contribute to difficulties in treating chronic infections (Subramaniyan et al., 2025). While bioavailable iron in the human body is strictly limited, the pulmonary environment exposes P. aeruginosa to varying levels of iron, an environmental factor that may impact its virulence and pathogenicity (Haas et al., 2023). Therefore, it is necessary to understand the precise role of iron in P. aeruginosa-mediated persistent pulmonary infections. Current research combining in vitro and in vivo experiments to investigate the role of iron in P. aeruginosa persistent pulmonary infection remains scarce. Our study aims to assess, using both in vivo and in vitro models, whether varying iron concentrations affect the growth, biofilm formation, and virulence of P. aeruginosa that causes persistent pulmonary infection.
Materials and methods
Bacterial strains
Two clinical isolates of P. aeruginosa (designated PA1 and PA3) obtained from sputum samples of patients with persistent pulmonary infections at The First Affiliated Hospital of Fujian Medical University in 2025, along with the reference strain PAO1 purchased from China Bosa Biological Co., Ltd., were selected for this study. These strains are capable of producing most recognized virulence determinants, including siderophores, proteases, hemolysins, pyocyanin, among others.
Effect of iron
We referenced previous studies on the influence of iron on the pathogenicity of Klebsiella pneumoniae (Chen et al., 2020). As shown in the Supplementary Figure 1, we first confirmed the growth curve of the P. aeruginosa reference strain PAO1 under different iron conditions using a microplate reader. We created varying iron-deficient conditions by supplementing TSB with 0 μM, 100 μM, 200 μM, 300 μM, 400 μM, and 500 μM of the iron chelator dipyridyl (DP). With the increasing concentration of DP, the growth of PAO1 gradually slowed down. At 500 μM DP, PAO1 exhibited almost no growth. Therefore, we selected TSB supplemented with 400 μM DP as the most stringent iron-deficient condition. To confirm that the growth inhibition of P. aeruginosa PAO1 in TSB supplemented with 400 μM DP, representing the most stringent iron-deficient condition, was specifically due to iron limitation, we added FeCl₃ at concentrations of 0 μM, 0.1 μM, 1 μM, 5 μM, and 10 μM. As shown in Supplementary Figure 2, the growth of PAO1 gradually recovered with increasing FeCl₃ supplementation. When 10 μM FeCl₃ was added, the growth of PAO1 did not show a significant further increase compared to 5 μM. Therefore, for further experiments, we selected the condition of TSB supplemented with 400 μM DP (the most stringent iron-deficient medium) with an additional 5 μM FeCl₃. We measured the iron concentration and growth curves of the collected P. aeruginosa in TSB medium supplemented with 0 μM DP, 400 μM DP, and 400 μM DP + 5 μM FeCl₃.
Determination of iron concentrations
The iron concentration in TSB media containing 0 μM DP, 400 μM DP, and 400 μM DP + 5 μM FeCl₃ was determined using a colorimetric chelation assay. Briefly, ascorbic acid was added to the medium and reacted with Ferene to form a blue complex, which was quantified spectrophotometrically.
Growth curves
The assessment of iron’s influence on P. aeruginosa growth was conducted with modifications based on a previously established methodology (Chen et al., 2020). Briefly, PA1, PA3, and PAO1 strains were cultured overnight in TSB and harvested. The overnight cultures were diluted 1:100 in TSB media containing different iron concentrations: 0 μM DP, 400 μM DP, and 400 μM DP supplemented with 5 μM FeCl₃. Cultures were incubated at 37 °C with constant shaking at 220 rpm. Optical density at 600 nm (OD600) was measured at 2-h intervals over a 24-h period. Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Biofilm formation assay
The effect of iron on P. aeruginosa biofilm formation was evaluated using a modified established methodology (Chen et al., 2020). Overnight cultures of strains PA1, PA3, and PAO1 in TSB medium were diluted 1:100 in TSB containing different iron conditions (0 μM DP, 400 μM DP, and 400 μM DP + 5 μM FeCl₃). A 200 μL aliquot of each dilution was added to a 96-well polystyrene microtiter plate and incubated at 37 °C for 24 h, with sterile TSB serving as a negative control. Following incubation, the wells were washed thrice with PBS (pH 7.0), air-dried, fixed with methanol for 20 min, stained with 1% crystal violet for 15 min, and then rinsed with PBS until colorless. After drying, the bound dye was solubilized with 200 μL absolute ethanol, transferred to a new plate, and the absorbance was measured at 570 nm. Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Cell-free supernatant and bacterial pellet
Overnight cultures of P. aeruginosa strains PA1, PA3, and PAO1 grown in TSB medium were diluted 1:100 in TSB media containing varying iron concentrations (0 μM DP, 400 μM DP, and 400 μM DP + 5 μM FeCl₃). The diluted bacterial suspensions were incubated at 37 °C with constant shaking at 180 rpm for 48 h. After adjusting the cultures to an OD600 of 1.0, the samples were centrifuged at 5,000 rpm for 5 min to collect both cell-free supernatants and bacterial pellets.
Pyocyanin production
A 5 mL aliquot of the collected P. aeruginosa cell-free supernatant was mixed with 3 mL of chloroform. The resulting mixture was vortexed and centrifuged. The upper aqueous layer was then treated with 1 mL of 0.2 M hydrochloric acid for acidification. Following thorough mixing and subsequent centrifugation, the upper pink phase was carefully collected, and its absorbance was measured at 520 nm. Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Hemolysin production
Hemolytic activity was measured using a modified version of the method described by previous Research (Alamu et al., 2022; Wang et al., 2022). Briefly, a 5% suspension of sheep red blood cells (obtained from Nanjing Senbeijia Biological Technology Co., Ltd.) was incubated with an equal volume of cell-free supernatant harvested from P. aeruginosa culture. The mixture was incubated at 37 °C for 1 h, followed by centrifugation at 500 × g for 5 min. The absorbance of the resulting supernatant was measured at 450 nm. Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Siderophore production
Following the manufacturer’s instructions, the Chrome Azurol S (CAS) assay solution (Beijing Coolaber Technology Co., Ltd.) was mixed with an equal volume of either the collected P. aeruginosa cell-free supernatant or deionized water. The mixtures were protected from light and incubated at room temperature for 1 h. The absorbance of the supernatant mixture (A) and the deionized water mixture (Ar) was then measured at 630 nm. Siderophores induce a color change in the CAS assay solution, resulting in a decrease in OD630. Therefore, siderophore production was quantified using the following formula: 1 − A/Ar. Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Elastase production
Briefly, P. aeruginosa cells cultured under different iron conditions were collected by centrifugation (5,000 rpm, 5 min). The supernatant was discarded, and any residual liquid was thoroughly removed. The corresponding extraction buffer was added at a ratio of 10 μL per 10^7 cells, followed by gentle mixing via pipetting. The cells were then lysed using ice-bath sonication (power 300 W, 3 s pulse on, 7 s pulse off, total duration 3 min). The lysate was centrifuged at 8000 g for 10 min at 4 °C, and the resulting supernatant was collected and kept on ice for subsequent detection. According to the manufacturer’s instructions, the elastase activity in the supernatant was measured using the EnzChek Elastase Assay Kit. Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Protease production
Briefly, P. aeruginosa cells cultured under different iron conditions were collected by centrifugation (5,000 rpm, 5 min). The supernatant was discarded, and any residual liquid was thoroughly removed. The corresponding extraction buffer was added at a ratio of 10 μL per 10^7 cells, followed by gentle mixing via pipetting. The cells were then lysed using ice-bath sonication (power 300 W, 3 s pulse on, 7 s pulse off, total duration 3 min). The lysate was centrifuged at 8000 g for 10 min at 4 °C, and the resulting supernatant was collected and kept on ice for subsequent detection. According to the manufacturer’s instructions, the protease activity in the supernatant was measured using the Protease Assay Kit from Beyotime Biotechnology Co., Ltd. Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Alkaline protease production
Briefly, P. aeruginosa cells cultured under different iron conditions were collected by centrifugation (5,000 rpm, 5 min). The supernatant was discarded, and any residual liquid was thoroughly removed. The corresponding extraction buffer was added at a ratio of 10 μL per 10^7 cells, followed by gentle mixing via pipetting. The cells were then lysed using ice-bath sonication (power 300 W, 3 s pulse on, 7 s pulse off, total duration 3 min). The lysate was centrifuged at 8000 g for 10 min at 4 °C, and the resulting supernatant was collected and kept on ice for subsequent detection. According to the manufacturer’s instructions, the alkaline protease activity in the supernatant was measured using the Alkaline Protease Assay Kit from Box Biotechnology Co., Ltd. The results were normalized to the total protein concentration in each sample using an Enhanced BCA Protein Assay Kit (Beyotime). Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Phospholipase C production
Briefly, P. aeruginosa cells cultured under different iron conditions were collected by centrifugation (5,000 rpm, 5 min). The supernatant was discarded, and any residual liquid was thoroughly removed. The corresponding extraction buffer was added at a ratio of 10 μL per 10^7 cells, followed by gentle mixing via pipetting. The cells were then lysed using ice-bath sonication (power 300 W, 3 s pulse on, 7 s pulse off, total duration 3 min). The lysate was centrifuged at 8000 g for 10 min at 4 °C, and the resulting supernatant was collected and kept on ice for subsequent detection. According to the manufacturer’s instructions, the phospholipase C activity in the supernatant was measured using the Phospholipase C Assay Kit from Elabscience Biotechnology Co., Ltd., and the total protein concentration in each sample was normalized using the Enhanced BCA Protein Assay Kit (Beyotime). Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Exotoxin A production
Briefly, P. aeruginosa cells cultured under different iron conditions were collected by centrifugation (5,000 rpm, 5 min). The supernatant was discarded, and any residual liquid was thoroughly removed. The corresponding extraction buffer was added at a ratio of 10 μL per 10^7 cells, followed by gentle mixing via pipetting. The cells were then lysed using ice-bath sonication (power 300 W, 3 s pulse on, 7 s pulse off, total duration 3 min). The lysate was centrifuged at 8000 g for 10 min at 4 °C, and the resulting supernatant was collected and kept on ice for subsequent detection. According to the manufacturer’s instructions, the production of exotoxin A in the supernatant was measured using the Exotoxin A Assay Kit from Wuhan Fine Biotech Co., Ltd. Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Galleria mellonella infection model
The larval lethality assay using Galleria mellonella was performed with modifications based on a previously established method (Chen et al., 2020). Larvae were anesthetized on ice for 5 min. Bacterial pellets of P. aeruginosa, prepared as described above, were resuspended in phosphate-buffered saline to achieve a concentration of 108 CFU/mL. A 25 μL Hamilton microsyringe was used to inject 10 μL of the bacterial suspension (108 CFU/mL) into the last left proleg of each larva. Inoculated larvae were incubated in darkness at 25 °C and monitored at 24, 48, and 72 h post-infection. Larvae were considered dead when they displayed no response to repeated physical stimulation. Results were analyzed using the Kaplan–Meier method.
Lung epithelial cell adhesion assay
The Lung epithelial cell adhesion assay was conducted with modifications based on a previously described method (Liu et al., 2023). Human Normal Lung Epithelial Cells (BEAS-2B cells) were purchased from Shanghai Biyuntian Biotechnology Co., Ltd. BEAS-2B cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C under 5% CO₂. The prepared P. aeruginosa bacterial pellets were resuspended in DMEM and added to 24-well plates at a multiplicity of infection (MOI) of 10. After 2 h of incubation, the supernatant was discarded, and the cells were washed with sterile PBS to remove non-adherent bacteria. The cells were then lysed with 0.01% Triton X-100, and the lysates were plated on blood agar plates for bacterial enumeration. Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Mice infection model
The mice lung infection model was established with modifications based on a previously described methodology (Nielsen et al., 2018). Male C57BL/6 mice (6–8 weeks old) were obtained from Beijing Huafukang Biological Technology Co., Ltd. The mice were anesthetized using isoflurane (Shanghai Yuyan Instruments Co. Ltd., Shanghai, China), and their upper incisors were secured with fine thread to suspend them in a vertical position on a rack. The tongue was carefully extended from the oral cavity using sterile blunt forceps and gently immobilized. The nasal passages were temporarily occluded to maintain oral breathing. Concurrently, a second operator administered 50 μL of a bacterial suspension containing P. aeruginosa pellets resuspended in PBS (1 × 109 CFU/mL) at the oropharyngeal junction using a micropipette. Control mice received an equal volume of PBS buffer. Successful pulmonary inoculation was confirmed by a characteristic crackling sound upon reflex inhalation, indicating aerosolization and lung deposition of the inoculum. At 24 h post-infection, mice were anesthetized with isoflurane and sacrificed by cervical dislocation for subsequent lung tissue isolation.
Lung tissue isolation
Mice lung tissues were aseptically harvested and rinsed with PBS, followed by weighing with an analytical balance to record tissue mass. The left lung was placed in a microcentrifuge tube containing 500 μL of pre-cooled saline. The tube was maintained on ice, and tissue homogenization was performed using a mechanical homogenizer with five cycles of 60-s grinding interspersed with 10-s intervals. The resulting homogenate was utilized for bacterial enumeration and ELISA-based quantification of inflammatory cytokine levels. The right lung was transferred to a 2 mL microcentrifuge tube and fixed with 4% paraformaldehyde at room temperature for 48 h in preparation for pathological examination.
Lung bacterial load
A 100 μL aliquot of the lung tissue homogenate was serially diluted in sterile PBS, and 50 μL of the appropriate dilution was plated onto blood agar plates. After spreading with a sterile cell spreader and air-drying, the plates were incubated at 37 °C overnight. Bacterial colonies were enumerated, and the bacterial load in lung tissue was calculated as CFU per gram of tissue. All experiments were performed in triplicate.
Lung tissue cytokine analysis
The levels of interleukin-6 (IL-6), interleukin-10 (IL-10), and procalcitonin (PCT) in the lung homogenates were quantified using commercial ELISA kits (Elabscience Biotechnology Co., Ltd.) according to the manufacturer’s protocols. Analysis was based on the average absorbance value obtained from triplicate measurements for each sample.
Lung histopathological examination
The fixed lung tissues were embedded in paraffin after overnight fixation in 4% paraformaldehyde. Sections of 5 μm thickness were prepared and stained with hematoxylin and eosin (H&E) for assessment of pulmonary damage severity (Sen-Kilic et al., 2019).
Statistical analysis
Data were analyzed using analysis of variance (ANOVA), Fisher’s exact test, and Student’s t-test. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed with SPSS version 19.0.
Results
Determination of iron concentrations in culture media
To investigate the effects of iron on growth, biofilm formation, and virulence traits, Pseudomonas aeruginosa was cultured in media containing varying concentrations of the iron chelator dipyridyl (DP) (0–400 μM) and FeCl₃ (0–5 μM). Tryptic Soy Broth (TSB) supplemented with 0 μM DP served as the iron-replete environment, with an iron content of 0.65 μg/mL, while TSB with 400 μM DP served as the iron-deficient environment, with an iron content of 0.04 μg/mL. In the environment of TSB supplemented with 400 μM DP and 5 μM FeCl₃, the iron content was 0.37 μg/mL.
Effect of iron on Pseudomonas aeruginosa growth
All P. aeruginosa strains demonstrated optimal growth in iron-replete environments (0 μM DP TSB medium), with significantly better growth compared to both the iron-restricted (400 μM DP) and iron-supplemented (400 μM DP + 5 μM FeCl₃) conditions (p < 0.05) (Figure 1).
Figure 1. Growth curve of P. aeruginosa. All P. aeruginosa isolates grew optimally in TSB broth, superior to the growth in TSB broth containing 400 μM iron chelator +(−) 5 μM iron (p < 0.05). DP: 2,2′-Dipyridyl, added as an iron chelator. TSB + 400 M DP group was used as control.
Effect of iron on Pseudomonas aeruginosa biofilm formation
All P. aeruginosa strains exhibited significantly enhanced biofilm formation in the iron-replete environment (0 μM DP TSB medium) compared to both the iron-supplemented (400 μM DP + 5 μM FeCl₃) and iron-restricted (400 μM DP TSB) conditions (p < 0.05) (Figure 2).
Figure 2. Biofilm formation of P. aeruginosa. All P. aeruginosa isolates exhibited the strongest biofilm formation ability in TSB broth, which was superior to the biofilm formation ability of the respective strains in TSB broth containing 400 μM iron chelator +(−)5 μM iron (p < 0.05). DP: 2,2′-ipyridyl, added as an iron chelator. TSB + 400 M DP group was used as control. Data are presented as mean ± s.d. A student’s t-test was used for between-group comparisons, and multi-group comparisons were performed using analysis of variance (ANOVA). The results are representative of three independent experiments.*p < 0.05; **p < 0.01; ***p < 0.001.
Effect of iron on virulence determinants of Pseudomonas aeruginosa
All P. aeruginosa strains exhibited significantly reduced production of virulence determinants—including protease, pyocyanin, exotoxin A, phospholipase C, alkaline protease, elastase, siderophore, and hemolysin—in the iron-replete TSB medium (0 μM DP) compared to both the iron-supplemented (400 μM DP + 5 μM FeCl₃) and iron-restricted (400 μM DP) conditions (p < 0.05) (Table 1). Furthermore, the production of these virulence determinants were significantly lower under the iron-supplemented condition (400 μM DP + 5 μM FeCl₃) compared to the iron-restricted condition (400 μM DP).
Table 1. Virulence factors of P. aeruginosa under iron-restricted and iron-replete culture conditions.
Galleria mellonella infection model
Galleria mellonella larvae infected with P. aeruginosa cultured in iron-replete TSB medium (0 μM DP) demonstrated significantly higher survival rates than those infected with bacteria grown in iron-restricted TSB medium (400 μM DP) (Figure 3).
Figure 3. Models of Galleria mellonella infected with P. aeruginosa. The virulence of PA1, PA3, and PAO1 supplemented with an iron chelator was significantly higher than that of strains without it (p < 0.05). DP: 2,2′-Dipyridyl, added as an iron chelator. The PBS was control group. Galleria mellonella mortality was analyzed by Kaplan–Meier and log-rank tests.
Pulmonary epithelial cell adhesion assay
P. aeruginosa strains cultured under iron-replete conditions exhibited significantly enhanced adhesion to pulmonary epithelial cells compared to those grown in iron-restricted environments (Figure 4).
Figure 4. Adhesion capacity of P. aeruginosa to pulmonary epithelial cells. All strains exhibited the strongest adhesion capacity in TSB broth, which was superior to the respective strains in TSB broth containing 400 μM iron chelator (p < 0.05). DP: 2,2′-Dipyridyl, added as an iron chelator. TSB + 400 M DP group was used as control. A Fisher’s exact test was used for between-group comparisons. Data are presented as mean ± s.d. (n = 3).*p < 0.05; **p < 0.01.
Mice pulmonary infection model
Bacterial load
Consistent with the in vitro growth observations, mice infected with P. aeruginosa cultured in iron-replete TSB medium exhibited significantly higher pulmonary bacterial loads compared to those infected with bacteria grown in iron-restricted TSB medium (400 μM DP) (Figure 5).
Figure 5. Bacterial load in the lungs of mice infected with P. aeruginosa. All strains exhibited a higher bacterial load in mice lungs when cultured in TSB broth than in TSB broth supplemented with 400 μM iron chelator (p < 0.05). DP: 2,2′-Dipyridyl, added as an iron chelator. TSB + 400 μM DP group was used as control. A student’s t-test was used for between-group comparisons. Data are presented as mean ± s.d. (n = 3).*p<0.05; **p<0.01.
Inflammatory cytokines
Mice infected with P. aeruginosa cultured under iron-replete conditions demonstrated significantly lower levels of pulmonary PCT, IL-6, and IL-10 compared to those infected with bacteria grown in iron-restricted medium (Figure 6).
Figure 6. Inflammatory cytokines in the lungs of mice infected with P. aeruginosa. All strains exhibited lower levels of inflammatory factors in mice lungs when cultured in TSB broth than in TSB broth supplemented with 400 μM iron chelator (p < 0.05). DP: 2,2′-dipyridyl, added as an iron chelator. TSB + 400 μM DP group was used as control. A student’s t-test was used for between-group comparisons. Data are presented as mean ± s.d. (n = 3). *p<0.05; **p<0.01; ***p<0.001.
Pathological injury
Mice infected with P. aeruginosa cultured in iron-replete TSB medium showed minimal granulocyte infiltration in alveolar walls (yellow arrows) and mildly irregular bronchiolar epithelium with luminal exfoliated cells and eosinophilic material (green arrows) (Figure 7B), with black boxes indicating magnified view locations (Figure 7A); whereas those infected with bacteria from iron-restricted medium (400 μM DP) exhibited extensive pathology including alveolar narrowing/collapse (Figure 7C), significant lymphocyte/granulocyte/macrophage infiltration (yellow arrows), bronchiolar irregularities with eosinophilic material (purple arrows), perivascular edema with loose connective tissue (blue arrows), occasional perivascular hemorrhage (brown arrows), and interstitial vascular congestion (pink arrows) (Figure 7D).
Figure 7. Lung tissues from mice infected with P. aeruginosa cultured under different iron conditions were sectioned and subjected to histopathological examination with H&E staining. For mice infected with P. aeruginosa cultured in iron-replete TSB medium, overview of lung tissue sections (magnification ×2.0) showed mild overall inflammation (A). The black box indicates the area of the magnified view. At higher magnification (×20.0), a small number of granulocytes were observed infiltrating the alveolar walls (yellow arrows). Minor irregularities were noted in the bronchiolar epithelial cell arrangement, with exfoliated epithelial cells and eosinophilic material visible in the lumen (green arrows) (B). In contrast, lung tissues from mice infected with P. aeruginosa cultured in iron-restricted TSB medium (with 400 μM DP) exhibited alveolar narrowing or even obliteration, occasional perivascular hemorrhage (brown arrows), and some interstitial vascular congestion (pink arrows) (C). The black box indicates the area of the magnified view. At higher magnification (×20.0), substantial infiltration of lymphocytes, granulocytes, and macrophages was evident (yellow arrows). Additionally, infiltration by a smaller number of lymphocytes and granulocytes was observed (blue arrows), and eosinophilic material was visible in the bronchiolar lumen (purple arrows) (D). DP: 2,2′-Dipyridyl, added as an iron chelator.
Discussion
The complex pulmonary environment is known to contain varying levels of electrolytes, particularly iron, which has been demonstrated to play a crucial role in determining the ultimate outcome of infections (Chen et al., 2020). Iron exhibits extremely low solubility, and the host restricts its availability to pathogens through complexation with proteins such as transferrin and lactoferrin. This mechanism represents an important host defense strategy (Mayneris-Perxachs et al., 2022). The iron concentration in the pulmonary environment of healthy individuals ranges from 8.6 to 21.5 μg/L, which is relatively close to the iron-restricted condition established in this study. This indicates that the lung is indeed an iron-limited environment under normal physiological conditions, and our study has realistically simulated the pulmonary iron milieu (Gray et al., 2010). Researcher has found that the iron content in the sputum of patients with chronic inflammatory lung diseases (such as cystic fibrosis and bronchiectasis) is significantly elevated compared to that of healthy controls (Gray et al., 2010). Similarly, other study has reported that the iron concentration in the bronchoalveolar lavage fluid of cystic fibrosis patients is also significantly higher than in healthy controls (Ghio et al., 2013). Elevated sputum iron concentration is a characteristic alteration of the pulmonary microenvironment in cystic fibrosis patients. During stable periods, it strongly correlates with the burden of P. aeruginosa, suggesting that iron accumulation may promote the persistence of infection (Reid et al., 2007). In the present study, iron-restricted conditions were established by supplementing Tryptic Soy Broth (TSB) with 400 μM dipyridyl, while unsupplemented TSB served as the iron-replete condition. P. aeruginosa was cultured separately in iron-restricted and iron-replete media, and its growth, biofilm formation, and pathogenicity under these two conditions were evaluated both in vitro and in vivo. Previously, study has demonstrated the crucial role of iron in Klebsiella pneumoniae-induced liver abscesses by pre-culturing the bacteria under two conditions: iron-replete conditions in TSB and iron-restricted conditions established by adding 300 μM dipyridyl to TSB (Chen et al., 2020).
In this study, we demonstrated that adequate iron availability promoted the growth of P. aeruginosa, whereas the isolates exhibited slower growth under iron-restricted conditions. Furthermore, iron enhanced biofilm formation by P. aeruginosa, while iron limitation significantly compromised this capacity. Existing studies have demonstrated that iron chelators, including ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, ethylenediamine-N, N′-diacetic acid, lactoferrin, deferoxamine, 5-hydroxy-2-hydroxymethyl-4H-pyran-4-one, 3-hydroxy-2-methyl-4-pyrone, 1,2,3,4,6-penta-O-galloyl-β-D-glucopyranose, and deferiprone, can significantly inhibit the growth and biofilm formation of P. aeruginosa and/or other bacteria and fungi (O’May et al., 2009; Temel and Aksoyalp, 2023; Moon et al., 2013; Nazik et al., 2015; Brilhante et al., 2021; Saha et al., 2025; Guedes et al., 2023; Ramage et al., 2007; Lin et al., 2012; Coraça-Huber et al., 2018; De Sá Almeida et al., 2020; Qiao et al., 2018; Leitão et al., 2025). Furthermore, iron chelators such as deferiprone and deferoxamine can enhance the antibacterial activity of conventional antibiotics like clindamycin, gentamicin, vancomycin, amoxicillin-clavulanic acid, and meropenem by disrupting bacterial biofilms (Temel and Aksoyalp, 2023). The reduced biofilm formation by P. aeruginosa under iron-restricted conditions may be associated with the decreased biosynthesis of Psl exopolysaccharide, a key component of its biofilm, which is elevated under iron-replete conditions (Yu et al., 2016). Previous studies have confirmed that Acinetobacter baumannii similarly shows markedly diminished biofilm formation in iron-limited environments, with significant restoration upon iron supplementation, indicating iron’s essential role in A. baumannii biofilm development (Saha et al., 2025). The presence of sufficient iron also facilitates biofilm formation in Staphylococcus epidermidis, Staphylococcus aureus, and Klebsiella pneumoniae (Chen et al., 2020; Oliveira et al., 2017; Lin et al., 2012). Collectively, these findings and our results substantiate that iron significantly modulates bacterial growth and biofilm formation processes.
Previous studies have established that iron promotes Klebsiella pneumoniae virulence through enhanced serum resistance, potentially mediated by iron uptake genes sitA/C/D, prompting our investigation into iron’s role in modulating P. aeruginosa virulence during pulmonary infection (Chen et al., 2020; Chen et al., 2025). Our systematic assessment under defined iron conditions revealed maximal production of all virulence determinants (proteases, exotoxin A, phospholipase C, alkaline protease, elastase, siderophores, and hemolysins) under iron restriction, with minimal yields in iron-replete TSB, while observing enhanced BEAS-2B pulmonary epithelial adhesion in iron-rich conditions. These findings align with Kim and Michael, who similarly reported upregulated protease, exotoxin A, and pyochelin production in P. aeruginosa under iron-depleted conditions (TSB + DP) (Kim et al., 2003; Bjorn et al., 1979). Additionally, we found that under iron-deficient conditions, hemolysin production in P. aeruginosa was significantly increased. Mechanistically, iron deficiency induces adaptive stress responses in strain PAO1, including significantly elevated secretion of elastase, hemolysin, and siderophores, which facilitates bacterial iron acquisition (Schaible et al., 2017). Furthermore, Fur, the master regulator of iron homeostasis in P. aeruginosa, controls the expression of hundreds of genes, including those involved in iron acquisition and virulence. The Fur protein represses the expression of genes responsible for the biosynthesis and transport of the siderophores pyoverdine and pyochelin. As a result, these iron-chelating compounds are produced exclusively under iron-limited conditions (Sánchez-Jiménez et al., 2023). Similarly, under iron-sufficient conditions, Fur represses the expression of the hemophore HasAp and the heme uptake systems Phu, Has, and Hxu. Furthermore, Fur-mediated repression encompasses other key virulence factors, such as exotoxin A, the proteases PIV/PrpL and IcmP, the extracellular lipase LipA, and the hemolytic phospholipase C PlcH (Ochsner et al., 2002). Galleria mellonella larvae, an invertebrate model, are commonly used for assessing bacterial virulence (Wang et al., 2022; Insua et al., 2013). Complementary Galleria mellonella infection models confirmed significantly higher larval mortality with iron-restricted (TSB + DP) versus iron-replete (TSB) cultures, collectively substantiating that iron availability critically governs P. aeruginosa virulence expression.
To further evaluate in vivo the characteristics of enhanced adhesion and attenuated virulence of P. aeruginosa under iron-replete conditions, we employed a murine pulmonary infection model, which shares structural and genetic similarities with human lungs (Mural et al., 2002). Mice were infected with P. aeruginosa cultured in either iron-restricted or iron-replete media. Infection with bacteria grown under iron-replete conditions resulted in significantly higher pulmonary bacterial loads, alongside markedly reduced pathological damage and inflammatory cytokine levels. Conversely, mice infected with P. aeruginosa from iron-restricted medium exhibited significantly lower bacterial counts but intensified pathological injury and inflammatory responses. These in vivo results may be attributed to the fact that, under in vitro iron-restricted conditions, P. aeruginosa exhibits reduced adhesion to pulmonary epithelial cells while concurrently increasing the production of siderophores, pyocyanin, alkaline protease, exotoxin A, elastase, protease, phospholipase C, and hemolysins. Existing study has demonstrated that the siderophores pyoverdine and pyochelin of P. aeruginosa play distinct roles in the virulence of acute pulmonary infections. Pyoverdine induces cell death by modulating rhamnolipid secretion, independently of host iron chelation or known toxins. Pyochelin can penetrate cell membranes, activating pro-inflammatory pathways (such as NLRP3 inflammasome and IL-8 secretion) by chelating intracellular iron, with pyoverdine enhancing this pro-inflammatory effect (Donghoon et al., 2024). Furthermore, in a mouse pneumonia model, it has been confirmed that siderophores (enterobactin, salmochelin, and yersiniabactin) secreted by Klebsiella pneumoniae can independently induce the secretion of inflammatory cytokines including IL-6, CXCL1, and CXCL2, promote bacterial dissemination to the spleen, and stabilize host hypoxia-inducible factor-1α (HIF-1α), which in alveolar epithelial cells is a prerequisite for bacterial dissemination (Holden et al., 2016).
Previous studies have demonstrated that iron availability in the in vitro culture environment can alter the renal pathogenicity of Escherichia coli in mice (S et al., 1991). However, there remains a paucity of correlated research examining P. aeruginosa under different iron conditions in the context of pulmonary infection. To address this gap, we pre-cultured P. aeruginosa in iron-restricted and iron-replete media prior to inducing murine pulmonary infection, thereby elucidating its iron-dependent pathogenicity in vivo. Our findings indicate that iron levels influence the growth, biofilm formation, and virulence traits of P. aeruginosa, establishing its crucial role in pathogenicity.
Currently, the siderophore-based antibacterial agent cefiderocol, which targets bacterial iron metabolism, has demonstrated remarkable efficacy against resistant Gram-negative bacteria such as P. aeruginosa (Cruz-López et al., 2022). Additionally, the FDA-approved iron chelators deferoxamine and deferasirox, by restricting P. aeruginosa’s ability to acquire iron from the environment, have been shown to reduce existing P. aeruginosa biofilm production by approximately 90% and significantly decrease viable bacterial counts when used in combination with tobramycin (Moreau-Marquis et al., 2009). Based on the findings of this study, iron is demonstrated to be essential for the growth, biofilm formation, and pathogenicity of P. aeruginosa in the context of pulmonary infection. However, attempting to treat these infections by creating an iron-restricted condition solely through the use of iron chelators such as deferoxamine and deferasirox may potentially exacerbate lung damage in patients. This is because our study also revealed that under iron-deficient conditions, key virulence determinants of P. aeruginosa—including siderophores, pyocyanin, proteases, hemolysins, phospholipase C, and exotoxin A—were significantly elevated (Table 1). Additionally, inflammatory markers such as PCT, IL-6, and IL-10 showed significant increases (Figure 6). Most critically, lung injury in mice was more pronounced under these conditions (Figure 7).
Pyoverdine, a major siderophore and a key virulence factor in P. aeruginosa, plays a critical functional role. It not only facilitates iron acquisition but also regulates the secretion of exotoxin A, endoprotease, and pyoverdine itself (Lamont et al., 2002). Bacterial efflux pumps, such as those from the RND family, typically mediate cellular adaptive responses to various environmental stimuli, including iron. Hannauer et al. discovered that one of the pathways by which P. aeruginosa secretes the siderophore pyoverdine is via efflux pumps (Hannauer et al., 2010). Efflux pump inhibitors, by interfering with iron uptake and simultaneously reducing the secretion of key virulence factors like pyoverdine, may represent a promising therapeutic strategy for managing persistent pulmonary infections (Mudgil et al., 2024). Gallium nitrate, an iron metabolism disruptor, has been shown to inhibit the growth and biofilm formation of P. aeruginosa. However, pyoverdine can sequester gallium nitrate within the periplasmic space through irreversible chelation, significantly diminishing its antibacterial activity. 5-fluorocytosine can overcome this antagonism by blocking either the synthesis or function of pyoverdine, thereby enabling synergistic efficacy with gallium nitrate for potent antibacterial and virulence-attenuating effects, while maintaining a low selective pressure for resistance development (Kang et al., 2021). Additionally, alternative anti-virulence strategies employing natural compounds, such as Manuka honey and allicin, target siderophore systems. By suppressing pyoverdine production, they disrupt bacterial iron acquisition, concurrently inhibiting biofilm formation and the expression of virulence factors (Kronda et al., 2013; Xu et al., 2019). Owing to their lower cytotoxicity to host cells, these strategies hold promise for the clinical treatment of P. aeruginosa infections.
Limitations
This study has several limitations. First, it was conducted at a single center, which may limit the diversity and representativeness of the bacterial strains included. Additionally, all experiments assessing growth, biofilm formation, and virulence were performed exclusively on P. aeruginosa in the planktonic mode; these results may not fully reflect the responses of P. aeruginosa in the biofilm mode. The specific pathways through which iron affects the growth, biofilm formation, and pathogenicity of P. aeruginosa and contributes to persistent pulmonary infections remain to be further investigated. Furthermore, in this study, the use of the iron chelator to create iron-restricted conditions in tryptic soy broth (TSB), and the use of TSB alone as the iron-replete condition, may not fully recapitulate the complexity of the lung environment. Despite these limitations, our findings establish a foundation for a deeper understanding of the role of iron in the pathogenesis of P. aeruginosa pulmonary infection.
Conclusion
In summary, environmental iron facilitates the growth and biofilm formation of P. aeruginosa causing pulmonary infections, while attenuating its virulence. This iron-mediated adaptation may be associated with the persistence of P. aeruginosa pulmonary infections. To better understand these findings, a further in-depth evaluation is warranted.
Data availability statement
The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.
Ethics statement
Ethical approval was not required for the studies on humans in accordance with the local legislation and institutional requirements because only commercially available established cell lines were used. The animal study was approved by the Committee on Ethical Use of Animals of The First Affiliated Hospital of Fujian Medical University. It is affiliated with Fujian Medical University. The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
CA: Investigation, Writing – original draft, Visualization, Validation. RC: Writing – review & editing, Investigation, Validation, Visualization. BW: Visualization, Writing – review & editing, Investigation. SC: Visualization, Writing – review & editing, Investigation. SZ: Writing – review & editing, Investigation, Visualization. YL: Visualization, Investigation, Writing – review & editing. BY: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. CL: Writing – review & editing, Conceptualization, Funding acquisition, Supervision.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This study was financially supported by the National Natural Science Foundation of China (grant no. 82172327), The National Natural Youth Science Foundation of China (grant no. 82402686).
Conflict of interest
The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that Generative AI was not used in the creation of this manuscript.
Any alternative text (alt text) provided alongside figures in this article has been generated by Frontiers with the support of artificial intelligence and reasonable efforts have been made to ensure accuracy, including review by the authors wherever possible. If you identify any issues, please contact us.
Publisher’s note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
Supplementary material
The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb.2025.1742683/full#supplementary-material
References
Alamu, J., Kakithakara Vajravelu, L., Venkatesan, B., and Thulukanam, J. (2022). Correlation of phenotypic and genotypic virulence markers, antimicrobial susceptibility pattern, and outcome of Pseudomonas aeruginosa sepsis infection. Microb. Pathog. 170:105716. doi: 10.1016/j.micpath.2022.105716,
Banin, E., Vasil, M. L., and Greenberg, E. P. (2005). Iron and Pseudomonas aeruginosa biofilm formation. Proc. Natl. Acad. Sci. USA 102, 11076–11081. doi: 10.1073/pnas.0504266102,
Bjorn, M. J., Sokol, P. A., and Iglewski, B. H. (1979). Influence of Iron on yields of extracellular products in Pseudomonas aeruginosa cultures. J. Bacteriol. 138, 193–200. doi: 10.1128/jb.138.1.193-200.1979,
Brilhante, R. S. N., Costa, A. D. C., Pereira, V. S., Fernandes, M. R., de Oliveira, J. S., Rodrigues, A. M., et al. (2021). Antifungal activity of deferiprone and EDTA against Sporothrix spp.: effect on planktonic growth and biofilm formation. Med. Mycol. 59, 537–544. doi: 10.1093/mmy/myaa073,
Chen, T., Dong, G., Zhang, S., Zhang, X., Zhao, Y., Cao, J., et al. (2020). Effects of iron on the growth, biofilm formation and virulence of Klebsiella pneumoniae causing liver abscess. BMC Microbiol. 20:36. doi: 10.1186/s12866-020-01727-5,
Chen, J., Li, H., Li, F., Kang, M., Wang, R., Chu, Y., et al. (2025). Iron metabolism contributes to virulence by enhancing serum resistance in the emerging high-risk clone ST15 CRKP. Virulence 16:2569700. doi: 10.1080/21505594.2025.2569700,
Coraça-Huber, D. C., Dichtl, S., Steixner, S., Nogler, M., and Weiss, G. (2018). Iron chelation destabilizes bacterial biofilms and potentiates the antimicrobial activity of antibiotics against coagulase-negative staphylococci. Pathogens Disease. 76:fty052. doi: 10.1093/femspd/fty052,
Cruz-López, F., Martínez-Meléndez, A., Morfin-Otero, R., Rodriguez-Noriega, E., Maldonado-Garza, H. J., and Garza-González, E. (2022). Efficacy and in vitro activity of novel antibiotics for infections with Carbapenem-resistant gram-negative pathogens. Front. Cell. Infect. Microbiol. 12:884365. doi: 10.3389/fcimb.2022.884365,
De Sá Almeida, J. S., De Oliveira Marre, A. T., Teixeira, F. L., Boente, R. F., Domingues, R. M. C. P., de Paula, G. R., et al. (2020). Lactoferrin and lactoferricin B reduce adhesion and biofilm formation in the intestinal symbionts Bacteroides fragilis and Bacteroides thetaiotaomicron. Anaerobe 64:102232. doi: 10.1016/j.anaerobe.2020.102232,
Donghoon, K., Qi, X., and Natalia V, K. (2024). In vitro lung epithelial cell model reveals novel roles for Pseudomonas aeruginosa siderophores. Microbiol. Spectr. 12:e0369323. doi: 10.1128/spectrum.03693-23,
Du, X., Youle, R. J., FitzGerald, D. J., and Pastan, I. (2010). Pseudomonas exotoxin A-mediated apoptosis is Bak dependent and preceded by the degradation of Mcl-1. Mol. Cell. Biol. 30, 3444–3452. doi: 10.1128/MCB.00813-09,
Faure, E., Kwong, K., and Nguyen, D. (2018). Pseudomonas aeruginosa in chronic lung infections: how to adapt within the host? Front. Immunol. 9:2416. doi: 10.3389/fimmu.2018.02416,
Ghio, A. J., Roggli, V. L., Soukup, J. M., Richards, J. H., Randell, S. H., and Muhlebach, M. S. (2013). Iron accumulates in the lavage and explanted lungs of cystic fibrosis patients. J. Cyst. Fibros. 12, 390–398. doi: 10.1016/j.jcf.2012.10.010,
Gray, R. D., Duncan, A., Noble, D., Imrie, M., O'Reilly, D. S. J., Innes, J. A., et al. (2010). Sputum trace metals are biomarkers of inflammatory and Suppurative lung disease. Chest 137, 635–641. doi: 10.1378/chest.09-1047,
Guedes, G. M. D. M., Ribeiro, K. V. C., Araújo, E. S. D., Pereira, V. C., Soares, A. C. d. C. F., Freitas, A. S., et al. (2023). In vitro effect of the iron chelator deferiprone on the antimicrobial susceptibility and biofilms of Burkholderia pseudomallei. Biofouling 39, 135–144. doi: 10.1080/08927014.2023.2192405,
Haas, A. L., Zemke, A. C., Melvin, J. A., Armbruster, C. R., Hendricks, M. R., Moore, J., et al. (2023). Iron bioavailability regulates Pseudomonas aeruginosa interspecies interactions through type VI secretion expression. Cell Rep. 42:112270. doi: 10.1016/j.celrep.2023.112270,
Hamza, E. H., El-Shawadfy, A. M., Allam, A. A., and Hassanein, W. A. (2023). Study on pyoverdine and biofilm production with detection of LasR gene in MDR Pseudomonas aeruginosa. Saudi J. Biol. Sci. 30:103492. doi: 10.1016/j.sjbs.2022.103492,
Hannauer, M., Yeterian, E., Martin, L. W., Lamont, I. L., and Schalk, I. J. (2010). An efflux pump is involved in secretion of newly synthesized siderophore by Pseudomonas aeruginosa. FEBS Lett. 584, 4751–4755. doi: 10.1016/j.febslet.2010.10.051,
Holden, V. I., Breen, P., Houle, S., Dozois, C. M., and Bachman, M. A. (2016). Klebsiella pneumoniae Siderophores induce inflammation, bacterial dissemination, and HIF-1α stabilization during pneumonia. MBio 7, e01397–e01316. doi: 10.1128/mBio.01397-16,
Insua, J. L., Llobet, E., Moranta, D., Pérez-Gutiérrez, C., Tomás, A., Garmendia, J., et al. (2013). Modeling Klebsiella pneumoniae pathogenesis by infection of the wax moth galleria mellonella. Infect. Immun. 81, 3552–3565. doi: 10.1128/IAI.00391-13,
Jones, C. J., and Wozniak, D. J. (2017). Psl produced by mucoid Pseudomonas aeruginosa contributes to the establishment of biofilms and immune evasion. MBio 8, e00864–e00817. doi: 10.1128/mBio.00864-17,
Kang, D., Revtovich, A. V., Deyanov, A. E., and Kirienko, N. V. (2021). Pyoverdine inhibitors and gallium nitrate synergistically affect Pseudomonas aeruginosa. mSphere 6, e00401–e00421. doi: 10.1128/mSphere.00401-21
Kim, E. J., Sabra, W., and Zeng, A. P. (2003). Iron deficiency leads to inhibition of oxygen transfer and enhanced formation of virulence factors in cultures of Pseudomonas aeruginosa PAO1. Microbiology 149, 2627–2634. doi: 10.1099/mic.0.26276-0,
Kronda, J. M., Cooper, R. A., and Maddocks, S. E. (2013). Manuka honey inhibits siderophore production in Pseudomonas aeruginosa. J. Appl. Microbiol. 115, 86–90. doi: 10.1111/jam.12222,
Lamont, I. L., Beare, P. A., Ochsner, U., Vasil, A. I., and Vasil, M. L. (2002). Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 99, 7072–7077. doi: 10.1073/pnas.092016999,
Lau, G. W., Ran, H., Kong, F., Hassett, D. J., and Mavrodi, D. (2004). Pseudomonas aeruginosa Pyocyanin is critical for lung infection in mice. Infect. Immun. 72, 4275–4278. doi: 10.1128/IAI.72.7.4275-4278.2004,
Leitão, M. M., Gonçalves, A. S. C., Moreira, J., Fernandes, C., Borges, F., Simões, M., et al. (2025). Unravelling the potential of natural chelating agents in the control of Staphylococcus aureus and Pseudomonas aeruginosa biofilms. Eur. J. Med. Chem. 283:117163. doi: 10.1016/j.ejmech.2024.117163,
Liao, C., Huang, X., Wang, Q., Yao, D., and Lu, W. (2022). Virulence factors of Pseudomonas aeruginosa and Antivirulence strategies to combat its drug resistance. Front. Cell. Infect. Microbiol. 12:926758. doi: 10.3389/fcimb.2022.926758,
Lin, M. H., Shu, J. C., Huang, H. Y., and Cheng, Y. C. (2012). Involvement of Iron in biofilm formation by Staphylococcus aureus. PLoS One 7:e34388. doi: 10.1371/journal.pone.0034388,
Liu, S., Chen, H., Chen, J., Wang, T., Tu, S., Zhang, X., et al. (2023). Transcriptome and proteome of methicillin-resistant Staphylococcus aureus small-Colony variants reveal changed metabolism and increased immune evasion. Microbiol Spectr. 11, e0189822–e0189822. doi: 10.1128/spectrum.01898-22,
Mayneris-Perxachs, J., Moreno-Navarrete, J. M., and Fernández-Real, J. M. (2022). The role of iron in host–microbiota crosstalk and its effects on systemic glucose metabolism. Nat. Rev. Endocrinol. 18, 683–698. doi: 10.1038/s41574-022-00721-3,
Minandri, F., Imperi, F., Frangipani, E., Bonchi, C., Visaggio, D., Facchini, M., et al. (2016). Role of Iron uptake Systems in Pseudomonas aeruginosa virulence and airway infection. Infect. Immun. 84, 2324–2335. doi: 10.1128/IAI.00098-16,
Moon, J. H., Kim, C., Lee, H. S., Kim, S. W., and Lee, J. Y. (2013). Antibacterial and antibiofilm effects of iron chelators against Prevotella intermedia. J. Med. Microbiol. 62, 1307–1316. doi: 10.1099/jmm.0.053553-0,
Moreau-Marquis, S., O’Toole, G. A., and Stanton, B. A. (2009). Tobramycin and FDA-approved Iron Chelators eliminate Pseudomonas aeruginosa biofilms on cystic fibrosis cells. Am. J. Respir. Cell Mol. Biol. 41, 305–313. doi: 10.1165/rcmb.2008-0299OC,
Mudgil, U., Khullar, L., Chadha, J., Prerna,, and Harjai, K. (2024). Beyond antibiotics: emerging antivirulence strategies to combat Pseudomonas aeruginosa in cystic fibrosis. Microb. Pathog. 193:106730. doi: 10.1016/j.micpath.2024.106730
Mural, R. J., Adams, M. D., Myers, E. W., Smith, H. O., Miklos, G. L., Wides, R., et al. (2002). A comparison of whole-genome shotgun-derived mouse chromosome 16 and the human genome. Science 296, 1661–1671. doi: 10.1126/science.1069193,
Nazik, H., Penner, J. C., Ferreira, J. A., Haagensen, J. A. J., Cohen, K., Spormann, A. M., et al. (2015). Effects of Iron Chelators on the formation and development of Aspergillus fumigatus biofilm. Antimicrob. Agents Chemother. 59, 6514–6520. doi: 10.1128/AAC.01684-15,
Nielsen, T. B., Yan, J., Luna, B., and Spellberg, B. (2018). Murine oropharyngeal aspiration model of ventilator-associated and hospital-acquired bacterial pneumonia. JoVE 136:57672. doi: 10.3791/57672,
O’May, C. Y., Sanderson, K., Roddam, L. F., Kirov, S. M., and Reid, D. W. (2009). Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions. J. Med. Microbiol. 58, 765–773. doi: 10.1099/jmm.0.004416-0,
Ochsner, U. A., Wilderman, P. J., Vasil, A. I., and Vasil, M. L. (2002). GeneChip® expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes. Mol. Microbiol. 45, 1277–1287. doi: 10.1046/j.1365-2958.2002.03084.x,
Oliveira, F., França, Â., and Cerca, N. (2017). Staphylococcus epidermidis is largely dependent on iron availability to form biofilms. Int. J. Med. Microbiol. 307, 552–563. doi: 10.1016/j.ijmm.2017.08.009,
Pont, S., Nilly, F., Berry, L., Bonhoure, A., Alford, M. A., Louis, M., et al. (2025). Intracellular Pseudomonas aeruginosa persist and evade antibiotic treatment in a wound infection model. PLoS Pathog. 21:e1012922. doi: 10.1371/journal.ppat.1012922,
Preda, V. G., and Săndulescu, O. (2019). Communication is the key: biofilms, quorum sensing, formation and prevention. Discover 7:e10. doi: 10.15190/d.2019.13,
Qiao, J., Purro, M., Liu, Z., and Xiong, M. P. (2018). Terpyridine-micelles for inhibiting bacterial biofilm development. ACS Infect Dis. 4, 1346–1354. doi: 10.1021/acsinfecdis.8b00091,
Ramage, G., Wickes, B. L., and López-Ribot, J. L. (2007). Inhibition on Candida albicans biofilm formation using divalent cation chelators (EDTA). Mycopathologia 164, 301–306. doi: 10.1007/s11046-007-9068-x,
Reid, D. W., Carroll, V., O’May, C., Champion, A., and Kirov, S. M. (2007). Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis. Eur. Respir. J. 30, 286–292. doi: 10.1183/09031936.00154006,
Ruksakiet, K., Stercz, B., Tóth, G., Jaikumpun, P., Gróf, I., Tengölics, R., et al. (2021). Bicarbonate evokes reciprocal changes in intracellular cyclic di-GMP and cyclic AMP levels in Pseudomonas aeruginosa. Biology 10:519. doi: 10.3390/biology10060519,
S, S., K, H., and R, M. (1991). Enhanced siderophore production and mouse kidney pathogenicity in Escherichia coli grown in urine. J. Med. Microbiol. 35, 325–329. doi: 10.1099/00222615-35-6-325
Saha, S., RoyChowdhury, D., Khan, A. H., Mandal, S., Sikder, K., Manna, D., et al. (2025). Harnessing the effect of iron deprivation to attenuate the growth of opportunistic pathogen Acinetobacter baumannii. Antimicrob. Agents Chemother. 69, e0168924–e0168924. doi: 10.1128/aac.01689-24,
Sánchez-Jiménez, A., Llamas, M. A., and Marcos-Torres, F. J. (2023). Transcriptional regulators controlling virulence in Pseudomonas aeruginosa. IJMS. 24:11895. doi: 10.3390/ijms241511895,
Sánchez-Jiménez, A., Marcos-Torres, F. J., and Llamas, M. A. (2023). Mechanisms of iron homeostasis in Pseudomonas aeruginosa and emerging therapeutics directed to disrupt this vital process. Microb. Biotechnol. 16, 1475–1491. doi: 10.1111/1751-7915.14241,
Schaible, B., Rodriguez, J., Garcia, A., von Kriegsheim, A., McClean, S., Hickey, C., et al. (2017). Hypoxia reduces the pathogenicity of Pseudomonas aeruginosa by decreasing the expression of multiple virulence factors. J. Infect. Dis. 215, 1459–1467. doi: 10.1093/infdis/jix139,
Sen-Kilic, E., Blackwood, C. B., Boehm, D. T., Witt, W. T., Malkowski, A. C., Bevere, J. R., et al. (2019). Intranasal Peptide-Based FpvA-KLH Conjugate Vaccine Protects Mice From Pseudomonas aeruginosa Acute Murine Pneumonia. Front. Immunol. 10:2497. doi: 10.3389/fimmu.2019.02497,
Sorbets, E., Evrevin, M., Jumas-Bilak, E., Masnou, A., Lotthé, A., Thuret, R., et al. (2019). An outbreak of Pseudomonas aeruginosa urinary tract infections following outpatient flexible cystoscopy. Am. J. Infect. Control 47, 1510–1512. doi: 10.1016/j.ajic.2019.05.005,
Subramaniyan, Y., Khan, A., Mujeeburahiman, M., Ashmitha,, Shivani, M., Patil, S., et al. (2025). Urological metabolic conditions alter regulation of quorum-sensing system and virulence genes expression in uropathogenic Pseudomonas aeruginosa. Microb. Pathog. 203:107502. doi: 10.1016/j.micpath.2025.107502,
Temel, A., and Aksoyalp, Z. Ş. (2023). A preliminary study on the effect of Deferoxamine on disruption of bacterial biofilms and antimicrobial resistance. tjps. 21, 267–273. doi: 10.4274/tjps.galenos.2023.23890,
Wang, X., Zhao, H., Wang, B., Zhou, Y., Xu, Y., Rao, L., et al. (2022). Identification of methicillin-resistant Staphylococcus aureus ST8 isolates in China with potential high virulence. Emerg. Microbes Infect. 11, 507–518. doi: 10.1080/22221751.2022.2031310,
Xu, Z., Zhang, H., Yu, H., Dai, Q., Xiong, J., Sheng, H., et al. (2019). Allicin inhibits Pseudomonas aeruginosa virulence by suppressing the rhl and pqs quorum-sensing systems. Can. J. Microbiol. 65, 563–574. doi: 10.1139/cjm-2019-0055,
Keywords: biofilm, growth, iron, Pseudomonas aeruginosa, virulence
Citation: An C, Chen R, Wu B, Chen S, Zou S, Lin Y, Yang B and Luo C (2026) Iron dictates the growth, biofilm formation, and virulence of Pseudomonas aeruginosa in pulmonary infections. Front. Microbiol. 16:1742683. doi: 10.3389/fmicb.2025.1742683
Edited by:
Giovanna Batoni, University of Pisa, ItalyReviewed by:
Donghoon Alex Kang, Houston Methodist Research Institute, United StatesMallika Somayajulu, Wayne State University, United States
Rosa Bellavita, University of Naples Federico II, Italy
Copyright © 2026 An, Chen, Wu, Chen, Zou, Lin, Yang and Luo. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Bin Yang, eWFuZ2JpbjI4NjRAMTYzLmNvbQ==; Chenshuo Luo, MTAxNzY1MjU3NUBxcS5jb20=