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Gallium nitrate inhibits multidrug-resistant Acinetobacter baumannii isolated from bloodstream infection by disrupting multiple iron-dependent metabolic processes

BMC Microbiology volume 25, Article number: 216 (2025) Cite this article

Abstract

Background

Acinetobacter baumannii is a major pathogen in hospitals, causing a notable rise in bloodstream infections among inpatients. Its growing resistance to multiple drugs limits treatment options. This study aims to examine the antibacterial effects of gallium nitrate [Ga(NO3)3] against A. baumannii and elucidate the underlying molecular mechanism.

Methods

40 strains of A. baumannii with different antimicrobials susceptibility patterns were isolated from bloodstream infections. The in vitro antibacterial activity of Ga(NO3)3 was analyzed by micro-dilution method and time-kill assay. The influence of ferric chloride/hemin on the antibacterial efficacy of Ga(NO3)3 was investigated. Transcriptome sequencing was performed to elucidate the antibacterial mechanism of Ga(NO3)3. A mouse infection model was conducted to assess its in vivo performance.

Results

Ga(NO3)3 exhibited a potent antibacterial effect in RPMI 1640 medium containing 10% human serum, with MICs ranging from 0.06 μg/mL to 0.125 μg/mL. The antibacterial activity of Ga(NO3)3 was found to be dose- and time- dependent. However, the antibacterial effect of Ga(NO3)3 was partially compromised in the presence of exogenous ferric chloride/hemin. Transcriptomics analysis revealed that Ga(NO3)3 exerted its antibacterial effect by up-regulating the expression of genes associated with siderophore biosynthesis and transport, while simultaneously disrupting multiple iron-dependent metabolic processes. Ga(NO3)3 treatment significantly reduced bacterial load in vivo using a neutropenic mouse thigh infection model.

Conclusion

This study sheds light on the antibacterial mechanisms of Ga(NO3)3 against A. baumannii, suggesting its potential as a promising antibacterial drug for treating bloodstream infections caused by multidrug-resistant A. baumannii.

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Introduction

Acinetobacter baumannii is a significant hospital-acquired pathogen and has become the primary cause of bloodstream infections among inpatients, particularly those in intensive care unit (ICU) [1]. Its ability to acquire multidrug, extensively drug and even pandrug resistance severely limits the treatment options [2]. The mortality rate associated with A. baumannii bloodstream infection exceeds 50% [3, 4]. In China, the regimen based on sulbactam, colistin and tigecycline is recommended as the main treatment for carbapenem-resistant A. baumannii [5]. However, concerning reports of increasing resistance to the last-resort antimicrobials in A. baumannii have emerged [6]. Therefore, there is an urgent need to explore new therapeutic options to effectively treat bloodstream infections caused by multidrug-resistant A. baumannii (MDR-AB).

Blocking bacterial access to essential nutrients has become a promising anti-infection strategy. Iron, a critical nutrient for the survival of various bacteria, including A. baumannii, serves as a cofactor for multiple vital enzymes [7]. It plays a significant role in essential processes, such as DNA synthesis, electron transfer, and defense against reactive oxygen species [8]. A critical factor contributing to the success of A. baumannii as a nosocomial pathogen lies in its diverse mechanisms for acquiring iron, a vital but limited nutrient in vivo [7]. To date, six gene cluster associated with iron acquisition systems have been identified in A. baumannii. These includes two heme uptake gene clusters: heme uptake cluster I, which encodes a TonB dependent outer membrane receptor (TonB/ExbB/ExbD), periplasmic heme binding protein and an inner membrane ATP-binding cassette transporter; and heme uptake cluster II, which encodes a TonB dependent receptor, an extra cytoplasmic function (ECF) sigma factor, its cognate anti-sigma factor, and a putative heme oxygenase (hemO). Additionally, there are three siderophore clusters for acinetobactin (bas/bau gene cluster), baumannoferrin(s) (bfn gene cluster), and fimsbactin(s) (fbs gene cluster), along with the ferrous uptake system Feo [9]. Given its critical role in bacterial physiology and pathogenicity, targeting iron uptake and metabolism has become attractive approach for developing new antimicrobials.

Gallium nitrate [Ga(NO3)3], an FDA-approved intravenous drug used to treat hypercalcemia associated with malignancy, has recently gained attention in antimicrobial research. Researches have revealed that metallic gallium acts as a “Trojan horse” by disrupting bacterial iron metabolism through gallium ions (Ga3+), which share similarities with iron ions (Fe3+) in terms of atomic radius and electron affinity [10]. Bacteria cannot differentiate gallium from iron, allowing Ga3+ to compete with Fe3+ at the iron binding site of enzymes. However, Ga3+ cannot perform redox reactions like Fe3+ under physiological conditions, leading to the inhibition of essential physiological function [11]. Ga(NO3)3 has exhibited antibacterial activity against ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, A. baumannii, Pseudomonas aeruginosa, and Enterobacter species) [12]. Nonetheless, to our knowledge, there have been no reports of transcriptomics analysis to uncover the molecular mechanisms underlying antibacterial activity of Gallium against A. baumannii. Furthermore, in vivo studies of its antibacterial effect remain limited.

In this study, we demonstrated that Ga(NO3)3 had remarkable antibacterial effect against A. baumannii, both in a medium resembling human serum and in a neutropenic mouse thigh infection model. Our findings suggested that the antibacterial mechanism of gallium nitrate involved two main aspects: up-regulation of iron uptake and transport pathways and disrupting iron-dependent metabolisms of A. baumannii.

Results

The antibacterial activity of Ga(NO3)3 was influenced by different media

The antibacterial efficacy of Ga(NO3)3 was tested in four different media, of which MH broth represented an iron-rich environment, while the other three media provided iron-poor conditions. Ga(NO3)3 had no antibacterial activity in MH broth, with MICs exceeding 512 μg/mL. However, in M9CA medium, the MICs of Ga(NO3)3 against 40 A. baumannii strains ranged from 32 to 128 μg/mL. Similarly, in RPMI 1640 medium, the MICs of Ga(NO3)3 were observed to be between 32 and 64 μg/mL. The addition of 10% human serum to RPMI medium substantially enhanced the antibacterial activity of Ga(NO3)3, resulting in a remarkable reduction in MIC to the range of 0.0625 to 0.125 μg/mL. Notably, Ga(NO3)3 exhibited excellent antibacterial activity against both MDR strains and susceptible strains, indicating that its efficacy remained unaffected by the antimicrobial susceptibility patterns. The details of MICs in different media were listed in Table 1.

Table 1 Susceptibility of gallium nitrate against Acinetobacter baumannii in different medium and iron uptake-related genes carried by Acinetobacter baumannii
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Ga(NO3)3 inhibits A. baumannii growth in a dose- and time-dependent manner

Ga(NO3)3 displayed a dose- and time-dependent inhibition of A. baumannii growth. The growth curves of A. baumannii BM-2333 under different concentrations of Ga(NO3)3 were shown in Fig. 1. The MIC value of BM-2333 in M9CA medium was determined to be 64 μg/mL. The growth of BM-2333 was effectively inhibited within 24 h when exposed to drug concentrations of 2× and 4× MIC. However, when treated with 1×MIC Ga(NO3)3, the growth inhibition was only observed in the early stage, and BM-2333 resumed growth at approximately 10 h. To further investigate the long-term effects, we extended the experiment to 72 h and monitored the growth curve. At 2× and 4× MIC concentration, the growth of strain BM-2333 eventually recovered over time. Interestingly, as the Ga(NO3)3 increased concentration, the maximum OD600 decreased, indicating that the growth potential of BM-2333 was hampered under higher Ga(NO3)3 concentrations.

Fig. 1
figure 1

The effect of Ga(NO3)3 on the growth of A. baumannii within 72 h

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Ferric chloride/hemin can weaken the antibacterial activity of Ga(NO3)3

The heatmaps illustrate the interaction between Ga(NO3)3 and iron chloride/hemin in Fig. 2. For BM-2333 and BM-4795, the results of the interaction between ferric chloride and Ga(NO3)3 exhibited similar patterns. As the concentration of ferric chloride increased, the antibacterial activity of Ga(NO3)3 weakened. However, at higher concentrations of Ga(NO3)3, it still displayed inhibitory effects on bacterial growth. This interaction was characterized as a competitive effect. However, the interaction between hemin and Ga(NO3)3 demonstrated distinct behavior. In the case of BM-2333, lower concentrations of hemin were able to restore the growth of strains, rendering it unresponsive to the inhibition effects of Ga(NO3)3 as its concentration increased. However, for BM-4795, the recovery of strain growth due to hemin was not remarkable, and could be suppressed by enhancing the concentration of Ga(NO3)3.

Fig. 2
figure 2

The effect of exogenous iron chloride/heme on the antibacterial activity of Ga(NO3)3, x-axis: concentration of Ga(NO3)3, y-axis: concentration of iron chloride/heme. The growth of A. baumannii ranging from poor growth (white) to strong growth (red)

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We further recorded the growth curve of BM-2333 and BM-4795 at the MICs (both were 0.0625 μg/ml) (Fig. 3). In the case of BM-2333, the antibacterial activity of Ga(NO3)3 was found to weaken as the concentration of ferric chloride or hemin increases. Similarly, for BM-4795, the antibacterial activity of Ga(NO3)3 decreases with an increase in ferric chloride concentration. However unlike BM-2333, the growth of BM-4795 only showed partial restoration under high concentrations of hemin within 24 h.

Fig. 3
figure 3

Growth curves in A. baumannii under different concentrations of ferric chloride/hemin and MIC of Ga(NO3)3 interaction

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Detection of iron-uptake genes

Due to the distinct hemin utilization abilities of BM-2333 and BM-4795, we conducted a comprehensive analysis of iron-uptake gene clusters. Among 40 A. baumannii, the positive rate for exbD, hemO and basA were found to be 92.5%, 57.5% and 95%, respectively. Notably, the positive rate of hemO of carbapenem-resistant A. baumannii (CRAB) was significantly higher than carbapenem-susceptible A. baumannii (CSAB) (90.9% vs. 16.7%, p < 0.05) (Table 1).

Transcriptomics analysis

According to RNA-Seq analysis, we identified a total of 1563 differentially expressed genes (DEGs) (Table S2). Among these, 755 genes were found to be up-regulated, while 808 genes were down-regulated in the Ga(NO3)3 treatment group compared to the control group (Fig. 4). The distribution of DEGs in various subcategories was visualized in Fig. S1, revealing their involvement in essential biological processes such as cellular process, metabolic process, cellular anatomical entity, intracellular, binding, catalytic activity and transporter activity. The GO classification enrichment of DEGs highlighted several significant GO Terms, including structural constituent of ribosome, structural molecule activity, ribosome, rRNA binding and oxidoreductase activity (Fig. 5).

Fig. 4
figure 4

The volcano plot of DEGs between control and experimental group, The X-axis represents the difference multiple value, and the Y-axis represents the significance value. Red represents up-regulated DEG, blue represents down-regulated DEG, and gray represents non-DEG

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Fig. 5
figure 5

GO analysis of DEGs. Bubble chart of GO enrichment of differentially expressed genes, The X axis is the enrichment ratio [the ratio of the number of genes annotated to an entry in the selected gene set to the total number of genes annotated to the entry in this species, the calculation formula: Rich Ratio = Term Candidate Gene Num / Term Gene Num], Y axis for GO Term, the size of the bubble represents the number of differential genes annotated to a GO Term, the color represents the enriched Q value, and the darker the color represents the smaller the Q value. Only differentially transcribed genes with absolute fold changes > 1 (log2 scale) and Q value < 0.05 were shown

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Furthermore, KEGG enrichment analysis shed light on specific pathways influenced by Ga(NO3)3. Differentially up-regulated genes were found to be involved in ribosome and biosynthesis of siderophore group nonribosomal peptides pathway. Conversely, multiple pathways related to metabolism and degradation were differentially down-regulated (Fig. 6). As shown in Fig. 7, the expression levels of siderophore biosynthesis encoding gene entA, entB, entC and entE, and iron-siderophore transporters encoding gene bauB, bauE and bauCD, were significantly up-regulated. Concerning the oxidative phosphorylation pathway, the genes encoding NADH dehydrogenase and Succinate dehydrogenase were significantly down-regulated. Whereas, transcription of F-type ATPase was enhanced. In addition, the expressions of genes contributed to biofilm formation didn’t demonstrate consistent trends, suggesting a complex regulatory mechanism at play (Fig. 7).

Fig. 6
figure 6

Bubble chart of KEGG pathway enrichment of differentially expressed genes. The X-axis is the enrichment ratio, The Y-axis is KEGG Pathway, the size of the bubble represents the number of genes annotated to a KEGG Pathway, the color represents the enriched p value, and the darker the color, the smaller the p value. Only differentially transcribed genes with absolute fold changes > 1 (log2 scale) and Q value < 0.05 were shown

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Fig. 7
figure 7

Heatmap of differentially expressed genes involved in siderophore and transport, Oxidative phosphorylation and biofilm formation. The colors of the heatmap represent normalized gene expression levels (FPKM), ranging from blue (lower expression) to red (higher expression)

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In vivo efficacy of Ga(NO3)3 in neutropenic mouse thigh infection model

The in vivo efficacy of Ga(NO3)3 was evaluated using a neutropenic mouse thigh infection model and the results were depicted in Fig. 8. Notably, the bacterial load in the thigh of the treatment group was significantly lower than the control group (107.00 CFU/g vs. 105.39 CFU/g, p < 0.05), demonstrating the effectiveness of Ga(NO3)3 in reducing bacterial burden in the infected thighs of neutropenic mice.

Fig. 8
figure 8

Antibacterial effect of Ga(NO3)3 in the neutropenic mouse thigh infection model. * indicated p < 0.05

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Discussion

Bloodstream infections caused by multidrug-resistant A. baumannii (MDR-AB) have become a growing concern in hospitalized patients, particularly those in ICUs. However, the rise of MDR has severely restricted the availability of effective antimicrobials, posing a significant challenge in treating these infections [2]. It is worth noting that iron plays a crucial role in the growth and survival of A. baumannii, making it an attractive target for therapeutic intervention. Gallium, which is similar to iron, has the unique ability to disrupt proteins’ function when it forms complexes with them. Hence, targeting iron metabolism could offer a promising approach for antibacterial therapy against MDR-AB infections.

The antibacterial activity of Ga(NO3)3 exhibited a dependence on the iron content of the medium in vitro, being more effective in iron-limiting conditions and chemically defined media, but less effective in iron-rich complex media. In mammalian hosts, Fe3+ is predominantly sequestered in iron-transport proteins, stored in ferritin, or bound in heme molecules, resulting in very low concentrations (< 10− 18 M) of free Fe3+ [13]. Currently, there is lack of standard protocols or reference media for testing the sensitivity of Ga(NO3)3. MH broth is not suitable due to its high iron content. To address this, we followed the approach from the literature [14] and used an iron-deficient M9CA medium with constant chemical composition. Our data demonstrates that the MICs of Ga(NO3)3 against 40 A. baumannii strains ranged between 32 and 128 μg/mL in M9CA medium. Remarkably, the MICs of Ga(NO3)3 were similar between MDR and sensitive groups, indicating that the antibacterial activity of Ga(NO3)3 was not affected by the antimicrobials susceptibility patterns. Since there are few antimicrobials targeting iron metabolism in clinical practice, the mechanisms of action between Ga(NO3)3 and antimicrobials do not overlap. The growth curve demonstrated that Ga(NO3)3 inhibited A. baumannii growth in a dose- and time-dependent manner. Interestingly, despite concentration higher than the MIC, A. baumannii eventually recovered growth over time. However, the growth occurred at reduced maximum growth capacity as the concentration of Ga(NO3)3 increased. This observation points to a complex relationship between Ga(NO3)3 and bacterial growth, potentially involving adaptive responses to Ga(NO3)3 exposure.

In this study, the strains were isolated from bloodstream infections, prompting us to assess the antibacterial activity of Ga(NO3)3 in medium mimics human serum. Previous studies have proved that human serum created an iron-deficient environment for A. baumannii [15]. Therefore, it is crucial to investigate the practical significance of antibacterial effect of Ga(NO3)3 in human serum. To simulate the human blood environment and evaluate the MIC of Ga(NO3)3 at 18 h, we followed a method outlined in the literature [16]. Specifically, we used RPMI 1640 medium supplemented with 10% normal human serum. The results indicated a substantial reduction in the MIC of Ga(NO3)3 in the medium containing 10% serum compared to the pure RPMI 1640 medium, resulting in a range of 0.0625 to 0.125 μg/mL. Considering the peak plasma concentration of Ga(NO3)3 used to treat cancer-related hypercalcemia (300 mg/m2 intravenously), approximately 28 μM or 7.2 μg/ml [15]. This concentration was significantly higher than the MIC measured at 18 h. As Ga(NO3)3 exhibited dose-dependent inhibition of A. baumannii growth, we can infer that at the peak concentration, there will likely be a sustained antibacterial effect. Our findings support the notion that Ga(NO3)3 possesses potent antibacterial activity against A. baumannii, especially in an environment resembling human serum. The observed dose-dependent growth inhibition suggests that the peak plasma concentration of Ga(NO3)3 can potentially be effective in combating A. baumannii infections.

The antibacterial effect of Ga(NO3)3 was influenced by the environment, as supported by previous research [17]. We found that the presence of ferric chloride can competitively counteract the antibacterial activity of Ga(NO3)3. Similarly, heme, which is predominantly bound to hemoglobin in red blood cells and contains an iron ion in the center [18], can also weaken the antibacterial activity of Ga(NO3)3. However, the effect of hemin on Ga(NO3)3 varies among different strains, unlike the consistent effect observed with ferric chloride. A. baumannii had evolved a highly effective iron acquisition system to obtain iron from the iron-limited environment of the human host [19]. One such mechanism involves the release of siderophores, such as Acinetobactin, which acts as a high-affinity iron chelator to capture free or protein-bound iron, transporting it to the bacteria. Heme and heme-containing proteins constitute another significant source of iron in the body, accounting for approximately 70% of iron present. Antunes characterized two heme uptake clusters in A. baumannii, with the heme uptake cluster I being inherent, and heme uptake cluster II present in only two-thirds of the strains [20]. Upon capturing heme, the heme oxygenase of the bacteria degrades it, releasing free iron. Our data showed that the strains carrying only the heme uptake cluster I were less affected by exogenous heme. Only at high concentrations did exogenous hemin restore the growth of the strain. Conversely, strains carrying both heme uptake clusters were susceptible to the counteractive effects to low concentration of hemin on antibacterial effect of Ga(NO3)3. This finding aligned with a previous study suggesting that heme uptake cluster II played a major role in heme uptake [9]. Moreover, the heme uptake cluster II was crucial for optimal utilization of heme in A. baumannii hypervirulent strains [9]. The implications of our research are significant in clinical settings, as it helps identify situations where Ga(NO3)3 may not be suitable for use, such as in patients with hemolysis or conditions involving excessive release of heme. Understanding the interplay between Ga(NO3)3 and the iron acquisition system in A. baumannii contributes to improved treatment strategies and more targeted antimicrobial use in specific patient populations.

To gain deeper insights into the antibacterial mechanism of Ga(NO3)3, transcriptome sequencing was conducted on A. baumannii after exposure to gallium nitrate. The enrichment analysis of GO and KEGG pathways revealed a significant enrichment of ribosome-related genes. The ribosome plays a crucial role in protein synthesis and function as one of the largest molecular machines in the cell [21]. Recent studies on P. aeruginosa have shown that the gallium ion targets the two subunits of RNA polymerase, RpoB and RpoC, which are gallium-binding proteins. This targeting of RNA polymerase by gallium suppresses RNA synthesis, resulting in reduced metabolic rates and energy utilization [14]. We found that the expression of ribosome-related genes was up-regulated in response to gallium nitrate treatment, indicating a defect in the protein translation process. To counteract iron-limitation, bacteria have developed high-affinity iron-uptake strategies, such as production of siderophore to bind iron and transport iron into the cell, utilization the heme iron by heme-binding proteins, and active iron transport mechanisms [12]. Our transcriptomics analysis revealed that differentially up-regulated genes associated with biosynthesis and transport of siderophore, which may be stimulated by the iron-deficient environment caused by Ga(NO3)3 treatment. Ga3+ can compete with Fe3+  for binding to enzymes that essential for various metabolic process [22]. Consequently, multiple pathways involved in metabolism and degradation were differentially down-regulated, potentially explaining the antibacterial mechanism of Ga(NO3)3. It is worth noting that Ga(NO3)3 has been found to inhibit the biofilm formation [23]. However, in this study the transcription of biofilm associated genes were heterogeneous, suggesting that the antibacterial effect of Ga(NO3)3 on biofilm formation may vary depending on different metabolic processes and warrants further investigation.

Previous studies had demonstrated the beneficial impact of Ga(NO3)3 on the survival of Galleria mellonella larvae infected with A. baumannii [15]. Moreover, recent finding had illustrated the effectiveness of Ga(NO3)3 in the treating chronic P. aeruginosa airway infections both in a mouse infection model and in a phase I clinical trial involving individuals with cystic fibrosis, and exhibited a lower propensity for resistance development compared to antimicrobials [24]. In this study, the neutropenic mouse thigh infection model verifies the antibacterial efficacy of Ga(NO3)3 on treating A. baumannii infection in vivo and demonstrated its potential of clinical application.

Conclusion

This study demonstrated the remarkable antibacterial efficacy of Ga(NO3)3 against multidrug-resistant A. baumannii both in vivo and in vitro. Ga(NO3)3 achieved its antibacterial effect by influencing the genes related to ribosome and siderophore, thereby disrupting multiple metabolism pathways of A. baumannii. Notably, the antibacterial effect was affected by the presence ferric chloride and hemin. Taken together, these findings point to the potential of Ga(NO3)3 as a novel therapeutic approach for combating bloodstream infections caused by multidrug-resistant A. baumannii, with a focus on targeting iron metabolism.

Materials and methods

Bacterial strains and culture conditions

The 40 A. baumannii strains used in this study and their antimicrobials susceptibility data were listed in Table S1 of supplemental material. These strains were isolated from patients with A. baumannii bloodstream infection at the First Affiliated Hospital of Wenzhou Medical University in 2018. The isolates were identified by the matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS; bioMérieux, Lyons, France). 22 out of 40 strains were multidrug-resistance [25] and exhibited resistance to carbapenem.

Culture media used in this study were as follows: Mueller-Hinton (MH) broth, M9 minimal medium supplemented with 0.2% casein hydrolysate (hereby called M9CA) [14], and RPMI 1640 medium. To simulate human blood environment, when required, the media were supplemented with 10% human serum at appropriate concentrations. Human serum collected from 20 healthy donors was pooled, filtered, and inactivated (30 min, 56 °C) as described previously [26].

Ga(NO3)3 susceptibility testing

Antibacterial activity of Ga(NO3)3 was tested by microdilution method in four kinds of media, including MH broth, M9CA, RPMI 1640 medium and RPMI 1640 medium supplemented with 10% human serum (RPMI-HS). In brief, colonies were selected from a 24 h culture agar plate, suspended in 0.9% saline, and adjusted to a turbidity similar to that of a 0.5 McFarland standard. The saline suspension was diluted 1:200 in medium to obtain an inoculum density of approximately 5 × 105 CFU/mL. Ga(NO3)3 was 2-fold serially diluted in a 96-well microplate. The concentration range of Ga(NO3)3 was 0.03-32 μg/mL for RPMI-HS, while for the other three media, it was 0.5–512 μg/mL. Growth control without drug and negative control (media only) were included in all experiments. A volume of 100 μL of this adjusted suspension was added to each well of the microtiter plates. The microtiter plates was read after 18 h of incubation in an ambient-air incubator (except for RPMI 1640 medium, which was incubated in a 5% CO2 incubator). Experiments were conducted in three biological replicates.

Time-kill assay

The impact of Ga(NO3)3 on the growth of A. baumannii BM-2333, a carbapenem resistant strain, was assessed according to a previously described method with slight modifications [12]. M9CA medium was employed to generate growth curve. Initially, the overnight bacterial cultures were 100-fold diluted and added to the sterile tubes containing different concentrations of Ga(NO3)3 (4×MIC, 2×MIC, 1×MIC, and 1/2×MIC), as well as tubes with no Ga(NO3)3 added for the growth control. The tubes were incubated with shaking at 37 °C. Samples were periodically taken from the culture for analysis. The absorbance at OD600 was measured at 0, 2, 4, 6, 8,10, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66 and 72 h. Experiments were conducted in triplicate.

Exogenous ferric chloride/hemin addition assay

Carbapenem resistant strain BM-2333 and carbapenem susceptible strain BM-4795 were used in this experiment. The effect of exogenous ferric chloride/hemin on the antibacterial activity of Ga(NO3)3 was examined using the checkerboard assay. RPMI-HS was used for this test. Referring to the MICs of test isolates (0.0625 μg/mL), Ga(NO3)3 was 2-fold serially diluted along the x-axis (0–32 μg/mL), while ferric chloride/hemin was 2-fold serially diluted along the y-axis (0–16 μg/mL) to form a matrix, allowing each well to contain a combination of two drugs at varying concentrations. The single bacterial colony grown overnight was diluted to 0.5 McFarland turbidity in sterile saline, followed by 1:200 dilution in RPMI-HS. Subsequently, 100 μL of bacterial suspension was added to the corresponding well to achieve a final concentration of approximately 5 × 105 CFU /mL. The 96-well plate was incubated in a 5% carbon dioxide incubator for 24 h and the growth situation (OD600) was measured every 2 h. Relative growth of each well was defined as (OD600 measured-OD6000h). Experiments were conducted in three biological replicates.

Genomic DNA extraction and PCR

Genomic DNA from the 40 experimental strains was extracted using Bioflux bacterial DNA extraction kit following the instructions of the manufacturer. Polymerase chain reaction (PCR) was used to detect iron-uptake gene clusters (The primer sequences are listed in Table S3), including heme-uptake gene cluster I (exbD), heme-uptake gene cluster II (hemO) and acinetobactin synthesis gene cluster (basA). The positive PCR product was sent to Shanghai Majorbio Bio-Pharm Technology Co. (Shanghai, China) for sequencing.

Genomic and transcriptomics analysis

Whole-genome sequencing (WGS) of A. baumannii BM-2333 was performed using long-read platform PacBio RS by the Beijing Genomics Institute (Wuhan, China). The plasmid and chromosome sequences of A. baumannii BM-2333 were used as the reference sequences for the subsequent RNA-seq read alignment and statistical analyses. A. baumannii BM-2333 cultured in M9CA medium with and without Ga(NO3)3 were defined as the experimental group and the control group, respectively. The cells were harvested at the exponential phase of growth for total RNAs extraction, following the Trizol/chloroform protocol, and then subjected to transcriptome sequencing analysis. The experimental group and the control group were carried out as three biological replicates. The RNA-seq data of six samples were filtered using SOAP nuke (v1.5.2). The RNA-seq reads were aligned to a reference coding gene set using Bowtie2 (v2.2.5), and the resulting alignments were used to calculate the expression levels of genes using RSEM (v1.2.12). Differential expression analysis was performed using the DESeq2(v1.4.5) with Q value ≤ 0.05. To gain insight into the changes of phenotype, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of annotated differential expressed genes was performed by Phyper based on Hypergeometric test. The significant levels of terms and pathways were corrected by applying a rigorous Q value threshold (Q value ≤ 0.05) by Bonferroni.

Neutropenic murine model

The neutropenic mouse thigh infection model was established to conduct in vivo studies [27]. Specific-pathogen-free (SPF) female ICR mice aged 5 to 6 weeks old (Charles river, Hangzhou, China), were used in this experiment. The mice were maintained in accordance with National Standards for Laboratory Animals of China (GB 14925 − 2010). To induce a neutropenia, we intraperitoneally injected cyclophosphamide (Yuanye Biotechnology Co., Ltd, Shanghai, China) at -4 day (150 mg/kg) and − 1 day (100 mg/kg) prior to thigh infection. 100 μL suspension of exponentially growing bacterial at a concentration of 1.5 × 107 CFU/mL were injected into each posterior thigh muscle. In vivo treatment studies commenced after 2 h following bacterial inoculation. Ga(NO3)3 was administered intraperitoneally at 300 mg/m2/24 h, based on the recommended dose [28]. The mice were euthanized by CO2 inhalation after 24 h of therapy. Bacterial burden was quantified by CFU counts obtained from posterior thigh homogenates. Both the treatment group and the control group contained 6 mice (12 thigh infections).

Statistical analysis

Log10-transformed CFU counts and log2-transformed values of gene expression were used to calculate means and standard deviations for each dataset. Differences between means were assessed by the Student’s t test. A p-value of < 0.05 was considered statistically significant. All statistical calculations were conducted with SPSS v.22.0 software (SPSS Inc., Chicago, IL, USA).

Data availability

The genome data supporting the results of this article are deposited in the NCBI as BioProject PRJNA799658. The transcriptome data are available in the Gene Expression Omnibus (GEO) data repository under accession number GSE194255.

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Acknowledgements

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Funding

This work was supported by: (i) the Zhejiang Province Natural Science Foundation of China (no. LY20H200004); (ii) the Major Projects of Wenzhou Science and Technology Bureau (no. ZY2019011); (iii) Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province (no. 2022E10022).

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Authors and Affiliations

  1. Department of Clinical Laboratory, Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, Zhejiang Province, China

    Zhuocheng Yao, Kaihang Yu, Changrui Qian, Beibei Zhou, Yishuai Lin, Xiaodong Zhang, Tieli Zhou, Weiliang Zeng & Yao Sun

  2. Department of Pathology, the Second Affiliated Hospital, School of Medical, Zhejiang University, Hangzhou, Zhejiang Province, China

    Kaihang Yu

  3. School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, 325000, Zhejiang Province, China

    Ying Zhang & Jianming Cao

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  1. Zhuocheng Yao
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Contributions

ZCY, KHY and BBZ carried out experiments. CRQ and YS performed the bioinformatics analysis. YSL, XDZ, and YZ analyzed the results and WLZ directed the drawing. ZCY wrote the manuscript. TLZ, JMC and YS designed the study and revised the manuscript. All authors reviewed and approved the final version of the manuscript.

Corresponding authors

Correspondence to Jianming Cao or Yao Sun.

Ethics declarations

Ethics approval and consent to participate

This study was approved by Ethics Committee in clinical research of the First Affiliated Hospital of Wenzhou Medical University (No. KY2022-R117), which waived the requirement for informed consent given the observational nature of this study, mainly focused on bacteria and involved no interventions with patients. Patient information was anonymized and de-identified during the entire process of data recording. All experiments were performed in compliance with the relevant laws and institutional guidelines in accordance with the ethical standards of the Declaration of Helsinki. Animal studies were approved by the Ethics Committee for Experimental Animals of the First Affiliated Hospital of Wenzhou Medical University (No. SYXK (Zhejiang) 2021-0017) and conducted in accordance with Wenzhou Laboratory Animal Welfare and ARRIVE guidelines.

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Yao, Z., Yu, K., Qian, C. et al. Gallium nitrate inhibits multidrug-resistant Acinetobacter baumannii isolated from bloodstream infection by disrupting multiple iron-dependent metabolic processes. BMC Microbiol 25, 216 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03950-4

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Keywords

BMC Microbiology

ISSN: 1471-2180

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