Maintaining Iron Homeostasis Is the Key Role of Lysosomal Acidity for Cell Proliferation.

The lysosome is an acidic multi-functional organelle with roles in macromolecular digestion, nutrient sensing, and signaling. However, why cells require acidic lysosomes to proliferate and which nutrients become limiting under lysosomal dysfunction are unclear. To address this, we performed CRISPR-Cas9-based genetic screens and identified cholesterol biosynthesis and iron uptake as essential metabolic pathways when lysosomal pH is altered. While cholesterol synthesis is only necessary, iron is both necessary and sufficient for cell proliferation under lysosomal dysfunction. Remarkably, iron supplementation restores cell proliferation under both pharmacologic and genetic-mediated lysosomal dysfunction. The rescue was independent of metabolic or signaling changes classically associated with increased lysosomal pH, uncoupling lysosomal function from cell proliferation. Finally, our experiments revealed that lysosomal dysfunction dramatically alters mitochondrial metabolism and hypoxia inducible factor (HIF) signaling due to iron depletion. Altogether, these findings identify iron homeostasis as the key function of lysosomal acidity for cell proliferation.

Iron deficiency-related symptoms in non-anemic whole blood donors.

INTRODUCTION: Whole blood donors are at increased risk for iron deficiency (ID). ID anemia is associated with several symptoms, such as fatigue, cognitive dysfunction, pica, and restless leg syndrome (RLS). However, it is unclear if these symptoms also occur when a donor has developed ID without anemia. This study aims to determine whether non-anemic ID (NAID) is associated with the occurrence of ID-related symptoms. STUDY DESIGNS AND METHODS: We combined data from three studies in whole blood donors (i.e., Donor Insight-III, FIND'EM, and FORTE) to create a substantial sample size (N = 12,143). The self-reported occurrence and severity of ID-related symptoms, such as physical and mental health, fatigue, cognitive functioning, pica, and RLS, was measured using validated questionnaires. Associations were studied using logistic regression modeling with ID-related symptoms derived from the questionnaires as the dependent variable and ferritin level group (0-15 µg/L, 15-30 µg/L, and >30 µg/L) as explanatory variable. RESULTS: After applying inclusion and exclusion criteria, 9829 donors were eligible for analysis. In the models corrected for age, body mass index, Hb level, and cohort, only fatigue was shown to be associated with ferritin levels in men, showing lower odds (OR 1.41, 95% CI 1.11-1.79) for fatigue with higher ferritin levels. CONCLUSION: In these studies, NAID was only associated with self-reported fatigue in male donors. Although selection bias may have led to underestimated associations, ferritin measurements in donors should be primarily considered as a measure to prevent anemia, rather than to prevent or mitigate NAID-related symptoms.

Donor knowledge and perceptions regarding donation-induced iron depletion and iron supplementation as a blood service policy.

BACKGROUND AND OBJECTIVES: Regular whole blood donations are associated with an increased risk of iron deficiency. Iron supplementation is an effective strategy to prevent donation-induced iron deficiency. However, research on donor perceptions towards such a policy is limited. Therefore, we aim to evaluate donors' knowledge on donation-induced iron depletion and their perceptions regarding iron supplementation as a blood service policy. MATERIALS AND METHODS: Three thousand Dutch whole blood donors were invited to complete a survey assessing their knowledge of donation-induced iron depletion and attitudes and perceptions towards iron supplementation as a policy. Linear regression modelling was used to evaluate associations between explanatory variables and perceptions. RESULTS: In total, 1093 (77.1%) donors were included in the analysis. Donors had poor knowledge of current iron management policies, but a better understanding of iron metabolism and supplementation. Iron supplementation as a policy was perceived mainly positive by donors, and the majority were willing to use iron supplements if provided. Iron supplementation was not perceived as invasive or negatively affecting donors' motivation to continue donating. Additional iron monitoring, information and donor physician involvement were regarded as important conditions for implementation. Male sex, trust in the blood service, prior experience with iron supplements and openness towards dietary supplements were strongly positively associated with willingness to use iron supplementation. CONCLUSION: Donors' knowledge regarding donation-induced iron depletion is limited, but not associated with their perceptions regarding iron supplementation. Donors do not consider iron supplementation as invasive, deterring or demotivating, and a majority are willing to take supplements if offered.

Iron Stress Affects the Growth and Differentiation of Toxoplasma gondii.

Iron is an indispensable nutrient for the survival of Toxoplasma gondii; however, excessive amounts can lead to toxicity. The parasite must overcome the host's "nutritional immunity" barrier and compete with the host for iron. Since T. gondii can infect most nucleated cells, it encounters increased iron stress during parasitism. This study assessed the impact of iron stress, encompassing both iron depletion and iron accumulation, on the growth of T. gondii. Iron accumulation disrupted the redox balance of T. gondii while enhancing the parasite's ability to adhere in high-iron environments. Conversely, iron depletion promoted the differentiation of tachyzoites into bradyzoites. Proteomic analysis further revealed proteins affected by iron depletion and identified the involvement of phosphotyrosyl phosphatase activator proteins in bradyzoite formation.

Iron deficiency-induced loss of skeletal muscle mitochondrial proteins and respiratory capacity; the role of mitophagy and secretion of mitochondria-containing vesicles.

Iron homeostasis is essential for mitochondrial function, and iron deficiency has been associated with skeletal muscle weakness and decreased exercise capacity in patients with different chronic disorders. We hypothesized that iron deficiency-induced loss of skeletal muscle mitochondria is caused by increased mitochondrial clearance. To study this, C2C12 myotubes were subjected to the iron chelator deferiprone. Mitochondrial parameters and key constituents of mitophagy pathways were studied in presence or absence of pharmacological autophagy inhibition or knockdown of mitophagy-related proteins. Furthermore, it was explored if mitochondria were present in extracellular vesicles (EV). Iron chelation resulted in an increase in BCL2/Adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) and BNIP3-like gene and protein levels, and the appearance of mitochondria encapsulated by lysosome-like vesicular structures in myotubes. Moreover, mitochondria were secreted via EV. These changes were associated with cellular mitochondrial impairments. These impairments were unaltered by autophagy inhibition, knockdown of mitophagy-related proteins BNIP3 and BNIP3L, or knockdown of their upstream regulator hypoxia-inducible factor 1 alpha. In conclusion, mitophagy is not essential for development of iron deficiency-induced reductions in mitochondrial proteins or respiratory capacity. The secretion of mitochondria-containing EV could present an additional pathway via which mitochondria can be cleared from iron chelation-exposed myotubes.

Iron deficiency as therapeutic target in heart failure: a translational approach.

Heart failure (HF) is a potentially debilitating condition, with a prognosis comparable to many forms of cancer. It is often complicated by anemia and iron deficiency (ID), which have been shown to even further harm patients' functional status and hospitalization risk. Iron is a cellular micronutrient that is essential for oxygen uptake and transportation, as well as mitochondrial energy production. Iron is crucially involved in electrochemical stability, maintenance of structure, and contractility of cardiomyocytes. There is mounting evidence that ID indeed hampers the homeostasis of these properties. Animal model and stem cell research has verified these findings on the cellular level, while clinical trials that treat ID in HF patients have shown promising results in improving real patient outcomes, as electromechanically compromised cardiomyocytes translate to HF exacerbations and arrhythmias in patients. In this article, we review our current knowledge on the role of iron in cardiac muscle cells, the contribution of ID to anemia and HF pathophysiology and the capacity of IV iron therapy to ameliorate the patients' arrhythmogenic profile, quality of life, and prognosis.

Validity of plasma collection cards for ferritin assessment-A proof-of-concept study.

OBJECTIVES: Iron depletion is common around the world and among certain risk groups in developed countries. The overall purpose was to test the suitability of a novel plasma collection card for minimally invasive iron status assessment. METHODS: Twenty participants (10 f/10 m) participated in this cross-sectional study. Ferritin and hemoglobin were measured from blood collected from a forearm vein, serving as reference method. Blood was also collected from the fingertip using the NoviplexTM Plasma Prep Card as well as capillary collection tubes. RESULTS: There was substantial concordance between ferritin measured from samples collected via NoviplexTM and venous ferritin (concordance correlation coefficient (CCC) = 0.96) with a mean bias of -0.8 ng/mL. Storing NoviplexTM cards at room temperature for 2 weeks resulted in slightly lower but good concordance when compared to venous ferritin (CCC = 0.95). Capillary hemoglobin (CCC = 0.42) and hematocrit (CCC = 0.25) were in poor agreement with venous data. CONCLUSIONS: NoviplexTM cards offer a suitable alternative for a minimally invasive ferritin screening in the field when compared to capillary collection tubes. Despite overall substantial concordance with the reference method, findings indicative of iron status abnormalities should be confirmed in venous samples.

New polyazamacrocyclic 3-hydroxy-4-pyridinone based ligands for iron depletion antitumor activity.

Iron depletion is an efficient strategy for the development of anticancer agents. In an effort to develop efficient chelators, two new 3-hydroxy-4-pyridinone based polyazamacrocycles 1e and 2e were designed and synthesized. A preliminary study of the ligands was carried out to investigate their iron chelating capability and anti-tumor activity. Chelating kinetics revealed that the ligands exhibited excellent iron depletion capacity in neutral and acidic NH4OAc buffer solutions. Moreover, MTT assay showed that the new ligands displayed potent inhibitory activity in the proliferation of HepG2 cells. The attachment of hydroxypyridione units on the polyazamacrocycles promoted iron chelating capability and improved the anti-tumor activity by offering additional chelating sites and lipophilicity. These results indicate that two novel compounds may possess the therapeutic potential in the treatment of cancer through depleting cellular iron.

Serum iron deficiency was associated with lower cognitive development in the children of the Santal tribe of West Bengal.

AIM: Poor cognitive scores and low serum iron have been reported among chronically undernourished children from the Santal tribe, West Bengal. Our aim was to investigate the association between iron status and non-verbal cognitive development. METHODS: We randomly selected 170 children (52.9% boys) aged 5-12 years from the Purulia district of West Bengal during 2007-2008 and assessed their iron status: haemoglobin concentration, serum concentration of iron, ferritin, transferrin, total iron-binding capacity and transferrin saturation. Their non-verbal cognitive development was assessed using the Raven's Coloured Progressive Matrices. RESULTS: The haemoglobin concentration, serum iron, serum ferritin and transferrin saturation levels of the 27 children with an intellectual deficit and the 32 who had a below average intelligence quotient (IQ) were significantly lower (P < .05) than the 65 children with an average IQ. A large number of boys (55.6%) and girls (41.7%) who have an intellectual deficit had stage III iron depletion. The cognitive scores of children with stage II and III iron depletion were significantly lower (P < .05) than those with a normal IQ. CONCLUSION: The iron depletion stage was associated with the severity of non-verbal cognitive impairment and serum ferritin appeared to be a sensitive biomarker for predicting non-verbal cognitive development.

Biocontrol ability and action mechanism of Metschnikowia citriensis against Geotrichum citri-aurantii causing sour rot of postharvest citrus fruit.

This study investigated the biocontrol efficiency of Metschnikowia citriensis strain FL01 against Geotrichum citri-aurantii, and evaluated possible mechanisms. The results showed that M. citriensis could effectively control the development of sour rot, and significantly inhibit the mycelial growth and spore germination of G. citri-aurantii. The population dynamics results and Scanning electron microscopy (SEM) analysis indicated that M. citriensis could rapidly colonize wounds and tightly adhere to the surface of the wounds to compete with G. citri-aurantii for nutrition and space. M. citriensis also showed the biofilm formation action in vitro. The response of G. citri-aurantii to different components of M. citriensis culture showed that only the yeast cells but not the extracellular metabolites and the volatile organic compounds (VOCs) exhibited inhibitory effect on the growth of G. citri-aurantii. M. citriensis adhered to the hyphae of G. citri-aurantii loosely and sparsely, and the production of lytic enzymes ß-1, 3-glucanase (GLU) and Chitinase (CHI) could not be induced by G. citri-auranti. Iron affected the pulcherrimin pigment production and antagonism of M. citriensis indicating iron depletion as the most important antagonistic mechanism. Besides, M. citriensis also induced resistance of fruit against sour rot. These results suggested that M. citriensis could be used as the potential alternative of fungicides to control postharvest pathogens on citrus fruit.

Regulation of the Synthesis and Secretion of the Iron Chelator Cyclodipeptide Pulcherriminic Acid in Bacillus licheniformis.

The cyclodipeptide pulcherriminic acid synthesized by Bacillus licheniformis is an iron chelator that antagonizes certain pathogens by removing iron from the environment. But since the insoluble iron-pulcherriminic acid complex cannot act as an iron carrier as siderophores do, excessive synthesized pulcherriminic acid causes iron starvation for the producer cells. At present, the regulation of pulcherriminic acid synthesis and the mechanism by which B. licheniformis strikes a balance between biocontrol and self-protection from excessive iron removal remain unclear. This study provides insights into the regulatory network and explains the mechanism of pulcherriminic acid biosynthesis. The yvmC-cypX synthetic gene cluster was directly negatively regulated by three regulators: AbrB, YvnA, and YvmB. Within the regulatory network, YvnA expression was repressed not only by AbrB but also by iron-limiting environments, while YvmB expression was repressed by YvnA. The transporter gene yvmA is repressed by YvmB and is required for pulcherriminic acid secretion. The biosynthesis window is determined by the combined concentration of the three regulators in an iron-rich environment. Under iron-limiting conditions, cells close the pulcherriminic acid synthesis pathway by downregulating YvnA expression.IMPORTANCE The cyclodipeptides are widespread in nature and exhibit a broad variety of biological and pharmacological activities. The cyclodipeptide scaffold is synthesized by nonribosomal peptide synthetases (NRPSs) and cyclodipeptide synthases (CDPSs). At present, it is clear that CDPSs use aminoacyl tRNAs as substrates to synthesize the two peptide bonds, and the pulcherriminic acid synthase YvmC is a member of the eight identified CDPSs. However, little is known about the regulation of cyclodipeptide synthesis and secretion. In this study, we show that AbrB, which is considered to be the main regulator of NRPS-dependent pathways, is also involved in the regulation of CDPS genes. However, AbrB is not the decisive factor for pulcherriminic acid synthesis, as the expression of YvnA determines the fate of pulcherriminic acid synthesis. With this information on how CDPS gene transcription is regulated, a clearer understanding of cyclodipeptide synthesis can be developed for B. licheniformis Similar approaches may be used to augment our knowledge on CDPSs in other bacteria.

Systematic review and meta-analysis to determine the impact of iron depletion in dysmetabolic iron overload syndrome and non-alcoholic fatty liver disease.

AIMS: Iron reduction has been proposed as treatment for dysmetabolic iron overload syndrome (DIOS) and non-alcoholic fatty liver disease (NAFLD), but results of published trials are conflicting. We undertook a systematic review and meta-analysis to determine the impact of phlebotomy in DIOS and NAFLD. METHODS: We searched multiple databases systematically for studies evaluating the impact of phlebotomy in DIOS and NAFLD. We calculated weighted summary estimates using the inverse variance method. Study quality was assessed using the Cochrane collaboration tool. RESULTS: We identified nine studies with 820 patients (427 had phlebotomy, 393 lifestyle changes alone). Iron depletion did not improve the Homeostasis Model Assessment (HOMA) index (mean difference [MD] -0.6; confidence interval (CI), -1.7, 0.5; P = 0.3), insulin level (MD -0.8 mU/L; CI, -5.3, 3.7; P = 0.73), or aspartate aminotransferase (AST) (MD -0.7 IU/L; CI, -3.2, 1.8; P = 0.6) in DIOS and/or NAFLD patients as compared to lifestyle changes alone (five studies, 626 patients). There was mild improvement in alanine aminotransferase (ALT) (MD -6.6 IU/L; CI, -11, -2.1); P < 0.01), but the effect size was very small (Cohen's d, 0.15; r statistic, 0.07). Even in the subgroup of patients with NAFLD and hyperferritinemia, phlebotomy did not improve the HOMA index, insulin level, ALT, or AST. Additionally, no study showed significant improvement in liver inflammation or fibrosis with iron reduction. CONCLUSIONS: Phlebotomy does not bring about significant improvement in indices of insulin resistance, liver enzymes, or liver histology in patients with DIOS and/or NAFLD compared to lifestyle changes alone. Current evidence does not support the use of phlebotomy in patients with DIOS or NAFLD.

KCa(H2 O)2 [FeIII (CN)6 ]⋠H2 O Nanoparticles as an Antimicrobial Agent against Staphylococcus aureus.

Biocompatible nanoparticles based on a calcium analogue of Prussian blue were designed and synthesized to take advantage of their ability to penetrate the cell membrane in Staphylococcus aureus and to undergo selective ion exchange with intracellular iron to disrupt iron metabolism in such pathogenic bacteria for antibacterial applications. KCa(H2 O)2 [FeIII (CN)6 ]⋅H2 O nanoparticles penetrate the bacterial cell membrane and sequester intracellular iron by ion exchange to form insoluble Prussian blue, thus inhibiting bacterial growth.

Iron depletion induces hepatic secretion of biliary lipids and glutathione in rats.

Iron depletion (ID) has been shown to induce the liver expression of Cyp7a1, the rate-limiting enzyme initiating conversion of cholesterol to bile acids (BA), although the effect on bile acids metabolism and bile production is unknown. Therefore, we investigated changes in bile secretion and BA synthesis during diet-induced iron depletion (ID) in rats. ID increased bile flow along with augmented biliary excretion of bile acids, glutathione, cholesterol and phospholipids. Accordingly, we found transcriptional upregulation of the Cyp7a1, Cyp8b1, and Cyp27a1 BA synthetic enzymes, as well as induction of the Abcg5/8 cholesterol transporters in ID rat livers. In contrast, intravenous infusion of 3H-taurocholate failed to elicit any difference in biliary secretion of this compound in the ID rats. This corresponded with unchanged expression of canalicular rate-limiting transporters for BA as well as glutathione. We also observed that ID substantially changed the spectrum of BA in bile and decreased plasma concentrations of BA and cholesterol. Experiments with differentiated human hepatic HepaRG cells confirmed human CYP7A1 orthologue upregulation resulting from reduced iron concentrations. Results employing a luciferase reporter gene assay suggest that the transcriptional activation of the CYP7A1 promoter under ID conditions works independent of farnesoid X (FXR), pregnane X (PXR) and liver X (LXRα) receptors activation. It can be concluded that this study characterizes the molecular mechanisms of modified bile production as well as cholesterol as along with BA homeostasis during ID. We propose complex upregulation of BA synthesis, and biliary cholesterol secretion as the key factors affected by ID.

Alterations in sympathetic nerve traffic in genetic haemochromatosis before and after iron depletion therapy: a microneurographic study.

AIMS: Haemochromatosis (HH) displays a number of circulatory alterations concurring at increase cardiovascular risk. Whether these include sympathetic abnormalities in unknown. METHODS AND RESULTS: In 18 males with primary HH (age: 42.3 ± 10.4 years, mean ± SD), clinic and beat-to-beat blood pressure (BP, Finapres), heart rate (HR, EKG), and muscle sympathetic nerve activity (MSNA, microneurography) traffic were measured in the iron overload state and after iron depletion therapy. Haemochromatosis patients displayed elevated serum iron indices while other haemodynamic and metabolic variables were superimposable to ones seen in 12 healthy subjects (C). Muscle sympathetic nerve activity was significantly greater in HH than C (64.8 ± 13.3 vs. 37.8 ± 6.7 bs/100 hb, P < 0.01). Iron depletion caused a significant reduction in serum ferritin, transferrin saturation, and MSNA (from 64.8 ± 13.3 to 39.2 ± 9.2 bs/100 hb, P < 0.01) and a significant improvement in baroreflex-MSNA modulation. This was paralleled by a significant increase in the high-frequency HR variability and by a significant reduction in the low-frequency systolic BP variability components. Before after iron depletion therapy, MSNA was significantly and directly related to transferrin saturation, liver iron concentration, and iron removed, while the MSNA reductions observed after the procedure were significantly and inversely related to the baroreflex-MSNA increases detected after iron depletion. In C, all variables remained unchanged following 1 month observation. CONCLUSION: These data provide the first evidence that in HH iron overload is associated with an hyperadrenergic state and a baroreflex alteration, which are reversed by iron depletion. These findings underline the importance of iron overload in modulating sympathetic activation, possibly participating at the elevated cardiovascular risk reported in HH.

Iron depletion has different consequences on the growth and survival of Toxoplasma gondii strains.

Toxoplasma gondii is an obligate intracellular parasite responsible for a pathology called toxoplasmosis, which primarily affects immunocompromised individuals and developing foetuses. The parasite can scavenge essential nutrients from its host to support its growth and survival. Among them, iron is one of the most important elements needed to sustain basic cellular functions as it is involved in a number of key metabolic processes, including oxygen transport, redox balance, and electron transport. We evaluated the effects of an iron chelator on the development of several parasite strains and found that they differed in their ability to tolerate iron depletion. The growth of parasites usually associated with a model of acute toxoplasmosis was strongly affected by iron depletion, whereas cystogenic strains were less sensitive as they were able to convert into persisting developmental forms that are associated with the chronic form of the disease. Ultrastructural and biochemical characterization of the impact of iron depletion on parasites also highlighted striking changes in both their metabolism and that of the host, with a marked accumulation of lipid droplets and perturbation of lipid homoeostasis. Overall, our study demonstrates that although acute iron depletion has an important effect on the growth of T. gondii, it has a more profound impact on actively dividing parasites, whereas less metabolically active parasite forms may be able to avoid some of the most detrimental consequences.

Maternal hemoglobin and iron status in early pregnancy and childhood cardiac outcomes.

BACKGROUND & AIMS: Dysregulation of iron homeostasis is associated with cardiac alterations in a sex-dependent manner in adults. It is unknown whether iron status during pregnancy has long-term impact on cardiovascular health, and if this association is influenced by sex. Therefore, this study aimed to evaluate sex-specific association between maternal iron status during early pregnancy and cardiac outcomes in children aged 10 years. METHODS: In a population-based cohort study among 1972 mother-child pairs, hemoglobin and ferritin were measured in early pregnancy (<18 weeks) and categorized into anemia (hemoglobin<11 g/dL), elevated hemoglobin (hemoglobin≥13.2 g/dL), iron deficiency (ferritin<15 µg/L), and iron overload (ferritin>150 µg/L). At 10 years of age, cardiac MRI was performed to measure right and left cardiac outcomes of function (ventricular end-diastolic volume (RVEDV and LVEDV) and ejection fraction (RVEF and LVEF)), and structure (left ventricular mass (LVM), and left ventricular mass-to-volume ratio (LMVR)). Results are presented for boys and girls separately and models were adjusted for confounders and multiple testing. RESULTS: In boys, one standard deviation score (SDS) increase in maternal hemoglobin was associated with lower RVEDV and LVEDV (difference (95%CI) -0.10 (-0.17, -0.03) SDS and -0.09 (-0.16, -0.03) SDS, respectively). In boys, maternal anemia, as compared to normal hemoglobin levels, was associated with higher LVEDV (difference 0.34 (0.10, 0.59) SDS). No associations were observed for other cardiac outcomes and for ferritin in boys. No associations were observed in girls. CONCLUSION: In boys, dysregulated iron status during early pregnancy might permanently alter cardiovascular RVEDV and LVEDV function. Underlying mechanisms need further study.

Iron status and developmental scores in 6-year-olds highlights ongoing need to tackle iron deficiency in infants.

AIM: To investigate iron status and developmental scores at 6 years of age in a population with decreased prevalence of iron deficiency in infancy. Iron status at 6 years and tracking from 12 months were also studied. METHODS: Children (n = 143) born in Iceland in 2005 were followed up at the age of six. Motor and verbal development was assessed by a parental questionnaire, and iron status was assessed by Hb, MCV and serum ferritin (SF). Iron depletion was defined as SF <15 µg/L and deficiency as MCV <76 fL and SF <15 µg/L. RESULTS: Iron depletion was observed in 5.6% of 6-year-olds, and 1.4% were iron deficient. Self-help (subset in motor development) differed by -4.14 (95% CI = -7.61, -0.67), between those iron depleted at 12 months (n = 6) and those nondepleted (n = 102), adjusted for maternal education. The combined motor developmental score seemed lower in iron depleted infants, although of borderline significance (p = 0.066). MCV concentration tracked from 12 months to 6 years (r = 0.31, p < 0.002), but Hb and SF did not. CONCLUSION: Improved iron status at 12 months and 6 years has diminished the public health threat associated with iron depletion in the population studied, but iron depletion and development still associate weakly. Action to prevent iron depletion in infancy remains important.

Comparison of Disk Diffusion, E-Test, and Broth Microdilution Methods for Testing In Vitro Activity of Cefiderocol in Acinetobacter baumannii.

The reference method for cefiderocol antimicrobial susceptibility testing is broth microdilution (BMD) with iron-depleted-Mueller-Hinton (ID-MH) medium, whereas breakpoints recommended for disk diffusion (DD) are based on MH-agar plates. We aimed to compare the performance of the commercial BMD tests ComASP (Liofilchem) and UMIC (Bruker), and DD and E-test using MH- and ID-MH-agar plates with the reference BMD method using 100 carbapenem-resistant-A. baumannii isolates. Standard BMD was performed according to the EUCAST guidelines; DD and E-test were carried out using two commercial MH-agar plates (BioMérieux and Liofilchem) and an in-house ID-MH-agar plate, while ComASP and UMIC were performed according to the manufacturer's guidelines. DD performed with the ID-MH-agar plates led to a higher categorical agreement (CA, 95.1%) with standard BMD and fewer categorization errors compared to the commercial MH-agar plates (CA BioMérieux 91.1%, Liofilchem 89.2%). E-test on ID-MH-agar plates exhibited a significantly higher essential agreement (EA, 75%) with standard BMD compared to the two MH-agar plates (EA BioMérieux 57%, Liofilchem 44%), and showed a higher performance in detecting high-level resistance than ComASP and UMIC (mean log2 difference with standard BMD for resistant isolates of 0.5, 2.83, and 2.08, respectively). In conclusion, DD and E-test on ID-MH-agar plates exhibit a higher diagnostic performance than on MH-agar plates and the commercial BMD methods. Therefore, we recommend using ID-MH-agar plates for cefiderocol susceptibility testing of A. baumannii.