Potential additional effects of iron chelators on antimicrobial- impregnated central venous catheters

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Introduction
Central venous catheters (CVCs) play a clinically important role in various treatments, including venous pressure monitoring, the infusion of drugs, such as anti-cancer chemotherapy and antibiotics, parenteral nutrition, and blood transfusion (Saugel et al., 2017). However, compared with peripheral venous catheters, patients with indwelling CVCs are at increased risk of thrombosis, embolism, and infection (Ball and Singh, 2023). Catheter-related infections (CRIs) are thought to be associated with the formation of bacterial and fungal colonies on the catheter (Hanna et al., 2004). Microorganisms adhere to device surfaces, produce an extracellular polymeric matrix, and form biofilms. The biofilm-forming microorganisms then become a source of medical device-associated infection (Donlan, 2002). CRI is defined as a positive catheter tip culture and systemic inflammatory findings, while catheter-related bloodstream infection (CRBSI) is defined as CRI-criteria plus detection of the same organisms as in the catheter tip culture in peripheral blood cultures (Garner et al., 1988;Rockholt et al., 2023). CRBSIs have a reported mortality rate of 12%−40% depending on a variety of factors, such as comorbidities, the type of CVC used, and the type of pathogen, and are associated with longer hospital stays and increased medical costs (O'Grady et al., 2011;Böll et al., 2021;Ball and Singh, 2023).
Several preventive measures exist for CRIs, such as thorough hand hygiene, sterilization of the CVC site, and avoidance of catheter insertion at the femoral site. It is widely accepted that CVCs, which are foreign substances that can be a source of infection, should be removed as soon as possible. However, in clinical practice, situations arise in which long-term indwelling is required. Therefore, in such special situations, the Centers for Disease Control and Prevention (CDC) recommends the use of antimicrobial agents or antiseptic-impregnated catheters with comprehensive and adequate infection control measures, as shown Supplementary Figure S1 (O'Grady et al., 2011). Antimicrobial-impregnated catheters are a promising option for cases that require long-term CVC use (Böll et al., 2021). The purpose of this article was to .
summarize previous attempts to use antimicrobial/antiseptic impregnated catheters and provide a new perspective.

Antimicrobial/antiseptic-impregnated catheters
The control of CRI remains a major challenge to overcome; hence, a multifaceted attempt should be conducted. Certainly, antimicrobial-impregnated catheters have achieved some success to date, but no established clinical efficacy has been achieved with antiseptic substances (e.g., silver), which have been studied for many years (Viola et al., 2017). One such catheter is the chlorhexidine/silver sulfadiazine catheter, but most studies have found that its effect on reducing CRBSIs is not significant (Viola et al., 2017). Minocycline/rifampicin and miconazole/rifampicin catheters have also been shown to reduce the incidence of CRBSIs in several studies, where the incidence of antibiotic resistance, which remains a concern, was low (Viola et al., 2017;Reitzel et al., 2020;Böll et al., 2021). In clinical trials of patients with cancer, minocycline/rifampicin-impregnated catheters have also been shown to reduce the incidence of CRBSIs when used for longer than 2 months (Hanna et al., 2004). The combination of chlorhexidine with minocycline and rifampicin on catheters has been reported to have enhanced antimicrobial activity (Raad et al., 2012). Chlorhexidine acts on cell membranes, minocycline on protein synthesis, and rifampin on RNA synthesis. Thus, the mechanism of action of the combination of these three drugs may be additive. However, the confirmed enhancement of activity is only the result of in vitro studies, and the possibility of clinical application needs to be confirmed in clinical trials (Viola et al., 2017). As discussed above, a combined approach with different mechanisms of action may enhance antimicrobial activity and broaden the spectrum. Therefore, new approaches that are not antimicrobial agents or antiseptic substances may produce additional benefits.

Hypothesis
From a different perspective, attempts have been made to inhibit iron, which is essential for the growth and proliferation of pathogenic microorganisms, as a strategy to increase antimicrobial activity (Schwarz et al., 2019;Scott et al., 2020). Specifically, the strategy is to use iron chelators in combination with antibacterial and antifungal agents. We hypothesize that the impregnation of catheters with antimicrobial agents and iron chelators would increase their antimicrobial and anti-biofilm activity and inhibit microbial colony and biofilm formation, resulting in reduced CRI incidence and longer catheter use, as shown in Supplementary Figure S2. The surface and lumen of the catheter are impregnated with antimicrobial and ironchelating agents, which are assumed to inhibit bacterial and fungal colony and biofilm formation by exerting antimicrobial and iron-chelating activities on the bacteria and fungi that attach to the surface.

Support for the hypothesis Antimicrobial activity of iron chelators
Because of its redox potential, iron is involved in many biochemical reactions. Iron is an essential element for all living organisms, including bacterial pathogens (Dev and Babitt, 2017). It also plays an important role as a cofactor in various intracellular pathways, such as DNA synthesis and repair, electron transfer, oxygen transport, and immune response (Scott et al., 2020). Iron levels in the human body are strictly regulated to protect against the toxic effects of free iron, which catalyzes the generation of reactive oxygen species (ROS), and the utilization of iron by invading microorganisms (Dixon and Stockwell, 2014). Through nutritional immunity, humans inhibit microbial growth by starving microorganisms of the metal ions they need (Hood and Skaar, 2012;Palmer and Skaar, 2016). Bacteria and fungi bind iron using high-affinity chelating compounds, called siderophores, as a strategy to acquire iron. Numerous types of siderophores have been reported. Siderophores are categorized into three main structural families-carboxylates, catecholates, and hydroxamates-with the catecholate siderophore, enterobactin, showing the highest affinity for iron, even higher than that for the host iron-binding protein, transferrin (Ellermann and Arthur, 2017). Iron chelators may be useful in the treatment of infectious diseases by limiting the availability of iron, thereby inhibiting the production of ROS, and by inhibiting microbial growth due to nutrient restriction (Lehmann et al., 2021;Paterson et al., 2022).
To date, the combinations of vancomycin and deferasirox against methicillin-resistant Staphylococcus aureus (Luo et al., 2014), doxycycline, and the iron chelator CP762 against Pseudomonas aeruginosa (Faure et al., 2021), and the triple combination of doxycycline, deferasirox, and thiostrepton (an antibiotic against gram-positive bacteria) against P. aeruginosa and Acinetobacter baumannii (Chan et al., 2020) have shown promising effects both in vitro and in animal models. For fungi, animal model experiments of combination therapy with liposomal amphotericin B (L-AMB), micafungin, and deferasirox against Aspergillus and Mucor (Ibrahim et al., 2011), animal experiments of combination therapy with L-AMB and deferasirox against Aspergillus fumigatus (Ibrahim et al., 2010), and in vitro experiments of combination therapy with AMB and deferoxamine against Cryptococcus spp. (Chayakulkeeree et al., 2020) have indicated that these combinations are useful. Despite promising experimental data for the combination of deferasirox and L-AMB for mucormycosis (Ibrahim et al., 2007(Ibrahim et al., , 2011Schwarz et al., 2019), unexpectedly, a higher mortality rate at 90 days was seen with the combination than with L-AMB alone in a randomized controlled trial (Spellberg et al., 2012). Patients were enrolled at multiple centers with heterogeneous patient populations, resulting in an imbalance in underlying diseases and risk factors, with more patients in the deferasirox group having hematologic malignancies, neutropenia, and pulmonary infections. This imbalance in the patient's background may have influenced the patient's prognosis (Spellberg et al., 2012). Therefore, clinical trials are inconclusive regarding the efficacy of iron chelation assisted therapy.
Nevertheless, iron-chelation therapy still has potential for clinical applications. Different types of iron-chelating agents .
/fmicb. . have been shown to have different antimicrobial activities. Of those tested, a water-soluble agent containing iron-selective copolymers (denying iron to bacterial infections; DIBI) was the most effective (Iron Binding Polymers | Fe Pharma, 2021; Lehmann et al., 2021). Recent studies have reported the promising finding that a combination of DIBI and antimicrobials may overcome drug-resistant bacteria and fungi. DIBI in combination with ciprofloxacin has been shown to be successful in treating ciprofloxacin-resistant A. baumannii-infected mice (Parquet et al., 2019). Moreover, the combination of DIBI and fluconazole was found to inhibit the growth of fluconazole-resistant Candida albicans in an in vitro study (Savage et al., 2018). These findings suggest that the appropriate selection of a type of iron chelator and its combination with an antimicrobial agent may be useful for dealing with a broad spectrum of infectious pathogens, and may overcome antibiotic resistance mechanisms.

Anti-biofilm activity of iron chelators
One notable problem related to CVC implantation is biofilm formation. Here, we further explain the relationship between iron chelators and biofilm formation. It has already been mentioned that iron is essential for bacterial and fungal growth and proliferation, but it is also required for biofilm formation (Nazik et al., 2015;Coraça-Huber et al., 2018;Firoz et al., 2021). Microorganisms produce biofilms that protect them against antimicrobials, making them drug resistant (Høiby et al., 2010;Singh et al., 2010;Brauner et al., 2016;Thi et al., 2020). Hence, methods are needed to inhibit the formation of biofilms and attack the microorganisms that have formed within them. Iron-chelation therapy has been tested in P. aeruginosa to explore adjunctive therapy for chronic P. aeruginosa infection. Deferiprone, a synthetic iron chelator, has been shown to inhibit biofilm formation (Houshmandyar et al., 2021). Furthermore, the combination of iron chelators and antimicrobial agents has shown enhanced biofilm inhibitory activity; examples include combination therapy with the iron (VI) chelator N, N'bis (2-hydroxybenzyl) ethylenediamine-N, N'-diacetic acid and colistin in an in vitro experiment (Mettrick et al., 2020), and with the iron chelators deferoxamine or deferasirox and tobramycin (Moreau-Marquis et al., 2009). Lactoferrin, a component of human secretions, also has iron-chelating activity (Singh, 2004), and ALX-109, an investigational agent containing lactoferrin and hypothiocyanite (a bactericidal agent), has shown activity against P. aeruginosa biofilms in combination with tobramycin and aztreonam (Moreau-Marquis et al., 2015). Deferasirox has shown biofilm inhibitory effects on the periodontal bacterium Prevotella intermedia (Moon et al., 2013), and lactoferrin treatment has significantly reduced proinflammatory cytokines production by gingival fibroblasts infected with P. intermedia. Moreover, an observational clinical trial of patients with periodontitis showed that treatment with lactoferrin reduced proinflammatory cytokines such as interleukin 6, edema, bleeding, pocket depth, and gingival and plaque indices in the crevicular fluid and improved .
/fmicb. . Fourteen compounds and one iron analog reported to have iron chelating activity were selected and tested for synergism with thiostrepton. The triple combination of thiostrepton, deferasirox, and doxycycline was the most effective against P. aeruginosa and A. baumannii isolates.

Ibrahim et al. (2010)
In vitro and in vivo study

Deferasirox L-AMB
A. fumigatus Deferasirox demonstrated in vitro fungicidal activity against A. fumigatus and prolonged survival in mice with invasive pulmonary aspergillosis. Deferasirox plus L-AMB synergistically improved survival and reduced pulmonary fungal burden compared to either agent alone.

R. oryzae; A. fumigatus
Triple therapy with L-AMB, micafungin, and deferasirox improved survival and reduced tissue fungal burden in mice with mucormycosis, but to a lesser extent in aspergillosis.

Parquet et al. (2019)
In vitro and in vivo study

DIBI Ciprofloxacin
A. baumannii DIBI inhibited clinical A. baumanii isolates at MICs below those of typical antibiotics; low-dose nasal administration of DIBI after intranasal challenge with ciprofloxacin-resistant A. baumanii LAC-4 significantly reduced the bacterial burden in mice and DIBI also inhibited the spread of infection to the spleen. Given the ciprofloxacin resistance of LAC-4, treatment of infected mice with ciprofloxacin alone was ineffective, but treatment with DIBI greatly enhanced the therapeutic effect. Savage et al. (2018) In vitro and in vivo study DIBI Fluconazole C. albicans DIBI inhibited the growth of C. albicans in vitro at relatively low concentrations, and this inhibition was reversed by the addition of iron; the combination of DIBI and various azoles showed stronger growth inhibition than azoles alone, as shown in fluconazole-resistant C. albicans. In an experimental model of C. albicans vaginitis, DIBI in combination with fluconazole significantly improved clearance of infection in mice inoculated with fluconazole-sensitive strains.

(Continued)
Frontiers in Microbiology frontiersin.org . /fmicb. .  Houshmandyar et al. (2021) In vitro study Deferiprone P. aeruginosa Deferiprone showed moderate activity against P. aeruginosa cells grown on plankton and was effective in inhibiting biofilm formation. Mettrick et al. (2020) In vitro study HBED Colistin P. aeruginosa HBED, a synthetic hexadentate iron chelator, inhibited the growth and biofilm formation of all clinical strains of P. aeruginosa under aerobic and anaerobic conditions; HBED in combination with colistin significantly enhanced the microcolony killing effect of colistin, resulting in almost complete biofilm removal.

Moreau-Marquis et al. (2009)
In vitro study Deferoxamine; Deferasirox Tobramycin P. aeruginosa Tobramycin in combination with deferoxamine or deferasirox reduced the P. aeruginosa biomass of established biofilms by approximately 90% and reduced viable bacteria by 7 log units; neither tobramycin, deferoxamine, nor deferasirox alone had such a significant effect. The combination of tobramycin and iron chelators also prevented biofilm formation on human respiratory cells.

Moreau-Marquis et al. (2015)
In vitro study ALX-109 [combination of an investigational drug containing lactoferrin and hypothiocyanite (a bactericidal agent)] Tobramycin; Aztreonam P. aeruginosa ALX-109 inhibited P. aeruginosa biofilm formation but did not affect established biofilms; ALX-109 enhanced the ability of tobramycin and aztreonam to inhibit P. aeruginosa biofilm formation and reduce established biofilms. Moon et al. (2013) In vitro study Deferasirox P. intermedia Deferasirox exhibited potent antibacterial activity against P. intermedia, partially inhibited bacterial growth, and significantly prolonged bacterial doubling time. Deferasirox also significantly reduced the biofilm-forming activity and biofilm formation of P. intermedia. In the ATP bioluminescence assay, Deferasirox significantly reduced biofilm bioactivity. Lin et al. (2012) In vitro study PGG S. aureus PGG may be a candidate for the development of anti-biofilm products for clinical use due to its anti-biofilm activity and low cytotoxicity. Impairment of S. aureus biofilm formation by PGG was shown to be due to iron chelation. Iron supplementation complemented the effect of PGG and restored biofilm formation. Richter et al. (2017) In vitro and in vivo study Deferiprone Gentamicin; Ciprofloxacin

S. aureus
The combination of deferiprone-GaPP significantly reduced bacterial load and increased survival of S. aureus small colony variant-infected C. elegans in an artificial wound model. When deferiprone-GaPP was combined with gentamicin or ciprofloxacin in a colony biofilm model, deferiprone-GaPP was able to enhance the activity of gentamicin or ciprofloxacin.

Coagulasenegative staphylococci
Deferiprone alone had only a moderate inhibitory effect on biofilm growth, but the combination of deferiprone and the respective antibiotics significantly reduced bacterial counts by 2-3 logs compared to the effect of the antibiotic alone. In addition, the combination of deferiprone and antibiotics showed severe to complete destruction of the biofilm, which was not seen when antibiotics were administered alone. Nazik et al. (2015) In vitro study Deferoxamine; Deferiprone

A. fumigatus
Deferoxamine concentrations below 1,250 µM had no effect, while 2,500 µM increased biofilm formation or preform biofilms of A. fumigatus. 156 to 2,500 µM deferiprone inhibited biofilm formation in a dose-response manner. In preform biofilms, deferiprone above 625 to 1,250 µM showed an inhibitory effect compared to controls. The results of deferoxamine-induced enhancement of biofilm formation were attributed to A. fumigatus acquiring many iron chelator complexes through a siderophore-like mechanism.
A. baumannii, Acinetobacter baumannii; A. fumigatus, Aspergillus fumigatus; DIBI, denying iron to bacterial infections; GaPP, gallium protoporphyrin; HBED, N, N'-bis ( clinical adhesion levels (Berlutti et al., 2011). Several studies were conducted on Staphylococcus spp. 1,2,3,4,6-Penta-O-galloylβ-D-glucopyranose, a plant-derived ingredient with iron-chelating properties commonly used in Chinese medicine, has shown biofilm inhibitory effects on S. aureus (Lin et al., 2012). The combination of deferiprone and the heme analog gallium protoporphyrin in combination with gentamicin or ciprofloxacin increased the biofilm inhibitory effects in an S. aureus colony biofilm model (Richter et al., 2017). Additionally, for coagulase-negative staphylococci, deferiprone showed enhanced antibacterial activity in combination with clindamycin, gentamicin, or vancomycin by disrupting biofilms (Coraça-Huber et al., 2018). Regarding fungi, deferiprone inhibited biofilm formation by A. fumigatus, whereas deferoxamine had the opposite effect, and promoted biofilm formation. The result of the promotion of biofilm formation by deferoxamine may be due to A. fumigatus acquiring more iron chelator complexes through a siderophore-like mechanism (Nazik et al., 2015). In addition, deferoxamine is used by zygomycetes and A. fumigatus as a siderophore to promote bacterial growth (Boelaert et al., 1993). Thus, depending on the combination of the type of microorganism and iron chelator, the microorganism has a tolerance mechanism and takes advantage of iron starvation. However, iron chelators have a different point of action than do antibacterial and antifungal agents, and thus, may carry less risk of inducing drug resistance in microorganisms. This is a major advantage of combining drugs with different mechanisms of action.

Mechanisms of antimicrobial and anti-biofilm activity of iron chelators and antimicrobial-impregnated catheters
The iron chelator CP762 in combination with tetracyclines against P. aeruginosa showed synergistic effects. Among the tetracyclines, doxycycline showed particularly high synergism (Faure et al., 2021). CP762 is a hexadentate hydroxypyridinone iron chelator that has high affinity and selectivity for iron and does not utilize many of the bacterial iron siderophore receptors, making it unlikely to donate iron to pathogenic microbes (Piyamongkol et al., 2005;Zhou et al., 2011). Tetracycline binds to the 30S bacterial ribosome via a magnesium bridge (White and Cantor, 1971;Pioletti et al., 2001), but iron can inhibit this mechanism by binding to the magnesium binding site (Faure et al., 2021). Sequestration of iron by iron chelators may minimize iron binding to tetracycline, which would facilitate its complexation with low-affinity ions such as magnesium, which is necessary for binding to the bacterial ribosome (Faure et al., 2021).

Conclusion
Most iron chelators examined in the past have been effective in inhibiting biofilm formation by bacteria and fungi, and their antimicrobial activity has been shown to be enhanced when used in combination with antimicrobial agents (Figure 1, Table 1). Therefore, we believe that the inclusion of iron chelators in antimicrobial-or antifungal-impregnated catheters could help overcome microbial resistance mechanisms and create CVCs with a lower risk of CRIs. Further research on iron chelators and antimicrobial-impregnated catheters, and confirmation of their effects on reducing the incidence of CRIs in clinical trials, are warranted.