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Article

Synthetic Human Lactoferrin Peptide hLF(1-11) Shows Antifungal Activity and Synergism with Fluconazole and Anidulafungin Towards Candida albicans and Various Non-Albicans Candida Species, Including Candidozyma auris

by
Carlo Brouwer
1,2,
Youp van der Linden
3,
Maria Rios Carrasco
3,
Saleh Alwasel
2,
Tarad Abalkhail
2,
Fatimah O. Al-Otibi
2,
Teun Boekhout
2,3 and
Mick M. Welling
4,*
1
CBMR Scientific Inc., Edmonton, AB T6J4V9, Canada
2
College of Sciences, King Saud University, Riyadh 11451, Saudi Arabia
3
Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands
4
Department of Radiology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
*
Author to whom correspondence should be addressed.
Antibiotics 2025, 14(7), 671; https://doi.org/10.3390/antibiotics14070671
Submission received: 21 May 2025 / Revised: 23 June 2025 / Accepted: 28 June 2025 / Published: 2 July 2025
(This article belongs to the Special Issue Bioactive Peptides and Their Antibiotic Activity)
Browse Figure
Review Reports Versions Notes

Abstract

Introduction: Candidozyma auris (Cz. auris) has emerged globally, and diseases caused by it are associated with a mortality rate of 30–72%. This yeast is often multidrug-resistant and challenging to treat. A synthetic peptide, consisting of 11 amino acids of human lactoferrin (hLF1-11), offers a new therapy that is active against Candida albicans, non-albicans Candida yeasts, as well as Cz. auris. The current study examined the susceptibility of clinically relevant Candida species to hLF(1-11) in vitro and investigated the synergistic interaction of this peptide with fluconazole (FLU) and anidulafungin (ANI). Methods: Susceptibility of the yeasts to hLF(1-11) was tested with a microdilution method to determine minimum inhibitory concentrations (MICs). A total of 59 strains belonging to 16 species of Candida or Candidozyma were tested. The treatment cohort included 20 strains of Cz. auris originating from six different countries. Results: Mean MIC values of all susceptible strains ranged from 16.66 ± 6.46 μg/mL to 45.83 ± 10.21 μg/mL. There were no statistical differences in the susceptibility of hLF(1-11) for Cz. auris across geographic origins. In the combinatory tests, drugs acting together, the fractional inhibitory concentration indexes [FIC] < 1.0, showed a synergistic or additive effect on the efficacy of FLU and ANI when used in combination with hLF(1-11). [FIC] indexes 1–2 were interpreted as intermediate. MIC values in combinatory use were 1–2 titer steps lower than when used alone. Conclusions: hLF(1-11) inhibits the growth of yeasts that belong to the genus Candida, including Cz. auris. The combinatory use may be further investigated to treat infections caused by resistant yeasts.

1. Introduction

Invasive fungal infections (IFIs) are characterized by late and difficult diagnosis and high mortality, affecting an increasing number of immunocompromised patients in hospitals [1]. Due to their public health importance, several of these species are listed on the WHO list of fungal pathogens (accessed on 30 June 2025). C. albicans, an opportunistic yeast that can cause mucosal or invasive infections when the host immune system is debilitated, remains the predominant species responsible for candidiasis [2]. However, recent clinical studies have shown an increasing incidence of candidemia due to non-albicans species such as C. parapsilosis, C. tropicalis, N. glabratus (also known as C. glabrata), Pichia kudriavsevii (also known as C. krusei), and, in recent years, Cz. auris (also known as C. auris) [2,3,4,5,6,7,8].
Many studies have reported the antifungal susceptibility of the clinically most common Candida and non-albicans Candida species, but limited data exist for the uncommon yeast species. Recently, several studies have investigated the susceptibility of antifungal drugs to several classes of uncommon but emerging yeast species [3,9,10]. Resistance against commonly used antifungal drugs is a well-known phenomenon, whether it is acquired, e.g., C. albicans becoming resistant to azoles, or intrinsically present, such as N. glabratus showing poor susceptibility to azole and echinocandin drugs, as is likely also the case for several emerging yeast species [2,3,11,12]. Prolonged treatment and prophylactic use strongly increase the likelihood of developing the resistance of various Candida species [13]. At present, the antifungal drug arsenal for the treatment of systemic infections is limited to five classes of antifungal drugs, namely the polyenes (amphotericin B), triazoles (e.g., itraconazole and fluconazole) [14], and echinocandins (e.g., caspofungin, micafungin, and anidulafungin) [15]. Flucytosine and allylamine are single classes. Hence, with the increased incidence of drug resistance, there is an urgent need for novel antifungal drugs.
Candida (now reclassified as Candidozyma) auris was described in 2009 based on a single isolate from the external ear canal of an inpatient in Japan [16], and has since then emerged around the globe. More than 10,000 infections with C. auris were reported in the USA from 2016 to 2023, and 22,931 people were found to be colonized (https://www.cdc.gov/candida-auris/tracking-c-auris/ (accessed on 27 June 2025)) without showing any symptoms.
Cz. auris has been detected in 61 nations spanning six continents. Reports indicate its presence in all subregions of Africa, with over 2500 cases. Significant outbreaks have been noted in specific healthcare environments in South Africa and India, where Cz. auris has been linked to as much as 25% and 40% of the candidemia occurrences.
It is argued that these figures are much higher due to a low identification rate [17]. Cz. auris mainly colonizes people without giving any symptoms, but an infection with this yeast can lead to various health problems. For example, infections of the ear, wounds, urinary tract, and primarily bloodstream infections are described (European Centre for Disease Prevention and Control. C. auris in healthcare settings—Europe—first update, 23 April 2018. Stockholm: ECDC; 2018) [3,15,16,18]. Cz. auris infections are related to several risk factors, such as long-term hospitalization, transplantation surgery, invasive procedures, and underlying diseases, such as diabetes or HIV infection. Consequently, ICU patients are most at risk [3,15,17,18,19,20,21]. The mortality rate of a Cz. auris infection varies between 30 and 72% [22], depending on the underlying comorbidities of the patients [18].
Treatment of infections with Cz. auris is challenging. Symptomatic infections are treated preferably with echinocandins, such as caspofungin, because of the relatively mild side effects [23]. Resistance of Cz. auris to commonly used antifungal drugs is caused by a variety of factors, such as mutations in the ERG11 gene (increased copy number of the ERG11 gene), overexpression of efflux pumps such as CDR1 and MDR1, and the excessive and prophylactic use of antifungal agents [3,15,23,24]. Almost all Cz. auris isolates are resistant to fluconazole [25]. An increasing problem is the development of drug resistance; Cz. auris is recognized for its intrinsic resistance to a broad spectrum of antifungal agents, encompassing those from the azole, echinocandin, and polyene categories. This resistance renders it a challenging pathogen to manage against fluconazole in combination with echinocandins and/or the more toxic polyene amphotericin B [3,17,18,25]. Some isolates of Cz. auris are even resistant to all three antifungal antibiotic classes. Thus, Cz. auris is an emerging, multidrug-resistant microbe.
In this view, antimicrobial peptides (AMPs) may provide an alternative to the commonly used antifungal treatment regimens for infections caused by Cz. auris [3,26,27,28]. Antimicrobial peptides act via different pathways and pose another possibility for reducing the effects of infections [29]. Lactoferrin (LF) is a peptide of 80 kDa that occurs a.o. in the milk and saliva of animals, including humans [30]. Human LF (hLF) has bactericidal activity against a range of pathogens and is suggested to be active against biofilms. A synthetic peptide of human lactoferrin-derived peptide hLF(1-11) (sequence: GRRRRSVQWCA), which has previously been shown to have a broad antimicrobial spectrum, was used in this study [3,28,30,31,32]. The hLF1-11 peptide’s antifungal mechanism involves disrupting fungal cell membranes and targeting mitochondrial function, leading to cell death. It interacts with and enhances immune cell function, fostering a more vigorous antifungal response.
The purpose of the current study was to examine the susceptibility of different, clinically relevant Candida species and species that until recently were classified in the genus Candida, including Cz. auris, to hLF(1-11) in vitro and, secondly, to investigate the interaction of this peptide with two commonly used antifungal agents, fluconazole and anidulafungin for those yeasts.

2. Results

2.1. Minimum Inhibitory Concentration (MIC) Values

This experiment was conducted to determine the susceptibility of several clinically relevant yeast species that belong to the genus Candida or were classified in this genus until recently to hLF(1-11). Emphasis was placed on Cz. auris. Fifty-nine strains belonging to 16 different yeast species were evaluated (Table 1).
hL(1-11) was effective against all 59 strains tested, with MIC values ranging from a mean of 25 μg/mL (6.25 μg/mL to 50 μg/mL) in one or two titer steps (Table 1), and all were regarded as susceptible. Negative controls that were incubated solely with cells or incubated with c-LF(1-11) yielded a significantly higher MIC of >100 μg/mL and showed no signs of growth inhibition (Table 2).
Various strains of Cz. auris showed MIC values of 12.5 or 25 μg/mL to hLF(1-11) (Table 3).
The range of MIC values of strains belonging to all species tested for FLU and ANI were between 0.25 and 128 μg/mL and between 0.025 and 8 μg/mL, respectively (Table 4). In the combinatory testing of hLF(1-11) with FLU or ANI, the MIC values for both the peptide and the antifungals were lower than when the compounds were tested alone. A comparison of these MIC values can be found in Table 4. The lowest MIC value from the combinatory testing was two dilutions lower (1.56 μg/mL compared to 6.25 μg/mL), and the highest was one dilution lower (25 μg/mL compared to 50 μg/mL) than when hLF(1-11) was tested alone.

2.2. Synergy Studies

Seventeen strains of sixteen species were tested in the synergy studies. For FLU, a synergistic effect ([FIC] ≤ 0.5) was present for one strain of D. rugosa CBS7138, and for ANI for two strains of Cz. auris CBS15279 and N. nivariensis CBS 9983. An additive effect ([FIC] = 0.5–1) for FLU was observed for 15 strains belonging to 14 species and for ANI for 13 strains of 12 species. Intermediate [FIC] values ([FIC] = 1–2), which reveal an indifferent effect, were observed for FLU tests against two strains of C. dubliniensis CBS 7987 and Cz. haemuli CBS 180 and for ANI, three strains: C. parapsilosis CBS604, Cyberlindnera jadinii CBS 1600, and Nakaseomyces bracarensis CBS 10154. An antagonistic effect ([FIC] ≥ 2) was not observed (Table 4).

3. Discussion

The main research objective in this study was to evaluate the susceptibility of yeasts that belong to the genus Candida or species formerly classified in this genus, emphasizing Cz. auris (Table 3). In line with previous studies, a strong antimicrobial effect of hLF(1-11) was found in all strains of all yeast species tested [4,35,41,42]. Furthermore, all the Cz. auris group values were either 12.5 μg/mL or 25 μg/mL and were regarded as susceptible. The hLF(1-11) peptide inhibited the growth of Cz. auris significantly if compared to a negative control peptide. No correlation was observed between the susceptibility for hLF(1-11) and the geographical origin of the Cz. auris strains, but it must be stressed that not all known genetic and geographic diversity was included in this study. Contrary to the strong resistance of Cz. auris against currently used antifungal agents, none of the Cz. auris strains showed resistance against hLF(1-11). This apparent lack of resistance to hLF(1-11) can be explained by the fact that hLF(1-11) has different working mechanisms than the commonly used antifungal agents. LF is a cationic, basic, and amphipathic peptide that induces Ca2+ influx in the mitochondria, leading to a release of adenosine triphosphate (ATP) production and reactive oxygen species (ROS) [41,42,43,44]. Azoles, echinocandins, and polyenes are uncharged molecules interacting with the cell wall and cell membranes; secondly, the yeast has not developed resistance mechanisms, probably because the peptide is not widely used yet [21,38]. It can be argued that it is less likely to develop because LF is present in breast milk and saliva, where it co-exists for longer times with pathogens, including yeasts. Although this study yielded no resistance to hLF(1-11), the literature implies that the long-term resistance to LF development cannot be entirely excluded, but long-term exposure studies are needed to investigate this further [45].
Antimicrobial peptides are ideal candidates for new antifungal therapies as their toxicity is minimal or absent [46], the resistance development rates are low, and they can be used with an antifungal drug. Here, we demonstrated that the synthetic antimicrobial membrane-disruptive and immunomodulatory peptide, consisting of the first amino acids of human antimicrobial lactoferrin peptide (hLF1-11), shows a synergistic or additive effect in the cases of species like Cz. auris when used in combination with FLU or ANI. Even for C. albicans and N. glabratus that is not inherently resistant to antifungals, we noticed, as in former studies, that the combinatory use of hLF(1-11) and the antifungal drug had an additive effect, resulting in a reduced drug dose needed to inhibit the growth of the yeast [6,41,42,47,48]. This additive effect was also true for other species, such as N. glabratus and Cz. auris that are inherently resistant to azole drugs. Thus, hLF(1-11) might be developed in a drug to combat those yeast species listed as priority fungal pathogens. Importantly, this may result in the decreased development of resistance to commonly used antifungals such as azoles. The results for the potential use of hLF(1-11) in combination with azole drugs and anidulafungin suggest that previously unsuitable antifungal treatment options could be reinstated due to the lower MIC values noted when the antifungal is used in combination with hLF(1-11). Such a treatment can potentially be a solution to treat patients infected with resistant isolates. For instance, Cz. auris has developed different kinds of resistance mechanisms. Fluconazole resistance is linked to three different genes. The first mechanism is the efflux of azole drugs through pumps mediated by MDR1/CDR1/CDR2 point mutation [6,11,41,42,49]. Secondly, the gene ERG 11 can be upregulated or mutated. Upregulation of ERG11 results in reduced plasma membrane fluidity and dysfunction of the cell membrane, and a point mutation ensures less binding opportunity between fluconazole and p450 14-α-lanosterol-demethylase [24,41,42,50]. The third mechanism is an ERG5 point mutation, which activates another ergosterol pathway that avoids interaction with azoles [41]. Echinocandin resistance can be caused by a point mutation in FSK1/FSK2 genes, inhibiting the target site [50]. Upcoming resistance against echinocandins and amphotericin B is also associated with biofilm formation, probably the consequence of forming an abiotic surface with phospholipase activity and efflux pumps and a biomechanistic inhibition [51,52]. The utterance of resistance can differ across strains and is associated with the clade they belong to [42,53,54]. Despite differences within a clade, there is a higher amount of genetic variation between clades. For instance, Cz. auris strains belonging to the South Asia and South Africa clades have different ERG11 mutations, while no mutation was reported for the strain from Japan (East Asia clade) [20]. Additionally, strains of the East Asia and South Asia (India) clades have a genetic similarity of 63.4% [25,50]. Human LF consists of two similarly sized (α and β) domains, of which the first domain is basic, cationic, and amphipathic [55]. The first 11 amino acids of hLF, i.e., hLF1-11, have the most potent antimicrobial activity as a membrane-disruptive and immunomodulatory peptide [56], and it was known to have a synergistic effect for C. albicans in combination with fluconazole therapy [4]. After passing the plasma membrane of yeast pathogens, hLF(1-11) releases Ca2+, which is transported to the mitochondria. Eventually, this in transport yields ROS and ATP release, causing a penetrable cell wall and cell death [57]. Additionally, hLF(1-11) enhances the immune system in vivo by stimulating macrophage maturation and activation of the complement system [42,58,59].
Antimicrobial tests using hLF(1-11) have been carried out by comparing logarithmic and stationary yeast stage cells [60]. A study using C. albicans supported that log phase cultures gave significant differences to stationary phase cells when adding ketoconazole or miconazole, resulting in a greater distribution of MICs than in the other growth phases [42,61,62]. In our tests with C. albicans, no remarkable differences were observed when using logarithmic or stationary phase inocula for this species.
This study showed reliable outcomes because of the low variation found across strains, as the MIC values varied only between 12.5 μg/mL and 25 μg/mL. The variation in the values between these two points can be explained by slight differences among the yeast strains tested or in the hLF(1-11) concentrations used, which may influence the oxidation reaction of AlamarBlue™ in single assays. Previous research has shown that the method of modified MIC tests used in this study is equally reliable compared to standard CLSI guidelines [42,63,64,65]. Another source of variation might be the slightly different growth rates between the isolates, as some strains grew more slowly during incubation in vitro. Slower growth leads to fewer cells and probably also diminished susceptibility [42,66,67].
EUCAST protocols are standardized guidelines used for antifungal testing. The methods recommended include RPMI 1640 [34,42,67], as this showed superior growth of Candida spp., regardless of the incubation conditions or the antimicrobial agent used. So far, the best results for antifungal and antibacterial liquid tests have been observed with this medium [42,68,69,70]. In general, MIC values obtained in the RPMI medium in this study were consistent with those obtained in previous studies using the same peptide [34], and the use of this medium has yielded good results for both yeasts and bacteria. Thus, these observations confirm the adequacy of RPMI 1640 for use in our experiments.
Synthetic hLF(1-11) is a safe drug, as shown by safely giving up to several doses of 5 mg/kg in hematopoietic stem cell transplantation patients [46]. Therefore, hLF(1-11) is a promising agent to fight yeast infections, including Cz. auris, but it could also play a role in cleaning surfaces and preventing the spread of yeasts. Besides resistance to antifungal agents, the spreading of Cz. auris is also a problem for its eradication. Transmission is mainly via contaminated surfaces, as Cz. auris attaches to different surfaces and materials [42,71,72]. The complete removal of Cz. auris from contaminated surfaces is difficult to treat due to adherence properties and biofilm formation. An infected environment could lead to an outbreak, especially in hospitals. For example, a hospital ward in London (UK) needed to be entirely broken down because the complete eradication of Cz. auris appeared to be impossible [73]. Therefore, it is crucial to diagnose and treat infected patients early and search for local sources of yeast. Early detection may also significantly decrease the spread of the disease to other patients.
More extensive studies with increased strains are needed to provide deeper insights into the mechanism of the observed synergistic antifungal effect of hLF(1-11) and azoles or anidulafungin. Also, pharmacodynamic studies are required to test the efficacy of hLF(1-11) alone and in combination with antifungal drugs in animal experiments. Finally, blinded clinical trials are needed to support this concept of synergism between hLF(1-11) and commonly used antifungals in patient and control cohorts. Examples of the use of hLF(1-11) against onychomycosis [74] exist, the data indicate that hLF(1-11) shows no toxicity in humans, and the development of resistance against it seems negligible [75]. The emergence of multidrug-resistant microbial pathogens has created a pressing want for brand new healing options, with LF and its derived peptides, lactoferricins (LFcins), rising as promising applicants because of their multifaceted roles in innate immunity and microbial pathogenesis. LF reveals bacteriostatic and bactericidal properties, promotes immune responses, and has been proven to inhibit viral access and counter microbial mechanisms of infection, making it a precious adjuvant in fighting antibiotic-resistant microorganisms and fungi.

4. Materials and Methods

4.1. Chemicals

All chemicals used for the experiments described in this manuscript were purchased from commercial sources and used without further purification.

4.2. Minimum Inhibitory Concentration (MIC) Values

The MIC of hLF(1-11) against different strains of the yeast species was analyzed using a modified EUCAST broth protocol for susceptibility (method for the determination of broth dilution minimum inhibitory concentrations of antifungal agents for yeasts, EUCAST document E, Def 7.4), (http://www.EUCAST.org, accessed at 30 June 2025) [39,42,76]. The yeast cells were stimulated to grow during incubation of the MIC tests since yeast cells are more susceptible to therapies when in the logarithmic growth phase [77]. AlamarBlueᵀᴹ (Thermo Fisher, Eugene, OR, USA, (https://www.thermofisher.com/order/catalog/product/DAL1025#/DAL1025 (accessed on 27 June 2025)) was used to visualize growth and cell viability [63], The MIC values were determined by measuring the absorbance at 570–600 nm, and the results were compared to equivalent concentrations with an inactive control peptide hLF(1-11) (c-hLF(1-11)) [34].

4.3. Candida Strains, Cell Viability Testing

Fifty-nine strains belonging to eight yeast species belonging to Candida or that were recently classified within the genus Candida were obtained from Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands. Six strains were obtained from Leiden University Medical Center (LUMC), Leiden, The Netherlands, and three strains from the American Type Culture Collection (ATCC, Rockville, MD, USA) (Table 1). C. auris strains from Japan and Korea originated from the East Asian clade, whereas strains from Kuwait, Oman, and India belonged to the South Asian clade. The strains were cultivated for 24–48 h on standard Sabouraud dextrose agar (SDA) plates. Subsequently, a single colony was transferred to a 50 mL sterile bottle containing 25 mL ¼ strength (25%) diluted RPMI 1640 medium (Invitrogen Corp., Thermo Fisher, Walthan, MA) with 10 mM NaPBS and a pH of 7.4, and incubated for 18–20 h (i.e., overnight) by 35 °C under vigorous shaking (150 rpm). From the overnight culture, 3 mL was transferred into a 50 mL sterile bottle containing 25 mL of ¼ strength RPMI 1640 medium. This subculture was pre-warmed (35 °C) and incubated again for 2.5 h under the same conditions to induce growth of the yeast cells. The cells were harvested from the pellet after centrifugation (10 min at 4 °C) at 4000 rpm. This pellet was washed twice with 10 mL of 10 mM NaPBS pH 7.4. Furthermore, the pellet was resuspended in 1 mL of PBS, and the concentrations were determined by reading OD 570–600 nm using a spectrophotometer (SPECTRO star Nano Absorbance Reader, BMG Labtech, Ortenberg, Germany). For the experiments, the concentration of all yeast suspensions was set to 0.5–2.5 × 105 forming units (CFU)/mL [34].

4.4. Antimicrobial Peptides

The peptides were purchased from Proteogenix, Schiltigheim, France. The synthetic hLF(1-11) consisted of (Ac-GRRRRSVQWCA-CONH2; MW 141,562 Da; purity > 98%). The negative control (c-hLF(1-11), consisted of the same amini acids as hLF-1-11 with substituted alanines at positions 2, 3, 6, 10 (Ac- GAARRAVQWAA-CONH2; MW 1156.4 Da; purity 96.3%) [34]. The purity of the peptides was determined by reverse-phase high-performance liquid chromatography (HPLC) using a Waters HPLC system with a 1525EF pump and a 2489 UV/VIS detector. For analytical HPLC, a Dr. Maisch GmbH Reprosil-Pur C18-AQ 5 µm (250 × 4.6 mm) or a Dr. Maisch GmbH Reprosil-Pur C18-AQ 5 µm (250 × 10 mm) column was used, with a gradient of 0.1% v/v trifluoroacetic acid (TFA) in H2O/CH3CN 95:5 to 0.1% TFA in H2O/CH3CN 5:95 in 40 min (1 mL/min−1). The sample size was 20 mL of a peptide solution of hLF(1-11) (i.e., 1.5 mg/mL water). Stocks of the peptides were diluted at a concentration of 2 mg/mL of 0.01% HAc; pH of 3.7), dried in a Speed-Vac, stored at −20 °C, and thawed immediately before use [26].

4.5. EUCAST Broth Microdilution Method

The strains shown in Table 4 were all previously tested according to a modified EUCAST broth protocol for susceptibility to fluconazole (FLU) and anidulafungin (ANI) as described in the Section 3. The two antifungal compounds were purchased from the following companies: fluconazole (FLU), Pfizer Central Research Sandwich, UKITC, Janssen Research Foundation, Beerse, Belgium, and anidulafungin (ANI), Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany.

4.6. Minimal Inhibitory Growth Assays

Yeast cultures were tested and treated as described in Section 4.3 above. For the experiments, the concentration of all yeast suspensions was set to 0.5–2.5 × 105 colony forming units (CFUs)/mL [34].
Afterward, concentrations were adjusted to 2 × 105 CFUs/mL into ¼ strength RPMI 1640 medium. Before adding peptides into the microtiter plate (96 wells, u-bottom, low bind, Greiner Bio-one), electrostatic pressure was avoided by placing the plate on top of a wet tissue. The hLF(1-11) working stock solution (2 mg/mL) was prepared as described above. The peptides were pipetted in the microtiter plate in a concentration range of 0–200 μg/mL, supplemented with ¼ strength RPMI 1640 up to a total volume of 100 μL. After adding the peptides, 100 μL of yeast suspensions 2 × 105 CFU/mL dilutions and 1 μL of AlamarBlueᵀᴹ were added to the wells. The negative controls contained only 200 μL ¼ strength RPMI medium or a yeast dilution with AlamarBlueᵀᴹ for growth monitoring. Plates were incubated at 35 °C for 24–28 h under vigorous shaking (120 rpm). Growth was detected when a red/pink color appeared, and inhibition of growth as blue/purple. Color changes by AlamarBlueᵀᴹ were interpreted by the eye and confirmed at OD 570–600 nm with a microplate reader (Bio-Rad, Hercules, CA, USA). MIC values below 100 μg/mL were considered susceptible [27]. The c-LF(1-11) should not show any activity in RPMI media. The substitution of arginine into alanine results in a change in the charge, hydrophobicity, and amphipathic structure of the peptide, which alters the antimicrobial characteristics [10,27,33,78]. As controls, reference strains were incubated for 24–48 h at 35 °C without an agent, and then growth was monitored using a spectrophotometer at OD 600 nm. Results are means of at least five independent experiments (Table 2).

4.7. Synergy Studies

For the antifungal synergy studies, the cultures were incubated overnight at 35 °C in 25% RPMI 1640 (¼ strength), and the yeast cells were suspended to 2 × 105 CFUs/mL as described above. The hLF(1-11) working stock solution (2 mg/mL) was prepared as described previously. First, hLF(1-11) was dispensed in a 96-well microtiter plate with concentrations ranging from 800 to 0 μg/mL. A horizontal gradient was prepared on the plate with a high concentration on the left side. Secondly, two separate 96-well plates were prepared, one with fluconazole and one with anidulafungin, with concentrations ranging from 512 to 0 and 32 to 0 μg/mL, respectively, in a vertical gradient with the highest concentration at the top side (Figure 1) [79].
Finally, 150 μL of yeast suspension with a concentration of 2 × 105 CFUs/mL was dispensed in all wells of the checkerboard plate, and 25 μL of the peptide was added. Plates were briefly incubated on a shaker at 35 °C for 15 min [34]. Then, 25 μL of the antifungal drug, either fluconazole or anidulafungin, was added to each well. The peptide and the antifungal drug were placed at the same positions as in the original and antifungal drug plates to give a final volume of 200 μL per well [80]. Endpoints were determined by measuring the OD 600 nm at 0, 24, and 48 h after incubating the plates at 35 °C. The plates were agitated before reading to ensure the contents were resuspended. The fractional inhibitory concentration [FIC] index for combinations of two antimicrobials was calculated according to the equation: [FIC] index = [FIC]A + [FIC]B = A/MICA + B/MICB, where A and B are the MICs of drug A and drug B in the combination; MICA and MICB are the MICs of drug A and drug B alone; and [FIC]A and [FIC]B are the [FIC] of drug A and drug B [42]. The [FIC] indexes were interpreted as follows: ≤0.5, synergistic; >0.5–1, additive; 1–2 intermediate; and ≥2.0, antagonistic [34].

5. Conclusions

The research on the potential of LF peptides to inhibit Candida and similar yeasts, including Cz. auris, shows promising results. The LF peptides have strong antifungal properties, making them valuable candidates for developing new treatment options. The small LF peptide hLF(1-11) is considered a promising drug in antifungal interventions but testing its efficacy in animals and eventually in humans is needed. As the prevalence of Cz. auris infections continue to rise, and antifungal resistance becomes a growing concern, exploring alternative therapies, such as those using LF-based peptides, could be a significant step forward in addressing this challenge. In the combinatory tests, the fractional inhibitory concentration indexes [FIC] for the tested strains were up to 1.0, showing that there is a synergistic or additive effect on the efficacy of fluconazole and anidulafungin when used in combination with hLF(1-11). MIC values in combinatory use were one or two titer steps lower than when applied alone. The [FIC] indexes of the checkerboard assays were all <1, showing that hLF(1-11) and the antifungal drugs fluconazole or anidulafungin have a synergistic-additive relationship. hLF(1-11) inhibits the growth of Cz. auris and other ascomycetous yeasts of clinical relevance and, therefore, has a promising medical application. The combinatory use may have promise in treating infections caused by resistant isolates.

Author Contributions

Conceptualization, C.B., M.M.W., and T.B.; methodology, C.B., M.M.W., and T.B.; investigation and resources; C.B., S.A., M.M.W., and T.B.; technical support, Y.v.d.L., M.R.C., T.A., and F.O.A.-O.; writing—original draft preparation, C.B., T.B., S.A., and M.M.W.; writing—review and editing, C.B., M.M.W., S.A., and T.B.; supervision, C.B., T.B., and M.M.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Acknowledgments

The authors thank Ferry Hagen, Bert Gerrits van den Ende, Aimi Stavrou, and Laura Wiercx, the latter two are students at Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands, for providing Candida isolates and providing technical support. The authors would like to thank the Researchers Supporting Project, and the Distinguished Scientist Follow Program (DSFP) from King Saud University, Saudi Arabia.

Conflicts of Interest

C.B. is a co-founder of CBMR Scientific Inc. Other authors declare no competing interests. The research in this manuscript has no underlying commercial activity.

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Antibiotics 14 00671 g001
Figure 1. An example of a checkerboard assay is combining two compounds to test increased effectiveness [49].
Figure 1. An example of a checkerboard assay is combining two compounds to test increased effectiveness [49].
Antibiotics 14 00671 g001
Table 1. Minimum inhibitory concentration (MIC) values of the antimicrobial compound hLF(1-11) against selected species of Candida or species that were formerly classified in the genus Candida [33,34,35,36,37,38,39,40].
Table 1. Minimum inhibitory concentration (MIC) values of the antimicrobial compound hLF(1-11) against selected species of Candida or species that were formerly classified in the genus Candida [33,34,35,36,37,38,39,40].
NameOld NameStrainCountry of OriginSourceMIC Values
MeanSt. Dev
Candida albicans AA (LUMC)NetherlandsBlood22.925.10
Candida albicans ATCC 90028United StatesBlood18.756.85
Candida albicans ATCC 10231unknownBronchomycosis23.214.73
Candida albicans CBS 562UruguaySkin20.466.31
Candida albicans CMC 1968ItalyHuman23.214.73
Candida albicans Y01-19unknownunknown16.676.46
Candida dubliniensis CBS 7987Icelandunknown22.925.10
Candida parapsilosis ATCC 22019United StatesCase of sprue23.448.99
Candida parapsilosis CBS 604Puerto Ricounknown20.836.46
Candida parapsilosis CMC 2039ItalyHuman18.756.85
Candida parapsilosis LUMCNetherlandsHuman18.756.85
Candida tropicalis CBS 1920unknownunknown21.887.66
Candida tropicalis CBS 94unknownunknown45.8310.21
Candida tropicalis CMC 2041ItalyHuman41.6712.91
Candida tropicalis LUMCNetherlandsHuman23.214.73
Candidozyma aurisCandida auris2MG-1491unknownunknown21.256.04
Candidozyma aurisCandida aurisCBS 10913JapanEar21.255.88
Candidozyma aurisCandida aurisCBS 12372KoreaBlood19.386.38
Candidozyma aurisCandida aurisCBS 12373KoreaBlood21.885.55
Candidozyma aurisCandida aurisCBS 12805IndiaBlood20.006.46
Candidozyma aurisCandida aurisCBS 12806IndiaBlood21.256.04
Candidozyma aurisCandida aurisCBS 12807IndiaBlood20.006.46
Candidozyma aurisCandida aurisCBS 12874IndiaBlood20.006.46
Candidozyma aurisCandida aurisCBS 12875IndiaBlood20.006.46
Candidozyma aurisCandida aurisCBS 12883IndiaBlood20.006.46
Candidozyma aurisCandida aurisCBS 12884IndiaBlood21.256.04
Candidozyma aurisCandida aurisCBS 12885IndiaHuman22.505.27
Candidozyma aurisCandida aurisCBS 14144KuwaitBlood18.756.59
Candidozyma aurisCandida aurisCBS 1491unknownunknown20.006.85
Candidozyma aurisCandida aurisCBS 14916OmanBlood18.756.59
Candidozyma aurisCandida aurisCBS 14918OmanBlood20.006.46
Candidozyma aurisCandida aurisCBS 1492unknownunknown20.006.85
Candidozyma aurisCandida aurisCBS 15108Omanunknown20.006.46
Candidozyma aurisCandida aurisCBS 15109OmanHuman20.006.46
Candidozyma aurisCandida aurisCBS 15279BelgiumKuwaiti patient18.186.53
Candidozyma duobushaemuliCandida pseudohaemuloniiCBS 10004Thailandunknown19.798.31
Candidozyma duobushaemuliCandida pseudohaemuloniiCBS 12371Koreaunknown20.836.46
Candidozyma duobushaemuliCandida pseudohaemuloniiCBS 7798United Statesunknown22.505.59
Candidozyma duobushaemuliCandida pseudohaemuloniiCBS 7800United Statesunknown20.006.85
Candidozyma duobusheamuliCandida duobushaemuloniiCBS 7798United Statesunknown21.886.25
Candidozyma duobusheamuliCandida duobushaemuloniiCBS 7800United Statesunknown40.0013.69
Candidozyma haemuliCandida haemuloniiCBS 12437Spainunknown22.925.10
Candidozyma haemuliCandida haemuloniiCBS 12439Spainunknown20.836.46
Candidozyma haemuliCandida intermediaCBS 572Puerto Ricounknown22.925.10
Clavispora lusitaniaeCandida lusitaniaeCBS 6936Israelunknown20.836.46
Clavispora lusitaniaeCandida lusitaniaeCMC 1944ItalyHuman45.8310.21
Diutina rugosaCandida rugosaCBS 613unknownunknown22.505.59
Diutina rugosaCandida rugosaCBS 7138Netherlandsunknown22.505.59
Meyerozyma guilliermondiiCandida guilliermondiiCBS 2030United Statesunknown45.0011.18
Nakaeomyces glabratusCandida glabrataCBS 138Italyunknown22.735.06
Nakaeomyces glabratusCandida glabrataLUMCNetherlandsHuman22.925.10
Nakaseomyces bracarensisCandida bracarensisCBS 10154Portugalunknown21.887.66
Nakaseomyces bracarensisCandida glabrataCMC 1933ItalyHuman23.214.73
Nakaseomyces nivariensisCandida nivariensisCBS 9983Spainunknown22.925.10
Pichia inconspicuaCandida inconspicuaCBS 180Netherlandsunknown27.0812.29
Pichia inconspicuaCandida inconspicuaCBS 1735Norwayunknown20.836.46
Pichia kudriavseviiCandida kruseiLUMCNetherlandsHuman23.214.73
Pichia kudriavseviiCandida kruseiCMC 2002ItalyHuman22.925.10
Pseudolindnera jadiniiCandida jadiniiCBS 1600Franceunknown22.925.10
Table 2. Minimum inhibitory concentration values of the negative control peptide, c-hLF(1-11), containing alanine substitutions at positions 2, 3, 6, and 10 against 11 selected yeast strains. Negative controls with only selected strains dilution or c-hLF(1-11) yielded a significantly higher MIC of >100 μg/mL and showed no signs of growth inhibition [34,36,37,38,41]. * = will be soon renamed.
Table 2. Minimum inhibitory concentration values of the negative control peptide, c-hLF(1-11), containing alanine substitutions at positions 2, 3, 6, and 10 against 11 selected yeast strains. Negative controls with only selected strains dilution or c-hLF(1-11) yielded a significantly higher MIC of >100 μg/mL and showed no signs of growth inhibition [34,36,37,38,41]. * = will be soon renamed.
SpeciesStrainMIC-Mean
Candida albicansATCC 90028>100
Candida albicansATTC 10231>100
Candida albicansY01-19>100
Candida parapsilosis *ATTC 22019>100
Candida parapsilosis *LUMC>100
Candida tropicalisLUMC>100
Candidozyma aurisCBS 10913>100
Candidozyma aurisCBS 12372>100
Candidozyma aurisCBS 15279>100
Nakaseomyces glabratusLUMC>100
Pichia kudriavseviiLUMC>100
Table 3. MIC (μg/mL) values of the antimicrobial compound hLF1-11 against 19 strains from five different countries of selected Candidozyma strains.
Table 3. MIC (μg/mL) values of the antimicrobial compound hLF1-11 against 19 strains from five different countries of selected Candidozyma strains.
SpeciesStrainCountryMeanSt. Dev
hLF1-11 MIC
Cz. auris2MG-1491unknown21.256.04
Cz. aurisCBS 10913Japan21.255.88
Cz. aurisCBS 12372Korea19.386.38
Cz. aurisCBS 12373Korea21.885.55
Cz. aurisCBS 12805India20.006.45
Cz. aurisCBS 12806India21.256.04
Cz. aurisCBS 12807India20.006.45
Cz. aurisCBS 12874India20.006.45
Cz. aurisCBS 12875India20.006.45
Cz. aurisCBS 12883India20.006.45
Cz. aurisCBS 12884India21.256.04
Cz. aurisCBS 12885India22.505.27
Cz. aurisCBS 14144Kuwait18.756.59
Cz. aurisCBS 1491unknown20.006.85
Cz. aurisCBS 14916Oman18.756.59
Cz. aurisCBS 14918Oman20.006.45
Cz. aurisCBS 1492unknown20.006.85
Cz. aurisCBS 15108Oman20,006.45
Cz. aurisCBS 15109Oman20.006.45
Table 4. Strains of Candida species or those classified in this genus until recently were used for checkerboard combination testing. The MIC values obtained for the fluconazole and anidulafungin with hLF(1-11) from the checkerboard microdilution testing are shown. The [FIC] index for every strain for each antifungal was between 0.5 and 1.0 and 1.0 and 2.0, revealing a synergistic or additive effect between the antifungal drugs and hLF(1-11) [36,37,38,39].
Table 4. Strains of Candida species or those classified in this genus until recently were used for checkerboard combination testing. The MIC values obtained for the fluconazole and anidulafungin with hLF(1-11) from the checkerboard microdilution testing are shown. The [FIC] index for every strain for each antifungal was between 0.5 and 1.0 and 1.0 and 2.0, revealing a synergistic or additive effect between the antifungal drugs and hLF(1-11) [36,37,38,39].
SpeciesStrainFLU MICANI MIChLF
(1-11) MIC		FLU
+ hLF MIC 		ANI
+ hLF MIC 		hLF + FLU MIChLF + ANI MICFIC
hLF
+ FLU		FIC hLF + ANI
EucastCombination
Antifungal + Peptide		Combination Peptide + Antifungal
Candida
albicans		CBS 562 10.25500.50.1312.512.50.750.63
Candida
dubliniensis		CBS 79871280.0325640.0312.56.25>11
Candida
parapsilosis		CBS 6042812.5183.12512.50.75>1
Candida
tropicalis		CBS 19200.250.56.250.250.133.136.250.751
Candidozyma
auris		CBS 152791612540.2512.53.130.750.5
Candidozyma duobushaemuliCBS 780012845032212.512.50.750.75
Candidozyma duobushaemuliCBS 1237164212.5160.253.136.250.750.63
Candidozyma haemuliCBS 12439128412.51280.512.53.13>10.63
Candidozyma haemuliCBS 180 64112.5160.256.256.2510.63
Clavispora
lusitaniae		CBS 69364112.510.253.133.130.750.63
Cyberlindnera jadiniiCBS 160080.0112.5403.136.250.75–1.0>1
Diotuna
intermedia		CBS 572 160.52580.256.2512.50.751
Diutina rugosaCBS 7138160.52540.251.566.250.51
Meyerozyma guilliermondiiCBS 203032250160.253.133.130.750.63
Nakaeomyces glabratusCBS 138 80.2512.5226.256.250.751
Nakaseomyces bracarensisCBS 10154486.25143.1256.250.75>1
Nakaseomyces nivariensisCBS 998316112.540.056.253.130.750.5
Pichia
kudriavsevii		CMC 2002320.062580.0312.512.50.751
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Brouwer, C.; van der Linden, Y.; Carrasco, M.R.; Alwasel, S.; Abalkhail, T.; Al-Otibi, F.O.; Boekhout, T.; Welling, M.M. Synthetic Human Lactoferrin Peptide hLF(1-11) Shows Antifungal Activity and Synergism with Fluconazole and Anidulafungin Towards Candida albicans and Various Non-Albicans Candida Species, Including Candidozyma auris. Antibiotics 2025, 14, 671. https://doi.org/10.3390/antibiotics14070671

AMA Style

Brouwer C, van der Linden Y, Carrasco MR, Alwasel S, Abalkhail T, Al-Otibi FO, Boekhout T, Welling MM. Synthetic Human Lactoferrin Peptide hLF(1-11) Shows Antifungal Activity and Synergism with Fluconazole and Anidulafungin Towards Candida albicans and Various Non-Albicans Candida Species, Including Candidozyma auris. Antibiotics. 2025; 14(7):671. https://doi.org/10.3390/antibiotics14070671

Chicago/Turabian Style

Brouwer, Carlo, Youp van der Linden, Maria Rios Carrasco, Saleh Alwasel, Tarad Abalkhail, Fatimah O. Al-Otibi, Teun Boekhout, and Mick M. Welling. 2025. "Synthetic Human Lactoferrin Peptide hLF(1-11) Shows Antifungal Activity and Synergism with Fluconazole and Anidulafungin Towards Candida albicans and Various Non-Albicans Candida Species, Including Candidozyma auris" Antibiotics 14, no. 7: 671. https://doi.org/10.3390/antibiotics14070671

APA Style

Brouwer, C., van der Linden, Y., Carrasco, M. R., Alwasel, S., Abalkhail, T., Al-Otibi, F. O., Boekhout, T., & Welling, M. M. (2025). Synthetic Human Lactoferrin Peptide hLF(1-11) Shows Antifungal Activity and Synergism with Fluconazole and Anidulafungin Towards Candida albicans and Various Non-Albicans Candida Species, Including Candidozyma auris. Antibiotics, 14(7), 671. https://doi.org/10.3390/antibiotics14070671

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