Ethical statement

Approval for the participation of human subjects was granted by the General Directorate of Health Affairs at Hamadan University of Medical Sciences, Iran, under ethics approval number IR.UMSHA.REC.1401.809. All clinical strains used in this study was obtained from routine medical testing or existing strain collections, ensuring that only bacterial cultures from these approved sources were utilized.

This research was conducted at Besat Hospital in Hamadan city and involved 48 patients diagnosed with type II diabetes mellitus, aged between 43 and 61 years, who were admitted with diabetic foot infections to the Internal Ward from January to April 2023. Additionally, consent was obtained from all participating patients before the commencement of the experiment. Swab samples (comprising aspirates and/or pus) were carefully gathered from heavily infected ulcers, employing sterile bottles and adhering to rigorous hygienic protocols.

Materials

Fucoidan, a sulfated polysaccharide, along with gentamicin and imipenem, was purchased from Sigma-Aldrich, USA. The fucoidan used in this study was extracted from the brown algae Sargassum angustifolium. Its structure is primarily composed of L-fucose and sulfate ester groups, with minor components of other monosaccharides such as galactose, mannose, xylose, and glucuronic acid.

Collection of clinical isolates of S. aureus and A. baumannii

A total of thirty-one isolates of S. aureus and twenty-seven isolates of A. baumannii were collected from clinical samples of DFU patients. These clinical samples were transferred to the microbiology laboratory at Hamadan University using transport media, including trypticase soy agar (TSA) and brain heart infusion (BHI).

Upon arrival at the laboratory, the initial identification of S. aureus was carried out using established protocols, followed by genetic confirmation through polymerase chain reaction (PCR), specifically targeting the amplification of the nuc gene [24]. A standard strain of S. aureus ATCC 25923 was employed as a positive control to ensure experimental consistency. Similarly, for A. baumannii, chemical identification was conducted using oxidase-catalase tests, motility tests, triple sugar iron (TSI) tests, and microscopic examination. Genetic confirmation of the Acinetobacter genus, as well as species determination, involved PCR amplification of the blaoxa51-like gene, with A. baumannii ATCC 19606 serving as the positive control. The blaoxa51-like gene products were subsequently sent for sequencing [25].

After the final identification, all S. aureus and A. baumannii isolates were preserved in trypticase soy broth (TSB) containing 20% glycerol at -70 °C for further analysis. All culture media and reagents used in this study were obtained from Merck, Germany.

Screening of S. aureus based on methicillin resistance

Isolates of S. aureus diagnosed for methicillin resistance were screened using the disk diffusion method with Cefoxitin (FOX; 30 µg) (MAST, UK) disk on Mueller-Hinton agar (MHA) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, employing standard zone size inhibition criteria. The confirmation was performed using the PCR method for the mecA gene [26]. S. aureus ATCC 33591 was used as a positive control, and Escherichia coli ATCC 25922 had served as a negative control in the PCR test.

Biofilm formation assay

The biofilm-forming ability of S. aureus and A. baumannii strains was assessed using the microtiter plate method, as previously described depending on the bacterial type [26]. To investigate dual-species biofilms formation, the biofilms of S. aureus were allowed to grow before the addition of A. baumannii to facilitate the survival of S. aureus. Colonies of S. aureus were inoculated in 5 mL TSB supplemented with 1% glucose (1% Glu TSB) and cultured overnight. Cultures were diluted to 5 × 105 CFU/mL in 1% Glu TSB, and 1.5 mL of the diluted S. aureus suspension was seeded onto 24-well plates (JET Biofil, China). The S. aureus biofilms were allowed to form for 24 h on a shaker (37 °C, 220 rpm). After washing and discarding planktonic cells, 1.5 mL of A. baumannii suspension with a concentration of 5 × 105 CFU/mL was added to the S. aureus biofilms. The dual-species biofilms were then incubated for an additional 18 h on a shaker (37 °C, 220 rpm). The wells were washed with 1.5 mL 1% Glu TSB, and planktonic cells from both species were removed. Adherent bacteria were fixed with 200 µL 99% methanol for 25 min. After removing the methanol, the wells were dried, and 200 µL of 2% crystal violet was added for 20 min to allow staining of biofilm-forming strains. The plates were emptied, and excess stain was washed off with sterile distilled water. Next, 200 µL of 33% glacial acetic acid was added to dissolve remaining colors completely. The plates were held at room temperature for 15 min, and finally, the optical density (OD) of the wells was read at 570 nm wavelength using an ELISA reader (BioTek, Germany). The experiment was performed in triplicate for each bacterial sample, using a well containing 1% Glu TSB culture medium as a negative control. Biofilm formation was assessed by comparing the OD of each test well with that of the control well, and the results were classified into the following categories [27]:

Strong biofilm

OD greater than four times the cut-off value (ODc).

Moderate biofilm

OD between two and four times the ODc.

Weak biofilm

OD between the ODc and two times the ODc.

Non-biofilm

OD equal to or less than the ODc.

To examine the formation of dual-species biofilms prior to methanol addition, we cultured 10 µL of dual-species biofilms were cultured on selective Leeds media for A. baumannii and mannitol salt agar (MSA) media for S. aureus [8, 28]. This step occurred subsequent to the initial biofilm formation and before fixation with 99% methanol.

Antibiotic susceptibility pattern and MDR isolates

To establish the antibiotic sensitivity pattern, all isolates of S. aureus and A. baumannii were subjected to the Disk Diffusion method, following the CLSI recommendations for the year 2023 [29]. For S. aureus, the antibiotic susceptibility testing encompassed Gentamicin (GM; 10 µg), Clindamycin (CD; 2 µg), trimethoprim-sulfamethoxazole (TS; 1.25/23.75 µg), erythromycin (E; 15 µg), linezolid (LZD; 30 µg), imipenem (IMP; 10 µg), and vancomycin (VAN; 30 µg) (MAST, UK). Similarly, susceptibility testing for A. baumannii comprised imipenem (IMP; 10 µg), meropenem (MER; 10 µg), gentamicin (GM; 10 µg), Piperacillin (PIP; 100 µg), ampicillin (AMP; 10 µg), ceftriaxone (CEF; 30 µg), amoxicillin/clavulanic acid (AUG; 20/10 µg), tetracycline (TET; 30 µg), and ciprofloxacin (CIP; 5 µg) (MAST, UK). Subsequently, isolates that showed resistance to a minimum of three classes of different antibiotics were considered as multidrug-resistant (MDR) based on the defined criteria [30].

Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) and MBC/MIC

The broth microdilution technique, as per the CLSI guidelines, was employed to ascertain the MIC and MBC values for 10 strains of S. aureus and 10 strains of A. baumannii exhibiting resistance to two antibiotics, namely gentamicin and imipenem [29]. In brief, fresh bacterial isolates were cultured overnight in cation-adjusted Mueller-Hinton broth (caMHB) at 37 °C with shaking at 180 rpm. The following day, the bacterial cells were adjusted to the mid-logarithmic phase at a turbidity standard of 0.5 McFarland (equivalent to 10⁸ CFUs/mL). The bacterial concentration was then adjusted to 10⁶ CFUs/mL in the same medium. Simultaneously, two-fold serial dilutions of antibiotics and fucoidan were prepared in caMHB in a volume of 100 µL per well in a 96-well flat-bottom microplate (Jet Biofil, Guangzhou, China). The concentration ranges were as follows: gentamicin and imipenem (0.5 to 1024 µg/mL), and fucoidan (3.9 to 1000 µg/mL). Subsequently, 100 µL of the bacterial suspension containing 106 CFUs/mL was added to each well containing the serially diluted antibiotics or fucoidan. The microplates were incubated at 37 °C for 18–24 h. For co-culture contexts, 50 µL of each S. aureus and A. baumannii bacterial suspension, totaling 5 × 104 CFU/mL, were added in equal proportions (1:1) to the wells. Following incubation at 37 °C for 24 h, the MIC was determined as the lowest concentration of the antimicrobial agent capable of inhibiting bacterial growth. Additionally, to ascertain MBC, 10 µL from the last wells displaying no visible growth were streaked onto MHA plates and incubated at 37 °C for 24 h. MBC was defined as the minimum concentration of the antimicrobial substance capable of eradicating 100% of the initial bacterial inoculum (105 CFU). The process of determining MIC and MBC for antibiotics was repeated at least three times for accuracy and consistency.

Determination of minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC)

The MBIC and MBEC were evaluated for the aforementioned 20 isolates. In summary, fresh bacterial colonies were cultivated overnight in 5 mL of 1% Glu TSB at 37 °C with shaking at 180 rpm. The bacterial cells were first adjusted to a turbidity standard of 0.5 McFarland (equivalent to 10⁸ CFU/mL). The concentration was then further diluted to 10⁶ CFU/mL in the same medium. Subsequently, an inoculum of 106 CFUs was added to the wells of U-shaped microplates filled with 1% Glu TSB medium. The method mentioned was used in dual-species biofilm cases. The plates were incubated for 24 h at 37 °C. Wells containing bacteria grown in 1% Glu TSB without exposure to antimicrobial agents were considered positive controls, while wells containing 1% Glu TSB without bacteria served as negative controls. MBIC was calculated as the lowest concentration of the antibiotic that caused 90% inhibition of biofilm formation in the tested isolate according to the formula [31]:

$$}\,}\,\left[ }\, - \,\left( }\,}\,}\,}\,}} \right)} \right]\,}\,}$$

To assess the eradication effects MBEC, biofilms were first formed using the above method. Subsequently, the wells were washed three times with PBS solution (200 µL) and air-dried. Serial dilutions of gentamicin, imipenem and fucoidan were added to the wells containing the formed biofilm and incubated for 24 h at 37 °C. In the next step, after discarding the liquid, the wells were washed three times with PBS, and after scraping the well contents and mixing with physiological saline, 10 µL of the well contents were cultured on MHA medium and incubated at 37 °C for 48 h. MBEC of gentamicin and imipenem was considered as the minimum amount of antimicrobial agent required to eliminate 100% of bacteria [32]. The positive control for this test consisted of standard isolates forming biofilm in 1% Glu TSB without exposure to antimicrobial agents, and the negative control was 1% Glu TSB without bacteria.

Determination of the synergistic effects in the planktonic state of isolates

The synergistic effects of gentamicin, imipenem and fucoidan were evaluated for six selected isolates, including S. aureus 6, S. aureus 7, S. aureus 22, A. baumannii 1, A. baumannii 8, and A. baumannii 20, utilizing the checkerboard microdilution method. These approaches were applied in individual culture studies, drawing from established methodologies [33]. Briefly, serial dilutions of gentamicin (0.25 to 1024 µg/mL), imipenem (0.25 to 1024 µg/mL), and fucoidan (3.9 to 1000 µg/mL) were prepared in 100 µL volumes per well within 96-well plates. Columns 1 to 11 of the plates, each containing 2-fold serial dilutions of gentamicin, were paired with rows A to G, housing 2-fold serial dilutions of imipenem. Column 12 featured serial dilutions of gentamicin alone, while row H contained serial dilutions of imipenem. Following the generation of 0.5 McFarland turbidity and dilution, 100 µL equivalent to 105 CFU/mL of bacterial suspension was added to the wells. In the co-cultivation experiments performed for the first time in our study, 50 µL (equivalent to 5 × 104 CFU/mL) of both S. aureus and A. baumannii bacteria were concurrently introduced in equal proportions (1:1) to the wells.

The fractional inhibitory concentration index (FICi) for the combined antibacterial agents was calculated as follows [34]:

$$\eqalign}\,}\, & \left( }\,}\,}\,}\,}\,}\,}\,}} \right)} \cr & }\,\left( }\,}\,}\,}\,}\,}\,}\,}} \right) \cr}$$

The obtained value was interpreted as follows: less than 0.5 indicated synergy, between 0.5 and 1 indicated relative synergy, equal to 1 indicated an additive effect, between 1 and 4 indicated no interaction, and more than 4 indicated antagonism [35].

Moreover, to explore the interactions among antibacterial drugs, the MBC values were assessed using the broth microdilution checkerboard technique, referred to as the fractional bactericidal concentration index (FBCi). This method for evaluating antibacterial interactions was similar to the FIC method. A 100 µL aliquot of diluted bacterial stock at 105 CFUs/mL was added to each well, followed by incubation at 37 °C for 24 h. After incubation, 10 µL from each well was plated on MHA, allowing for the determination of the MBC values for gentamicin, imipenem, and fucoidan, defined as the lowest concentrations needed to eliminate 100% of the cultured isolates. The FBCi was then calculated using the formula:

$$\eqalign}\,} & \,\left( }\,}\,}\,}\,}\,}\,}\,}\,}\,}} \right) \cr } & }\,\left( }\,}\,}\,}\,}\,}\,}\,}\,}\,}} \right) \cr}$$

These FBCi indices were employed to identify the type of interaction between the antibacterial agents, analogous to the FICi method [36].

Determining the synergistic effects in the biofilm state of isolates

Utilizing MBIC, the fractional biofilm inhibitory concentration index (FBICi) for six isolates, including S. aureus 6, S. aureus 7, S. aureus 22, A. baumannii 1, A. baumannii 8, and A. baumannii 20, was determined. Briefly, preformed dual-species biofilms were established in 96 U-shape microplates over 24 h. Subsequently, serial dilutions of antibiotics ranging from 1 to 1024 µg/mL (100 µL each) and fucoidan (3.9 to 1000 µg/mL) were added to the wells, followed by overnight incubation at 37 °C. The FBICi for combined agents was calculated using the formula [36]:

$$\eqalign}}_}}}\, = & \,\left( }\,}\,}\,}\,}\,}\,}\,}\,}} \right) \cr } & }\,\left( }\,}\,}\,}\,}\,}\,}\,}\,}} \right) \cr}$$

The resultant values indicated the nature of drug interaction as described above.

Additionally, employing MBEC, a novel formula, termed Fractional biofilm eradication concentration index (FBECi), was introduced to assess the interaction between antimicrobial agents against the biofilm form. FBECi for two combined anti-biofilm agents was calculated using the formula:

$$\eqalign}}_}}}\,} & \,\left( }\,}\,}\,}\,}\,}\,}\,}\,}} \right)} \cr & }\,\left( }\,}\,}\,}\,}\,}\,}\,}\,}} \right) \cr}$$

The resulting values conveyed the type of drug interaction akin to the FBICi calculations mentioned earlier.

Field emission scanning electron microscopy (FE-SEM)

Field emission scanning electron microscopy (FE-SEM) was employed to visualize the synergistic effects of gentamicin, imipenem, and fucoidan at their FBIC amounts on dual-species biofilms of S. aureus and A. baumannii. Two strains were used in this study: S. aureus 6 and A. baumannii 1. Sample preparation was conducted according to the protocol described by Shams Khozani et al., with some modifications [37]. Briefly, fresh bacterial cultures were grown in 1% Glu TSB at 37 °C for 24 h. The biofilms were then treated with combinations of gentamicin-imipenem, gentamicin-fucoidan, and imipenem-fucoidan, each incubated with a bacterial suspension at a concentration of 1.5 × 10⁷ CFU/mL at 37 °C for 24 h, according to their respective FBIC doses. Prior to incubation, sterile slides were cut and placed into the wells. Following incubation, the slides were gently washed three times with sterile distilled water and then fixed in 2.5% glutaraldehyde in 1x PBS for 3 h at room temperature. The slides were subsequently rinsed three times in distilled water and post-fixed in 1.5% osmium tetroxide for 1 h. Following three additional rinses in distilled water, the samples were dehydrated using graded alcohol concentrations of 20%, 30%, 50%, 70%, 80%, 90%, and 100%, with each step lasting 10 min. The specimens were then mounted on conductive copper SEM tape, coated with gold nanoparticles, and examined using an FE-SEM instrument (MIRA3, TESCAN Co., Czechia).

Effect of antimicrobials on the biofilm encoding genes

Biofilm-forming strains were chosen for real-time PCR analysis to examine the expression of biofilm-related genes icaA in S. aureus and bap in A. baumannii. After creating single-species biofilm and dual-species biofilms, these strains were treated overnight with 1/2 FBIC concentrations of synergistic gentamicin, imipenem and fucoidan. The following day, total RNA was extracted using a RNX-plus Mini Kit (Sinaclon, Iran), according to the manufacturer’s guidelines. The RNA concentration, purity, and integrity were evaluated. Subsequently, 1 µg of RNA was used for a cDNA Synthesis kit (Parstous Biotechnology, Iran), following the manufacturer’s instructions. Gene expression was then assessed using the 2X Q-PCR Master Mix with 2 µL of cDNA and 1 µL of each icaA, bap, and 16 S rRNA primers in a 20 µL volume on a real-time PCR device (LightCycler® 96 Instrument, Roche, United States). The primers for icaA, bap, and 16 S rRNA were obtained from previous studies [38, 39]. A standard curve, created using serial dilutions of mRNA from untreated S. aureusATCC 29213 for icaA and untreated A. baumannii ATCC 19606 for bap, was used to ensure amplification efficiency [40]. Gene expression was ultimately calculated using the Ct assay, with 16 S rRNA genes serving as internal controls for each bacterium.

Toxicity assays

The cytotoxicity of fucoidan on host cells was assessed using the MTT assay [41]. Human Skin Fibroblast cells (HSF-PI 16) were cultivated in DMEM supplemented with 10% fetal calf serum (FCS) and antibiotics (100 U/mL penicillin and 100 U/mL streptomycin). The cells were maintained at 37 °C with 5% CO2 and 95% relative humidity until they reached a density of 4 × 104 cells per well and were then cultured overnight. Subsequently, various concentrations of fucoidan, ranging from 1000 to 3.9 µg, were prepared. Additionally, combinations of fucoidan at doses of 125 µg and 62.5 µg with imipenem at concentrations of 32 µg and 64 µg, as well as fucoidan at 125 µg and 62.5 µg with gentamicin at 32 µg and 64 µg, were added to a 96-well microplate. The plates were then incubated for 24 h at 37 °C. The supernatant was aspirated, and 100 µL of DMSO was included in each well to dissolve the formazan crystals. The absorbance at 570 nm was determined using a spectrophotometer (BioTek, USA). Cell viability was computed using the formula: Percentage survival = (OD test / OD control) × 100.

Statistical analysis

The statistical analyses were performed using GraphPad Prism 9 software (GraphPad Software, Inc., La Jolla, CA, United States). A t-test was used to evaluate the significance of the findings regarding the anti-biofilm effect of the combination of antibiotics. Additionally, the ANOVA test was employed to compare the survival rates of the HSF-PI 16 cell line exposed to different concentrations of fucoidan relative to the control group and the gene expression between the single-species biofilm isolates and the dual-species biofilms, as well as the FBIC values. All assays were conducted with a confidence level of 95%, and a p-value of less than 0.05 was considered statistically significant. To describe the correlation between the examined concentrations and the percentage of activities, a non-linear regression test was performed. All experiments were conducted in triplicate.