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The antimicrobial and antibiofilm effects of gentamicin, imipenem, and fucoidan combinations against dual-species biofilms of Staphylococcus aureus and Acinetobacter baumannii isolated from diabetic foot ulcers

Annals of Clinical Microbiology and Antimicrobials volume 23, Article number: 101 (2024) Cite this article

Abstract

Introduction

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycemia due to impaired insulin production or utilization, leading to severe health complications. Diabetic foot ulcers (DFUs) represent a major complication, often exacerbated by polymicrobial infections involving Staphylococcus aureus and Acinetobacter baumannii. These pathogens, notorious for their resistance to antibiotics, complicate treatment efforts, especially due to biofilm formation, which enhances bacterial survival and resistance. This study explores the synergistic effects of combining gentamicin, imipenem, and fucoidan, a sulfated polysaccharide with antimicrobial properties, against both planktonic and biofilm forms of S. aureus and A. baumannii.

Methods

Isolates of S. aureus and A. baumannii were collected from DFUs and genetically confirmed. Methicillin resistance in S. aureus was identified through disk diffusion and PCR. Biofilm formation, including dual-species biofilms, was analyzed using the microtiter plate method. The antimicrobial efficacy of gentamicin, imipenem, and fucoidan was assessed by determining the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), minimum biofilm inhibitory concentration (MBIC), and minimum biofilm eradication concentration (MBEC). Synergistic interactions were evaluated using the fractional inhibitory concentration index (FICi) and fractional bactericidal concentration index (FBCi). The expression of biofilm-associated genes (icaA in S. aureus and bap in A. baumannii) was analyzed, and the cytotoxicity of fucoidan was assessed.

Results

The study revealed that 77.4% of S. aureus and all A. baumannii isolates showed multidrug resistance. Among 837 tested conditions for dual-species biofilm formation, 72 resulted in strong biofilm formation and 67 in moderate biofilm formation. The geometric mean MIC values for gentamicin were 12.2 µg/mL for S. aureus, 22.62 µg/mL for A. baumannii, and 5.87 µg/mL for their co-culture; for imipenem, they were 19.84, 9.18, and 3.70 µg/mL, respectively, and for fucoidan, 48.50, 31.20, and 19.65 µg/mL, respectively. The MBC values for gentamicin were 119.42, 128, and 11.75 µg/mL; for imipenem, they were 48.50, 14.92, and 8 µg/mL; and for fucoidan, they were 88.37, 62.62, and 42.48 µg/mL. The MBIC values were 55.71, 119.42, and 18.66 µg/mL for gentamicin; 68.59, 48.50, and 25.39 µg/mL for imipenem; and 153.89, 101.49, and 53.53 µg/mL for fucoidan. The MBEC values were 315.17, 362.03, and 59.25 µg/mL for gentamicin; 207.93, 157.58, and 74.65 µg/mL for imipenem; and 353.55, 189.46, and 99.19 µg/mL for fucoidan. When cultured in planktonic form, the geometric mean FICi and FBCi values indicated additive effects, while co-culture showed FICi values of ≤ 0.5, suggesting a synergistic interaction. Treatment with gentamicin and fucoidan led to significant downregulation of the icaA and bap genes in both single-species and dual-species biofilms of S. aureus and A. baumannii. The reductions in gene expression were more pronounced in dual-species biofilms compared to single-species biofilms. Additionally, treatment with imipenem and fucoidan also resulted in significant downregulation of these genes in both biofilm types. Cytotoxicity assessments indicated that higher concentrations of fucoidan were toxic, yet no harmful effects were noted at the optimal synergistic concentrations used with antibiotics.

Conclusion

In our investigation, we found that combining gentamicin, imipenem, and fucoidan had a synergistic effect on dual-species biofilms of S. aureus and A. baumannii, suggesting potential benefits for treating such infections effectively. This underscores the importance of understanding microbial interactions, antibiotic susceptibility, and biofilm formation in DFUs.

Introduction

Diabetes mellitus is identified as a chronic metabolic disorder marked by ongoing hyperglycemia, stemming from the body’s inability to produce or effectively utilize insulin [1]. The increasing global prevalence of diabetes presents considerable health, social, and economic challenges [2]. Among the various complications that arise from diabetes, diabetic foot ulcers (DFUs) stand out as particularly debilitating, often leading to severe infections, extended hospitalizations, and even amputations [3]. These chronic, non-healing wounds play a major role in the mortality rates among diabetic patients, emphasizing the critical necessity for effective management strategies [4].

Bacterial infections complicate DFUs and can often be polymicrobial [5]. Staphylococcus aureus and Acinetobacter baumannii are two of the most concerning pathogens associated with these ulcers [6]. Known for its ability to evade the immune response, S. aureus, especially its methicillin-resistant strains (MRSA), can cause a variety of infections [7]. A. baumannii, a highly adaptable Gram-negative bacterium, is infamous for its resistance to multiple antibiotics and its role in hospital-acquired infections [8]. The presence of these pathogens not only aggravates the severity of the wounds but also complicates treatment efforts due to their resilience.

Biofilm formation plays a crucial role in the persistence of these infections [9]. Biofilms consist of structured groups of bacteria that are enclosed in a self-produced extracellular matrix, allowing them to adhere to surfaces, including tissues and medical devices [10]. Within biofilms, bacteria demonstrate enhanced resistance to antibiotics and immune responses from the host [11]. The coexistence of S. aureus and A. baumannii within biofilms creates an even more significant challenge, as their interactions may bolster their survival mechanisms and resistance, complicating efforts to eradicate these infections [12].

Fucoidan, a type of sulfated polysaccharide, is abundantly produced by diverse algae species, and its biological properties have been extensively investigated [13]. The antimicrobial activity of fucoidan is attributed to its ability to hinder bacterial nutrient exchange and exert inhibitory effects on bacterial pathogens by modulating the bacterial cell wall permeability or forming a gel-like barrier on the bacterial membrane [14]. The escalating resistance of bacteria to conventional antibiotics has prompted the exploration of novel antimicrobial agents. Natural compounds, such as fucoidan, offer promise as potential sources of antimicrobial drugs due to their perceived lower side effects and cost-effectiveness.

Interactions among microbes that confer mutual benefits, particularly within mixed biofilms, play a significant role in increasing resistance to antimicrobial agents [15]. Combination therapy involves the simultaneous use of two or more active antibiotics [16]. This approach lowers the likelihood of drug resistance, reduces the need for high doses of potentially toxic drugs, and often produces more potent effects in biochemical activity compared to monotherapy [17, 18]. The majority of retrospective studies have indicated that combination therapy yields greater effectiveness compared to monotherapy [19,20,21]. The efficacy of combination therapy remains a subject of scientific debate. However, concurrent cultivation has shown a potential for heightened sensitivity in S. aureus and P. aeruginosa, prompting speculation regarding similar behavior in S. aureus and A. baumannii [22, 23]. Research on the optimal antibiotic combinations for addressing infections caused by these isolates is currently limited. In this study, we explored the synergistic effects of combining gentamicin and imipenem antibiotics with fucoidan against both individual and co-culture planktonic conditions of S. aureus and A. baumannii, as well as in single-species biofilm and dual-species biofilm conditions.

Materials and methods

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]:

$${\rm{MBIC}}\,{\rm{ = }}\,\left[ {{\rm{1}}\, - \,\left( {{\rm{OD}}\,{\rm{test}}\,{\rm{/}}\,{\rm{OD}}\,{\rm{control}}} \right)} \right]\,{\rm{ \times }}\,{\rm{100\% }}$$

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]:

$$\eqalign{{\rm{FICiA/B}}\,{\rm{ = }}\, & \left( {{\rm{MIC}}\,{\rm{drug}}\,{\rm{A}}\,{\rm{in}}\,{\rm{combination/MIC}}\,{\rm{drug}}\,{\rm{A}}\,{\rm{alone}}} \right){\rm{ }} \cr & {\rm{ + }}\,\left( {{\rm{MIC}}\,{\rm{drug}}\,{\rm{B}}\,{\rm{in}}\,{\rm{combination/MIC}}\,{\rm{drug}}\,{\rm{B}}\,{\rm{alone}}} \right) \cr}$$

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:

$$\eqalign{{\rm{FBCiA/B}}\,{\rm{ = }} & \,\left( {{\rm{MBC}}\,{\rm{of}}\,{\rm{drug}}\,{\rm{A}}\,{\rm{in}}\,{\rm{combination/MBC}}\,{\rm{of}}\,{\rm{drug}}\,{\rm{A}}\,{\rm{alone}}} \right) \cr {\rm{ }} & {\rm{ + }}\,\left( {{\rm{MBC}}\,{\rm{of}}\,{\rm{drug}}\,{\rm{B}}\,{\rm{in}}\,{\rm{combination/MBC}}\,{\rm{of}}\,{\rm{drug}}\,{\rm{B}}\,{\rm{alone}}} \right) \cr}$$

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]:

$$\eqalign{{\rm{FBIC}}{{\rm{i}}_{{\rm{A/B}}}}\, = & \,\left( {{\rm{MBIC}}\,{\rm{drug}}\,{\rm{A}}\,{\rm{in}}\,{\rm{combination/}}\,{\rm{MBIC}}\,{\rm{drug}}\,{\rm{A}}\,{\rm{alone}}} \right) \cr {\rm{ }} & {\rm{ + }}\,\left( {{\rm{MBIC}}\,{\rm{drug}}\,{\rm{B}}\,{\rm{in}}\,{\rm{combination/}}\,{\rm{MBIC}}\,{\rm{drug}}\,{\rm{B}}\,{\rm{alone}}} \right) \cr}$$

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:

$$\eqalign{{\rm{FBEC}}{{\rm{i}}_{{\rm{A/B}}}}\,{\rm{ = }} & \,\left( {{\rm{MBEC}}\,{\rm{drug}}\,{\rm{A}}\,{\rm{in}}\,{\rm{combination/}}\,{\rm{MBEC}}\,{\rm{drug}}\,{\rm{A}}\,{\rm{alone}}} \right){\rm{ }} \cr & {\rm{ + }}\,\left( {{\rm{MBEC}}\,{\rm{drug}}\,{\rm{B}}\,{\rm{in}}\,{\rm{combination/}}\,{\rm{MBEC}}\,{\rm{drug}}\,{\rm{B}}\,{\rm{alone}}} \right) \cr}$$

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.

Result

MRSA and biofilm production analysis

The findings from both antibacterial sensitivity assessments and molecular analyses, focusing on the FOX disc and the mecA gene, revealed that 80.6% (n = 25) of S. aureus isolates exhibited characteristics consistent with Methicillin-Resistant S. aureus (MRSA). Notably, a considerable proportion of both S. aureus and A. baumannii isolates demonstrated variable levels of biofilm formation, as evidenced by OD values ranging from 0.2 to 2.8 across all isolates. Furthermore, based on these findings, the isolates’ biofilm-forming capacity was classified into strong, intermediate, and weak producers, as detailed in both Tables 1 and 2.

Table 1 Characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates, multiple drug-resistance (MDR) isolates, antibiotic resistance patterns, and biofilm formation types in Staphylococcus aureus
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Table 2 Characteristics of multiple drug-resistance (MDR) isolates, antibiotic resistance patterns, and biofilm formation types in Acinetobacter baumannii
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Antibiotic susceptibility patterns and MDR isolates

An examination of disk diffusion data from clinical isolates was undertaken to evaluate antibiotic resistance rates. In S. aureus, resistance rates to IMP, TS, E, CD, GM, LZD, and VAN were determined as 80.6%, 67.7%, 64.5%, 58.5%, 51.6%, 0%, and 0%, respectively. Similarly, antibiotic resistance rates for selected clinical isolates of A. baumannii, based on disk diffusion data, were as follows: PIP 100%, CEF 100%, AM 100%, TET 92.6%, AUG 85.1%, GM 77.7%, MER 77.7%, CIP 74.0%, and IMP 55.5%. Overall, 77.4% of S. aureus isolates and 100% of A. baumannii isolates exhibited MDR. Further information regarding the antimicrobial susceptibility testing of antibiotics against the isolates is provided in Tables 1 and 2. The statistical analysis is essential in assessing the relationship between MDR and biofilm formation (P < 0.05). Our findings reveal a noteworthy association between antibiotic resistance and the presence of strong biofilm formations among S. aureus clinical isolates (P < 0.05). Refer to Table 3 for further details.

Table 3 Comparison of biofilm phenotypic patterns between MDR and non-MDR Staphylococcus aureus clinical isolates
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Ability to formation of dual-species biofilms

We investigated various conditions for all isolates (31 S.aureus and 27 A. baumanii), revealing that 72 conditions formed strong dual-species biofilms, while 67 formed moderate dual-species biofilms out of a total 837 conditions. Detailed information for all isolates is presented in Table 4; Fig. 1. In some cases, despite the fact that OD was higher than the negative control, the growth of both bacteria after biofilm formation was not observed in culture on MSA and Leeds media, in which case it was reported as non-biofilm formation.

Fig. 1
figure 1

Biofilm formation classification based on optical density (OD) values in single-species and dual-species biofilms of Staphylococcus aureus and Acinetobacter baumannii

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Table 4 Data regarding the formation of dual-species biofilm
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MIC and MBC ranges and MBC/MIC values

In isolated individual cultures of S. aureus, the geometric mean MIC values were 12.12 µg/mL for gentamicin, 19.84 µg/mL for imipenem, and 48.50 µg/mL for fucoidan. For isolated individual cultures A. baumannii, these values were 22.62 µg/mL for gentamicin, 9.18 µg/mL for imipenem, and 31.20 µg/mL for fucoidan. Similarly, the geometric mean MBC values for gentamicin, imipenem and fucoidan in S. aureus were 119.42, 48.50, and 88.37 µg/mL, respectively, while for A. baumannii, they were 128, 14.92 and 62.62 µg/mL, respectively. Additionally, the geometric mean values for MBC/MIC for gentamicin, imipenem and fucoidan were 9.84, 2.46, and 2.14 respectively, in S. aureus, and 5.65, 1.74, and 2 respectively, in A. baumannii. For further details, refer to Table 5.

Table 5 Ranges of MIC, MBC, and MBC/MIC ratio for gentamicin, imipenem and fucoidan
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In the co-culture condition, we selected six isolates for investigation: S. aureus 6, S. aureus 7, S. aureus 22, A. baumannii 1, A. baumannii 8, and A. baumannii 20. These isolates are capable of forming dual-species biofilms under nine different conditions (3 × 3). Among these, eight conditions lead to strong dual-species biofilms, while one condition results in a moderate dual-species biofilm. MIC and MBC values were assessed in co-culture contexts where inhibition or eradication of both bacterial species occurred, as detailed in Table 5; Fig. 2A. The geometric mean MIC and MBC values, as well as the MBC/MIC ratios for gentamicin, imipenem and fucoidan, were calculated as follows: 5.87 µg/mL, 11.75 µg/mL, and 1.85 for gentamicin, 3.70 µg/mL, 8 µg/mL, and 2.16 for imipenem, and 19.65 µg/mL, 42.48 µg/mL, and 2.16 for fucoidan respectively.

Fig. 2
figure 2

A) The geometric mean values of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for both Staphylococcus aureus and Acinetobacter baumannii, under planktonic conditions. These values are shown for each species individually, as well as when the two bacteria are co-cultured. B) The geometric mean values of the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) are depicted for S. aureus and A. baumannii in both single-species biofilms and in dual-species biofilms, where both bacteria are present together

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Evaluation of synergy in the planktonic state

In the present study, the geometric mean values for the most effective synergistic concentrations of gentamicin and imipenem, as determined by FICi, against isolates of S. aureus and A. baumannii across co-culture conditions of three selected isolates were 0.46, 0.34, and 0.21, respectively. For the combination of gentamicin and fucoidan, the geometric mean values were 0.63, 0.50, and 0.22, respectively. Additionally, for imipenem and fucoidan, the values were 0.71, 0.47, and 0.32, respectively.

Furthermore, the geometric mean values for the optimal synergistic concentrations, assessed by FBCi, revealed that for gentamicin and imipenem against S. aureus and A. baumannii isolates, the values were 0.42, 0.30, and 0.27, respectively. For gentamicin and fucoidan, the geometric mean values were 0.66, 0.59, and 0.22, respectively. Lastly, for imipenem and fucoidan, the values were 0.43, 0.45, and 0.33, respectively. Detailed information is provided in Table 6.

Table 6 The optimal synergistic concentrations of gentamicin-imipenem, gentamicin-fucoidan, and imipenem-fucoidan against planktonic cultures
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MBIC and MBEC ranges

In isolated samples of S. aureus, the geometric mean MBIC values were 55.71 µg/mL for gentamicin, 68.59 µg/mL for imipenem, and 153.89 µg/mL for fucoidan. For isolated A. baumannii samples, the corresponding values were 119.42 µg/mL for gentamicin, 48.50 µg/mL for imipenem, and 101.49 µg/mL for fucoidan.

Additionally, the geometric mean MBEC values for S. aureus were 315.17 µg/ml for gentamicin, 207.93 µg/mL for imipenem, and 353.55 µg/mL for fucoidan. For A. baumannii, the values were 362.03 µg/mL for gentamicin, 157.58 µg/mL for imipenem, and 189.46 µg/mL for fucoidan.

In dual-species biofilms, the geometric mean MBIC and MBEC values were 18.66 µg/mL and 59.25 µg/mL for gentamicin, 25.39 µg/mL and 74.65 µg/mL for imipenem, and 53.53 µg/mL and 99.19 µg/mL for fucoidan, respectively. These results are further detailed in Table 7; Fig. 2B.

Table 7 Ranges of MBIC and MBEC for gentamicin, imipenem, and fucoidan
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Evaluation of synergy in the Biofilm condition

In our investigation, we determined the geometric mean values for the most potent synergistic concentrations of various antibiotic combinations against isolates of S. aureus and A. baumannii under dual-species biofilm conditions. Using the FBICi, we found that for gentamicin and imipenem, the values were 0.82, 0.59, and 0.29 across three selected isolates. For gentamicin and fucoidan, the values were 0.47, 0.44, and 0.28, respectively. Similarly, the combination of imipenem and fucoidan yielded values of 0.47, 0.46, and 0.37.

Additionally, we evaluated the optimal synergistic concentrations using the FBECi. For gentamicin and imipenem, the geometric mean values were 0.98, 0.69, and 0.24. For gentamicin and fucoidan, they were 0.55, 0.58, and 0.36. Lastly, for imipenem and fucoidan, the values were 0.52, 0.48, and 0.39 across the same isolates. Further details of these findings are presented in Table 8.

Table 8 The optimal synergistic concentrations of gentamicin-imipenem, gentamicin-fucoidan, and imipenem-fucoidan against biofilm condition
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FE-SEM

The synergistic potential of gentamicin, imipenem, and fucoidan in eradicating or neutralizing dual-species biofilms of S. aureus and A. baumannii was evaluated using FE-SEM. The results demonstrated that the combination of fucoidan with antibiotics at their FBIC concentrations had a significant degradative effect on the biofilm structures. Specifically, the synergy between fucoidan and the antibiotics at these concentrations led to substantial disruption of the dual-species biofilm, causing bacterial lysis and a marked reduction in the overall biofilm biomass. The cohesive biofilm structures were broken down into smaller, less organized fragments. Moreover, the combination of gentamicin and imipenem also showed a strong synergistic effect, leading to the reduction of large biofilm biomass, bacterial lysis, and the formation of detached bacterial cells (Fig. 3).

Fig. 3
figure 3

Effect of combination gentamicin, imipenem, and, fucoidan on dual-species biofilms of Staphylococcus aureus and Acinetobacter baumannii. (A) single-species biofilm S. aureus (Untreated), (B) single-species biofilm A. baumannii (Untreated), (C) dual-species biofilm of S. aureus and A. baumannii (Untreated), (D) dual-species biofilm treated with 32 μg/mL gentamicin and 64 μg/mL imipenem, (E) dual-species biofilm treated with 32 μg/mL gentamicin and 31.2 μg/mL fucoidan, (F) dual-species biofilm treated with 31.2 μg/mL fucoidan and 64 μg/mL imipenem. LBB, large biofilm biomass; SBB, small biofilm biomass

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Activity of synergistic effects on the biofilm encoding genes

Expression levels of the icaA gene in S. aureus and the bap gene in A. baumannii were evaluated after 24-hour exposure to 1/2 MBIC concentrations of gentamicin combined with imipenem, gentamicin combined with fucoidan, and imipenem combined with fucoidan. In single-species biofilms, icaA expression was downregulated by 2.98- to 1.52-fold, and bap expression by 3.15- to 1.94-fold. For dual-species biofilms, icaA in S. aureus decreased by 5.92- to 3.64-fold, while bap in A. baumannii was reduced by 5.78- to 4.17-fold. With gentamicin and fucoidan, icaA was downregulated by 3.32- to 1.90-fold and bap by 2.86- to 1.79-fold in single-species biofilms. In dual-species biofilms, icaA decreased by 6.12- to 4.20-fold in S. aureus, and bap by 5.68- to 3.79-fold in A. baumannii. After treatment with imipenem and fucoidan, icaA was downregulated by 3.1- to 1.72-fold, and bap by 3.12- to 1.68-fold in single-species biofilms. In dual-species biofilms, icaA decreased by 5.53- to 3.75-fold in S. aureus, and bap by 6.87- to 4.48-fold in A. baumannii. ANOVA showed significant differences in gene expression between single-species and dual-species biofilms (p < 0.05) (Fig. 4).

Fig. 4
figure 4

Reduction in expression of biofilm-related genes icaA (A) in Staphylococcus aureus and Bap (B) in Acinetobacter baumannii when exposed to sub-FBIC of gentamicin, imipenem and fucoidan (GEN, gentamicin; IMP, imipenem, FUC, fucoidan; mean ± SD, n = 3, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001)

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Cytotoxicity of fucoidan

Cytotoxicity assessments revealed that fucoidan concentrations of 1000, 500, and 250 µg resulted in cytotoxicity levels of 78.3%, 42.1%, and 11%, respectively, in HSF-PI 16 cells. Importantly, when fucoidan was combined with the antibiotics imipenem (32 and 64 µg) and gentamicin (32 and 64 µg) at optimal synergistic concentrations of 125, 62.5, 32.1, 15.6, and 7.8 µg, no cytotoxic effects were detected in HSF-PI 16 cells. Furthermore, statistical analysis using a t-test indicated no significant differences in survival rates between the fucoidan treatments at these concentrations and the control sample (p = 0.083).

Discussion

The DFUs represent one of the most severe complications of diabetes mellitus, often leading to chronic infections, delayed wound healing, and in extreme cases, limb amputation [42]. Among the microorganisms commonly involved in DFU infections, S. aureus and A. baumannii are particularly problematic due to their ability to form biofilms and develop resistance to antibiotics [9]. Biofilms, a structural mode of bacterial growth, significantly enhance bacterial survival and protect the organisms from antimicrobial agents, making infections exceedingly difficult to treat [43]. The increasing prevalence of MDR strains further exacerbates this challenge, emphasizing the urgent need for alternative therapeutic strategies [43].

Regrettably, a significant proportion of the S. aureus strains isolated in our study demonstrated resistance to methicillin, exceeding the rates reported in other studies [44,45,46]. Additionally, 77.41% (n = 24) of the isolates were capable of forming strong and moderate biofilms. This indicates a significant association between this type of biofilm and the MDR of S. aureus isolates, a relationship we had previously demonstrated in P. aeruginosa studies [47]. Additionally, all isolates exhibited susceptibility to two antibiotics, linezolid and vancomycin. This observation underscores the continued efficacy of these drugs as preferred choices for treatment [48,49,50]. In hospital settings, upon detecting S. aureus in diabetic wounds, vancomycin is typically prescribed promptly. However, administering intravenous vancomycin to hospitalized patients can result in considerable expenses due to extended hospital stays, staffing requirements, and potential adverse reactions [51]. Of particular concern is vancomycin-associated acute kidney injury, especially for diabetic patients who are already at a higher risk of kidney complications [52]. Furthermore, the emergence of vancomycin-resistant strains of S. aureus highlights the need to explore alternative treatment strategies [53]. Therefore, employing a combination of two antibiotics with synergistic effects offers a sensible approach to treatment. In this study, upon investigating the presence of A. baumannii in diabetic wounds, we discovered that all isolates were MDR, and 88.8% of the bacteria demonstrated the ability to form strong and moderate biofilms, a concerning finding. Additionally, given the weakened immune system of diabetic individuals, the likelihood of developing a mixed infection, particularly with S. aureus, is notably high, which can cause an increase in infection severity may occur [54]. This significantly diminishes the prospects of successful treatment.

In polymicrobial infections, inherent resistance mechanisms of pathogens can influence the efficacy of certain antibiotics, sometimes resulting in enhanced performance during polymicrobial formation. Previous studies have shown that Pseudomonas spp. can alter the activity of antistaphylococcal agents by releasing endopeptidases, rhamnolipids, and 2-heptyl-4-hydroxyquinolone N-oxide [55, 56]. Our study found that co-culturing S. aureus and A. baumannii resulted in a lower geometric mean MIC and MBC for gentamicin, imipenem, and fucoidan compared to individual cultures, indicating increased sensitivity in co-culture. These findings align with previous research by Smith et al., which demonstrated that S. aureus enhances the sensitivity of A. baumannii to meropenem in co-culture [57]. However, Castellanos et al. observed no significant growth reduction or change in antibiotic susceptibility when co-culturing clinical strains of A. baumannii and S. aureus from a diabetic patient’s skin and soft tissues [54]. This discrepancy might be due to the small sample size in the study by Castellanos et al. The observed reduction in resistance to imipenem, gentamicin, and fucoidan in our study can be attributed to several factors. Firstly, synergistic interactions between the bacterial species might enhance antibiotic susceptibility through metabolic interactions, gene expression changes, or alterations in the bacterial cell envelope [58]. Secondly, resource competition in the co-culture environment might induce physiological changes, such as metabolic shifts or altered growth rates, making the pathogens more susceptible to antibiotics [59]. Lastly, bacterial communication via quorum sensing and signaling molecules might affect antibiotic susceptibility by influencing the production and perception of these molecules in co-culture conditions [60, 61]. Moreover, the reduced geometric mean ratio of MBC to MIC in the co-culture of S. aureus and A. baumannii with imipenem, gentamicin, and fucoidan suggests an increase in the bactericidal potency of these antibiotics compared to their efficacy in individual cultures. This enhanced bactericidal effect underscores the potential benefits of considering polymicrobial interactions in antibiotic treatment strategies.

Rewrite the following text:

In our study, based on OD values, we observed that 16.6% (139/837) of the various conditions for the tested isolates were capable of forming moderate to strong dual-species biofilms. Notably, certain strains of S. aureus (e.g., S. aureus 2, S. aureus 10, and S. aureus 26) demonstrated an inability to form dual-species biofilms when paired with any of the A. baumannii isolates. However, this pattern was not seen with A. baumannii, suggesting that S. aureus may have a more substantial role in influencing dual-species biofilm formation compared to A. baumannii. It is also noteworthy, as demonstrated in previous studies, that when S. aureus and A. baumannii were co-cultured, there was no significant reduction in the growth of either clinical strain, indicating that both bacteria can coexist within the same infection site [54, 62]. This coexistence may further complicate treatment strategies and contribute to the persistence of infections in clinical settings.

In dual-species biofilms, S. aureus showed a marked increase in susceptibility to imipenem and gentamicin. Prior studies have underscored the role of siderophores produced by A. baumannii in increasing S. aureus susceptibility to various antibiotics, offering a potential explanation for the observed effect in our study [63]. Moreover, treatment of dual-species biofilms containing S. aureus and A. baumannii with broad-spectrum imipenem and gentamicin led to nearly fivefold lower inhibitory concentrations and eradication of both bacteria compared to monoculture treatment. This phenomenon mirrors findings from previous research indicating reduced S. aureus resistance to aminoglycosides in dual-species biofilms with P. aeruginosa [64]. Additionally, our investigation revealed that S. aureus impacts the susceptibility of A. baumannii biofilm-embedded cells to antimicrobials, especially aminoglycosides. However, the precise mechanism underlying the enhanced susceptibility of A. baumannii remains uncertain, suggesting the involvement of a specific metabolite produced by S. aureus, although the exact active molecule has yet to be identified. Additionally, in our study, the anti-biofilm effect of fucoidan was observed against both A. baumannii and S. aureus, in both single-species biofilm and dual-species biofilms. This finding aligns with the results of Chmit et al., who demonstrated the anti-biofilm activity of fucoidan against Gram-positive bacteria (S. aureus, Enterococcus faecalis, and S. epidermidis) as well as two Gram-negative bacteria (E. coli and P. aeruginosa) [65]. Furthermore, Jun et al. also reported an anti-biofilm effect of fucoidan on dental plaque bacteria, specifically targeting biofilms formed by Streptococcus mutans and S. sobrinus [66].

The analysis of the synergistic effects of gentamicin, imipenem, and fucoidan against S. aureus and A. baumannii revealed distinct outcomes based on the bacterial state. When these bacteria were in their planktonic form and cultured individually, the geometric mean FICi and FBCi values generally indicated additive effects, with values between 0.5 and 1.0. However, when the bacteria were co-cultured, the FICi values dropped to ≤ 0.5, indicating a synergistic interaction [35]. This suggests that the combined effect of these antibiotics is significantly enhanced under co-culture conditions. These findings align with previous research by Lee et al., who observed synergistic effects of fucoidan when combined with ampicillin and gentamicin against oral pathogens [67]. Similarly, Choi et al. reported that fucoidan improved the effectiveness of oxacillin and ampicillin against MRSA [68]. The observed synergy in planktonic cultures extends to dual-species biofilms of S. aureus and A. baumannii, suggesting that the simultaneous administration of gentamicin and imipenem, in the presence of fucoidan, could improve the treatment outcomes for infections involving dual-species biofilms.

The synergistic potential of gentamicin, imipenem, and fucoidan in eradicating or neutralizing dual-species biofilm of S. aureus and A. baumannii was assessed through the use of FE-SEM. The experimental outcomes revealed that when fucoidan was combined with antibiotics at the concentration of FBICs, a remarkable and potent degradative effect was observed in biofilm structures. According to the results, fucoidan and antibiotics at the concentration of FBICs produced high destruction effects on the dual-species biofilm layer and interbacterial biofilm and lysis of bacteria and it reduced the large biofilm biomass and turned it into small biofilm biomass and destroyed the cohesive structures of the biofilm. Synergism of gentamicin-imipenem also decreased large biofilm biomass, caused bacterial lysis, and led to the formation of detached bacteria (Fig. 3).

To explore the anti-biofilm efficacy of the synergistic combination of gentamicin, imipenem, and fucoidan and to elucidate its underlying mechanisms, we conducted real-time PCR analysis to evaluate the expression of biofilm-associated genes in both single-species biofilm and dual-species biofilms. Our findings revealed a significant downregulation of the icaA gene in S. aureus and the bap gene in A. baumannii within dual-species biofilms compared to single-species biofilms. This observation is consistent with the study by Mani et al., which also demonstrated that their treatment not only exhibited antibiofilm and antimicrobial effects against Proteus vulgaris and Salmonella enterica but also effectively suppressed the expression of biofilm-related genes in these pathogens [69].

Conclusion

Our investigation into the combined effect of gentamicin, imipenem and fucoidan revealed synergistic interactions in dual-species biofilms of S. aureus and A. baumannii, presenting promising implications for treatment efficacy. Leveraging the synergistic effects of these antimicrobials holds the potential to improve treatment outcomes for dual-species biofilm infections. These findings highlight the intricate interplay between microbial interactions, antibiotic susceptibility, and biofilm formation in DFUs, emphasizing the necessity of tailored therapeutic approaches to effectively manage these complex infections. Prospective studies could center on exploring the precise antibacterial and anti-biofilm mechanisms of the gentamicin, imipenem and fucoidan combination via in vivo and molecular analyses. Furthermore, from a broader perspective, we propose that introducing antagonistic bacteria into preexisting monoculture biofilms could enhance their antimicrobial treatment efficacy. While our study demonstrated the synergy of interbacterial antagonism with antimicrobials using S. aureus and A. baumannii model system, we believe that numerous other bacteria from the normal body microflora could serve as antagonists against nosocomial pathogens. This suggests that microbial transplantation strategies could be explored to enhance the treatment of microbial infections.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

MDR:

Multidrug-resistant

MRSA:

methicillin-resistant S. aureus

DFU:

diabetic foot ulcer

S.a:

S. aureus

MSA:

mannitol Salt Agar

TSA:

trypticase soy agar

TSB:

trypticase soy broth

BHI:

brain heart infusion

MHB:

Mueller-Hinton broth

PCR:

polymerase chain reaction

OD:

optical density

CFUs:

colony-forming units

MIC:

minimum inhibitory concentration

MBC:

minimum bactericidal concentration

MBIC:

minimum biofilm inhibitory concentration

MBEC:

minimum biofilm eradication concentration

FICi:

fractional inhibitory concentration index

FBCi:

fractional bactericidal concentration index

FBICi:

fractional biofilm inhibitory concentration index

FBECi:

fractional biofilm eradication concentration index

SEM:

Scanning electron microscopy

References

  1. Ojo OA, Ibrahim HS, Rotimi DE, Ogunlakin AD, Ojo AB. Diabetes mellitus: from molecular mechanism to pathophysiology and pharmacology. Med Novel Technol Devices. 2023;19:100247.

    Article  Google Scholar 

  2. Motala AA, Mbanya JC, Ramaiya K, Pirie FJ, Ekoru K. Type 2 diabetes mellitus in sub-saharan Africa: challenges and opportunities. Nat Reviews Endocrinol. 2022;18(4):219–29.

    Article  Google Scholar 

  3. Raja JM, Maturana MA, Kayali S, Khouzam A, Efeovbokhan N. Diabetic foot ulcer: a comprehensive review of pathophysiology and management modalities. World J Clin Cases. 2023;11(8):1684.

    Article  PubMed  PubMed Central  Google Scholar 

  4. DesJardins-Park HE, Gurtner GC, Wan DC, Longaker MT. From chronic wounds to scarring: the growing health care burden of under-and over-healing wounds. Adv Wound Care. 2022;11(9):496–510.

    Article  Google Scholar 

  5. Baig MS, Banu A, Zehravi M, Rana R, Burle SS, Khan SL, Islam F, Siddiqui FA, Massoud EES, Rahman MH. An overview of diabetic foot ulcers and associated problems with special emphasis on treatments with antimicrobials. Life. 2022;12(7):1054.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Du F, Ma J, Gong H, Bista R, Zha P, Ren Y, Gao Y, Chen D, Ran X, Wang C. Microbial infection and antibiotic susceptibility of diabetic foot ulcer in China: literature review. Front Endocrinol. 2022;13:881659.

    Article  Google Scholar 

  7. Cheung GY, Bae JS, Otto M. Pathogenicity and virulence of Staphylococcus aureus. Virulence. 2021;12(1):547–69.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Nazari M, Azizi O, Solgi H, Fereshteh S, Shokouhi S, Badmasti F. Emergence of carbapenem resistant Acinetobacter baumannii clonal complexes CC2 and CC10 among fecal carriages in an educational hospital. Int J Environ Health Res. 2022;32(7):1478–88.

    Article  CAS  PubMed  Google Scholar 

  9. Pouget C, Dunyach-Remy C, Pantel A, Schuldiner S, Sotto A, Lavigne J-P. Biofilms in diabetic foot ulcers: significance and clinical relevance. Microorganisms. 2020;8(10):1580.

    Article  PubMed  PubMed Central  Google Scholar 

  10. Yadav MK, Vidal JE, Song J-J. Microbial biofilms on medical indwelling devices. New and Future developments in Microbial Biotechnology and Bioengineering: Microbial Biofilms. edn.: Elsevier; 2020. pp. 15–28.

  11. Nataraj BH, Mallappa RH. Antibiotic Resistance Crisis: an update on antagonistic interactions between Probiotics and Methicillin-Resistant Staphylococcus aureus (MRSA). Curr Microbiol. 2021;78(6):2194–211.

    Article  CAS  PubMed  Google Scholar 

  12. Anju V, Busi S, Imchen M, Kumavath R, Mohan MS, Salim SA, Subhaswaraj P, Dyavaiah M. Polymicrobial infections and biofilms: clinical significance and eradication strategies. Antibiotics. 2022;11(12):1731.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zayed A, El-Aasr M, Ibrahim A-RS, Ulber R. Fucoidan characterization: determination of purity and physicochemical and chemical properties. Mar Drugs. 2020;18(11):571.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Ayrapetyan ON, Obluchinskaya ED, Zhurishkina EV, Skorik YA, Lebedev DV, Kulminskaya AA, Lapina IM. Antibacterial properties of fucoidans from the brown algae Fucus vesiculosus L. of the barents sea. Biology. 2021;10(1):67.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Liu HY, Prentice EL, Webber MA. Mechanisms of antimicrobial resistance in biofilms. Npj Antimicrobials Resist. 2024;2(1):27.

    Article  Google Scholar 

  16. Sullivan GJ, Delgado NN, Maharjan R, Cain AK. How antibiotics work together: molecular mechanisms behind combination therapy. Curr Opin Microbiol. 2020;57:31–40.

    Article  CAS  PubMed  Google Scholar 

  17. Zhu M, Tse MW, Weller J, Chen J, Blainey PC. The future of antibiotics begins with discovering new combinations. Ann N Y Acad Sci. 2021;1496(1):82–96.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Siedentop B, Kachalov VN, Witzany C, Egger M, Kouyos RD, Bonhoeffer S. The effect of combining antibiotics on resistance: A systematic review and meta-analysis. medRxiv 2023.

  19. Nabarro L, Veeraraghavan B. Combination therapy for carbapenem-resistant Enterobacteriaceae: increasing evidence, unanswered questions, potential solutions. Eur J Clin Microbiol Infect Dis. 2015;34:2307–11.

    Article  CAS  PubMed  Google Scholar 

  20. Bassetti M, Peghin M, Pecori D. The management of multidrug-resistant Enterobacteriaceae. Curr Opin Infect Dis. 2016;29(6):583–94.

    Article  CAS  PubMed  Google Scholar 

  21. Wang X, Wang Q, Cao B, Sun S, Zhang Y, Gu B, Li B, Liao K, Zhao F, Jin L. Retrospective observational study from a Chinese network of the impact of combination therapy versus monotherapy on mortality from carbapenem-resistant Enterobacteriaceae bacteremia. Antimicrob Agents Chemother. 2019;63(1):01511–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/aac.

    Article  Google Scholar 

  22. Rüger M, Ackermann M, Reichl U. Species-specific viability analysis of Pseudomonas aeruginosa, Burkholderia cepacia and Staphylococcus aureus in mixed culture by flow cytometry. BMC Microbiol. 2014;14:1–15.

    Article  Google Scholar 

  23. Mashburn LM, Jett AM, Akins DR, Whiteley M. Staphylococcus aureus serves as an iron source for Pseudomonas aeruginosa during in vivo coculture. J Bacteriol. 2005;187(2):554–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ren Q, Liao G, Wu Z, Lv J, Chen W. Prevalence and characterization of Staphylococcus aureus isolates from subclinical bovine mastitis in southern Xinjiang, China. J Dairy Sci. 2020;103(4):3368–80.

    Article  CAS  PubMed  Google Scholar 

  25. Abhari SS, Badmasti F, Modiri L, Aslani MM, Asmar M. Circulation of imipenem-resistant Acinetobacter baumannii ST10, ST2 and ST3 in a university teaching hospital from Tehran, Iran. J Med Microbiol. 2019;68(6):860–5.

    Article  CAS  PubMed  Google Scholar 

  26. Mirzaei R, Esmaeili Gouvarchin Ghaleh H, Ranjbar R. Antibiofilm effect of melittin alone and in combination with conventional antibiotics toward strong biofilm of MDR-MRSA and-Pseudomonas aeruginosa. Front Microbiol. 2023;14:1030401.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Hemmati J, Nazari M, Abolhasani FS, Ahmadi A, Asghari B. In vitro investigation of relationship between quorum-sensing system genes, biofilm forming ability, and drug resistance in clinical isolates of Pseudomonas aeruginosa. BMC Microbiol. 2024;24(1):99.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Han Z, Lautenbach E, Fishman N, Nachamkin I. Evaluation of mannitol salt agar, CHROMagar Staph Aureus and CHROMagar MRSA for detection of meticillin-resistant Staphylococcus aureus from nasal swab specimens. J Med Microbiol. 2007;56(1):43–6.

    Article  PubMed  Google Scholar 

  29. Wayne P. Clinical and Laboratory Standards Institute: Performance standards for antimicrobial susceptibility testing: 20th informational supplement. CLSI document M100-S20 2010.

  30. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, Harbarth S, Hindler JF, Kahlmeter G, Olsson-Liljequist B, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infection: Official Publication Eur Soc Clin Microbiol Infect Dis. 2012;18(3):268–81.

    Article  CAS  Google Scholar 

  31. Bardbari AM, Arabestani MR, Karami M, Keramat F, Aghazadeh H, Alikhani MY, Bagheri KP. Highly synergistic activity of melittin with imipenem and colistin in biofilm inhibition against multidrug-resistant strong biofilm producer strains of Acinetobacter baumannii. Eur J Clin Microbiol Infect Dis. 2018;37:443–54.

    Article  CAS  PubMed  Google Scholar 

  32. Mirzaei R, Alikhani MY, Arciola CR, Sedighi I, Yousefimashouf R, Bagheri KP. Prevention, inhibition, and degradation effects of melittin alone and in combination with Vancomycin and rifampin against strong biofilm producer strains of methicillin-resistant Staphylococcus epidermidis. Biomed Pharmacother. 2022;147:112670.

    Article  CAS  PubMed  Google Scholar 

  33. Lorian V. Antibiotics in laboratory medicine. Lippincott Williams & Wilkins; 2005.

  34. Yang B, Lei Z, Zhao Y, Ahmed S, Wang C, Zhang S, Fu S, Cao J, Qiu Y. Combination susceptibility testing of common antimicrobials in vitro and the effects of sub-MIC of antimicrobials on Staphylococcus aureus biofilm formation. Front Microbiol. 2017;8:2125.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Jalalifar S, Razavi S, Mirzaei R, Irajian G, Pooshang Bagheri K. A hope for ineffective antibiotics to return to treatment: investigating the anti-biofilm potential of melittin alone and in combination with penicillin and oxacillin against multidrug resistant-MRSA and-VRSA. Front Microbiol. 2024;14:1269392.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Mirzaei R, Alikhani MY, Arciola CR, Sedighi I, Irajian G, Jamasbi E, Yousefimashouf R, Bagheri KP. Highly synergistic effects of melittin with Vancomycin and Rifampin against Vancomycin and rifampin resistant Staphylococcus epidermidis. Front Microbiol. 2022;13:869650.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Shams Khozani R, Shahbazzadeh D, Harzandi N, Feizabadi MM, Pooshang Bagheri K. Kinetics study of antimicrobial peptide, melittin, in simultaneous biofilm degradation and eradication of potent biofilm producing MDR Pseudomonas aeruginosa isolates. Int J Pept Res Ther. 2019;25:329–38.

    Article  CAS  Google Scholar 

  38. Kot B, Sytykiewicz H, Sprawka I. Expression of the Biofilm-Associated genes in Methicillin-Resistant Staphylococcus aureus in Biofilm and Planktonic conditions. Int J Mol Sci 2018, 19(11).

  39. Akhgari Z, Nazari R, Zargar M, Tanomand A. Antibacterial and antibiofilm properties of Acroptilon repens (L.) Dc extract and its effect on exotoxin A gene expression of Pseudomonas aeruginosa. Gene Rep. 2021;25:101357.

    Article  CAS  Google Scholar 

  40. Azizi O, Shahcheraghi F, Salimizand H, Modarresi F, Shakibaie MR, Mansouri S, Ramazanzadeh R, Badmasti F, Nikbin V. Molecular analysis and expression of bap gene in biofilm-forming multi-drug-resistant Acinetobacter baumannii. Rep Biochem Mol Biology. 2016;5(1):62.

    CAS  Google Scholar 

  41. Elizondo-Gonzalez R, Cruz-Suarez LE, Ricque-Marie D, Mendoza-Gamboa E, Rodriguez-Padilla C, Trejo-Avila LM. In vitro characterization of the antiviral activity of fucoidan from Cladosiphon okamuranus against Newcastle Disease Virus. Virol J. 2012;9:1–9.

    Article  Google Scholar 

  42. Hurlow JJ, Humphreys GJ, Bowling FL, McBain AJ. Diabetic foot infection: a critical complication. Int Wound J. 2018;15(5):814–21.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Afonso AC, Oliveira D, Saavedra MJ, Borges A, Simões M. Biofilms in diabetic foot ulcers: impact, risk factors and control strategies. Int J Mol Sci. 2021;22(15):8278.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Anwar K, Hussein D, Salih J. Antimicrobial susceptibility testing and phenotypic detection of MRSA isolated from diabetic foot infection. Int J Gen Med 2020:1349–57.

  45. Tsige Y, Tadesse S, Tefera MM, Amsalu A, Menberu MA, Gelaw B. Prevalence of methicillin-resistant Staphylococcus aureus and associated risk factors among patients with wound infection at referral hospital, northeast Ethiopia. Journal of pathogens 2020, 2020.

  46. Arfaoui A, Sallem RB, Fernández-Fernández R, Eguizábal P, Dziri R, Abdullahi IN, Sayem N, Ben Khelifa Melki S, Ouzari H-I, Torres C. Methicillin-Resistant Staphylococcus aureus from Diabetic Foot Infections in a Tunisian Hospital with the First Detection of MSSA CC398-t571. Antibiotics 2022, 11(12):1755.

  47. Hemmati J, Nazari M, Ahmadi A, Bayati M, Jalili M, Taheri M, Mohammadi Y, Asghari B. In vitro evaluation of biofilm phenotypic and genotypic characteristics among clinical isolates of Pseudomonas aeruginosa in Hamadan, West of Iran. J Appl Genet 2023:1–10.

  48. John J Jr. The treatment of resistant staphylococcal infections. F1000Research 2020, 9.

  49. Jatin V, Jayanthi M, Ramu R. Safety and efficacy of linezolid treatment in comparison with Vancomycin for methicillin-resistant Staphylococcus aureus nosocomial infection: a prospective study in Mysore City, South India. Int J Health Allied Sci. 2021;10(1):23–23.

    Article  Google Scholar 

  50. Savon IL, Shapoval SD, Maksymova OO, Sofilkanych MM, Sheremet MI, Shidlovskyi OV, Lazaruk OV. Alternative antibacterial therapy of Methicillin-resistant Staphylococcus aureusin patients with diabetic foot and sepsis. Romanian J Diabetes Nutr Metabolic Dis. 2021;28(1):11–7.

    CAS  Google Scholar 

  51. Roszell S, Jones C. Intravenous administration issues: a comparison of intravenous insertions and complications in Vancomycin versus other antibiotics. J Infus Nurs. 2010;33(2):112–8.

    Article  PubMed  Google Scholar 

  52. Joseph WS, Kosinski MA, Rogers LC. Parenteral vancomycin in the treatment of MRSA-Associated Diabetic Foot infections: an unnecessary risk. Int J Low Extrem Wounds 2023:15347346231207553.

  53. Cong Y, Yang S, Rao X. Vancomycin resistant Staphylococcus aureus infections: a review of case updating and clinical features. J Adv Res. 2020;21:169–76.

    Article  PubMed  Google Scholar 

  54. Castellanos N, Nakanouchi J, Yüzen DI, Fung S, Fernandez JS, Barberis C, Tuchscherr L, Ramirez MS. A study on Acinetobacter baumannii and Staphylococcus aureus strains recovered from the same infection site of a diabetic patient. Curr Microbiol. 2019;76:842–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Radlinski L, Rowe SE, Kartchner LB, Maile R, Cairns BA, Vitko NP, Gode CJ, Lachiewicz AM, Wolfgang MC, Conlon BP. Pseudomonas aeruginosa exoproducts determine antibiotic efficacy against Staphylococcus aureus. PLoS Biol. 2017;15(11):e2003981.

    Article  PubMed  PubMed Central  Google Scholar 

  56. Orazi G, Ruoff KL, O’Toole GA. Pseudomonas aeruginosa increases the sensitivity of biofilm-grown Staphylococcus aureus to membrane-targeting antiseptics and antibiotics. MBio 2019, 10(4):https://doiorg.publicaciones.saludcastillayleon.es/10.1128/mbio. 01501–01519.

  57. Smith NM, Ang A, Tan F, Macias K, James S, Sidhu J, Lenhard JR. Interaction of Staphylococcus aureus and Acinetobacter baumannii during in vitro β-lactam exposure. Antimicrob Agents Chemother. 2021;65(4):02414–20. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/aac.

    Article  Google Scholar 

  58. Mitosch K, Bollenbach T. Bacterial responses to antibiotics and their combinations. Environ Microbiol Rep. 2014;6(6):545–57.

    Article  PubMed  Google Scholar 

  59. Tong C, Honda K, Derek C. A review on microalgal-bacterial co-culture: the multifaceted role of beneficial bacteria towards enhancement of microalgal metabolite production. Environ Res 2023:115872.

  60. Jiang B, Wu C, Liang Y, Li X, Li J, Song G. Bacillus co-culture inhibits Quorum sensing in Pseudomonas aeruginosa. Curr Microbiol. 2023;80(4):123.

    Article  CAS  PubMed  Google Scholar 

  61. Zhao X, Yu Z, Ding T. Quorum-sensing regulation of antimicrobial resistance in bacteria. Microorganisms. 2020;8(3):425.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Fernandez JS, Tuttobene MR, Montaña S, Subils T, Cantera V, Iriarte A, Tuchscherr L, Ramirez MS. Staphylococcus aureus α-toxin effect on Acinetobacter baumannii behavior. Biology. 2022;11(4):570.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Orazi G, O’Toole GA. Pseudomonas aeruginosa alters Staphylococcus aureus sensitivity to Vancomycin in a biofilm model of cystic fibrosis infection. MBio. 2017;8(4). https://doiorg.publicaciones.saludcastillayleon.es/10.1128/mbio. 00873 – 00817.

  64. Trizna EY, Yarullina MN, Baidamshina DR, Mironova AV, Akhatova FS, Rozhina EV, Fakhrullin RF, Khabibrakhmanova AM, Kurbangalieva AR, Bogachev MI, et al. Bidirectional alterations in antibiotics susceptibility in Staphylococcus aureus-Pseudomonas aeruginosa dual-species biofilm. Sci Rep. 2020;10(1):14849.

    Article  PubMed  PubMed Central  Google Scholar 

  65. Chmit M, Kanaan H, Habib J, Abbass M, Mcheik A, Chokr A. Antibacterial and antibiofilm activities of polysaccharides, essential oil, and fatty oil extracted from Laurus nobilis growing in Lebanon. Asian Pac J Trop Med. 2014;7:S546–52.

    Article  Google Scholar 

  66. Jun J-Y, Jung M-J, Jeong I-H, Yamazaki K, Kawai Y, Kim B-M. Antimicrobial and antibiofilm activities of sulfated polysaccharides from marine algae against dental plaque bacteria. Mar Drugs. 2018;16(9):301.

    Article  PubMed  PubMed Central  Google Scholar 

  67. Lee K-Y, Jeong M-R, Choi S-M, Na S-S, Cha J-D. Synergistic effect of fucoidan with antibiotics against oral pathogenic bacteria. Arch Oral Biol. 2013;58(5):482–92.

    Article  CAS  PubMed  Google Scholar 

  68. Choi S-M, Jang E-J, Cha J-D. Synergistic effect between Fucoidan and Antibiotics against Clinic Methicillin-Resistant Staphylococcus aureus Synergistic Effect between Fucoidan and Antibiotics against Clinic Methicillin-Resistant Staphylococcus aureus. Adv Bioscience Biotechnol. 2015;6(04):275–85.

    Article  Google Scholar 

  69. Mani G, Rajendran I, Jayakumar T, Mani A, Govindaraju R, Dhayalan S. Evaluation of antibiofilm and antiquorum sensing activities of fucoidan characterized from Padina boryana against nosocomial pathogens. Appl Biochem Biotechnol 2023:1–18.

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Acknowledgements

The authors express their gratitude to the Vice-chancellor of Research and Technology of Hamadan University of Medical Sciences, Hamadan, Iran, for advocating this research. The authors would like to thank the Clinical Research Development Unit of Besat Hospital, Hamadan, Iran, for guidance and advice.

Funding

This work was supported by the Research Centre of Hamadan University of Medical Sciences on the grant number 140110279171. This funding has been devoted just to purchase materials used in our study.

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Authors and Affiliations

  1. Department of Microbiology, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

    Mohsen Nazari, Mohammad Taheri & Mohammad Yousef Alikhani

  2. Department of Pharmaceutical Biotechnology, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran

    Fatemeh Nouri

  3. Department of Anatomical Sciences, School of Medicine, Hamadan University of Medical Sciences, Hamadan, Iran

    Maryam Bahmanzadeh

  4. Fertility and Infertility Research Center, Avicenna Institute of Clinical Sciences, Avicenna Health Research Institute , Hamadan University of Medical Sciences, Hamadan, Iran

    Maryam Bahmanzadeh

  5. Infectious Disease Research Center, Avicenna Institute of Clinical Sciences, Avicenna Health Research Institute , Hamadan University of Medical Sciences, P.O. Box: 6517838678, Hamadan, Iran

    Mohammad Yousef Alikhani

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Contributions

MN performed all experiments and analyses, and also wrote the manuscript. MYA served as advisor. MT, MB and FN contributed as a supervisor and also in the revision of the manuscript. All authors contributed to the article and approved the submitted version.

Corresponding author

Correspondence to Mohammad Yousef Alikhani.

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Ethics approval and consent to participate

The Ethics Review Board of the Hamadan University of Medical Sciences, Hamadan, Iran approved the present study (Ethical approval code: IR.UMSHA.REC.1401.809). Ethical Review Board approved informed consent taken from all the participants, and all experiments were performed in accordance with relevant guidelines and regulations. We reported our findings according to the STROBE guidelines.

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Not applicable.

Competing interests

The authors declare no competing interests.

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Nazari, M., Taheri, M., Nouri, F. et al. The antimicrobial and antibiofilm effects of gentamicin, imipenem, and fucoidan combinations against dual-species biofilms of Staphylococcus aureus and Acinetobacter baumannii isolated from diabetic foot ulcers. Ann Clin Microbiol Antimicrob 23, 101 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12941-024-00760-w

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Keywords

Annals of Clinical Microbiology and Antimicrobials

ISSN: 1476-0711

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