Diabetes is a chronic condition involving metabolism, defined by ongoing elevated blood glucose that results in significant health issues. Diabetic foot ulcers, often known as DFUs, represent one of the significant complications faced by patients and are frequently worsened by infections involving multiple types of microbes, such as Staphylococcus aureus and Acinetobacter baumannii.

This blog post features the research by Mohsen Nazari and colleagues titled “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.”

First, results from antimicrobial susceptibility testing and molecular analysis focusing on oxacillin disks and the mecA gene revealed that 80.6% of the isolated Staphylococcus aureus strains exhibited characteristics consistent with methicillin-resistant Staphylococcus aureus (MRSA). Many of the S. aureus and A. baumannii isolates demonstrated varying degrees of biofilm-forming ability, with optical density (OD) values ​​across all isolates ranging from 0.2 to 2.8. Furthermore, based on these results, the biofilm-forming capacity of each isolate was classified as “strong,” “intermediate,” or “weak.”

Data acquired from the disk diffusion method for clinical isolates were analysed to evaluate antimicrobial resistance rates. The resistance rates for S. aureus against IMP, TS, E, CD, GM, LZD, and VAN were 80.6%, 67.7%, 64.5%, 58.5%, 51.6%, 0%, and 0%, respectively. Similarly, based on disk diffusion data, the antimicrobial resistance rates for A. baumannii clinical isolates were: 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 the S. aureus isolates and 100% of the A. baumannii isolates exhibited multidrug resistance (MDR). The study findings revealed a notable association between antimicrobial resistance and strong biofilm formation in S. aureus clinical isolates.

Upon examining various conditions for all isolates (31 strains of S. aureus and 27 strains of A. baumanii), it was revealed that, out of a total of 837 tested conditions, robust dual-species biofilms were formed under 72 conditions, while moderate dual-species biofilms were formed under 67 conditions (see Figure1). In some instances, although OD values ​​exceeded those of the negative control, no growth of either bacterium was observed upon subsequent culture on MSA and Leeds media; consequently, these cases were classified as “non-biofilm-forming.”

The geometric meaning MIC values ​​for S. aureus in monoculture was 12.12 µg/mL for gentamicin, 19.84 µg/mL for imipenem, and 48.50 µg/mL for fucoidan. For A. baumannii in monoculture, 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 were 119.42, 48.50, and 88.37 µg/mL, respectively, for S. aureus, and 128, 14.92, and 62.62 µg/mL, respectively, for A. baumannii. Furthermore, the geometric mean MBC/MIC ratios were 9.84, 2.46, and 2.14 for gentamicin, imipenem, and fucoidan, respectively, in S. aureus, and 5.65, 1.74, and 2, respectively, in A. baumannii.

The geometric mean MIC (minimum inhibitory concentration) values ​​for S. aureus in monoculture were 12.12 µg/mL for gentamicin, 19.84 µg/mL for imipenem, and 48.50 µg/mL for fucoidan. For A. baumannii in monoculture, 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 (minimum bactericidal concentration) values ​​for gentamicin, imipenem, and fucoidan were 119.42, 48.50, and 88.37 µg/mL, respectively, for S. aureus, and 128, 14.92, and 62.62 µg/mL, respectively, for A. baumannii. Furthermore, the geometric mean MBC/MIC ratios were 9.84, 2.46, and 2.14 for gentamicin, imipenem, and fucoidan, respectively, in S. aureus, and 5.65, 1.74, and 2, respectively, in A. baumannii.

Six isolates—S. aureus 6, S. aureus 7, S. aureus 22, A. baumannii 1, A. baumannii 8, and A. baumannii 20—were selected for study under co-culture conditions. These isolates were capable of forming dual-species biofilms under nine different conditions (3 × 3). Robust dual-species biofilms were formed under eight of these conditions, while moderate dual-species biofilms were formed under one condition. In addition, MIC and MBC values were measured under co-culture conditions that demonstrated growth suppression or killing of both bacterial species (see Figure 2A). The calculated geometric mean values ​​for MIC and MBC, as well as the MBC/MIC ratios, for gentamicin, imipenem, and fucoidan were as follows: for gentamicin, 5.87 µg/mL, 11.75 µg/mL, and 1.85, respectively; for imipenem, 3.70 µg/mL, 8 µg/mL, and 2.16; and for fucoidan, 19.65 µg/mL, 42.48 µg/mL, and 2.16.

In this study, using three selected isolates under co-culture conditions, the geometric mean values ​​of the most effective synergistic concentrations of gentamicin and imipenem against S. aureus and A. baumannii 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 the combination of imipenem and fucoidan, the respective values ​​were 0.71, 0.47, and 0.32.

Furthermore, an examination of the geometric mean values ​​of the optimal synergistic concentrations—evaluated using FBCi—revealed that for the combination of gentamicin and imipenem against S. aureus and A. baumannii isolates, the values ​​were 0.42, 0.30, and 0.27, respectively. For the combination of gentamicin and fucoidan, the geometric mean values ​​were 0.66, 0.59, and 0.22, respectively. Finally, for the combination of imipenem and fucoidan, the respective values ​​were 0.43, 0.45, and 0.33.

For S. aureus isolates, the geometric mean MBIC (Minimum Biofilm Inhibitory Concentration) values ​​were 55.71 µg/mL for gentamicin, 68.59 µg/mL for imipenem, and 153.89 µg/mL for fucoidan. For A. baumannii isolates, the respective values ​​were 119.42 µg/mL for gentamicin, 48.50 µg/mL for imipenem, and 101.49 µg/mL for fucoidan.

Furthermore, the geometric mean MBEC (Minimum Biofilm Eradication Concentration) 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.

The geometric mean MBIC and MBEC values ​​for dual-species biofilms 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 (Figure 2B).

In this study, they determined the geometric mean values ​​of concentrations exhibiting the strongest synergistic effects for various antimicrobial combinations against dual-species mixed biofilms formed by S. aureus and A. baumannii isolates. Analysis using FBICi yielded values ​​of 0.82, 0.59, and 0.29 for the gentamicin-imipenem combination across the three selected isolates. For the gentamicin-fucoidan combination, the values ​​were 0.47, 0.44, and 0.28, respectively. Similarly, the imipenem-fucoidan combination showed values ​​of 0.47, 0.46, and 0.37.

Furthermore, they evaluated the optimal synergistic concentrations using FBECi. For the gentamicin-imipenem combination, the geometric mean values ​​were 0.98, 0.69, and 0.24. For the gentamicin-fucoidan combination, the values ​​were 0.55, 0.58, and 0.36, respectively. Finally, the imipenem-fucoidan combination yielded values ​​of 0.52, 0.48, and 0.39 for the same isolates.

Using FE-SEM, we evaluated the potential synergistic effects of gentamicin, imipenem, and fucoidan in eliminating or neutralizing mixed-species biofilms of S. aureus and A. baumannii. The results demonstrated that the combination of fucoidan and antibiotics at FBIC (biofilm formation inhibitory concentration) levels caused significant disruption of the mixed-species biofilms, resulting in bacterial lysis and a marked reduction in total biofilm mass. The robust biofilm structures were broken down into smaller, disorganized fragments. Furthermore, a strong synergistic effect was also observed with the combination of gentamicin and imipenem, resulting in reduced biofilm mass, bacterial lysis, and the release of bacterial cells from the biofilm.

The expression levels of the S. aureus icaA gene and the A. baumannii bap gene was evaluated after 24-hour exposure to combined treatments (each at 1/2 MBIC concentration): gentamicin plus imipenem, gentamicin plus fucoidan, or imipenem plus fucoidan. In single-species biofilms, icaA expression decreased by 2.98–1.52-fold and bap expression by 3.15–1.94-fold. In dual-species biofilms, S. aureus icaA decreased by 5.92–3.64-fold, and A. baumannii bap decreased by 5.78–4.17-fold. With the combination of gentamicin and fucoidan, icaA decreased by 3.32–1.90-fold and bap by 2.86–1.79-fold in single-species biofilms; in dual-species biofilms, S. aureus icaA decreased by 6.12–4.20-fold and A. baumannii bap by 5.68–3.79-fold. Following treatment with imipenem and fucoidan, icaA decreased by 3.1–1.72-fold and bap by 3.12–1.68-fold in single-species biofilms; in dual-species biofilms, S. aureus icaA decreased by 5.53–3.75-fold and A. baumannii bap by 6.87–4.48-fold. ANOVA results indicated a significant difference in gene expression between single-species and dual-species biofilms (Figure 3).

Cytotoxicity test results for HSF-PI 16 cells showed cytotoxicity levels of 78.3%, 42.1%, and 11% at fucoidan concentrations of 1000, 500, and 250 µg, respectively. Notably, no cytotoxicity was observed in HSF-PI 16 cells when fucoidan was used in combination with the antibiotics imipenem (32 and 64 µg) or gentamicin (32 and 64 µg) at concentrations (125, 62.5, 32.1, 15.6, and 7.8 µg) selected for their potential synergistic effects.

The research revealed that the combined use of gentamicin, imipenem, and fucoidan exhibited a synergistic interaction against mixed-species biofilms created by Staphylococcus aureus and Acinetobacter baumannii, indicating promising implications for treatment effectiveness. Leveraging the synergistic effects of these antimicrobial agents could potentially improve treatment outcomes for infections involving such mixed-species biofilms. The results emphasize that personalized approaches to treatment are necessary to effectively handle complex infections such as diabetic foot ulcers, where there is a complex relationship between microbial activity, antibiotic resistance, and biofilm development.

Source: Ann Clin Microbiol Antimicrob. 2024 Nov 15;23:101. doi: 10.1186/s12941-024-00760-w