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Staphylococcus aureus is a predominant cause of infections in individuals with spinal cord stimulation (SCS) devices. Biofilm formation complicates these infections, commonly requiring both surgical and antibiotic treatments. This study explored the biofilm matrix composition and antimicrobial susceptibility of planktonic and biofilm-growing S. aureus isolates from individuals with SCS-related infections. Whole-genome sequencing (WGS) examined genotypes, virulome, resistome, and the pan-genome structure. The study also analyzed biofilm matrix composition, early surface adhesion, hemolytic activity, and antibiotic-susceptibility testing. WGS revealed genetic diversity among isolates. One isolate, though oxacillin susceptible, contained the mec A gene. The median number of virulence factor genes per isolate was 58. All isolates harbored the biofilm-related ica A/D genes. When assessing phenotypic characteristics, all strains demonstrated the ability to form biofilms in vitro . The antimicrobial susceptibility profile indicated that oxacillin, rifampin, and teicoplanin showed the highest efficacy against S. aureus biofilm. Conversely, high biofilm tolerance was observed for vancomycin, trimethoprim/sulfamethoxazole, and levofloxacin. These findings suggest that S. aureus isolates are highly virulent and produce robust biofilms. In cases of suspected biofilm infections caused by S. aureus , vancomycin should not be the primary choice due to its low activity against biofilm. Instead, oxacillin, rifampin, and teicoplanin appear to be more effective options to manage SCS infections.IMPORTANCESCS devices are increasingly used to manage chronic pain, but infections associated with these devices, particularly those caused by Staphylococcus aureus , present significant clinical challenges. These infections are often complicated by biofilm formation, which protects bacteria from immune responses and antibiotic treatments, making them difficult to eradicate. Understanding the genetic diversity, virulence, and biofilm characteristics of S. aureus isolates from SCS infections is critical to improving treatment strategies. Our study highlights the need to reconsider commonly used antibiotics like vancomycin, which shows reduced activity against biofilm-growing cells. Identifying more effective alternatives, such as oxacillin, rifampin, and teicoplanin, provides valuable insight for clinicians when managing biofilm-related S. aureus infections in patients with SCS implants. This research contributes to the growing evidence that biofilm formation is crucial in treating device-related infections, emphasizing the importance of tailoring antimicrobial strategies to the biofilm phenotype.

Atopic dermatitis (AD) is a chronic inflammatory skin disorder exacerbated by Staphylococcus aureus colonization. The specific factors that drive S. aureus overgrowth and persistence in AD remain poorly understood. This study analyzed skin barrier functions and microbiome diversity in lesional (LE) and non-lesional (NL) forearm sites of individuals with severe AD compared to healthy control subjects (HS). Notable differences were found in transepidermal water loss, stratum corneum hydration, and microbiome composition. Cutibacterium was more prevalent in HS, while S. aureus and S. lugdunensis were predominantly found in AD LE skin. The results highlighted that microbial balance depends on inter-species competition. Specifically, network analysis at the genus level demonstrated that overall bacterial correlations were higher in HS, indicating a more stable microbial community. Notably, network analysis at the species level revealed that S. aureus engaged in competitive interactions in NL and LE but not in HS. Whole-genome sequencing (WGS) showed considerable genetic diversity among S. aureus strains from AD. Despite this variability, the isolates exhibited convergence in key phenotypic traits such as adhesion and biofilm formation, which are crucial for microbial persistence. These common phenotypes suggest an adaptive evolution, driven by competition in the AD skin microenvironment, of S. aureus and underscoring the interplay between genetic diversity and phenotypic convergence in microbial adaptation., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Sodium hypochlorite (NaOCl) is widely recognized for its broad-spectrum antimicrobial efficacy in skin wound care. This study investigates the effectiveness of NaOCl against a range of bacterial and fungal isolates from pressure ulcer (PU) patients. We analyzed 20 bacterial isolates from PU patients, comprising carbapenem-resistant Klebsiella pneumoniae (CRKP), multidrug-resistant Acinetobacter baumannii (MDRAB), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), along with 5 Candida albicans isolates. Antibiotic resistance profiles were determined using standard susceptibility testing. Whole-genome sequencing (WGS) was employed to identify antimicrobial resistance genes (ARGs) and disinfectant resistance genes (DRGs). Genetic determinants of biofilm formation were also assessed. The antimicrobial activity of NaOCl was evaluated by determining the minimum inhibitory concentration (MIC) and the minimal biofilm eradication concentration (MBEC) for both planktonic and biofilm-associated cells. CRKP and MDRAB showed resistance to fluoroquinolones and carbapenems, while MRSA exhibited resistance to β-lactams and levofloxacin. MSSA displayed a comparatively lower resistance profile. WGS identified significant numbers of ARGs in CRKP and MDRAB, with fewer DRGs compared to MRSA and MSSA. All isolates possessed genes associated with fimbriae production and adhesion, correlating with pronounced biofilm biomass production. NaOCl demonstrated substantial antimicrobial activity against both planktonic cells and biofilms. The MIC 90 for planktonic bacterial cells was 0.125 mg/mL, and the MBEC 90 ranged from 0.225 to 0.5 mg/mL. For planktonic C. albicans , the MIC 90 was 0.150 mg/mL, and the MBEC 90 was 0.250 mg/mL. These results highlight the challenge in treating biofilm-associated infections and underscore the potential of NaOCl as a robust antimicrobial agent against difficult-to-treat biofilm infections at concentrations lower than those typically found in commercial disinfectants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Fabrizio, Sivori, Cavallo, Truglio, Toma, Sperati, Francalancia, Obregon, Pamparau, Kovacs, Pimpinelli and Di Domenico.)

Wound repair and skin regeneration is a very complex orchestrated process that is generally composed of four phases: hemostasis, inflammation, proliferation, and remodeling. Each phase involves the activation of different cells and the production of various cytokines, chemokines, and other inflammatory mediators affecting the immune response. The microbial skin composition plays an important role in wound healing. Indeed, skin commensals are essential in the maintenance of the epidermal barrier function, regulation of the host immune response, and protection from invading pathogenic microorganisms. Chronic wounds are common and are considered a major public health problem due to their difficult-to-treat features and their frequent association with challenging chronic infections. These infections can be very tough to manage due to the ability of some bacteria to produce multicellular structures encapsulated into a matrix called biofilms. The bacterial species contained in the biofilm are often different, as is their capability to influence the healing of chronic wounds. Biofilms are, in fact, often tolerant and resistant to antibiotics and antiseptics, leading to the failure of treatment. For these reasons, biofilms impede appropriate treatment and, consequently, prolong the wound healing period. Hence, there is an urgent necessity to deepen the knowledge of the pathophysiology of delayed wound healing and to develop more effective therapeutic approaches able to restore tissue damage. This work covers the wound-healing process and the pathogenesis of chronic wounds infected by biofilm-forming pathogens. An overview of the strategies to counteract biofilm formation or to destroy existing biofilms is also provided.

Seborrheic dermatitis (SD) affects 2-5% of the global population, with imbalances in the skin microbiome implicated in its development. This study assessed the impact of an oily suspension containing Lactobacillus crispatus P17631 and Lacticaseibacillus paracasei I1688 (termed EUTOPLAC) on SD symptoms and the skin mycobiome-bacteriome modulation. 25 SD patients were treated with EUTOPLAC for a week. Symptom severity and skin mycobiome-bacteriome changes were measured at the start of the treatment (T0), after seven days (T8), and three weeks post-treatment (T28). Results indicated symptom improvement post-EUTOPLAC, with notable reductions in the Malassezia genus. Concurrently, bacterial shifts were observed, including a decrease in Staphylococcus and an increase in Lactobacillus and Lacticaseibacillus. Network analysis highlighted post-EUTOPLAC instability in fungal and bacterial interactions, with increased negative correlations between Malassezia and Lactobacillus and Lacticaseibacillus genera. The study suggests EUTOPLAC's potential as a targeted SD treatment, reducing symptoms and modulating the mycobiome-bacteriome composition., (© 2024. The Author(s).)

The photoantibacterial properties of titania nanoparticles (TiO 2 NPs) are attracting much interest, but the separation of their suspension limits their application. In this study, the encapsulation of commercial TiO 2 NPs within self-assembling tripeptide hydrogels to form hgel-TiO 2 NP composites with significant photoantibacterial properties is reported. The Fmoc-Phe 3 hydrogelator was synthesized via an enzymatic method. The resulting composite was characterized with DLS, ζ-potential, SAXS, FESEM-EDS and rheological measurements. Two different concentrations of TiO 2 NPs were used. The results showed that, by increasing the TiO 2 NP quantity from 5 to 10 mg, the value of the elastic modulus doubled, while the swelling ratio decreased from 63.6 to 45.5%. The antimicrobial efficacy of hgel-TiO 2 NPs was tested against a laboratory Staphylococcus aureus ( S. aureus ) strain and two methicillin-resistant S. aureus (MRSA) clinical isolates. Results highlighted a concentration-dependent superior antibacterial activity of hgel-TiO 2 NPs over TiO 2 NPs in the dark and after UV photoactivation. Notably, UV light exposure substantially increased the biocidal action of hgel-TiO 2 NPs compared to TiO 2 NPs. Surprisingly, in the absence of UV light, both composites significantly increased S. aureus growth relative to control groups. These findings support the role of hgel-TiO 2 NPs as promising biocidal agents in clinical and sanitation contexts. However, they also signal concerns about TiO 2 NP exposure influencing S. aureus virulence.

Acinetobacter baumannii is increasingly associated with various epidemics, representing a serious concern due to the broad level of antimicrobial resistance and clinical manifestations. During the last decades, A. baumannii has emerged as a major pathogen in vulnerable and critically ill patients. Bacteremia, pneumonia, urinary tract, and skin and soft tissue infections are the most common presentations of A. baumannii , with attributable mortality rates approaching 35%. Carbapenems have been considered the first choice to treat A. baumannii infections. However, due to the widespread prevalence of carbapenem-resistant A. baumannii (CRAB), colistin represents the main therapeutic option, while the role of the new siderophore cephalosporin cefiderocol still needs to be ascertained. Furthermore, high clinical failure rates have been reported for colistin monotherapy when used to treat CRAB infections. Thus, the most effective antibiotic combination remains disputed. In addition to its ability to develop antibiotic resistance, A. baumannii is also known to form biofilm on medical devices, including central venous catheters or endotracheal tubes. Thus, the worrisome spread of biofilm-producing strains in multidrug-resistant populations of A. baumannii poses a significant treatment challenge. This review provides an updated account of antimicrobial resistance patterns and biofilm-mediated tolerance in A. baumannii infections with a special focus on fragile and critically ill patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cavallo, Oliva, Pages, Sivori, Truglio, Fabrizio, Pasqua, Pimpinelli and Di Domenico.)

The recognized antibacterial properties of silver nanoparticles (AgNPs) characterize them as attractive nanomaterials for developing new bioactive materials less prone to the development of antibiotic resistance. In this work, we developed new composites based on self-assembling Fmoc-Phe3 peptide hydrogels impregnated with in situ prepared AgNPs. Different methodologies, from traditional to innovative and eco-sustainable, were compared. The obtained composites were characterized from a hydrodynamic, structural, and morphological point of view, using different techniques such as DLS, SEM, and rheological measurements to evaluate how the choice of the reducing agent determines the characteristics of AgNPs and how their presence within the hydrogel affects their structure and properties. Moreover, the antibacterial properties of these composites were tested against S. aureus , a major human pathogen responsible for a wide range of clinical infections. Results demonstrated that the hydrogel composites containing AgNPs (hgel@AgNPs) could represent promising biomaterials for treating S. aureus -related infections.

Acne vulgaris is a common inflammatory disorder affecting more than 80% of young adolescents. Cutibacterium acnes plays a role in the pathogenesis of acne lesions, although the mechanisms are poorly understood. The study aimed to explore the microbiome at different skin sites in adolescent acne and the role of biofilm production in promoting the growth and persistence of C. acnes isolates. Microbiota analysis showed a significantly lower alpha diversity in inflammatory lesions (LA) than in non-inflammatory (NI) lesions of acne patients and healthy subjects (HS). Differences at the species level were driven by the overabundance of C. acnes on LA than NI and HS. The phylotype IA1 was more represented in the skin of acne patients than in HS. Genes involved in lipids transport and metabolism, as well as potential virulence factors associated with host-tissue colonization, were detected in all IA1 strains independently from the site of isolation. Additionally, the IA1 isolates were more efficient in early adhesion and biomass production than other phylotypes showing a significant increase in antibiotic tolerance. Overall, our data indicate that the site-specific dysbiosis in LA and colonization by virulent and highly tolerant C. acnes phylotypes may contribute to acne development in a part of the population, despite the universal carriage of the microorganism. Moreover, new antimicrobial agents, specifically targeting biofilm-forming C. acnes, may represent potential treatments to modulate the skin microbiota in acne., (© 2022. The Author(s).)

The present paper investigated the synthesis of peptide-based hydrogel composites containing photo-generated silver nanoparticles (AgNPs) obtained in the presence and absence of honey as tensile strength enhancer and hydrogel stabilizer. Fmoc-Phe and diphenylalanine (Phe 2 ) were used as starting reagents for the hydrogelator synthesis via an enzymatic method. In particular, we developed an in situ one-pot approach for preparing AgNPs inside peptide hydrogels using a photochemical synthesis, without any toxic reducing agents, with reaction yields up to 30%. The structure and morphology of the nanohybrids were characterized with different techniques such as FESEM, UV-Vis, DLS, SAXS and XPS. Moreover, the antibacterial activity of these hybrid biomaterials was investigated on a laboratory strain and on a clinical isolate of Staphylococcus aureus . Results demonstrated that honey increased both swelling ability and also mechanical stability of the hydrogel. Finally, a higher antibacterial effect of AgNPs in the hybrid was observed in the presence of honey. In particular, AgNPs/hgel and AgNPs/hgel-honey showed an enhanced antibacterial activity (3.12 mg/L) compared to the free form of AgNPs, alone or in combination with honey (6.25 mg/L) for both S. aureus strains.

Inflammation and biofilm-associated infection are common in chronic venous leg ulcers (VU), causing deep pain and delayed healing. Albeit important, clinical markers and laboratory parameters for identifying and monitoring persistent VU infections are limited. This study analyzed 101 patients with infected (IVU) and noninfected VUs (NVU). Clinical data were collected in both groups. The serum homocysteine (Hcys) and inflammatory cytokines from the wound fluid were measured. In addition, microbial identification, antibiotic susceptibility, and biofilm production were examined. IVU were 56 (55.4%) while NVU were 45 (44.5%). IVUs showed a significant increase in the wound's size and depth compared to NVUs. In addition, significantly higher levels of interleukin (IL)-6, IL-10, IL17A, and tumor necrosis factor-alpha (TNF-α) were found in patients with IVUs compared to those with NVUs. Notably, hyperhomocysteinemia (HHcy) was significantly more common in patients with IVUs than NVUs. A total of 89 different pathogens were identified from 56 IVUs. Gram-negative bacteria were 51.7%, while the Gram-positives were 48.3%. At the species level, Staphylococcus aureus was the most common isolate (43.8%), followed by Pseudomonas aeruginosa (18.0%). Multidrug-resistant organisms (MDROs) accounted for 25.8% of the total isolates. Strong biofilm producers (SBPs) (70.8%) were significantly more abundant than weak biofilm producers (WBP) (29.2%) in IVUs. SBPs were present in 97.7% of the IVUs as single or multispecies infections. Specifically, SBPs were 94.9% for S. aureus , 87.5% for P. aeruginosa , and 28.6% for Escherichia coli . In IVU, the tissue microenvironment and biofilm production can support chronic microbial persistence and a most severe clinical outcome even in the presence of an intense immune response, as shown by the high levels of inflammatory molecules. The measurement of local cytokines in combination with systemic homocysteine may offer a novel set of biomarkers for the clinical assessment of IVUs caused by biofilm-producing bacteria.

Methicillin-resistant Staphylococcus aureus (MRSA) has become the leading cause of skin and soft tissue infections (SSTIs). Biofilm production further complicates patient treatment, contributing to increased bacterial persistence and antibiotic tolerance. The study aimed to explore the efficacy of different antibiotics on biofilm-producing MRSA isolated from patients with SSTI. A total of 32 MRSA strains were collected from patients with SSTI. The MIC and minimal biofilm eradication concentration (MBEC) were measured in planktonic and biofilm growth. The study showed that dalbavancin, linezolid, and vancomycin all inhibited MRSA growth at their EUCAST susceptible breakpoint. Of the MRSA strains, 87.5% ( n  = 28) were strong biofilm producers (SBPs), while only 12.5% ( n  = 4) were weak biofilm producers (WBPs). The MBEC 90 values for dalbavancin were significantly lower than those of linezolid and vancomycin in all tested strains. We also found that extracellular DNA (eDNA) contributes to the initial microbial attachment and biofilm formation. The amount of eDNA differed among MRSA strains and was significantly higher in those isolates with high dalbavancin and vancomycin tolerance. Exogenously added DNA increased the MBEC 90 and protection of biofilm cells from dalbavancin activity. Of note, the relative abundance of eDNA was higher in MRSA biofilms exposed to MBEC 90 dalbavancin than in untreated MRSA biofilms and those exposed to sub-MIC 90 . Overall, dalbavancin was the most active antibiotic against MRSA biofilms at concentrations achievable in the human serum. Moreover, the evidence of a drug-related increase of eDNA and its contribution to antimicrobial drug tolerance reveals novel potential targets for antibiofilm strategies against MRSA. IMPORTANCE Staphylococcus aureus is the most common cause of skin and soft tissue infections (SSTIs) worldwide. In addition, methicillin-resistant S. aureus (MRSA) is increasingly frequent in postoperative infections and responsible for a large number of hospital readmissions and deaths. Biofilm formation by S. aureus is a primary risk factor in SSTIs, due to a higher antibiotic tolerance. Our study showed that the biofilm-forming capacity varied among MRSA strains, although strong biofilm producers were significantly more abundant than weak biofilm producer strains. Notably, dalbavancin demonstrated a potent antibiofilm activity at concentrations achievable in human serum. Nevertheless, dalbavancin activity was affected by an increased concentration of extracellular DNA in the biofilm matrix. This study provides novel insight for designing more targeted therapeutic strategies against MRSA and to prevent or eradicate harmful biofilms.

Bacterial bloodstream infection (BSI) represents a significant complication in hematologic patients. However, factors leading to BSI and progression to end-organ disease and death are understood only partially. The study analyzes host and microbial risk factors and assesses their impact on BSI development and mortality. A total of 96 patients with hematological malignancies and BSI were included in the study. Host-associated risk factors and all causes of mortality were analyzed by multivariable logistic regression at 30 days after BSI onset of the first neutropenic episode. The multidrug-resistant profile and biofilm production of bacterial isolates from primary BSI were included in the analysis. Median age was 60 years. The underlying diagnoses were acute leukemia (55%), lymphoma (31%), and myeloma (14%). A total of 96 bacterial isolates were isolated from BSIs. Escherichia coli was the most common isolate (29.2%). Multidrug-resistant bacteria caused 10.4% of bacteremia episodes. Weak biofilm producers (WBPs) were significantly ( P  < 0.0001) more abundant (72.2%) than strong biofilm producers (SBPs) (27.8%). Specifically, SBPs were 7.1% for E. coli, 93.7% for P. aeruginosa, 50% for K. pneumoniae, and 3.8% for coagulase-negative staphylococci. Mortality at day 30 was 8.3%, and all deaths were attributable to Gram-negative bacteria. About 22% of all BSIs were catheter-related BSIs (CRBSIs) and mostly caused by Gram-positive bacteria (79.0%). However, CRBSIs were not correlated with biofilm production levels ( P  = 0.75) and did not significantly impact the mortality rate ( P  = 0.62). Conversely, SBP bacteria were an independent risk factor ( P  = 0.018) for developing an end-organ disease. In addition, multivariate analysis indicated that SBPs ( P  = 0.013) and multidrug-resistant bacteria ( P  = 0.006) were independent risk factors associated with 30-day mortality. SBP and multidrug-resistant (MDR) bacteria caused a limited fraction of BSI in these patients. However, when present, SBPs raise the risk of end-organ disease and, together with an MDR phenotype, can independently and significantly concur at increasing the risk of death. IMPORTANCE Bacterial bloodstream infection (BSI) is a significant complication in hematologic patients and is associated with high mortality rates. Despite improvements in BSI management, factors leading to sepsis are understood only partially. This study analyzes the contribution of bacterial biofilm on BSI development and mortality in patients with hematological malignancies (HMs). In this work, weak biofilm producers (WBPs) were significantly more abundant than strong biofilm producers (SBPs). However, when present, SBP bacteria raised the risk of end-organ disease in HM patients developing a BSI. Besides, SBPs, together with a multidrug-resistant (MDR) phenotype, independently and significantly concur at increasing the risk of death in HM patients. The characterization of microbial biofilms may provide key information for the diagnosis and therapeutic management of BSI and may help develop novel strategies to either eradicate or control harmful microbial biofilms.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a prominent cause of nosocomial infections associated with high rates of morbidity and mortality, particularly in oncological patients. The hypermucoviscous (HMV) phenotype and biofilm production are key factors for CRKP colonization and persistence in the host. This study aims at exploring the impact of CRKP virulence factors on morbidity and mortality in oncological patients. A total of 86 CRKP were collected between January 2015 and December 2019. Carbapenem resistance-associated genes, antibiotic susceptibility, the HMV phenotype, and biofilm production were evaluated. The median age of the patients was 71 years (range 40-96 years). Clinically infected patients were 53 (61.6%), while CRKP colonized individuals were 33 (38.4%). The most common infectious manifestations were sepsis (43.4%) and pneumonia (18.9%), while rectal surveillance swabs were the most common site of CRKP isolation (81.8%) in colonized patients. The leading mechanism of carbapenem resistance was sustained by the KPC gene (96.5%), followed by OXA-48 (2.3%) and VIM (1.2%). Phenotypic CRKP characterization indicated that 55.8% of the isolates were strong biofilm-producers equally distributed between infected (54.2%) and colonized (45.8%) patients. The HMV phenotype was found in 22.1% of the isolates, which showed a significant (P<0.0001) decrease in biofilm production as compared to non-HMV strains. The overall mortality rate calculated on the group of infected patients was 35.8%. In univariate analysis, pneumoniae significantly correlated with death (OR 5.09; CI 95% 1.08-24.02; P=0.04). The non-HMV phenotype (OR 4.67; CI 95% 1.13-19.24; P=0.03) and strong biofilm-producing strains (OR 5.04; CI95% 1.39-18.25; P=0.01) were also associated with increased CRKP infection-related mortality. Notably, the multivariate analysis showed that infection with strong biofilm-producing CRKP was an independent predictor of mortality (OR 6.30; CI 95% 1.392-18.248; P=0.004). CRKP infection presents a high risk of death among oncological patients, particularly when pneumoniae and sepsis are present. In infected patients, the presence of strong biofilm-producing CRKP significantly increases the risk of death. Thus, the assessment of biofilm production may provide a key element in supporting the clinical management of high-risk oncological patients with CRKP infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer AA declared a shared affiliation with the authors to the handling editor at time of review., (Copyright © 2020 Di Domenico, Cavallo, Sivori, Marchesi, Prignano, Pimpinelli, Sperduti, Pelagalli, Di Salvo, Celesti, Paluzzi, Pronesti, Koudriavtseva, Ascenzioni, Toma, De Luca, Mengarelli and Ensoli.)

Infections are among the most frequent and challenging events in diabetic foot ulcers (DFUs). Pathogenic bacteria growing in biofilms within host tissue are highly tolerant to environmental and chemical agents, including antibiotics. The present study was aimed at assessing the use of silver sulfadiazine (SSD) for wound healing and infection control in 16 patients with DFUs harboring biofilm-growing Staphylococcus aureus and Pseudomonas aeruginosa . All patients received a treatment based on a dressing protocol including disinfection, cleansing, application of SSD, and application of nonadherent gauze, followed by sterile gauze and tibio-breech bandage, in preparation for toilet surgery after 30 days of treatment. Clinical parameters were analyzed by the T.I.M.E. classification system. In addition, the activity of SSD against biofilm-growing S. aureus and P. aeruginosa isolates was assessed in vitro. A total of 16 patients with S. aureus and P. aeruginosa infected DFUs were included in the study. Clinical data showed a statistically significant ( p < 0.002) improvement of patients' DFUs after 30 days of treatment with SSD with significant amelioration of all the parameters analyzed. Notably, after 30 days of treatment, resolution of infection was observed in all DFUs. In vitro analysis showed that both S. aureus and P. aeruginosa isolates developed complex and highly structured biofilms. Antibiotic susceptibility profiles indicated that biofilm cultures were significantly ( p ≤ 0.002) more tolerant to all tested antimicrobials than their planktonic counterparts. However, SSD was found to be effective against fully developed biofilms of both S. aureus and P. aeruginosa at concentrations below those normally used in clinical preparations (10 mg/mL). These results strongly suggest that the topical administration of SSD may represent an effective alternative to conventional antibiotics for the successful treatment of DFUs infected by biofilm-growing S. aureus and P. aeruginosa .