Microbiology and Antimicrobial Resistance in Diabetic Foot Infections

Abstract

Diabetic foot infections (DFIs) can be caused by a wide range of microorganisms, including mainly bacteria and fungi. This class of infections poses significant medical challenges, affecting a large proportion of individuals with diabetic foot ulcers (DFUs). This chapter aims to provide a comprehensive overview of the primary microorganisms responsible for diabetic foot infections, elucidating their virulence factors, pathogenesis, and susceptibility to antimicrobial agents. It also explores the impact of antimicrobial resistance (AMR) on diabetic foot infections (DFIs), highlighting the role of biofilms in chronic diabetic foot infections and the resulting treatment difficulties, all with the goal of reducing complications. By gaining insight into the diverse spectrum of microorganisms involved in diabetic foot infections, healthcare professionals can customize treatment plans for individual patients, ultimately improving patient outcomes and quality of life.

1. Introduction

Diabetic foot infections (DFIs) are considerable medical problems, as approximately half to six in every ten individuals with diabetic foot ulcers (DFUs) eventually contract an infection [1]. These infections can stem from a wide array of microorganisms, encompassing bacteria and fungi. Owing to the intricate nature of diabetic foot ulcers, the microbiology of these infections can exhibit diversity and present difficulties in both diagnosis and treatment. This chapter aims to offer a comprehensive overview of the major types of microorganisms responsible for diabetic foot infections, elucidating their virulence factors, pathogenesis, and susceptibility to antimicrobial agents. Additionally, it addresses the impact of antimicrobial resistance (AMR) on diabetic foot infections (DFIs), shedding light on the role of biofilms in chronic diabetic foot infections and the ensuing treatment challenges, all in a concerted effort to mitigate implications. By gaining insight into the heterogeneous spectrum of microorganisms implicated in diabetic foot infections, healthcare providers can tailor treatment plans to individual patients, thereby enhancing patient outcomes and quality of life.

2. Epidemiology of DFU pathogens and their antimicrobial resistance

Across various countries, the microbiology of DFU exhibits notable diversity, yet specific species consistently play significant roles. Staphylococcus aureus (S. aureus), for instance, emerges as a frequently encountered pathogen in several nations, including Pakistan, Ethiopia, Egypt, Jordan, and Lebanon, with prevalence rates of 46, 25.19, 22, 14.2, and 9% respectively (Figure 1) [3, 4, 5, 6, 7]. There is an evident tendency toward Gram-negative species in multiple countries. In Egypt, Gram-negative bacilli (rods) prevalence was reported to be 56.1% of pathogens causing DFI, where K. pneumoniae accounted for 26.8% of infections. Whereas Gram-positive bacteria such as coagulase-negative Staphylococci accounted for 22% [5]. Similarly, in Lebanon, the prevalence of Gram-negative rods is higher than Gram-positive cocci 55 vs. 39%. The most prevalent isolates in Lebanon include Escherichia coli (15%), followed by Enterococcus (14%) and Pseudomonas aeruginosa (11%) [7]. A similar pattern is also observed in Kuwait, where Gram-negative strains are more prevalent in ischemic ulcers, although S. aureus dominates in non-ischemic ulcers infections [8]. Meanwhile, in Mexico, S. aureus is the most commonly isolated pathogen in DFU infections, with higher prevalence of aerobic Gram-positive cocci (47%) [9]. In Pakistan, Gram-negative bacilli are predominant where the prevalence is estimated for E. coli (28%), Pseudomonas aeruginosa (6%), and Klebsiella (3.5%) [3]. Iranian DFU patients, especially of older age with repeated hospitalizations, were also more affected by Gram-negative bacilli [10].

Figure 1.

Prevalence of S. aureus among DFU infections across countries in the Middle East and Asia. S. aureus Emerged as a notably prevalent pathogen associated with diabetic foot ulcers (DFIs) across numerous countries. Its prevalence exhibits considerable variation, ranging from approximately 48% in Pakistan to around 8% in Iran [2]. Additional research is imperative to ascertain its prevalence in other nations.

Additionally, the prevalence of polymicrobial and monomicrobial DFU infections was variable among countries. In Kuwait, for instance, monomicrobial DFI was more common than polymicrobial DFI (57.3 vs. 34.8%) [8]. However, the trend is rather unclear in Lebanon, where only 38% of Lebanese patients were affected by polymicrobial DFI in 2018 [11], while between 2008 and 2017 the prevalence rate was 54% [7].

A variability in antibiotic sensitivity is also prominently evident across different countries. In Mexico, Penicillin and Dicloxacillin showed the lowest efficacy against Gram-positive bacteria, while Cephalothin and Penicillin were found to be least effective against Gram-negative bacteria. In contrast, Levofloxacin, Cephalothin, and Amikacin emerged as the most effective antibiotics against Gram-positive and Gram-negative bacteria, respectively [9]. Whereas in Egypt, Amikacin, Tigecycline, and Meropenem exhibited the highest activity against Gram-negative bacteria, while Linezolid and Vancomycin demonstrated effectiveness against Staphylococci [5]. In Lebanon, Ciprofloxacin sensitivity was observed in 72% of Pseudomonas spp., but for Enterobacteriaceae, 63.6% were sensitive to either Amoxicillin/Clavulanate or Ciprofloxacin, and 91% were susceptible to Piperacillin/Tazobactam [11]. In Pakistan, Vancomycin demonstrated sensitivity against 92% of S. aureus, while Clindamycin exhibited sensitivity in 67% of cases. Among E. coli isolates, 81% displayed sensitivity to Imipenem, 69% to aminoglycosides, and 31% to quinolones.

Multidrug resistance is a significant challenge across various nations, characterized by the diverse array of resistance mechanisms exhibited by bacterial strains [3]. The estimated prevalence of AMR among DFI pathogens in Ethiopia is 92% [4]. The presence of extended-spectrum beta-lactamase (ESBL) was identified in 52.6% of Enterobacteriaceae isolates, with the blaCTX-M gene being the most prevalent (90%), followed by blaTEM (65%) and blaSHV (35%) [5]. Similarly, a study conducted in Egypt showed that 78 out of the 82 isolates (95%) tested were multidrug resistant organism [5]. The prevalence of AMR among pathogens causing DFI in Iran is estimated to be 48.4%. A study reported that 37.5% of Enterococcus spp. were identified as Vancomycin-resistant (VRE), 48.8% of Staphylococcus spp. exhibited methicillin resistance (MRSA), 77.8% of isolated E. coli demonstrated extended-spectrum beta-lactamase (ESBL) production, and 66.7% of isolated Pseudomonas strains showcased multidrug resistance (MDR) [10]. In Jordan, over 75% of E. coli and P. aeruginosa isolates were resistant to Ampicillin, Cefotaxime, Ceftazidime, and Ertapenem. Among the E. coli and P. aeruginosa tested, Imipenem was effective against all strains, while Gentamicin and Sulfamethoxazole-trimethoprim were effective against 87.5% of P. aeruginosa and 75% of E. coli, respectively. As for MRSA, almost 50% of the isolates were resistant to aminoglycosides, fluoroquinolones, and macrolides, while all were susceptible to Vancomycin, Mupirocin, and Linezolid with the exception of one isolate found to be Vancomycin resistant [6].

3. Microbiology of DFU infections

Diabetic foot infection (DFI) is a common and serious complication of diabetes mellitus. It occurs when the protective skin barrier is compromised, allowing pathogens to proliferate in the soft tissues. Depending on the severity, DFIs are classified into mild, moderate, and severe cases. These infections are typically polymicrobial, involving Gram-positive cocci like Staphylococcus aureus, MRSA, Enterococcus spp, and Streptococcus spp.

3.1 Gram-positive bacteria

3.1.1 Staphylococcus aureus

Staphylococcus aureus is a coagulase positive cocci arranged in tetrads and clusters, resembling a bunch of grapes when viewed under a microscope. It is a facultative anaerobic bacteria [12]. S. aureus is known for its aggressive nature in complicating diabetic foot wounds. It accounts for 20–25% of all isolated bacteria in patients with diabetic foot infections (DFIs), making it the most prevalent bacterial pathogen associated with DFIs in Occidental countries [13, 14].

3.1.1.1 Virulence factors of S. aureus

S. aureus produces wide range of virulence factors including various toxins and degrading enzymes as shown in Figure 2.

Figure 2.

Schematic depicting Staphylococcus aureus virulence factors and mechanism of action for soluble pore-forming toxin leukocidin.

Coagulase: Clinical isolates of the human pathogen Staphylococcus aureus produce coagulase (Coa), a crucial enzyme for pathogenesis. Coagulase, existing in two forms - bound coagulase (clumping factor) and free coagulase, serves as a virulence factor by interacting with the host’s blood-clotting system. Bound coagulase binds to host proteins, notably fibrinogen, leading to cell aggregation and evasion of phagocytosis by immune cells [15]. Additionally, it triggers fibrinogen conversion to fibrin, promoting blood clot formation that shields the bacterium from the host’s defenses and antibiotics. Free coagulase directly activates prothrombin, further enhancing blood clot formation and aiding the bacterium in evading host defenses, facilitating localized infections [16].

Panton-Valentine leukocidin (PVL) comprises two distinct protein components, LukS-PV (slow) and LukF-PV (fast), which are chromatographically separate. These proteins collectively form a highly potent cytotoxin. The active toxin functions by creating pores in the membrane of neutrophils, leading to their lysis. Strains carrying PVL can cause chronic soft skin tissue infections (SSTI) and necrotizing pneumonia, even in healthy individuals. Studies have shown that isolates carrying the gene responsible for PVL production can exacerbate wound conditions. Nonetheless, PVL-encoding strains are relatively rare, with less than 10% of methicillin-sensitive S. aureus (MSSA) clinical isolates found to possess the PVL gene in the community [17].

α-toxin: Among different types of hemolysins (α-, β-, γ-, and δ-) encoded by S. aureus, α-hemolysin is the most extensively studied [18]. It is a pore forming toxin, produced by most S. aureus strains, that can cause host cell lysis. It is regulated by multiple global regulatory loci, including the accessory gene regulator, the Staphylococcal accessory gene regulator, and the Staphylococcal accessory protein effector, which control its expression in vitro [19]. It acts on a wide range of cells, but mainly on red blood cells and leukocytes [14]. Several studies have shown that S. aureus USA300 α-hemolysin contributes to severe infections, including pneumonia, osteomyelitis, and bacteremia [18].

Exfoliative toxins: The primary cause of Staphylococcal scalded skin syndrome (SSSS) is attributed to exfoliative toxins A (ETA) and B (ETB), which function as epidermolytic proteases. This syndrome is characterized by fever and widespread rapid skin desquamation, without affecting mucosal tissues [20]. The study conducted by Dunyach-Remy et al. (2016) mentions that exfoliative toxins A (ETA), B (ETB), and D (ETD) are linked to human infections. The distribution of these toxins varies among clinical grades of DFU infections, being more common in Grade 4 (13.8%) compared to Grade 1 (4%) or Grades 2–3 (3.5%). However, they do not follow a similar representation pattern across different grades [14].

Enterotoxin: Superantigens, which include Staphylococcal enterotoxins (SEs), Staphylococcal enterotoxin-like toxins (SEls), and toxic shock syndrome toxin 1 (TSST-1), can cause an excessive production of cytokines, resulting in cell apoptosis by mechanisms that are not yet fully understood. Among these superantigens, the most studied is the toxic shock syndrome toxin 1 (TSST-1), responsible for inducing toxic shock syndrome (TSS) [14]. In complicated DFUs, most S. aureus strains produce superantigens (Sags), particularly SEs and SEls. These toxins activate T cells, leading to the production of cytokines and causing a state of chronic inflammation, this chronic inflammation delays the wound healing process in DFIs.

3.1.1.2 Pathogenesis of S. aureus

Attachment and Adhesion: The initial event in diabetic foot infections is the attachment of S. aureus to surface components, including fibrinogen, fibronectin, and epidermal keratinocytes [14]. The adhesion of S. aureus to diabetic foot ulcer surfaces relies on the expression of microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) [14], such as clumping factor A (CIfA) and clumping factor B (CIfB) [21]. Both proteins are present on the surface of S. aureus and are covalently anchored to the cell wall peptidoglycan layer. They bind to fibrinogen using variants of the ‘dock, lock and latch’ (DLL) mechanism. For docking, the described clumping factors has two subdomains called N2 and N3. These two subdomains are positioned in a particular configuration that forms a groove between them, providing a docking site for ligands. For locking, ligand docking causes a change in the shape of the protein called DEv-IgG, specifically in the N3 subdomain. The N3 subdomain has an extension that is normally disordered, but ligand docking causes this extension to move and interact with the ligand, effectively locking it in place. For latching, the resulting change in shape facilitates the formation of an extra beta-strand referred to as the G” beta-strand within the C-terminal region of the N3 extension. This newly formed beta-strand aligns itself with the E beta-strand located in the N2 subdomain which results in a “latch” that serves to enhance the stability of the DEv-IgG-ligand complex even further [21].

Invasion: Fibronectin-binding proteins A and B (FnBPA and FnBPB) mediate S. aureus invasion into epithelial and endothelial cells [18]. S. aureus is capable of invading osteoblasts, fibroblasts, and endothelial cells, forming small-colony variants (SCVs) within the intracellular compartment. SCVs enable the bacteria to survive in a metabolically inactive state while preserving the integrity of the host cell [14]. FnBPs have high affinity fibronectin-binding repeats that at least one of them is required for internalization of the bacteria into the cells. For example, arginine–glycine–aspartate sequence is one of these sequences, when FnBPs bind to fibronectin, α5β1 integrin recognizes them, so it clusters on the host cell surface, this triggers the activating of an intracellular signaling cascade that ends up with endocytosis of bacterial cells [21].

Evasion: One of the main mechanisms by which S. aureus evades the host immune response is protein A mediated. S. aureus protein A binds to the Fc region of IgGs leads to disorientation of the IgGs coating the bacterial cell, this prevents not only neutrophil receptors recognition of the bacteria but also classical pathway activation of the complement system [21].

Other virulence factors: S. aureus possesses various virulence factors involved in soft tissue and bone infections. It can secrete toxins leading to tissue necrosis. It also can secrete glycocalyx, which starts after adhesion, forming a crucial component for ‘biofilm’ development [14].

3.1.1.3 Antimicrobial sensitivity profile

In a study conducted by Mamdoh et al. in 2023, Staphylococci demonstrated a significant level of resistance to amoxiclav, cefoxitin, and oxacillin. Although, most Staphylococci spp. exhibited susceptibility to linezolid, chloramphenicol, and rifampicin [22]. Another study conducted in 2022, Woldeteklie et al. found a high resistance in S. aureus to methicillin, with 81.3% (26/32) identified as MRSA, and 18.7% (6/32) were MSSA [23]. This study reported that 50% of the screened MRSA isolates were sensitive to amikacin and chloramphenicol. While previously done reports showed MSSA’s susceptibility to Clindamycin, Vancomycin, and Ciprofloxacin, the findings of Woldeteklie et al.’s study remain consistent with studies conducted in Ethiopia, Egypt, and Sudan.

Methicillin-resistant S. aureus (MRSA) strains resist not only all β-lactam antibiotics but also a wide range of other antimicrobials, leading to a challenging management of MRSA infections Ceftaroline and Ceftobiprole are the only cephalosporins that inhibit PBP2a, making them effective against MRSA skin and skin structure infections [24].

There are two major types of MRSA infections: hospital-acquired ([HA]-MRSA) and community-acquired MRSA (CA-MRSA). Individuals with diabetes face an increased risk of contracting both variants due to their high susceptibility of developing sores and ulcerations [12]. MRSA tends to be more frequently isolated from patients with a history of hospitalization or residing in chronic care facilities, those who have recently undergone antibiotic treatment or individuals who have had a previous amputation [12, 14].

3.1.2 Enterococcus spp.

Enterococci are Gram-positive cocci that can survive in both aerobic and anaerobic environments, forming short to medium chains [25]. Enterococci, are typically found in the gastrointestinal tract as commensal organisms, but can cause various infections, such as urinary tract infection, bacteremia, and endocarditis [26]. DFIs with Enterococci were found to involve not only a polymicrobial infection but also showed positive associations with extended-spectrum β-lactamase (ESBL)-producing Gram-negative organisms and anaerobes. Conversely, DFIs with Enterococci were negatively associated with methicillin-susceptible or methicillin-resistant S. aureus and other Streptococci [27].

3.1.2.1 Virulence factors and pathogenesis

Aggregation substance (Agg): Formation of large aggregates has been shown to contribute to E. feacalis pathogenesis in an in vivo evidence. Agg that is present on the surface of the enterococcal cells is a pheromone-inducible surface glycoprotein, which means that it is produced in response to specific chemical signals. Agg is a multifunctional factor that contributes to the pathogenicity and adaptability of E. faecalis by multiple mechanisms. Firstly, evasion of host immune response. Agg has the ability to promote bacterial aggregation, which means that it encourages individual bacterial cells to come together and form clusters or aggregates. When these Enterococci cells aggregate, it increase the hydrophobicity of their cell surfaces which leads to the localization of cholesterol to phagosomes [28]. This cholesterol localization is believed to interfere with or delay the fusion of phagosomes with lysosomes, where bacteria are typically broken down. This delay may help E. faecalis evade the host’s immune defenses and persist within host cells, potentially leading to infections that are challenging to eliminate [28]. Secondly, Agg aids in plasmid transfer, a vital mechanism for the exchange of genetic material among bacteria. This genetic exchange can contribute to the acquisition of new traits, including antibiotic resistance, enhancing the bacterium’s adaptability and survival in various environments. Thirdly, Agg assists E. faecalis in adhering to a wide range of eukaryotic surfaces, such as those found in host tissues. This adhesive property can promote colonization within the host, potentially leading to infections [28].

Extracellular surface protein (Esp): esp gene is believed to play a multifaceted role in bacterial pathogenicity. It contributes to adhesion, colonization, and evasion of the host immune system, while also being implicated in antibiotic resistance to Ampicillin, Ciprofloxacin and Imipenem, as demonstrated by research conducted by Foulquie Moreno et al. in 2006 [28]. Additionally, Esp plays a crucial role in the formation of enterococcal biofilms, which confer resistance to environmental stresses and facilitate adhesion to eukaryotic cells, such as those found in the urinary tract [28].

Cytolysin: It exhibits β-hemolytic properties when in contact with human red blood cells, causing them to rupture. The genes that code for cytolysin production are located on plasmids. The production of cytolysin is regulated by a quorum-sensing mechanism. This communication involves a two-component system, which consists of two proteins that work together to detect the presence of other bacteria and activate cytolysin production. The cylLs group of genes are part of the cytolysin operons, which are clusters of genes that work together to produce cytolysin. These genes are non-regulatory, which means that they do not control the production of cytolysin but are necessary for its function. Clinical isolates of Enterococcus species have a higher incidence of cylLs genes compared to food isolates, which suggests that these genes may be important for causing disease in humans [28].

Hyaluronidase: Hyaluronidase is an enzyme that acts on hyaluronic acid, a component of connective tissue, and it functions as a degradative enzyme. This enzyme is associated with tissue damage because it depolymerizes the mucopolysaccharide part of connective tissue. By breaking down hyaluronic acid, hyaluronidase facilitates the spread of Enterococci bacteria, as well as their toxins, through host tissue. This enzymatic action makes it easier for the bacteria to move within the host and potentially cause infections [28].

3.1.2.2 Antimicrobial sensitivity profile

In a multicenter study conducted in selected Hospitals in Addis Ababa, Ethiopia, all Enterococcus species exhibited resistance to Oxacillin, Penicillin, Cefoxitin, and Bacitracin. Moreover, a high level of resistance was observed among all Enterococcus isolates to Gentamycin, Doxycycline, Erythromycin, and Cotrimoxazole and it was found that all isolated Enterococcus strains exhibited sensitivity to Chloramphenicol (100%). However, two out of four Enterococcus species (50%) were resistant to Vancomycin [4]. Interestingly, no association was found between previous hospitalization within the last 6 months and antibiotic treatment within the last 3 months before admission with a higher prevalence of Enterococcal infection [27].

3.1.3 Streptococcus pyogenes—group a streptococcus (GAS)

Streptococcus pyogenes is a Gram-positive, β-hemolytic bacteria. Bacterial cells are sphericaland usually occur in chains. Complicated skin and skin structure infections (cSSSIs) encompass a range of infections, with major abscesses and diabetic foot infections (DFIs) being the predominant subtypes. S. pyogens and aureus are the primary causative agents responsible for cSSSIs [29].

3.1.3.1 Virulence and pathogenesis

Virulence factors: It possesses several virulence factors that contribute to its pathogenicity. Some of the main virulence factors of S. pyogenes include M Protein, which is a cell surface protein that helps the bacterium evade the host immune system by inhibiting phagocytosis. It also plays a role in adherence to host cells. S. pyogenes produces two types of hemolysins streptolysin O (SLO) and streptolysin S (SLS). These toxins can damage host cells and contribute to tissue destruction. Hyaluronic Acid Capsule expressed by some strains of S. pyogenes helps the bacterium evade the immune system by mimicking host tissues. In addition, streptokinase is an enzyme produced by the bacterium that can activate plasmin, leading to the breakdown of blood clots, which facilitate the spread of the infection. Streptococcal Cysteine Protease (SpeB) is another virulence factors that can degrade host proteins, aiding in tissue invasion and immune evasion. Streptococcal Pyrogenic Exotoxins (SPEs) are superantigens produced by some strains of S. pyogenes that can cause an excessive immune response, leading to conditions such as scarlet fever and toxic shock syndrome. These virulence factors work together to help S. pyogenes evade the host immune system, adhere to host tissues, invade and damage host cells, and cause a range of diseases, including pharyngitis (strep throat), skin infections, and more severe conditions like necrotizing fasciitis and streptococcal toxic shock syndrome [30].

Adherence and invasion: the bacteria adhere to host epithelial cells, including those of the skin. It binds to host extracellular matrix proteins (fibronectin) mainly via fibronectin binding proteins, which is reported to be an adhesion and invasion factor [31].

Immune evasion: S. pyogenes evades the immune system via inactivating the human complement system by cleaving C3 and C3b, which acts as an opsonin, by several proteases it possesses. It also has been reported that a protein on the surface of S. pyogenes, identified as GAPDH, binds to human C5a and enhances C5a cleavage on the bacterial surface. When C5a is cleaved, its activity is hindered, thereby inhibiting its function as a chemotaxin. One more mechanism this bacterium employs to evade the immune system is by binding to C6, which contributes to inhibiting the membrane attach complex polymerization [31].

3.1.3.2 Antimicrobial sensitivity profile

In a multicenter study conducted in selected Hospitals in Addis Ababa, Ethiopia, all S. pyogenes and Streptococcus viridans species were sensitive to the majority of antimicrobial agents [4].

3.1.4 Clostridium spp.

Clostridium species are a group of Gram-positive anaerobic bacteria, marked by their rod-shaped vegetative form, particularly when reproducing. In diabetic patients, Clostridium spp, specifically C. perfringens, take an active role in the development and succession of complications associated with diabetic foot infections and ulcers [32].

3.1.4.1 Virulence factors and pathogenesis

Agr-like Quorum Sensing system: C. perfringens employs density-sensing quorum sensing (QS) systems as a mechanism to regulate the production of virulence factors [33]. The most crucial of these QS systems is the Agr-like system, identified for controlling the production of CPA and PFO toxins [34]. This system is vital for C. perfringens in causing gas gangrene [35]. Additionally, the Agr-like QS system plays a significant role in biofilm formation by C. perfringens, which may have implications in infections, although this is not definitively proven [33].

Biofilms: C. perfringens can complicate DFI through the formation of biofilms [33]. In a mouse model of type 2 diabetes, E. coli, B. fragilis, and C. perfringens collectively formed a polymicrobial biofilm and demonstrated a synergistic effect on each other. This synergistic interaction resulted in an increased mortality rate among the type-2 diabetic mice compared to those who received an inoculation of single bacterial strains [36].

Toxins: C. perfringens has a significant capacity to produce a wide array of toxins, leading to the development of histotoxic, enteric, and/or enterotoxemic diseases. The production of these toxins varies considerably among different strains of C. perfringen [37]. While not specified in the literature, it is thought that the more significant toxins in the context of DFU are C. perfringens Alpha Toxin (CPA) and Perfringolysin O (PFO).

C. perfringens Alpha Toxin (CPA): CPA is an enzyme capable of degrading phosphatidylcholine and sphingomyelin found in cell membranes leading to membrane disruption [33, 38]. It has the ability to hinder the movement and development of neutrophils while also triggering the arachidonic acid metabolism process. This metabolic pathway ultimately results in vasoconstriction and the aggregation of platelets. As a result, this toxin establishes a localized environment characterized by limited tissue blood flow and a compromised innate immune response [33, 38]. In addition, lethal levels of CPA can lead to significant degradation of the plasma membrane and the subsequent release of lactate dehydrogenase (LDH), which is a hallmark of necrotic cell death [37, 39]. Therefore, CPA is the critical virulence factor contributing to gas gangrene, also known as clostridial myonecrosis [37].

Perfringolysin O (PFO): also known as theta toxin, is produced by most C. perfringens strains except type F strains. PFO belongs to the cholesterol-dependent cytolysins family and is a pore-forming toxin (PFT) [33]. It has four domains, with the fourth domain in the C-terminal region containing three loops responsible for binding to cholesterol on target cells. This high-affinity binding to cholesterol allows PFO to concentrate on the plasma membrane, leading to toxin oligomerization and pores formation. Large pores disrupt plasma membrane integrity, leading to cell lysis through a colloid osmotic mechanism [40].

Adhesins: It has been suggested that collagen adhesion protein (CNA) and fibrinogen-binding proteins FbpA and FbpB act as adhesins in the context of various diseases mediated by C. perfringens [33]. While CNA plays a role in enteritis, fibronectin (Fn) is as a possible extracellular matrix glycoprotein used by C. perfringens for binding. In the presence of Fn, C. perfringens can use FbpA and FbpB to adhere to collagen, particularly types II and III. C. perfringens may exploit Fn to facilitate contact with host cells and enhance colonization [33].

Degradative Enzymes: C. perfringens secretes a wide range of extracellular enzymes with degradative properties, including proteases such as clostripain, hyaluronidase (mu toxin), collagenase, and endoglycosidases. The role of these enzymes in C. perfringens virulence is still investigated in research. It has been recently shown that the endo-N-acetylgalactosaminidase (EngCP), which is an enzyme that has a role in tissue degradation, is significant in gas gangrene induced by type A strains [33, 41].

3.1.4.2 Antimicrobial sensitivity profile

Resistance to Lincomycin and Clindamycin was noticed among strains of all Clostridia species as well as to Tetracycline and Doxycycline among strains of C. perfringens [42].

3.2 Gram-negative bacteria

Diabetes Miletus patients are susceptible to developing diabetic foot ulcers which can be exacerbated by vicious infections that are accompanied by peripheral neuropathy, changes in foot architecture, and trauma. In addition to these diabetic complications, pathogenic infections also dictate disease management and affect quality of life [43, 44]. A major class of bacteria involved in DFU infections are Gram-negative bacteria classifying the infections by their causative agents is crucial for appropriate management and treatment of diabetic patients’ wounds. Most common Gram-negative bacteria include Pseudomonas aeruginosa, Escherichia coli, Klebsiella species, and Proteus species. Their prevalence differs according to geographical region [45, 46, 47].

3.2.1 Pseudomonas aeruginosa

Pseudomonas aeruginosa is a Gram-negative, encapsulated, facultative-aerobic, bacilli-shaped opportunistic pathogen. P. aeruginosa can cause infections in patients with cystic fibrosis, burn wounds, immunodeficiency, chronic obstructive pulmonary disorder (COPD), cancer, and healthcare-acquired infections such as ventilator-associated pneumonia [48]. P. aeruginosa also causes diabetic foot ulcer infections [47].

3.2.1.1 Virulence factors and pathogenesis

P. aeruginosa possesses various virulence factors that enables biofilm formation and successful initiation of infection (Figure 3). The main virulence factors are briefly described herein.

Figure 3.

Schematic depicting Pseudomonas aeruginosa virulence factors.

Lipopolysaccharides (LPS): LPS is a major component of the outer membrane of P. aeruginosa and plays a critical role in protecting the bacterial cell from host defenses and environmental stresses. However, the lipid A component of LPS is highly endotoxic and can cause tissue damage, inflammation, and sepsis [49]. It is a potent activator of the host immune response, triggering the release of pro-inflammatory cytokines and chemokines. However, P. aeruginosa can modify its LPS structure to evade host immune recognition and develop antibiotic resistance [50]. For example, P. aeruginosa can add a positively charged amino arabinose moiety to its LPS, which reduces its negative charge and decreases it’s binding to cationic antimicrobial peptides and complement proteins [51]. This modification also reduces the activation of TLR4 and the downstream immune response. LPS is also involved in bacterial adherence to host cells and biofilm formation [52].

Adherence factors: P. aeruginosa produces several adherence factors, such as pili and flagella, which enable the bacteria to attach to host cells and surfaces. Pili are hair-like structures that extend from the bacterial surface and can mediate attachment to host cells and biofilm formation. Flagella are whip-like structures that enable the bacteria to move toward nutrients and host cells. P. aeruginosa is characterized by a single unipolar flagellum that beats, granting the pathogen motility in pathogenesis [53].

Exotoxin A: Exotoxin A is a potent virulence factor of P. aeruginosa that can inhibit protein synthesis in host cells and cause tissue damage. Exotoxin A is a type of AB toxin that consists of two subunits: the enzymatic A subunit and the binding B subunit. The B subunit binds to host cell receptors, and the A subunit is internalized and cleaves elongation factor 2 (EF-2), a critical component of protein synthesis machinery. This results in inhibition of protein synthesis and cell death [54].

Deoxyribonuclease: DNase is an exoenzyme produced by P. aeruginosa that can degrade extracellular DNA, which can be released by host cells during tissue damage or by bacterial cells during biofilm formation. DNase can degrade extracellular DNA and promote bacterial dissemination and evasion of host immune responses. DNase can also modulate host immune responses by degrading neutrophil extracellular traps (NETs), which are web-like structures composed of DNA and antimicrobial proteins that can trap and kill bacteria. By degrading NETs, DNase can enable P. aeruginosa to evade host immune defenses and cause persistent infections [55].

Proteases: P. aeruginosa secretes a variety of proteases, such as elastase and alkaline protease, which can degrade host tissues and proteins and promote bacterial dissemination. Elastase is a zinc metalloprotease that can cleave elastin, collagen, and other extracellular matrix proteins. Alkaline protease is a serine protease that can degrade a wide range of host proteins, including immunoglobulins, complement factors, and cytokines [56].

Quorum sensing: Quorum sensing (QS) allows P. aeruginosa to coordinate the expression of virulence factors and biofilm formation in response to population density. This enables P. aeruginosa to form complex communities and persist in various environments, including host tissues. QS in P. aeruginosa is mediated by several autoinducers, such as N-acyl homoserine lactones (AHLs), which are synthesized and detected by the Las and Rhl systems [57]. Quorum sensing also regulates the production of elastase, alkaline protease, and exotoxin A, which are involved in host tissue damage and immune evasion. Inhibition of quorum sensing has been shown to reduce P. aeruginosa virulence and improve host survival in animal models [58, 59].

Two-component systems: Two-component systems are ubiquitous signaling regulators that play vital roles in bacterial survival, metabolism, and virulence. They allow P. aeruginosa to sense and respond to various environmental cues, such as pH, osmolarity, and antibiotic [60]. This enables P. aeruginosa to adapt to different host niches and develop antibiotic resistance. Two-component systems also regulate the expression of virulence factors including siderophores production such as pyocyanin, pyoverdine, and pyochelin, which are involved in host tissue damage and immune evasion [61]. Targeting two-component systems has been proposed as a potential strategy to control P. aeruginosa infections.

Secretion systems: P. aeruginosa has several secretion systems, such as the type III and type VI secretion systems, which can deliver effector proteins into host cells and modulate host immune responses. The type III secretion system (T3SS) is a needle-like structure that can inject effector proteins directly into host cells. The T3SS can modulate host cell signaling pathways and promote bacterial survival and dissemination. T3SS, like exoenzyme S also plays a role in the inhibition of wound repair in diabetic skin ulcers [62]. While T6SSs allow P. aeruginosa to deliver effector proteins into target cells, such as host cells and competing bacteria. This enables P. aeruginosa to manipulate host cell functions and compete for resources in various environments. T6SSs are involved in host tissue damage, immune evasion, and inter-bacterial competition [56]. The functional roles of some T6SSs are still unclear due to the limited substrates available for research.

Pyocyanin pigment and Pyoverdine siderophore: Pyocyanin and pyoverdine are two important virulence factors produced by P. aeruginosa and play significant roles in virulence and pathogenicity. Pyocyanin is a blue-green, water-soluble pigment that can cause oxidative damage. It generates reactive oxygen species (ROS) within host tissues, leading to tissue damage and inflammation. Pyocyanin also impairs the function of immune cells, including neutrophils and macrophages, making it more difficult for the host to mount an effective immune response against the infection. Pyocyanin also contributes to the formation and maintenance of biofilms, which are complex communities of bacteria encased in a protective matrix [63].

Pyoverdine is a siderophore, which is a molecule secreted by bacteria to scavenge iron from the host environment. It is a high-affinity iron-chelating compound with a characteristic fluorescent yellow-green color. Pyoverdine allows P. aeruginosa to acquire iron from the host, even in iron-restricted environments. This gives the pathogen a competitive advantage over the host in obtaining this essential nutrient [64].

Outer membrane vesicles (OMV): OMVs are small spherical structures released by P. aeruginosa that contain various bacterial components, including lipids, proteins, and nucleic acids. OMVs are involved in various bacterial functions, such as inter-bacterial communication, virulence, and host immune evasion [65]. OMVs are involved in P. aeruginosa virulence and host immune evasion by delivering virulence factors and toxins to host cells and modulating host immune responses. They can also be engineered to express specific antigens or drug molecules, which can be delivered to host cells and induce immune responses or kill bacterial pathogens [66].

3.2.1.2 Antimicrobial sensitivity profile

P. aeruginosa exhibits a high degree of resistance to a broad spectrum of antibiotics including Aminoglycosides, Piperacillin-Tazobactam, and Carbapenems [67]. Antimicrobial susceptibility of P. aeruginosa strains exhibited a varying degree of resistance to the antibiotics as described [68]. Multidrug resistance for about 8–11 antibiotics was observed among 55.5% of the isolates. Disk diffusion results show 100% resistance to Ampicillin, Cefoperazone, Erythromycin, Norfloxacin, and only Ceftazidime, Ciprofloxacin exhibited greater activity against P. aeruginosa [69]. However, some virulence factors have been linked to antibiotic resistance in specific strains. Pigment production was significantly associated with Ceftazidime susceptibility, phospholipase C production was significantly linked to sensitivity to Cefepime, and DNase production was significantly associated with intermediate resistance to Meropenem. When investigating alginate and the biofilm produced, Ambroxol showed a high anti-biofilm activity against it [70].

3.2.2 Escherichia coli

Escherichia coli (E. coli) is a Gram-negative, rod-shaped, facultative anaerobic bacteria. Non-pathogenic E. coli is considered the normal flora in the gastrointestinal tract. In diabetic foot ulcer infections, E. coli has been shown to be the leading cause of gram-negative diabetic foot infections in Tehran [71]. In some cases, E. coli may gain access to diabetic foot ulcers from urinary tract infections (UTIs), especially in patients with comorbid urinary problems.

3.2.2.1 Virulence factors and pathogenesis

Adhesins: E. coli can produce different types of adhesins such as type 1 fimbriae, P fimbriae, S fimbriae, and afimbrial adhesins that help in colonizing and attaching to the host cells [72, 73].

Iron-acquisition systems: E. coli can produce different types of iron-acquisition systems such as siderophores, hemophores, and iron-regulated outer membrane proteins that help in acquiring iron from the host cells. Siderophores are small molecules that can chelate iron and transport it into the bacterial cells. E. coli can produce different types of siderophores such as enterobactin, aerobactin, and yersiniabactin [74]. Hemophores are proteins that can bind to heme and transport it into the bacterial cells. E. coli can produce different types of hemophores such as HxuA and ChuA [75]. Iron-regulated outer membrane proteins are porins that can regulate the influx of iron into the bacterial cells. E. coli can produce different types of iron-regulated outer membrane proteins such as FepA and CirA [76]. Iron is an essential nutrient for bacterial growth and survival, and its acquisition can help in bacterial pathogenesis.

Capsule: E. coli can produce different types of capsules that help in evading the host immune system [77].

3.2.2.2 Antimicrobial sensitivity profile

E coli was found to be most susceptible to Meropenem, and least susceptible, meaning more resistant to Ciprofloxacin [46]. Other study found that the most prevalent antibiotic resistance pattern for E. coli was observed for ampicillin, Cotrimoxazole, and Ciprofloxacin [78].

3.2.3 Klebsiella species

Klebsiella is a Gram-negative, encapsulated, rod-shaped bacterium belongs to the Enterobacteriaceae family. It is an emerging pathogen in diabetic foot infections (DFIs) and has been reported to be the 3rd most prevalent Gram-negative bacteria in diabetic foot infections in Asian countries [79]. Klebiella has the ability to produce biofilms and its intrinsic resistance to various antibiotics contribute to its pathogenicity, which is challenging to treat [80].

3.2.3.1 Virulence factors and pathogenesis

Polysaccharide capsule: The capsule is a significant virulence factor that protects bacteria from phagocytosis and inhibits the host response. Several capsule types (K), especially K1, K2, K54, K57, K20, and K5, are commonly associated with the pattern of community-acquired invasive pyogenic liver abscess, septicemia, and pneumonia. Moreover, K1, K2, K20, K54, and K57 are predominantly detrimental to experimental infections in mice and are frequently associated with severe infections in humans [81].

Endotoxin: Lipopolysaccharides (LPS) aka endotoxin is formed from lipid A, oligosaccharide synopsis, O antigens, and are known as endotoxins based on all Gram-negative pathogens, including K. pneumoniae. The outermost subunit based on LPS, O antigen, is the primary constituent faced by the innate immune system and protects bacteria against complement-mediated inflammation [82]. In particular, the O antigens bind to the complement protein C3b, which involves pore arrangement before mediating the perforation of host tissue. At present, it is not clear whether the LPS hvKP strain endotoxin activity has an individual role in hypervirulence [81].

Siderophore: Pathogenic bacteria require iron for their replication. Siderophores (iron carriers) are composites buried by microorganisms (similar to bacteria and fungi) to transport iron in the cell membranes. They bind iron with higher affinity than the host transport protein transferrin [83]. K. pneumoniae produces siderophores to gain iron from host iron-chelating proteins or the terrain for survival and reduplication during mammalian infection. Enterobactin, yersiniabactin, salmochelin, and aerobactin are different types of siderophores expressed by K. pneumonia, with the latter one enabling growth and survival of more virulent strains of the pathogen [84]. Further, K. pneumoniae has an iron-scavenging system that helps survival in iron-limited environments. In addition to production of siderophores production, the expression of iron-regulated outer membrane proteins (IROMPs) facilitates the uptake of iron from host proteins.

Adhesins: Adhesins are proteins that help bacteria to adhere to host cells and tissues. K. pneumoniae expresses several types of adhesins, including fimbriae, which are hair-like structures that help bacteria to attach to surfaces. Fimbria types 1 and 3 are crucial virulence factors contributing to biofilm formation in K. pneumonia [85].

3.2.3.2 Antimicrobial sensitivity profile

Klebsiella species are known for their ability to develop resistance to many antibiotics, especially in environments of chronic wound infections like diabetic foot ulcers. Yoga et al. mentioned that Klebsiella pneumoniae isolates were resistant to Ampicillin and Ceftazidime in 83 and 50% respectively [86].

3.2.4 Proteus species

Proteus is a Gram-negative, rod-shaped, aerobic and motile bacteria that belongs to the Enterobacteriaceae family. It is able to migrate across surfaces due its “swarming” characteristic [87]. In diabetic foot ulcers, Proteus species have been identified as one of the significant causative infective agents, particularly in polymicrobial infections [13]. Proteus species are well known to cause community-acquired infections, and urinary tract infections in nosocomial status.

3.2.4.1 Virulence factors and pathogenesis

Biofilms: Proteus mirabilis has the ability to form biofilms, which are communities of microorganisms that adhere to surfaces and are embedded in a self-produced extracellular matrix. Biofilms protect bacteria from host defenses and antibiotics, making them difficult to eradicate [88].

Adhesion molecules: Adhesion virulence factors in Proteus mirabilis encompass a range of components crucial for its pathogenicity. One significant factor is the classical autotransporter, Proteus toxic agglutinin (pta), which functions as a serine protease and exhibits cytotoxic effects on bladder and kidney epithelial cells [89]. Additionally, fimbrial genes play a pivotal role in adhesion, with P. mirabilis possessing a diverse repertoire of at least 10 distinct fimbrial types that enhance its fitness during polymicrobial infections [90]. Furthermore, the flagellar cascade components contribute to P. mirabilis’ motility, facilitating its adhesion to host tissues. In addition, cell wall features are also associated with biofilm formation, which is closely linked to adhesion in P. mirabilis [91].

Urease: Proteus mirabilis produces urease, an enzyme that hydrolyzes urea to produce ammonia and carbon dioxide. This increases the pH of the urine, leading to the formation of struvite and calcium phosphate stones, which can cause tissue damage and inflammation [92].

Proteases: Proteus mirabilis produces several proteases that can degrade host proteins, leading to tissue damage and inflammation [87]. One of the proteases secreted by Proteus mirabilis is the extracellular metalloproteinase. This metalloproteinase cleaves serum immunoglobulin A (IgA) [93], which is an important immunoglobulin for the body’s defense against infections and pathogens. The cleavage of serum immunoglobulin A by the extracellular metalloproteinase produced by Proteus mirabilis can compromise the immune system’s ability to neutralize and protect against invading microorganisms, contributing to tissue damage and inflammation [94].

Siderophores: Proteus mirabilis produces siderophores, which are small molecules that bind to iron and transport it into the bacterial cell. One of the main siderophores secreted by Proteus mirabilis is Proteobactin. the production of proteobactin allows the bacterium to scavenge and acquire iron from its environment, even when iron is limited. Proteobactin can bind to iron ions in the surroundings, forming a complex that the bacterium can then take up into the cell [95]. This ability to obtain iron is crucial for the bacterium’s growth and survival, especially in iron-restricted conditions.

Toxins: Proteus mirabilis produces several toxins, including hemolysin and cytotoxic necrotizing factor, which can cause tissue damage and inflammation [87]. Hemolysin is a protein that can cause the lysis of red blood cells, leading to the release of hemoglobin and other cellular contents. This can cause tissue damage and inflammation and may contribute to the virulence of Proteus mirabilis [96]. Cytotoxic necrotizing factor is a protein that can cause cell death and tissue damage. It has been shown to induce apoptosis (programmed cell death) in various cell types, including epithelial cells and macrophages [97]. This can lead to tissue damage and inflammation and may contribute to the pathogenesis of Proteus mirabilis.

3.2.4.2 Antimicrobial sensitivity profile

Proteus isolates were found to be sensitive to Imipenem, Meropenem, and Amikacin, with some being sensitive and others being resistant to Ciprofloxacin and Gentamicin. However, tested isolated were found most resistant to Tetracycline, Ampicillin, Cefaroxin, Ceftixime, and Cefipime [98].

3.3 Non-bacterial infections (yeast/fungi)

3.3.1 Candida albicans

Candida albicans is a fungus (yeast) that is part of the human microbiota and present at different parts of human body, including the skin. This yeast can cause oral, vaginal, or dermal infections known as monilial thrush [99]. In a study conducted on a population in Delhi, Candida spp was identified as the most commonly isolated yeast from diabetic foot ulcers, with Candida albicans being reported as the most prevalent species [100].

3.3.1.1 Virulence and pathogenesis

The hallmark of C. albicans infections is biofilm formation [101]. This process can be summarized in the following steps:

Adherence: it begins with single fungal cells adhering to host substrate. Once the yeast cells have attached to the substrate, they start to multiply and form a layer of cells that serves as the foundation of the biofilm. This layer is called the basal yeast cell layer, and it provides a stable surface for the growth of additional layers of cells [101].

In addition, Quorum sensing, or cell-cell communication, is an important phenomenon in microbial biofilms, where cell density and secreted signaling molecules govern microbial behaviors [101]. Farnesol, a quorum-sensing molecule produced by C. albicans inhibits hyphal formation and promotes yeast cell formation, aiding in biofilm dispersal. At high concentrations, farnesol triggers apoptosis in C. albicans through oxidative stress and accumulation of reactive oxygen species [101].

3.3.1.2 Antifungal sensitivity profile

Although the highest susceptibility was observed in flucytosine and amphotericin B, Candida species in a study conducted at a tertiary hospital in Kenya demonstrated notable susceptibility to various antifungal agents, including voriconazole, micafungin, and fluconazole, while showing higher resistance to caspofungin. Among the Candida species, C. albicans exhibited resistance to several antifungal agents but remained susceptible to amphotericin B and flucytosine [102].

4. Polymicrobial infections

In complicated diabetes mellitus where DFU are more prevalent, polymicrobial infections are more common than mono-bacterial ones [45, 71, 103]. Polymicrobial infections can include bacterial and non-bacterial agents such as fungi or viruses, with bacteria being the most dominant infections [104]. The diverse bacteria present in these infections can synergistically enhance tissue damage, exacerbate inflammation, and impair the host immune response. Generally, DFU infections arise from skin microbiota, however the pathogenesis of these microorganisms chronically can create an environment for non-native bacterial infections [103]. It is difficult to predict the specific bacterial strains contributing to polymicrobial infection in DFU due to the variability in the bacterial composition and antimicrobial resistance patterns [103]. Chronic wound infections and ulcers often lead to osteomyelitis. This obstacle in recognizing the causative agents and treating wounds urgently results in progression to deeper tissue ending with lower-limb amputations as a lifesaving measure [105].

5. Correlation between various pathogens and the severity of DFU

The pathogenesis of DFU as a polymicrobial disease involves various types of pathogens as previously discussed. Among the major factors contributing to the prognosis and complications of DFU, some pathogens have been shown to correlate with more severe manifestations of the disease as discussed below.

5.1 Gram-positive bacteria

Gram-positive bacteria, particularly S. aureus, are frequently implicated in mild and moderate infections corresponding to grades 1 and 2 on the Wagner scale measuring the severity of DFU. However, the genome of S. aureus found in cases of poor wound healing outcomes contained a number of genes responsible for antibiotic resistance and genes encoding staphylococcal enterotoxins leading to more severe stages of DFU [106]. An important gene is the agr type III agrABCD operon, which contains genes responsible for the AGR quorum sensing system, which produces autoinducing peptides (AIPs). This system plays a crucial role in regulating various aspects, including biofilm formation and the expression of virulence factors such as toxins and degradative exoenzymes. Clinically, a study conducted at a central Malaysian tertiary care hospital indicated that Staphylococcus spp infections are associated with major amputations [45]. In a similar manner, DFU patients infected with Enterococci exhibited a significantly higher in-hospital amputation rate (72%) compared to those with non-Enterococci infections (50%) [27]. Furthermore, DFIs caused by MRSA demonstrated a comparable level of infection severity to MSSA, with the notable exception of the α-toxin’s heightened role in MSSA pathogenicity.

While various articles have presented evidence suggesting similar clinical presentations and outcomes between MRSA and other pathogens in DFIs, some authors maintain that MRSA detection in DFIs may still be associated with more severe infections [14]. In the realm of DFIs, experts continue to consider anaerobic bacteria as potential contributors to persistent and severe infections, even though their exact role in polymicrobial infections remains a subject of debate [107]. This is supported by studies indicating that anaerobic bacteria are often detected in DFIs characterized by deeper ulcers, chronicity, and associations with ischemia, necrosis, gangrene, or foul odor [108]. The presence of anaerobes may thus play a role in the severity and persistence of certain DFIs.

Additionally, Clostridium species presence is prominently linked to diabetic foot infection severity, indicated by severity-based grading of Wagner-Meggitt classification [109]. The presence of Clostridium perfringens for instance is found in mostly grade 5 diabetic foot ulcers [86]. This does not only reveal the seriousness of anaerobic infections but also highlights that their involvement is accompanied with more detrimental outcomes in DFU patients. Anaerobic infection, especially Clostridium spp, are present in more severe cases. As the Wagner-Meggitt classification grading advanced, there was a corresponding rise in the incidence of anaerobic pathogens. Notably, ulcers infected with anaerobic pathogens displayed an extended healing duration compared to those infected with aerobic pathogens [110].

5.2 Gram-negative bacteria

In a study conducted at a tertiary hospital in central Malaysia, P. aeruginosa emerged as the primary contributor to amputations, and notably, a majority of amputee patients were male [45]. The severity of infections caused by this opportunistic pathogen was closely linked to the specific strain of Pseudomonas and its antibiotic sensitivity.

While Escherichia coli is not commonly associated with DFIs, surprisingly it was identified as the fourth most frequent Gram-negative bacterium in DFIs in central Malaysia. All E. coli infections resulted in major amputations, underscoring E. coli’s aggressive capacity to infiltrate deep tissues, including bones, leading to osteomyelitis [45]. E. coli encompasses multiple strains, each exhibiting variations in virulence and antibiotic sensitivity. A study conducted in southern Iran reported that E. coli as the most prevalent infectious agent in DFIs leading to osteomyelitis [10].

Klebsiella infections are generally infrequent in DFUs. However, their occurrence often signals an immune-compromised state that allows Klebsiella species to invade DFU wounds. Klebsiella infections tend to be perilous and are typically associated with moderate to severe diabetic foot wound infections, as per the Infectious Diseases Society of America (IDSA) grading criteria [71].

Proteus species were found to be present across a spectrum of diabetic foot infections, encompassing mild, moderate, and severe cases, with varying prevalence rates. They were notably more prevalent in moderate to severe cases, corresponding to grades 3 and 4 of Wagner’s DFU wound classification. Additionally, in some instances, the severity of infection was positively correlated with a higher prevalence of Proteus spp. isolates [71].

5.3 Fungi and other pathogens

A study conducted on Delhi population revealed a concerning association between chronic non-healing ulcers, which often lead to amputations, and an increasing prevalence of fungal infections [34].

6. The role of biofilms in chronic diabetic foot infection

Biofilms play an important role in developing antimicrobial resistance in diabetic foot infections. Biofilms provide a protective layer for bacteria, allowing them to survive antibiotic treatment. These complex communities produce extracellular polymeric substances (EPS) to create a symbiotic environment for bacteria to thrive in the nutrient-rich environment, triggering chronic inflammatory responses that further worsen the infection. Macrophages and neutrophils at the infection site release Reactive Oxygen Species (ROS) and proteases, which deteriorate biofilms. However, in chronic infections, neutrophils damage surrounding tissue, allowing bacteria to colonize further surrounding tissue. Various studies have shown that chronic inflammatory responses, like the over-expression of matrix metalloproteinases (MMPs) and inflammatory cytokines are associated with the chronicity of diabetic foot ulcers. Lobmann et al. [111]. Demonstrated that MMP-1 levels were 65 times higher in chronic diabetic foot ulcers compared to normal healing ulcers. Furthermore, according to Mottola et al. [112], polymicrobial bacterial populations were found to produce larger biofilms than any individual bacteria.

7. Antimicrobial treatment of DFU

Empirical antibiotic treatment should be determined considering the infection’s seriousness and the probability of resistant pathogens being involved. Once culture and susceptibility results are available, antibiotic therapy should be adjusted accordingly. However, it’s not always essential to target every microorganism identified in cultures. Patients with non-infected ulcers should not be prescribed antibiotics. Instead, these patients typically benefit from localized wound management and measures aimed at alleviating pressure at the ulceration site [113, 114, 115]. Diabetic foot infections treatment with antimicrobial usually depends on the severity of infections, whether mild, moderate, or severe, and the causative pathogens involved as detailedin Table 1.

Recommended DFI Antimicrobial Regimen
Prevention of infections	No antibiotic therapy is needed for prevention of diabetic foot infections
Mild infections	Start oral antibiotics with activity against gram-positive organisms.; Prolonged antibiotic treatment (>14 days) is not recommended for mild soft tissue infections.
Moderate infections	Start antibiotics with activity against gram-positive and gram-negative organisms.; Moderate infections: choose the route of administration based on the clinical situation and antibiotic choice.
Severe infections	Antibiotics with activity against gram-positive and gram-negative organisms.; Begin treatment with intravenous antibiotics and then reassess based on the clinical situation.
Streptococci and Staphylococci (MSSA) infections	Cephalexin (500 mg every 6 hours); or; Dicloxacillin (500 mg every 6 hours); or; Amoxicillin-clavulanate (875/125 mg every 12 hours) or Moxifloxacin; + Trimethoprim-sulfamethoxazole (2 double-strength tablets (trimethoprim 160 mg and sulfamethoxazole 800 mg per tablet) every 12 hours); or; Ciprofloxacin or Levofloxacin; + Clindamycin (300 to 450 mg every 6 to 8 hours)
Additional antibiotics (in case MRSA coverage is warranted)	Vancomycin; or; Linezolid (600 mg every 12 hours); or; Daptomycin
Additional recommendations on dosing:	NICE recommends that patients with diabetes and osteomyelitis should be given prolonged antibiotic treatment (usually 6 weeks), taking into account local protocols; IDSA recommends considering prescribing antibiotic therapy for a short duration only e.g. 2–5 days if there is no infected tissue remaining. However, prolonged antibiotics are recommended e.g. 6 weeks if there is persistent infected bone or necrosis.

Table 1.

Antimicrobial regimen for the treatment of DFU as recommended by the IDSA, NICE, and the National Guidelines in Qatar [113, 114, 115].

Conflict of interest

The authors declare no conflict of interest.