Homocysteine and Inflammatory Cytokines in the Clinical Assessment of Infection in Venous Leg Ulcers

Abstract

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.

Bacterial Biofilm in Chronic Wounds and Possible Therapeutic Approaches

“…Chronic wounds are disposed to bacterial infection, leading to serious complications. Managing wounds in the presence of infection is still challenging due to prolonged healing and frequent reoccurrence [7]. The recent development of new-generation sequencing targeting the species-specific small subunit ribosomal RNA (16S rRNA) gene and shotgun metagenomics allows the characterization of the skin and wound microbiota thanks to the ability to identify anaerobic microorganisms [54].…”

“…Chronic wounds of different etiologies showed that Staphylococcus and Pseudomonas were the most common genera, 63% and 25%, respectively [61]. A study on chronic venous leg showed that Gram-negative bacteria constituted 51.7%, mostly represented by Pseudomonas aeruginosa, followed by Escherichia coli, Serratia marcescens, Enterobacter cloacae, and Morganella morganii, while the Gram-positive bacteria constituted 48.3%, with Staphylococcus aureus as the dominant species [7]. Indeed, the colonization of the chronic wound is characterized by the presence of multiple microorganisms, initially by Gram-positive belonging to Staphylococcus genera, which do not elicit strong immune responses [62], and subsequently, by Gram-negative bacteria such as Pseudomonas spp., E. coli, Klebsiella pneumoniae, and Enterobacter spp., which become the predominant species in the microenvironment.…”

“…This colonization leads to an increase in the bioburden, causing a delay in the healing process. Microbial products released by pathogenic bacteria can trigger various pathways of cytokine production to promote their survival and persistence within the host [7]. In particular, the deregulation of anti-inflammatory cytokine IL-10 is associated with virulence strategies leading to poor antimicrobial effector mechanisms, increased disease severity, and chronicity in S. aureus and P. aeruginosa infections [63].…”

“…The ability of pathogenic species to produce multicellular structures, called biofilms, is frequently associated with difficult-to-treat infections and high mortality rates [64]. Biofilms are defined as multicellular structures consisting of multiple bacterial species that adhere to a surface and are enclosed within an extracellular polymeric substance (EPS) [7,65]. This matrix is composed of polysaccharides, proteins, glycolipids, and extracellular DNA capable of protecting bacteria and facilitating intercommunication through chemical and physical signals, increasing both the integrity and strength of the biofilm [61,66].…”

“…Failure to proceed through the phases of the healing process in an orderly and timely manner leads to chronic wounds [6]. Chronic wounds represent a major health concern, and their management is particularly challenging, especially in the presence of infections [7].…”