Emerging Issues and Initial Insights into Bacterial Biofilms: From Orthopedic Infection to Metabolomics

Abstract

Bacterial biofilms, enigmatic communities of microorganisms enclosed in an extracellular matrix, still represent an open challenge in many clinical contexts, including orthopedics, where biofilm-associated bone and joint infections remain the main cause of implant failure. This study explores the scenario of biofilm infections, with a focus on those related to orthopedic implants, highlighting recently emerged substantial aspects of the pathogenesis and their potential repercussions on the clinic, as well as the progress and gaps that still exist in the diagnostics and management of these infections. The classic mechanisms through which biofilms form and the more recently proposed new ones are depicted. The ways in which bacteria hide, become impenetrable to antibiotics, and evade the immune defenses, creating reservoirs of bacteria difficult to detect and reach, are delineated, such as bacterial dormancy within biofilms, entry into host cells, and penetration into bone canaliculi. New findings on biofilm formation with host components are presented. The article also delves into the emerging and critical concept of immunometabolism, a key function of immune cells that biofilm interferes with. The growing potential of biofilm metabolomics in the diagnosis and therapy of biofilm infections is highlighted, referring to the latest research.

Keywords: bacterial dormancy; biofilm; canaliculo-lacunar network reservoir; immunometabolism; internalization; metabolomics; orthopedic implant infections.

Figure 1

The classical model of the biofilm cycle: the four-step process of biofilm formation in S. aureus. Planktonic cells adhere to the biomaterial surface through AtlA adhesin. Then, MSCRAMMs and SERAMs bacterial adhesins interact with the extracellular matrix (ECM) proteins coating the implant. Bacteria proliferate and produce an extracellular polymeric substance consisting of proteins (among which is the biofilm-associated protein Bap), the intercellular polysaccharide adhesin (PIA), and a series of other polymeric extracellular substances, among which is extracellular DNA (eDNA). Once a mature biofilm has formed, under the control of the quorum sensing system, the enzymes β-phenol-soluble modulins, proteases, and murein hydrolases dissolve biofilm and release planktonic bacteria to initiate a new biofilm cycle. The more recently emerged role of the microenvironment and host molecules in biofilm formation is also recalled. Abbreviations: AtlA, the major autolysin of S. aureus; EPS, extracellular polymeric substance; MSCRAMMs, Microbial Surface Components Recognizing Adhesive Matrix Molecules; SERAMs, Secretable Expanded Repertoire Adhesive Molecules.

Figure 2

S. aureus internalization into osteoblasts. Two entry mechanisms are depicted. One is mediated by fibronectin, which forms a bridge between staphylococcal FnBP adhesin and osteoblast α₁β₅ integrin. This interaction promotes expression of Tumor Necrosis Factor-Related Apoptosis Inducing Ligand (TRAIL), which induces caspase 8 activation and the consequent osteoblast apoptosis. The other way is mediated by the interaction of Staphylococcal protein A (SpA) and Tumor Necrosis Factor Receptor 1 (TNFR-1) on the osteoblast surface. This interaction promotes the expression of the Receptor Activator of NF Kappa B Ligand (RANKL), a key cytokine in promoting osteoclastogenesis. Bone destruction results from the apoptotic death of osteoblasts and from the activation of osteoclasts. Other, cytokines released by internalized osteoblasts recruit monocytes/macrophages and induce their differentiation into osteoclasts, thus corroborating osteoclastogenesis. Also depicted are bacteria nestled in the bone canaliculi and dormant bacteria within biofilm.

Figure 3

An example of the relationship between immune defenses and bacterial biofilm is summarized, in which c-di-NMPs are involved. Biofilms produce c-di-NMPs. c-di-NMPs stimulate the synthesis of biofilm matrix components. Host cells sense c-di-NMPs as PAMPs. As a result, an immune response is generated to eliminate pathogens. But, the biofilm matrix protects biofilm bacteria as a shield against phagocytosis and antibody deposition by plasma cells. Furthermore, biofilm induces the reprogramming of macrophages towards an M2 phenotype, characterized by poor microbicidal activity, and the recruitment of myeloid-derived suppressor cells (MDSCs) [142]. MDSCs produce the immunosuppressive molecule interleukin-10 (IL-10), which attenuates the immune response and contributes to biofilm persistence. Some bacteria within biofilms can be phagocytosed by immune cells but resist ROS and AMPs (such as defensins 3 and LL-37) thanks to the polysaccharides of their matrix [27,143,144,145]. They can survive intracellularly, thus contributing to the chronic nature of biofilm-associated infections. Abbreviations: c-di-NMPs, cyclic dinucleotides (intracellular signaling second-messenger systems); MØ, macrophages; IFN-γ, interferon-γ; NK, natural killer cells; PAMPs, pathogen-associated molecular patterns; T cells, T lymphocytes; B cells, B lymphocytes/plasma cells (plasma cells derive from B lymphocytes after encountering the antigen); Ab, antibodies; PMNs, polymorphonuclear neutrophils; AMPs, antimicrobial peptides; ROS, reactive oxygen species.