Breaking the Barrier of Chronic Infection Challenges: Multi-Species Biofilms Targeted via Complex Mechanisms and Customized Combination Therapies
Background
In clinical settings, recurring catheter infections, refractory chronic wounds, and severe respiratory infections are driven by biofilms—microbial communities that form protective structures. Traditional infection research and antibiotic susceptibility assessments have focused on single-strain culture environments. However, microbes present in actual in vivo lesions do not exist in isolation but form complex communities of various species.
Multi-species biofilms exhibit survival capabilities that differ physiologically from those of single-species biofilms. Different microorganisms within the community enhance their viability by exchanging metabolic byproducts and communicating via chemical signals to respond collectively to external attacks. They also reconfigure the local microenvironment, such as oxygen partial pressure or acidity, to their advantage. This is further reinforced by a physical defense matrix composed of extracellular polymeric substances (EPS), which blocks the penetration of conventional antibiotics. Horizontal gene transfer (HGT) occurring actively between bacteria within the community is a primary driver in drastically accelerating the acquisition of multi-drug resistance. This is why conventional single-antibiotic-centered dosing has repeatedly led to treatment failure and recurrence.
Key Findings
Multifaceted approaches to effectively disrupt pathogenic complex microbial communities are rapidly advancing. This review examines the formation mechanisms and refractory characteristics of multi-species biofilms and provides a comprehensive analysis of seven next-generation therapeutic strategies to neutralize them.
The control technologies presented by the researchers are precisely categorized by their mechanisms of action. First is quorum sensing (QS) inhibition technology, which disrupts the communication system between bacteria. It prevents aggregation by blocking autoinducer signals that induce community formation. Second is matrix-targeting therapy that attacks the EPS, the physical barrier; this involves using matrix-specific enzymes such as Dispersin B or alginate lyase to break down the structure, allowing antibiotics to penetrate. Third is bacteriophage therapy. This involves precisely targeting specific dominant bacterial species within the community or using endolysin enzymes secreted by phages to simultaneously degrade both the matrix and cell walls.
Approaches involving the delivery of CRISPR-Cas gene editors to directly disrupt specific resistance or toxin genes were also discussed as a major technology. Additionally, metabolic disruptors that block the uptake of essential nutrients like iron, natural antimicrobial peptides and plant-derived phytochemicals, and nanocarrier technologies that maximize drug penetration efficiency were identified as candidates for combination therapies. These strategies have been confirmed to significantly degrade already formed mature biofilms and restore susceptibility to antimicrobial agents. However, therapeutic efficacy varied considerably depending on the relative abundance of microbial community composition, biofilm maturity, and substrate component characteristics.
Significance and Outlook
Research into controlling multi-species biofilms has provided a foothold for shifting the paradigm of infectious disease treatment from the eradication of single pathogens to the disruption of complex ecosystems. Combination therapies, such as co-administering quorum sensing inhibitors or matrix-degrading enzymes with existing antibiotics, are considered promising alternatives for treating refractory infections while drastically reducing antibiotic usage. Technologies such as applying nanocomposite coatings to medical device surfaces or utilizing phage cocktails are also gaining high industrial value in the field of preventing implant and prosthetic joint infections.
However, several challenges remain before actual clinical application. The combination of species coexisting in patient lesions varies significantly, and the matrix composition also differs according to the patient's immune response. There is also a limitation where efficacy results obtained from simple co-culture models at the laboratory level may not be reproduced in the complex in vivo infection environment of actual patients. It is time for follow-up research to establish infection models that precisely simulate clinical environments and develop stepwise combination dosing protocols based on patient-specific strain analysis.
Mixed-species biofilm infections are important causes of persistent and difficult-to-treat infections. Compared with monospecies biofilms, mixed-species communities contain microorganisms with different physiological and genetic characteristics that interact through metabolic cooperation, chemical signaling, competition, and modification of the local environment. These interactions, together with the EPS matrix, physiological heterogeneity, and HGT, can increase antimicrobial tolerance, contributing to treatment failure and infection recurrence. Polymicrobial biofilms are associated with chronic wounds, respiratory infections, catheter- and device-associated infections, and other persistent infections. However, treatment of polymicrobial biofilms remains challenging because their complex structure and interspecies interactions can reduce antimicrobial efficacy. This review summarizes the clinical significance, formation, and persistence of mixed-species biofilms and discusses recent therapeutic strategies for their control. We also discuss recent approaches for controlling polymicrobial biofilms, including natural compounds, QS inhibition, CRISPR-Cas-based approaches, biofilm matrix targeting, metabolic and nutrient disruption, nanotechnology, and phage therapy. Recent studies show that these approaches can inhibit biofilm formation, disrupt established biofilms, enhance antimicrobial activity, and selectively target specific microbial populations. However, their effectiveness varies with microbial composition, biofilm maturity, matrix characteristics, and treatment conditions. Overall, current evidence supports the development of targeted and combination-based strategies for mixed-species biofilms. Further studies using clinically relevant models are needed to exactly define treatment efficacy and support the translation of these approaches into clinical practice.
This research presents new therapeutic pathways for patient groups where existing antibiotic treatments have failed, such as those with diabetic foot ulcers or chronic burn wounds. A representative example is the development of smart dressings that apply a matrix-degrading hydrogel patch to a wound to remove the protective barrier, followed by the sequential release of low-dose combination antimicrobials to degrade the complexes of Pseudomonas aeruginosa and Staphylococcus aureus proliferating at the wound site. For patients with ventilator-associated pneumonia or indwelling urinary catheters, the incidence of infection can be preemptively suppressed by coating medical device surfaces with nanomaterials that inhibit biofilm formation signals. The value of customized combination control technologies is expected to grow increasingly in the field of intensive care, where patients struggle against single-agent failures.