Microbial biofilms are complex three‐dimensional structures where sessile microbes are embedded in a polymeric extracellular matrix. Their resistance toward the host immune system as well as to a diverse range of antimicrobial treatments poses a serious health and development threat, being in the top 10 global public health threats declared by the World Health Organization. In an effort to combat biofilm‐related microbial infections, several strategies have been developed to independently eliminate biofilms or to complement conventional antibiotic therapies. However, their limitations leave room for other treatment alternatives, where the application of nanotechnology to biofilm eradication has gained significant relevance in recent years. Their small size, penetration efficiency, and the design flexibility that they present makes them a promising alternative for biofilm infection treatment, although they also present set‐backs. This review aims to describe the main possibilities and limitations of nanomedicine against biofilms, while covering the main aspects of biofilm formation and study, and the current therapies for biofilm treatment.This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Toxicology and Regulatory Issues in Nanomedicine > Toxicology of Nanomaterials Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine

Pathogenic bacteria form biofilms during infection, and polymicrobial biofilms are the most frequent manifestation. Biofilm attachment, maturation, and/or antibiotic sensitivity are mainly evaluated with microtiter plate assays, in which bacteria are stained to enable the quantification of the biomass by optical absorbance or fluorescence emission. However, using these methods to distinguish different species in dual-species or polymicrobial biofilms is currently impossible. Colony-forming unit counts from homogenized dual-species biofilms on selective agar medium allow species differentiation but are time-consuming for a high-throughput screening. Thus, reliable, feasible, and fast methods are urgently needed to study the behavior of polymicrobial and dual-species communities. This study shows that Pseudomonas aeruginosa and Burkholderia cenocepacia strains expressing specific fluorescent or bioluminescent proteins permit the more efficient study of dual-species biofilms compared to other methods that rely on measuring the total biomass. Combining fluorescence and bioluminescence measurements allows an independent analysis of the different microbial species within the biofilm, indicating the degree of presence of each one over time during a dual-species biofilm growth. The quantitative strategies developed in this work are reproducible and recommended for dual-species biofilm studies with high-throughput microtiter plate approaches using strains that can constitutively express fluorescent or bioluminescent proteins.

Multispecies biofilms are communities composed of different microorganisms embedded in an auto-synthesized polymeric matrix. Pseudomonas aeruginosa and Burkholderia cenocepacia are two multidrug-resistant and biofilm-forming opportunistic pathogens often found in the lungs of people living with cystic fibrosis. In this context, planktonic, static, and dynamic biofilms and in vivo models of both species were optimized in this work to understand their population dynamics, disposition, virulence, and antibiotic susceptibility. From the coculture models optimized in this work, we determined that B. cenocepacia grows in a clustered, aggregative manner at the bottom layers of biofilms, in close contact with P. aeruginosa, that tends to occupy the top layers. Their coexistence increases virulence-related gene expression in both species at early stages of coinfection and in in vivo models, while there was a general downregulation of virulence-related genes after longer coexistence periods as they eventually reach a non-competitive stage during chronic infections. When evaluating antimicrobial susceptibility, a decrease of antimicrobial tolerance was observed in both species when co-cultured. These findings shed light on the differential behavior of P. aeruginosa and B. cenocepacia in dual-species systems, stressing the relevance of multispecies studies in the clinical context.

Polymicrobial infections promote the appearance of a network of interactions that can lead to an increase in their antimicrobial tolerance or to the evasion of the host immune system. Pseudomonas aeruginosa and Burkholderia cenocepacia are two multidrug-resistant opportunistic pathogens that significantly influence host health and alter their antibiotic response when in coinfection. To characterize host-pathogen dynamics, we examined infection progression, immune responses, bacterial virulence gene expression, and antibiotic susceptibility in single and coinfections involving acute and chronic P. aeruginosa strains combined with B. cenocepacia . This work was entirely performed in vivo using G. mellonella larvae as a model. Larval survival and bacterial dissemination were monitored, revealing tissue-specific patterns of infection. Our findings indicated that coinfections increased larval lethality and worsened overall health. Notably, B. cenocepacia suppressed host melanization and immune responses, while P. aeruginosa triggered a strong immune activation. Coinfection also induced upregulation of virulence genes in both pathogens. Surprisingly, increased antibiotic susceptibility was observed in coinfected groups compared to single infections. This study advances understanding of host-pathogen interactions in polymicrobial infections and highlights the need for improved therapeutic strategies. Author summary: Bacterial coinfections, such as the ones with Pseudomonas aeruginosa and Burkholderia cenocepacia are especially critical in chronic lung diseases like cystic fibrosis, where persistent polymicrobial infections drive lung damage and health decline. Coinfection can enhance pathogen survival, increase drug tolerance, and disrupt host immunity, leading to worse clinical outcomes. Understanding coinfection dynamics can help improve treatment strategies, optimize antibiotic use and improve infection monitorization. Therefore, by studying and understanding microbial interactions, researchers can develop more effective therapies, ultimately improving patient care and combating the growing challenge of multidrug-resistant bacterial infections.

Treating bacterial biofilms remains a major clinical challenge, particularly in uropathogenic infections where the dense extracellular matrix impedes antib