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Chronic infections present a serious economic burden to health-care systems. The severity and prevalence of chronic infections are continuously increasing due to an aging population and an elevated number of lifestyle related diseases such as diabetes. Treatment of chronic infections has proven difficult, mainly due to the presence of biofilms that render bacteria more tolerant toward antimicrobials and the host immune response. Chronic infections have been described to harbor several different bacterial species and it has been hypothesized that microscale interactions and mixed-species consortia are present as described for most natural occurring biofilms i.e., aquatic systems and industrial settings, but also for some commensal human biofilms i.e., the mouth microbiota. However, the presence of mixed-species biofilms in chronic infections is most often an assumption based on culture-based methods and/or by means of molecular approaches, such as PCR and sequencing performed from homogenized bulk tissue samples. These methods disregard the spatial organization of the bacterial community and thus valuable information on biofilm aggregate composition, spatial organization, and possible interactions between different species is lost. Hitherto, only few studies have made visual  in situ  presentations of mixed-species biofilms in chronic infections, which is pivotal for the description of bacterial composition, spatial distribution, and interspecies interaction on the microscale. In order for bacteria to interact (synergism, commensalism, mutualism, competition, etc.) they need to be in close proximity to each other on the scale where they can affect e.g., solute concentrations. We argue that visual proof of mixed species biofilms in chronic infections is scarce compared to what is seen in e.g., environmental biofilms and call for a debate on the importance of mixed-species biofilm in chronic infections.

Do Mixed-Species Biofilms Dominate in Chronic Infections?–Need for in situ Visualization of Bacterial Organization