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Biofilms are spatially organized communities of microorganisms embedded in a self-produced organic matrix, conferring to the population emerging properties such as an increased tolerance to the action of antimicrobials. It was shown that some bacilli were able to swim in the exogenous matrix of pathogenic biofilms and to counterbalance these properties. Swimming bacteria can deliver antimicrobial agents in situ, or potentiate the activity of antimicrobial by creating a transient vascularization network in the matrix. Hence, characterizing swimmer trajectories in the biofilm matrix is of particular interest to understand and optimize this new biocontrol strategy in particular, but also more generally to decipher ecological drivers of population spatial structure in natural biofilms ecosystems. In this study, a new methodology is developed to analyze time-lapse confocal laser scanning images to describe and compare the swimming trajectories of bacilli swimmers populations and their adaptations to the biofilm structure. The method is based on the inference of a kinetic model of swimmer populations including mechanistic interactions with the host biofilm. After validation on synthetic data, the methodology is implemented on images of three different species of motile bacillus species swimming in a Staphylococcus aureus biofilm. The fitted model allows to stratify the swimmer populations by their swimming behavior and provides insights into the mechanisms deployed by the micro-swimmers to adapt their swimming traits to the biofilm matrix. Anyone who has ever cleaned a bathroom probably faced biofilms, the dark, slimy deposits that lurk around taps and pipes. These structures are created by bacteria which abandon their solitary lifestyle to work together as a community, secreting various substances that allow the cells to organise themselves in 3D and to better resist external aggression. Unwanted biofilms can impair industrial operations or endanger health, for example when they form inside medical equipment or water supplies. Removing these structures usually involves massive application of substances which can cause long-term damage to the environment. Recently, researchers have observed that a range of small rod-shaped bacteria – or ‘bacilli’ – can penetrate a harmful biofilm and dig transient tunnels in its 3D structure. These ‘swimmers’ can enhance the penetration of anti-microbial agents, or could even be modified to deliver these molecules right inside the biofilm. However, little is known about how the various types of bacilli, which have very different shapes and propelling systems, can navigate the complex environment that is a biofilm. This knowledge would be essential for scientists to select which swimmers could be the best to harness for industrial and medical applications. To investigate this question, Ravel et al. established a way to track how three species of bacilli swim inside a biofilm compared to in a simple fluid. A mathematical model was created which integrated several swimming behaviors such as speed adaptation and direction changes in response to the structure and density of the biofilm. This modelling was then fitted on microscopy images of the different species navigating the two types of environments. Different motion patterns for the three bacilli emerged, each showing different degrees of adapting to moving inside a biofilm. One species, in particular, was able to run straight in and out of this environment because it could adapt its speed to the biofilm density as well as randomly change direction. The new method developed by Ravel et al. can be redeployed to systematically study swimmer candidates in different types of biofilms. This would allow scientists to examine how various swimming characteristics impact how bacteria-killing chemicals can penetrate the altered biofilms. In addition, as the mathematical model can predict trajectories, it could be used in computational studies to examine which species of bacilli would be best suited in industrial settings.

Our research explores the mechanisms of bacterial adhesion within the dental plaque, focusing on Veillonella parvula , a key player in the oral microbiome. Dependent on lactate from streptococci, V. parvula plays a crucial bridging role in the formation of dental biofilms by co-aggregating with other bacteria. Despite its importance, the understanding of the underlying mechanisms of co-aggregation remains limited. Our study shows that V. parvula uses different trimeric autotransporters to adhere to oral Streptococci and Actinomyces. We additionally identify a novel adhesin from S. gordonii , VisA (SGO_2004) facilitating this interaction. We found that although co-aggregation does not affect cell-cell communication, it is critical for biofilm structure and species distribution. This research opens up new avenues for exploring microbial interactions in dental health and diseases.

Abstract Bacillus subtilis can form various types of spatially organised communities on surfaces, such as colonies, pellicles and submerged biofilms. These communities share similarities and differences, and phenotypic heterogeneity has been reported for each type of community. Here, we studied spatial transcriptional heterogeneity across the three types of surface-associated communities. Using RNA-seq analysis of different regions or populations for each community type, we identified genes that are specifically expressed within each selected population. We constructed fluorescent transcriptional fusions for 17 of these genes, and observed their expression in submerged biofilms using time-lapse confocal laser scanning microscopy (CLSM). We found mosaic expression patterns for some genes; in particular, we observed spatially segregated cells displaying opposite regulation of carbon metabolism genes ( gapA and gapB ), indicative of distinct glycolytic or gluconeogenic regimes coexisting in the same biofilm region. Overall, our study provides a direct comparison of spatial transcriptional heterogeneity, at different scales, for the three main models of B. subtilis surface-associated communities.

Bacteria grow in either planktonic form or as biofilms, which are attached to either inert or biological surfaces. Both growth forms are highly relevant states in nature and of paramount scientific focus. However, interchanges between bacteria in these two states have been little explored. We discovered that a subpopulation of planktonic bacilli is propelled by flagella to tunnel deep within a biofilm structure. Swimmers create transient pores that increase macromolecular transfer within the biofilm. Irrigation of the biofilm by swimmer bacteria may improve biofilm bacterial fitness by increasing nutrient flow in the matrix. However, we show that the opposite may also occur (i.e., swimmers can exacerbate killing of biofilm bacteria by facilitating penetration of toxic substances from the environment). We combined these observations with the fact that numerous bacteria produce antimicrobial substances in nature. We hypothesized and proved that motile bacilli expressing a bactericide can also kill a heterologous biofilm population, Staphylococcus aureus in this case, and then occupy the newly created space. These findings identify microbial motility as a determinant of the biofilm landscape and add motility to the complement of traits contributing to rapid alterations in biofilm populations.

Editorial on the Research Topic Methods in biofilms: 2022 Biofilms, complex communities of microorganisms adhering to surfaces, play a significant role in various fields, including healthcare, industry, and the environment (Flemming et al., 2016). As our understanding of biofilms expands, so does the need for advanced research methods and techniques to study their formation, composition, and physiological characteristics (Azeredo et al., 2017). This Research Topic aims to present a captivating collection of articles that highlight novel methodologies in biofilm research. Biofilms present researchers with a range of challenges and opportunities. The articles in this Research Topic address these challenges by focusing on the development and adaptation of cutting-edge methods for studying biofilms. By bridging the gap between different scientific disciplines, these contributions provide a comprehensive view of biofilm research. From investigating the dynamics of antibiotic resistance selection using microfluidic chips to the specific detection of multiple pathogens in co-infection and mixed biofilms, these articles offer valuable insights into the complex nature of biofilm formation and interactions. The Research Topic features articles that showcase innovative methodologies to study various aspects of biofilms. For instance, an article introduces a microfluidic chip for realtime monitoring of antibiotic resistance selection in bacterial biofilms (Tang et al.). This technology provides a valuable tool for understanding the dynamics of antibiotic resistance emergence and selection. Another article describes the development of a TaqMan duplex real-time PCR method for simultaneous detection of Streptococcus suis and Actinobacillus pleuropneumoniae in co-infection and mixed biofilms (Yi et al.). This method offers enhanced specificity and sensitivity for accurate quantification of these pathogens, facilitating disease prevention and control. Additionally, the Research Topic includes an upgraded repository of antimicrobial peptides (AMPs) for biofilm studies. This resource, B-AMP v2.0, provides a comprehensive collection of biofilm protein targets and AMPs, facilitating research into specific biofilm targets and anti-biofilm strategies (Ravichandran et al.). Furthermore, the utilization of fluorescence in situ hybridization (FISH) techniques is explored in another article, showcasing its applications for visualizing and quantifying microorganisms, genes, and metabolites within biofilms (Barbosa et al.). These advancements in FISH-based techniques offer valuable insights into biofilm structure Frontiers in Cellular and Infection Microbiology frontiersin.org 01

Pulcherriminic acid (PA) relay is a recently discovered phenomenon in which the Bacillus subtilis employs branching biofilms to relay the antimicrobial pigment, pulcherriminic acid towards the pathogen. PA interacts with the free iron in the environment to form the reddish-pink pigment, pulcherimin, which subsequently accumulates on the pathogen depriving them of the essential iron. In Staphylococcus aureus , the ferric uptake regulator (Fur) system plays a vital role in maintaining iron homeostasis, virulence, and biofilm formation. The perspective article discusses the plausible mechanistic insights on the impact of PA relay in hampering the Fur system. Taken together, these findings highlight PA and PA-producing Bacillus species as a promising alternative for mitigating drug resistant S. aureus infections.

Microalgae biofilms have been proposed as an alternative to suspended cultures in commercial and biotechnological fields. However, little is known about their architecture that may strongly impact biofilm behavior, bioprocess stability, and productivity. In order to unravel the architecture of microalgae biofilms, four species of commercial interest were cultivated in microplates and characterized using a combination of confocal laser scanning microscopy and FTIR spectroscopy. In all the species, the biofilm biovolume and thickness increased over time and reached a plateau after seven days; however, the final biomass reached was very different. The roughness decreased during maturation, reflecting cell division and voids filling. The extracellular polymeric substances content of the matrix remained constant in some species, and increased over time in some others. Vertical profiles showed that young biofilms presented a maximum cell density at 20 μm above the substratum co-localized with matrix components. In mature biofilms, the maximum density of cells moved at a greater distance from the substratum (30–40 μm), whereas the maximum coverage of matrix components remained in a deeper layer. Carbohydrates and lipids were the main macromolecules changing during biofilm maturation. Our results revealed that the architecture of microalgae biofilms is species-specific. However, time similarly affects the structural and biochemical parameters.

Background Multidrug resistant Acinetobacter baumannii is one of the major infection agents causing nosocomial pneumonia. Therefore, new therapeutic approaches against this bacterium are needed. Surface-exposed proteins from bacterial pathogens are implicated in a variety of virulence-related traits and are considered as promising candidates for vaccine development. Results We show in this study that a large Blp1 protein from opportunistic pathogen A. baumannii is encoded in all examined clinical strains of globally spread international clonal lineages I (IC I) and II (IC II). The two blp1 gene variants exhibit lineage-specific distribution profile. By characterization of blp1 deletion mutants and their complementation with blp1 alleles we show that blp1 gene is required for A. baumannii biofilm formation and adhesion to epithelial cells in IC I strain but not in the IC II strain. Nevertheless both alleles are functional in restoring the deficient phenotypes of IC I strain. Moreover, the blp1 gene is required for the establishing of A. baumannii virulence phenotype in nematode and murine infection models. Additionally, we demonstrate that C-terminal 711 amino acid fragment of Blp1 elicits an efficient protection to lethal A. baumannii infection in a murine model using active and passive immunization approaches. Antiserum obtained against Blp1-specific antigen provides opsonophagocytic killing of A. baumannii in vitro. Conclusions Lineage-specific variants of surface-exposed components of bacterial pathogens complicate the development of new therapeutic approaches. Though we demonstrated different impact of Blp1 variants on adherence of IC I and IC II strains, Blp1-specific antiserum neutralized A. baumannii strains of both clonal lineages. Together with the observed increased survival rate in vaccinated mice these results indicate that A. baumannii Blp1 protein could be considered as a new vaccine candidate.

Biofilm is a dynamic structure from which individual bacteria and microaggregates are released to subsequently colonize new niches by either detachment or dispersal. Screening of a transposon mutant library identified genes associated with the alteration of Klebsiella pneumoniae biofilm including fabR, which encodes a transcrip tional regulator involved in membrane lipid homeostasis. An isogenic ∆fabR mutant formed more biofilm than the wild-type (WT) strain and its trans-complemented strain. The thick and round aggregates observed with ∆fabR were resistant to extensive washes, unlike those of the WT strain. Confocal microscopy and BioFlux microfluidic observa tions showed that fabR deletion was associated with biofilm robustness and impaired erosion over time. The genes fabB and yqfA associated with fatty acid metabolism were significantly overexpressed in the ∆fabR strain, in both planktonic and biofilm conditions. Two monounsaturated fatty acids, palmitoleic acid (C16:1) and oleic acid (C18:1), were found in higher proportion in biofilm cells than in planktonic forms, whereas heptadece noic acid (C17:1) and octadecanoic acid, 11-methoxy (C18:0-OCH3) were found in higher proportion in the planktonic lifestyle. The fabR mutation induced variations in the fatty acid composition, with no clear differences in the amounts of saturated fatty acids (SFA) and unsaturated fatty acids for the planktonic lifestyle but lower SFA in the biofilm form. Atomic force microscopy showed that deletion of fabR is associated with decreased K. pneumoniae cell rigidity in the biofilm lifestyle, as well as a softer, more elastic biofilm with increased cell cohesion compared to the wild-type strain.

Objectives This study investigated the efficacy of daptomycin against adherent Staphylococcus aureus ( S. aureus ), a common colonizer of medical devices that leads to severe infections. For the first time, we evaluated the bactericidal effects of daptomycin on S. aureus immediately after adhesion, mimicking early-stage contamination of biomaterials. Time-kill curve assay and confocal laser scanning microscopy (CLSM) were used to analyze the process dynamics. In addition, atomic force microscopy (AFM) and scanning electron microscopy (SEM) were employed to elucidate daptomycin-induced structural changes in the bacterial cell wall. Results description Daptomycin, at clinically relevant concentrations, rapidly eradicated adherent bacteria in the exponential growth phase, demonstrating an efficiency comparable to its action against planktonic cells. Prolonged exposure to the antibiotic caused marked alterations in the bacterial cell wall, including surface roughening and perforation, as revealed by multimodal imaging. However, daptomycin effectiveness diminished as biofilm formation progressed, underscoring the need for further exploration of optimized clinical strategies.