Molecular mechanisms involved in Bacillus subtilis biofilm formation

Biofilms are the predominant lifestyle of bacteria in natural environments, and they severely impact our societies in many different fashions. Therefore, biofilm formation is a topic of growing interest in microbiology, and different bacterial models are currently studied to better understand the molecular strategies that bacteria undergo to build biofilms. Among those, biofilms of the soil‐dwelling bacterium Bacillus subtilis are commonly used for this purpose. Bacillus subtilis biofilms show remarkable architectural features that are a consequence of sophisticated programmes of cellular specialization and cell–cell communication within the community. Many laboratories are trying to unravel the biological role of the morphological features of biofilms, as well as exploring the molecular basis underlying cellular differentiation. In this review, we present a general perspective of the current state of knowledge of biofilm formation in B. subtilis and thereby placing a special emphasis on summarizing the most recent discoveries in the field.     

Proteomannans in biofilm of Helicobacter pylori ATCC 43504

Background: The human bacterial pathogen Helicobacter pylori forms biofilms. However, the constituents of the biofilm have not been extensively investigated. In this study, we analyzed the carbohydrate and protein components of biofilm formed by H. pylori strain ATCC 43504 (NCTC 11637). Materials and Methods: Development of H. pylori biofilm was analyzed using scanning electron microscopy (SEM) and quantified using crystal violet staining. The extracted extracellular polysaccharide (EPS) matrix was analyzed using GC‐MS and nuclear magnetic resonance (NMR) analyses. Proteomic profiles of biofilms were examined by SDS–PAGE while deletion mutants of upregulated biofilm proteins were constructed and characterized. Results: Formation of H. pylori biofilm is time dependent as shown by crystal violet staining assay and SEM. NMR reveals the prevalence of 1,4‐mannosyl linkages in both developing and mature biofilms. Proteomic analysis of the biofilm indicates the upregulation of neutrophil‐activating protein A (NapA) and several stress‐induced proteins. Interestingly, the isogenic mutant napA revealed a different biofilm phenotype that showed reduced aggregated colonial structure when compared to the wild type. Conclusions: This in vitro study shows that mannose‐related proteoglycans (proteomannans) are involved in the process of H. pylori biofilm formation while the presence of upregulated NapA in the biofilm implies the potency to increase adhesiveness of H. pylori biofilm. Being a complex matrix of proteins and carbohydrates, which are probably interdependent, the H. pylori biofilm could possibly offer a protective haven for the survival of this gastric bacterial pathogen in the extragastric environments.     

The role of the Listeria monocytogenes surfactome in biofilm formation

Listeria monocytogenes is a highly pathogenic foodborne bacterium that is ubiquitous in the natural environment and capable of forming persistent biofilms in food processing environments. This species has a rich repertoire of surface structures that enable it to survive, adapt and persist in various environments and promote biofilm formation. We review current understanding and advances on how L. monocytogenes organizes its surface for biofilm formation on surfaces associated with food processing settings, because they may be an important target for development of novel antibiofilm compounds. A synthesis of the current knowledge on the role of Listeria surfactome, comprising peptidoglycan, teichoic acids and cell wall proteins, during biofilm formation on abiotic surfaces is provided. We consider indications gained from genome-wide studies and discuss surfactome structures with established mechanistic aspects in biofilm formation. Additionally, we look at the analogies to the species L. innocua, which is closely related to L. monocytogenes and often used as its model (surrogate) organism.     

Predicting biofilm deformation with a viscoelastic phase-field model: Modeling and experimental studies

Biofilms commonly develop in flowing aqueous environments, where the flow causes the biofilm to deform. Because biofilm deformation affects the flow regime, and because biofilms behave as complex heterogeneous viscoelastic materials, few models are able to predict biofilm deformation. In this study, a phase-field (PF) continuum model coupled with the Oldroyd-B constitutive equation was developed and used to simulate biofilm deformation. The accuracy of the model was evaluated using two types of biofilms: a synthetic biofilm, made from alginate mixed with bacterial cells, and a Pseudomonas aeruginosa biofilm. Shear rheometry was used to experimentally determine the mechanical parameters for each biofilm, used as inputs for the model. Biofilm deformation under fluid flow was monitored experimentally using optical coherence tomography. The comparison between the experimental and modeling geometries, for selected horizontal cross sections, after fluid-driven deformation was good. The relative errors ranged from 3.2 to 21.1% for the synthetic biofilm and from 9.1 to 11.1% for the P. aeruginosa biofilm. This is the first demonstration of the effectiveness of a viscoelastic PF biofilm model. This model provides an important tool for predicting biofilm viscoelastic deformation. It also can benefit the design and control of biofilms in engineering systems.     

Microcolony and biofilm formation as a survival strategy for bacteria

Bacterial communities such as biofilms are widely recognized as being important for survival and persistence of bacteria in harsh environments. Mechanistic models of biofilm growth indicate that the way in which the surface is seeded can effect the morphology of simulated biofilms. Experimental studies indicate that genes which are important for chemotaxis also influence biofilm formation, perhaps by influencing aggregation on a surface. Understanding aggregation and microcolony formation could therefore help clarify factors influencing biofilm formation and illuminate how groups influence the fitness of bacteria. In this paper I develop an individual based model to examine how different behaviors involved in microcolony formation on a surface determine patterns of group sizes and link patterns to bacterial fitness.     

Prevention of Staphylococcus aureus biofilm formation and reduction in established biofilm density using a combination of phage K and modified derivatives

Aims: To investigate the ability of a mixture of phage K and six of its modified derivatives to prevent biofilm formation by Staphylococcus aureus and also to reduce the established biofilm density. Methods and Results: The bioluminescence‐producing Staph. aureus Xen29 strain was used in the study, and incubation of this strain in static microtitre plates at 37°C for 48 h confirmed its strong biofilm‐forming capacity. Subsequently, removal of established biofilms of Staph. aureus Xen29 with the high‐titre phage combination was investigated over time periods of 24 h, 48 h and 72 h. Results suggested that these biofilms were eliminated in a time‐dependant manner, with biofilm biomass reduction significantly greater after 72 h than after 24–48 h. In addition, initial challenge of Staph. aureus Xen29 with the phage cocktail resulted in the complete inhibition of biofilm formation over a 48‐h period with no appearance of phage resistance. Conclusions: In general, our findings demonstrate the potential use of a modified phage combination for the prevention and successful treatment of Staph. aureus biofilms, which are implicated in several antibiotic‐resistant infections. Significance and Impact of the Study: This study highlights the first use of phage K for the successful removal and prevention of biofilms of Staph. aureus.     

Utilisation of a packed-bed biofilm reactor for the determination of the potential of biofilm accumulation in water systems

Abstract

An experimental system has been developed that allows the monitoring of biofilm development on supports exposed to water of different characteristics. The system consists of a series of packed-bed reactors filled with glass beads, and by periodically removing biofilm attached to these beads for off-line analyses this provides a means for monitoring biofilm development. Despite its reduced dimensions (6.9 cm long and 1.58 cm in diameter), the experimental system used has a sampling surface of 90.3 cm² (including only the surface of the glass beads). This allows reproducible and representative samples to be taken from different water systems, providing a reliable and economic method for evaluating in situ the formation of biofilms from different environments. The set-up of the entire experimental system was constructed to meet the demands of field experiments in a well-defined hydrodynamic environment and to allow easy removal of samples for biomass quantification and microscopic observation. Data obtained using this device can be used as an indicator of the risk of biofilm formation in different water systems. This indicator, “the biofilm accumulation potential”, represents an effective and representative tool for the monitoring of biofilm development in an integrated antifouling strategy, in order to help keep biofouling, scaling and microbial risks under control. According to the experiments with the packed-bed reactors used with a high flow regime, the ratio TCN/HPC could provide an indication of the state of the biofilm, and lower ratios could indicate a higher biofilm accumulation potential.     

Formation of biofilms under phage predation: considerations concerning a biofilm increase

Bacteriophages are emerging as strong candidates for combating bacterial biofilms. However, reports indicating that host populations can, in some cases, respond to phage predation by an increase in biofilm formation are of concern. This study investigates whether phage predation can enhance the formation of biofilm and if so, if this phenomenon is governed by the emergence of phage-resistance or by non-evolutionary mechanisms (eg spatial refuge). Single-species biofilms of three bacterial pathogens (Pseudomonas aeruginosa, Salmonella enterica serotype Typhimurium, and Staphylococcus aureus) were pretreated and post-treated with species-specific phages. Some of the phage treatments resulted in an increase in the levels of biofilm of their host. It is proposed that the phenotypic change brought about by acquiring phage resistance is the main reason for the increase in the level of biofilm of P. aeruginosa. For biofilms of S. aureus and S. enterica Typhimurium, although resistance was detected, increased formation of biofilm appeared to be a result of non-evolutionary mechanisms.     

A new biofilm-associated colicin with increased efficiency against biofilm bacteria

Formation of bacterial biofilm communities leads to profound physiological modifications and increased physical and metabolic exchanges between bacteria. It was previously shown that bioactive molecules produced within the biofilm environment contribute to bacterial interactions. Here we describe new pore-forming colicin R, specifically produced in biofilms formed by the natural isolate Escherichia coli ROAR029 but that cannot be detected under planktonic culture conditions. We demonstrate that an increased SOS stress response within mature biofilms induces SOS-dependent colicin R expression. We provide evidence that colicin R displays increased activity against E. coli strains that have a reduced lipopolysaccharide length, such as the pathogenic enteroaggregative E. coli LF82 clinical isolate, therefore pointing to lipopolysaccharide size as an important determinant for resistance to colicins. We show that colicin R toxicity toward E. coli LF82 is increased under biofilm conditions compared with planktonic susceptibility and that release of colicin R confers a strong competitive advantage in mixed biofilms by rapidly outcompeting sensitive neighboring bacteria. This work identifies the first biofilm-associated colicin that preferentially targets biofilm bacteria. Furthermore, it indicates that the study of antagonistic molecules produced in biofilm and multispecies contexts could reveal unsuspected, ecologically relevant bacterial interactions influencing population dynamics in natural environments.     

Nutrients determine the spatial architecture of Paracoccus sp. biofilm

Bacterial biofilms adapt and shape their structure in response to varied environmental conditions. A statistical methodology was adopted in this study to empirically investigate the influence of nutrients on biofilm structural parameters deduced from confocal scanning laser microscope images of Paracoccus sp.W1b, a denitrifying bacterium. High concentrations of succinate, Mg⁺⁺, Ca⁺⁺, and Mn⁺⁺ were shown to enhance biofilm formation whereas higher concentration of iron decreased biofilm formation. Biofilm formed at high succinate was uneven with high surface to biovolume ratio. Higher Mg⁺⁺ or Ca⁺⁺ concentrations induced cohesion of biofilm cells, but contrasting biofilm architectures were detected. Biofilm with subpopulation of pillar-like protruding cells was distributed on a mosaic form of monolayer cells in medium with 10 mM Mg⁺⁺. 10 mM Ca⁺⁺ induced a dense confluent biofilm. Denitrification activity was significantly increased in the Mg⁺⁺- and Ca⁺⁺-induced biofilms. Chelator treatment of various biofilm ages indicated that divalent cations are important in the initial stages of biofilm formation.     

Contribution of rpoS and bolA genes in biofilm formation in Escherichia coli K-12 MG1655

Flexibility of gene expression in bacteria permits its survival in varied environments. The genetic adaptation of bacteria through systematized gene expression is not only important, but also clinically relevant in their ability to grow biofilms in stress environments. Stress responses enable their survival under more severe conditions, enhanced resistance and/or virulence. In Escherichia coli (E. coli), two of the possible important genes for biofilm growth are rpoS and bolA gene. RpoS is also called as a master regulator of general stress response. Even though many studies have revealed the importance of rpoS in planktonic cells, little is known about the functions of rpoS in biofilms. In contrast, bolA which is a morphogene in E. coli is overexpressed under stressed environments resulting in round morphology. The hypothesis is that bolA could be implicated in biofilm development. This study reviewed the literature with the aim of understanding the stress tolerance response of E. coli in relation with rpoS and bolA genes in different environmental conditions including heat shock, cold shock, and stress in response to oxidation, acidic condition and in presence of cadmium. Knowledge of the genetic regulation of biofilm formation may lead to the understanding of the factors that drive the bacteria to switch to the biofilm mode of growth.     

Mini-review: Microbial coaggregation: ubiquity and implications for biofilm development

Coaggregation is the specific recognition and adherence of genetically distinct microorganisms. Because most biofilms are polymicrobial communities, there is potential for coaggregation to play an integral role in spatiotemporal biofilm development and the moderation of biofilm community composition. However, understanding of the mechanisms contributing to coaggregation and the relevance of coaggregation to biofilm ecology is at a very early stage. The purpose of this review is to highlight recent advances in the understanding of microbial coaggregation within different environments and to describe the possible ecological ramifications of such interactions. Bacteria that coaggregate with many partner species within different environments will be highlighted, including oral streptococci and oral bridging organisms such as fusobacteria, as well as the freshwater sphingomonads and acinetobacters. Irrespective of environment, it is proposed that coaggregation is essential for the orchestrated development of multi-species biofilms.     

2-Hydroxy-4-methoxybenzaldehyde from Hemidesmus indicus is antagonistic to Staphylococcus epidermidis biofilm formation

Abstract

Staphylococcus epidermidis (SE) is an opportunistic nosocomial pathogen that accounts for recalcitrant device-related infections worldwide. Owing to the growing interest in plants and their secondary metabolites targeting bacterial adhesion, this study was intended to uncover the anti-biofilm potential of Hemidesmus indicus and its major constituent 2-hydroxy-4-methoxybenzaldehyde (HMB) against SE. The minimum biofilm inhibitory concentration (MBIC) of H. indicus root extract and HMB were found to be 500 and 250?µg ml^?1, respectively. The results of time-dependent biofilm inhibition and mature biofilm disruption assays confirmed that HMB targets initial cell adhesion. Furthermore, interference by HMB in the expression of adhesin genes (icaA, aap and bhp) and biofilm components was associated with an increased susceptibility of SE to oxidative stress and antibiotics. To conclude, this study reports for the first time HMB as a potential drug against SE biofilms.     

Aggregation of ammonia-oxidizing bacteria in microbial biofilm on oyster shell surface

Summary Formation and activity of bacterial nitrifying biofilms play an important role in the closed seawater systems for shrimp cultivation. The structure of microbial biofilm on empty oyster shells, used as a biofilm carrier in biofiltration of aquacultural water, was studied using fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy. FISH was performed with specific oligonucleotide probes for Bacteria and ammonia-oxidizing Nitrosomonas spp. The bacterial cells were arranged within the biofilm as a layer of vertically elongated aggregates. Aggregates of ammonia-oxidizing bacteria were embedded within the matrix formed by other bacteria. Vertically elongated cell aggregates may be ecologically important in bacterial biofilms because they have a higher surface-to-volume ratio than that of laminated biofilms.     

Identification of linoleic acid,a main component of the n-hexane fraction from Dryopteris crassirhizoma,as an anti-Streptococcus mutans biofilm agent

Dryopteris crassirhizoma is a semi-evergreen plant. Previous studies have shown the potential of this plant as an agent for the control of cariogenic biofilms. In this study, the main antibacterial components of the plant were identified by correlating gas chromatography–mass spectrometry data with the antibacterial activity of chloroform and n-hexane fractions and then evaluating the activity of the most potent antibacterial component against Streptococcus mutans UA159 biofilms. The most potent antibacterial component was linoleic acid, a main component of the n-hexane fraction. Linoleic acid reduced viability in a dose dependent manner and reduced biofilm accumulation during initial and mature biofilm formation. Furthermore, when the biofilms were briefly treated with linoleic acid (10?min/treatment, a total of six times), the dry weight of the biofilms was significantly diminished. In addition, the anti-biofilm activity of the n-hexane fraction was similar to that of linoleic acid. These results suggest that the n-hexane fraction of D. crassirhizoma and linoleic acid may be useful for controlling cariogenic biofilms.     

Unravelling interspecies interactions across heterogeneities in complex biofilm communities

The importance of microbial biofilms has been well-recognized for several decades, and focus is now shifting towards investigating multispecies biofilm communities rather than mono- or dual-species biofilms. Therefore, the demand for techniques that provide a sufficient amount of information at adequate resolution is increasing. One major challenge for multispecies studies is that diversity and spatial organization often lead to a high degree of spatial and chemical heterogeneity. Many current approaches do not account for such heterogeneity and therefore only provide average information (−omics techniques in particular), which could obscure important information about the community. Here, we bring attention to the issues of heterogeneity when analysing synthetic multi-species biofilms, in vitro, and the importance of multi-scale approaches. We provide an overview of current and newer approaches that can be applied to biofilm communities, in order to elucidate interactions at the appropriate scale.     

Identification of small molecules inhibiting diguanylate cyclases to control bacterial biofilm development

Biofilm formation by pathogenic bacteria is an important virulence factor in the development of numerous chronic infections, thereby causing a severe health burden. Many of these infections cannot be resolved, as bacteria in biofilms are resistant to the host’s immune defenses and antibiotic therapy. An urgent need for new strategies to treat biofilm-based infections is critically needed. Cyclic di-GMP (c-di-GMP) is a widely conserved second-messenger signal essential for biofilm formation. The absence of this signalling system in higher eukaryotes makes it an attractive target for the development of new anti-biofilm agents. In this study, the results of an in silico pharmacophore-based screen to identify small-molecule inhibitors of diguanylate cyclase (DGC) enzymes that synthesize c-di-GMP are described. Four small molecules, LP 3134, LP 3145, LP 4010 and LP 1062 that antagonize these enzymes and inhibit biofilm formation by Pseudomonas aeruginosa and Acinetobacter baumannii in a continuous-flow system are reported. All four molecules dispersed P. aeruginosa biofilms and inhibited biofilm development on urinary catheters. One molecule dispersed A. baumannii biofilms. Two molecules displayed no toxic effects on eukaryotic cells. These molecules represent the first compounds identified from an in silico screen that are able to inhibit DGC activity to prevent biofilm formation.     

Environmental determinants of Vibrio cholerae biofilm development

Vibrio cholerae is a versatile bacterium that flourishes in diverse environments, including the human intestine, rivers, lakes, estuaries, and the ocean. Surface attachment is believed to be essential for colonization of all of these natural environments. Previous studies have demonstrated that the vps genes, which encode proteins required for exopolysaccharide synthesis and transport, are required for V. cholerae biofilm development in Luria-Bertani broth. In this work, we showed that V. cholerae forms vps-dependent biofilms and vps-independent biofilms. The vps-dependent and -independent biofilms differ in their environmental activators and in architecture. Our results suggest that environmental activators of vps-dependent biofilm development are present in freshwater, while environmental activators of vps-independent biofilm development are present in seawater. The distinct environmental requirements for the two modes of biofilm development suggest that vps-dependent biofilm development and vps-independent biofilm development may play distinct roles in the natural environment.     

An expanded regulatory network temporally controls Candida albicans biofilm formation

Candida albicans biofilms are composed of highly adherent and densely arranged cells with properties distinct from those of free‐floating (planktonic) cells. These biofilms are a significant medical problem because they commonly form on implanted medical devices, are drug resistant and are difficult to remove. C. albicans biofilms are not static structures; rather they are dynamic and develop over time. Here we characterize gene expression in biofilms during their development, and by comparing them to multiple planktonic reference states, we identify patterns of gene expression relevant to biofilm formation. In particular, we document time‐dependent changes in genes involved in adhesion and metabolism, both of which are at the core of biofilm development. Additionally, we identify three new regulators of biofilm formation, Flo8, Gal4, and Rfx2, which play distinct roles during biofilm development over time. Flo8 is required for biofilm formation at all time points, and Gal4 and Rfx2 are needed for proper biofilm formation at intermediate time points.