Role In Biofilm Formation Research Articles

How Do Bacteria Recognize Their Environment? The Role of a Histidine Kinase in Initiating a Biofilm Program

Many bacteria form complex multicellular communities known as biofilms. While biofilms are a common cause of human infections, they can also have productive roles in ecology and agricultural settings. In contrast to the importance of these communities, less is known about how bacteria interpret and respond to environmental cues to form a biofilm. To help understand how bacteria recognize chemical signals from their environment, we studied the soil‐dwelling bacteria Bacillus subtilis, which is known to form beneficial biofilms on plant roots. We find extracts from many plant samples can stimulate B. subtilis biofilm formation. One protein that is likely important for the recognition of plant signals is a histidine kinase called KinD. KinD is known to have a role in biofilm formation and also to bind the molecule pyruvate. We find there are varying pyruvate concentrations in our different biofilm‐stimulating plant samples. Using transcriptional reporters of biofilm genes, we show that the binding of pyruvate to KinD can initiate the primary biofilm pathway of B. subtilis. However, the addition of pyruvate to bacterial cultures is not sufficient to trigger a biofilm phenotype. This research suggests that the binding of pyruvate to KinD is one of several signals that initiate the formation of a robust biofilm.

Update on diagnosis and management of refractory corneal infections.

Infectious keratitis is a medical emergency resulting in significant visual morbidity. Indiscriminate use of antimicrobials leading to the emergence of resistant or refractory microorganisms has further worsened the prognosis. Coexisting ocular surface diseases, delay in diagnosis due to inadequate microbiological sample, a slow-growing/virulent organism, or systemic immunosuppressive state all contribute to the refractory response of the ulcer. With improved understanding of these varied ocular and systemic factors contributing to the refractory nature of the microbes, role of biofilm formation and recent research on improving the bioavailability of drugs along with the development of alternative therapies have helped provide the required multidimensional approach to effectively diagnose and manage cases of refractory corneal ulcers and prevent corneal perforations or further dissemination of disease. In this review, we explore the current literature and future directions of the diagnosis and treatment of refractory keratitis.

Improving the Viability of Probiotics under Harsh Conditions by the Formation of Biofilm on Electrospun Nanofiber Mat.

For improving probiotics’ survivability under harsh conditions, this study used Lactiplantibacillus plantarum GIM1.648 as a model microorganism to investigate its ability to produce biofilms on electrospun ethyl cellulose nanofiber mats. SEM observations confirmed that biofilm was successfully formed on the nanofibers, with the latter being an excellent scaffold material. The optimal cultivation conditions for biofilm formation were MRS medium without Tween 80, a culture time of 36 h, a temperature of 30 °C, a pH of 6.5, and an inoculum concentration of 1% (v/v). The sessile cells in the biofilm exhibited improved gastrointestinal and thermal tolerance compared to the planktonic cells. Additionally, the RT-qPCR assay indicated that the luxS gene played a crucial role in biofilm formation, with its relative expression level being 8.7-fold higher compared to the planktonic cells. In conclusion, biofilm formation on electrospun nanofiber mat has great potential for improving the viability of probiotic cells under harsh conditions.

Exploration of bifurcated electron transfer mechanism in Bacillus cereus for enhanced power generation in double-chambered microbial fuel cells

Extracellular Electron Transfer (EET) is considered as the communication bridge between bacterial cells and outer electron acceptors. Apart from a few model microorganisms conventionally studied, the literature regarding the EET mechanism in Gram-positive bacteria and its conductive interaction is sparse. In the present study, proteolytic Gram-positive bacteria - Bacillus cereus is explored for enhancement of power generation in Microbial Fuel Cells (MFC). A low zeta potential (−0.1 V) of B. cereus, enables it to release electrons and protons much faster. We achieve a power density as high as 400 mW m−2 with a current density of ~5.8 mA cm−2 by inoculating an MFC with B. cereus. A genome wide transcriptome analysis by RNA-seq is used to categorize the genes and the significant pathways potentially involved in EET in B. cereus. The expression of gene cluster encodes for PTS (Phosphotransferase system) that catalyzes and transport phosphorylation of disaccharide present in the medium. This plays a regulatory role in biofilm formation. Simultaneously the secretion and assembly of extracellular flavin molecules by B. cereus, shuttle and mediate electron to the extracellular acceptor. In short, B. cereus uses bifurcated pathway i.e., directly by aligning the cytochrome complex and indirectly by secreting flavins molecules to promote electron transfer. These findings reveal its significant functional role in MFCs’ electrode community for sustainable bioenergy production.

Nonmotile Subpopulations of Pseudomonas aeruginosa Repress Flagellar Motility in Motile Cells through a Type IV Pilus- and Pel-Dependent Mechanism.

The downregulation of Pseudomonas aeruginosa flagellar motility is a key event in biofilm formation, host colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that on soft agar, swarming motility can be repressed by cells that are nonmotile due to the absence of a flagellum or flagellar rotation. Mutants that lack either flagellum biosynthesis or rotation, when present at as little as 5% of the total population, suppressed swarming of wild-type cells. Non-swarming cells required functional type IV pili and the ability to produce Pel exopolysaccharide to suppress swarming by the flagellated wild type. Flagellated cells required only type IV pili, but not Pel production, for their swarming to be repressed by non-flagellated cells. We hypothesize that interactions between motile and nonmotile cells may enhance the formation of sessile communities, including those involving multiple genotypes, phenotypically diverse cells, and perhaps other species. IMPORTANCE Our study shows that, under the conditions tested, a small population of non-swarming cells can impact the motility behavior of a larger population. The interactions that lead to the suppression of swarming motility require type IV pili and a secreted polysaccharide, two factors with known roles in biofilm formation. These data suggest that interactions between motile and nonmotile cells may enhance the transition to sessile growth in populations and promote interactions between cells with different genotypes.

Involvement of PhoP/PhoQ two-component system in biofilm formation in Cronobacter sakazakii

The aim of this study was to investigated the effect of PhoP/PhoQ two component system on the biofilm formation of Cronobacter sakazakii BAA-894. Crystal violet staining assay and XTT assay showed that the biofilm biomass and the viability of biofilm cells were significantly reduced in ΔphoPQ strain, while these defects were reversed in the complementation strain ΔphoPQC. Moreover, the phoPQ gene deletion weakened the ability of BAA-894 strain to form biofilms on glass and silicon wafers, and affected the content of extracellular polymeric substance (EPS) in biofilms formed on stainless-steel plates. In addition, qRT-PCR results showed that the PhoP/PhoQ system of C. sakazakii played roles in regulating the expression of several genes associated with flagella and cellulose biosynthesis, which are both involved in biofilm formation. These finding suggest that the PhoP/PhoQ system plays important roles in biofilm formation in C. sakazakii and the system could be considered as a potential target for developing novel alternative strategies for controlling biofilms.

Genome-wide analysis reveals a rhamnolipid-dependent modulation of flagellar genes in Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa is an opportunistic pathogen and an important model organism for the study of bacterial group behaviors, including cell motility and biofilm formation. Rhamnolipids play a pivotal role in biofilm formation and motility phenotypes in P. aeruginosa, possibly acting as wetting agents and mediating chemotactic stimuli. However, no biochemical mechanism or gene regulatory network has been investigated in regard to rhamnolipids' modulation of those group behaviors. Using DNA microarrays, we investigated the transcriptomic profiles in the stationary phase of growth of wild-type P. aeruginosa PAO1 and a rhlA-mutant strain, unable to produce rhamnolipids. A total of 134 genes were differentially expressed, comprising different functional categories, indicating a significant physiological difference between the rhamnolipid-producing and -non-producing strains. Interestingly, several flagellar genes are repressed in the mutant strain, which directly relates to the inability of the rhlA-minus strain to develop a swarming-motility phenotype. Supplementation with exogenous rhamnolipids has partially restored flagellar gene expression in the mutant strain. Our results show significant evidence that rhamnolipids, the major biosynthetic products of rhlABC pathway, seem to modulate gene expression in P. aeruginosa.

A Filamentous Bacteriophage Protein Inhibits Type IV Pili To Prevent Superinfection of Pseudomonas aeruginosa.

ABSTRACTPseudomonas aeruginosa is an opportunistic pathogen that causes infections in a variety of settings. Many P. aeruginosa isolates are infected by filamentous Pf bacteriophage integrated into the bacterial chromosome as a prophage. Pf virions can be produced without lysing P. aeruginosa. However, cell lysis can occur during superinfection, which occurs when Pf virions successfully infect a host lysogenized by a Pf prophage. Temperate phages typically encode superinfection exclusion mechanisms to prevent host lysis by virions of the same or similar species. In this study, we sought to elucidate the superinfection exclusion mechanism of Pf phage. Initially, we observed that P. aeruginosa that survive Pf superinfection are transiently resistant to Pf-induced plaquing and are deficient in twitching motility, which is mediated by type IV pili (T4P). Pf utilize T4P as a cell surface receptor, suggesting that T4P are suppressed in bacteria that survive superinfection. We tested the hypothesis that a Pf-encoded protein suppresses T4P to mediate superinfection exclusion by expressing Pf proteins in P. aeruginosa and measuring plaquing and twitching motility. We found that the Pf protein PA0721, which we termed Pf superinfection exclusion (PfsE), promoted resistance to Pf infection and suppressed twitching motility by binding the T4P protein PilC. Because T4P play key roles in biofilm formation and virulence, the ability of Pf phage to modulate T4P via PfsE has implications in the ability of P. aeruginosa to persist at sites of infection.

Quorum sensing inhibitors applications: A new prospect for mitigation of microbiologically influenced corrosion

Quorum sensing (QS) is a process of bacterial communication that involves the use of biochemical signals and adjusts the expression of specific genes as a response to the bacterial cell density within an environment. This process is employed by both Gram-positive and Gram-negative bacteria to regulate different physiological functions. In both cases, QS involves production, detection and responses to signalling chemicals, termed auto-inducers. Expression of virulence factors and formation of biofilms are the typical processes controlled by QS, which, therefore, inspires the exploration of QS as a plausible solution to mitigating the increasing microbial resistance to antibiotics. QS inhibitors (QSIs) from different origins have been recognised as a promising solution to biofilm related challenges in a large variety of applications. Though QSIs have demonstrated some strength in tackling biofouling, a key focus in the literature on QSIs based strategies has been to control microbially influenced corrosion. This article reviews the principles of QS, its mechanistic roles in biofilm formation and the feasibility of QSIs to mitigate biofilm related challenges in a number of commercial applications. The potential of QSIs in microbially influenced corrosion for future applications is also discussed.

Evaluation of trehalase as an enhancer for a green biocide in the mitigation of Desulfovibrio vulgaris biocorrosion of carbon steel.

Trehalase can biocatalyze the conversion of trehalose to glucose. It is an enzyme that plays an important role in biofilm formation. Thus, trehalase has been patented as a chemical for preventing and treating biofilms. Sulfate-reducing bacteria (SRB) biofilms are often found responsible for biocorrosion, also known as microbiologically infuenced corrosion (MIC), especially in the oil and gas industries and in water utilities. The MIC treatment process typically requires biocide treatment of biofilms, sometimes together with scrubbing. Owing to environmental concerns, a lower biocide dosage is desired in the treatment process. In this work, trehalase was tested as a green biocide enhancer to enhance tetrakis hydroxymethyl phosphonium sulfate (THPS) in the prevention of Desulfovibrio vulgaris MIC of C1018 carbon steel in ATCC 1249 culture medium at 37°C. THPS is one of the most popular industrial biocides owing to its broad-spectrum efficacy and green chemical status. After 7days of incubation in 50mL anaerobic vials containing 40mL culture medium at pH 7.0, the sessile cell counts indicated that 50ppm (w/w) THPS + 30ppm (w/w) trehalase led to an extra 5.7-fold sessile cell reduction when compared with the 50ppm THPS alone treatment. As a consequence, the combination treatment also resulted in an extra 54% in pit depth reduction and 30% in weight loss reduction when compared with the 50ppm THPS alone treatment (with 9.0μm and 1.0mg/cm2). The biofilm images corroborated the decreased sessile cell count and pitting corrosion.

Siderophore-Mediated Iron Acquisition Plays a Critical Role in Biofilm Formation and Survival of Staphylococcus epidermidis Within the Host.

Iron acquisition through siderophores, a class of small, potent iron-chelating organic molecules, is a widely spread strategy among pathogens to survive in the iron-restricted environment found in the host. Although these molecules have been implicated in the pathogenesis of several species, there is currently no comprehensive study addressing siderophore production in Staphylococcus epidermidis. Staphylococcus epidermidis is an innocuous skin commensal bacterium. The species, though, has emerged as a leading cause of implant-associated infections, significantly supported by an inherent ability to form biofilms. The process of adaptation from skin niche environments to the hostile conditions during invasion is yet not fully understood. Herein, we addressed the possible role of siderophore production in S. epidermidis virulence. We first identified and deleted a siderophore homolog locus, sfaABCD, and provided evidence for its involvement in iron acquisition. Our findings further suggested the involvement of siderophores in the protection against oxidative stress-induced damage and demonstrated the in vivo relevance of a siderophore-mediated iron acquisition during S. epidermidis infections. Conclusively, this study addressed, for the first time in this species, the underlying mechanisms of siderophore production, highlighting the importance of a siderophore-mediated iron acquisition under host relevant conditions and, most importantly, its contribution to survival within the host.

Staphylococcus lugdunensis.

Historically, staphylococci are grouped as coagulase-negative or coagulase-positive, according to their ability to induce clotting of mammalian serum through the activity of a secreted coagulase. S. lugdunensis belongs to the coagulase-negative staphylococci (CoNS) but it is in many ways unusual as it shares several features with the coagulase-positive and highly invasive opportunistic pathogen Staphylococcus aureus. Like S. aureus, many S. lugdunensis strains express a cell wall-anchored molecule (Fbl) promoting adhesion to fibrinogen [ 8. Geoghegan J.A. et al. Molecular characterization of the interaction of staphylococcal microbial surface components recognizing adhesive matrix molecules (MSCRAMM) ClfA and Fbl with fibrinogen. J. Biol. Chem. 2010; 285: 6208-6216 Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar ]. This mediates clumping of fibrinogen-coated latex beads, which are frequently used for the rapid identification of S. aureus. This can lead to misidentification of S. lugdunensis in clinical samples. Furthermore, S. lugdunensis is more virulent than other CoNS, a feature especially apparent in its ability to cause infective endocarditis. In fact, S. lugdunensis encodes several putative virulence factors, including the hemolytic SLUSH peptides, which allow the release of hemoglobin (Hb) from erythrocytes [ 9. Donvito B. et al. Synergistic hemolytic activity of Staphylococcus lugdunensis is mediated by three peptides encoded by a non-agr genetic locus. Infect. Immun. 1997; 65: 95-100 Crossref PubMed Google Scholar ], sortase A [ 4. Heilbronner S. et al. Sortase A promotes virulence in experimental Staphylococcus lugdunensis endocarditis. Microbiology. 2013; 159: 2141-2152 Crossref PubMed Scopus (30) Google Scholar ], a streptolysin-like toxin [ 10. Lebeurre J. et al. Comparative genome analysis of Staphylococcus lugdunensis shows clonal complex-dependent diversity of the putative virulence factor, ess/type VII locus. Front. Microbiol. 2019; 10: 2479 Crossref PubMed Scopus (10) Google Scholar ], and an ess/type VII locus [ 10. Lebeurre J. et al. Comparative genome analysis of Staphylococcus lugdunensis shows clonal complex-dependent diversity of the putative virulence factor, ess/type VII locus. Front. Microbiol. 2019; 10: 2479 Crossref PubMed Scopus (10) Google Scholar ]. Additionally, like many bacterial pathogens, S. lugdunensis possesses systems (Isd and Lha) for the acquisition of heme that is stripped from the host Hb, enabling its cytosolic degradation and the release of nutritional iron [ 11. Jochim A. et al. An ECF-type transporter scavenges heme to overcome iron-limitation in Staphylococcus lugdunensis. eLife. 2020; 9e57322 Crossref PubMed Scopus (8) Google Scholar ]. Further factors that might promote immune evasion, killing of immune cells, or adhesion to host tissues have been suggested but they have not been experimentally validated so far [ 3. Heilbronner S. Foster T.J. Staphylococcus lugdunensis: a skin commensal with invasive pathogenic potential. Clin. Microbiol. Rev. 2021; 34e00205-20 Crossref PubMed Scopus (8) Google Scholar ]. Finally, the extracellular matrix of S. lugdunensis biofilms is not enriched in polysaccharides but in proteinaceous factors [ 12. Frank K.L. Patel R. Poly-N-acetylglucosamine is not a major component of the extracellular matrix in biofilms formed by icaADBC-positive Staphylococcus lugdunensis isolates. Infect. Immun. 2007; 75: 4728-4742 Crossref PubMed Scopus (83) Google Scholar ], and biofilm formation depends on the action of the autolysin AtlL [ 13. Hussain M. et al. The adhesive properties of the Staphylococcus lugdunensis multifunctional autolysin AtlL and its role in biofilm formation and internalization. Int. J. Med. Microbiol. 2015; 305: 129-139 Crossref PubMed Scopus (32) Google Scholar ].

Capsules and Extracellular Polysaccharides in Escherichia coli and Salmonella.

Escherichia coli and Salmonella isolates produce a range of different polysaccharide structures that play important roles in their biology. E. coli isolates often possess capsular polysaccharides (K antigens), which form a surface structural layer. These possess a wide range of repeat-unit structures. In contrast, only one capsular polymer (Vi antigen) is found in Salmonella, and it is confined to typhoidal serovars. In both genera, capsules are vital virulence determinants and are associated with the avoidance of host immune defenses. Some isolates of these species also produce a largely secreted exopolysaccharide called colanic acid as part of their complex Rcs-regulated phenotypes, but the precise function of this polysaccharide in microbial cell biology is not fully understood. E. coli isolates produce two additional secreted polysaccharides, bacterial cellulose and poly-N-acetylglucosamine, which play important roles in biofilm formation. Cellulose is also produced by Salmonella isolates, but the genes for poly-N-acetylglucosamine synthesis appear to have been lost during its evolution toward enhanced virulence. Here, we discuss the structures, functions, relationships, and sophisticated assembly mechanisms for these important biopolymers.

The vicK gene of Streptococcus mutans mediates its cariogenicity via exopolysaccharides metabolism

Streptococcus mutans (S. mutans) is generally regarded as a major contributor to dental caries because of its ability to synthesize extracellular polysaccharides (EPS) that aid in the formation of plaque biofilm. The VicRKX system of S. mutans plays an important role in biofilm formation. The aim of this study was to investigate the effects of vicK gene on specific characteristics of EPS in S. mutans biofilm. We constructed single-species biofilms formed by different mutants of vicK gene. Production and distribution of EPS were detected through atomic force microscopy, scanning electron microscopy and confocal laser scanning microscopy. Microcosmic structures of EPS were analyzed by gel permeation chromatography and gas chromatography-mass spectrometry. Cariogenicity of the vicK mutant was assessed in a specific pathogen-free rat model. Transcriptional levels of cariogenicity-associated genes were confirmed by quantitative real-time polymerase chain reaction. The results showed that deletion of vicK gene suppressed biofilm formation as well as EPS production, and EPS were synthesized mostly around the cells. Molecular weight and monosaccharide components underwent evident alterations. Biofilms formed in vivo were sparse and contributed a decreased degree of caries. Moreover, expressional levels of genes related to EPS synthesis were down-regulated, except for gtfB. Our report demonstrates that vicK gene enhances biofilm formation and subsequent caries development. And this may due to its regulations on EPS metabolism, like synthesis or microcosmic features of EPS. This study suggests that vicK gene and EPS can be considered as promising targets to modulate dental caries.

A Polyclonal Antibody Raised against the Burkholderia cenocepacia OmpA-like Protein BCAL2645 Impairs the Bacterium Adhesion and Invasion of Human Epithelial Cells In Vitro.

Respiratory infections by bacteria of the Burkholderia cepacia complex (Bcc) remain a life threat to cystic fibrosis (CF) patients, due to the faster lung function decline and the absence of effective eradication strategies. Immunotherapies are regarded as an attractive alternative to control and reduce the damages caused by these infections. In this work, we report the cloning and functional characterization of the OmpA-like BCAL2645 protein, previously identified and found to be immunoreactive against sera from CF patients with a record of Bcc infections. The BCAL2645 protein is shown to play a role in biofilm formation, adherence to mucins and invasion of human lung epithelial cells. The expression of the BCAL2645 protein was found to be increased in culture medium, mimicking the lungs of CF patients and microaerophilic conditions characteristic of the CF lung. Moreover, a polyclonal antibody raised against BCAL2645 was found to inhibit, by about 75 and 85%, the ability of B. cenocepacia K56-2 to bind and invade in vitro CFBE41o- human bronchial epithelial cells. These results highlight the potential of anti-BCAL2645 antibodies for the development of passive immunization therapies to protect CF patients against Bcc infections.

Insulin Regulation of Escherichia coli Abiotic Biofilm Formation: Effect of Nutrients and Growth Conditions.

Escherichia coli plays an important role in biofilm formation across a wide array of disease and ecological settings. Insulin can function as an adjuvant in the regulation of biofilm levels. The modulation of insulin-regulated biofilm formation by environmental conditions has not been previously described. In the present study, the effects that various environmental growth conditions and nutrients have on insulin-modulated levels of biofilm production were measured. Micropipette tips were incubated with E. coli ATCC® 25922™ in a Mueller Hinton broth (MH), or a yeast nitrogen base with 1% peptone (YNBP), which was supplemented with glucose, lactose, galactose and/or insulin (Humulin®-R). The incubation conditions included a shaking or static culture, at 23 °C or 37 °C. After incubation, the biofilm production was calculated per CFU. At 23 °C, the presence of insulin increased biofilm formation. The amount of biofilm formation was highest in glucose > galactose >> lactose, while the biofilm levels decreased in shaking cultures, except for galactose (3-fold increase; 0.1% galactose and 20 μU insulin). At 37 °C, regardless of condition, there was more biofilm formation/CFU under static conditions in YNBP than in MH, except for the MH containing galactose. E. coli biofilm formation is influenced by aeration, temperature, and insulin concentration in combination with the available sugars.

Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium-Soybean Symbiosis.

ABSTRACTThe ubiquitous bacterial second messenger c-di-GMP is intensively studied in pathogens but less so in mutualistic bacteria. Here, we report a genome-wide investigation of functional diguanylate cyclases (DGCs) synthesizing c-di-GMP from two molecules of GTP in Sinorhizobium fredii CCBAU45436, a facultative microsymbiont fixing nitrogen in nodules of diverse legumes, including soybean. Among 25 proteins harboring a putative GGDEF domain catalyzing the biosynthesis of c-di-GMP, eight functional DGCs were identified by heterogenous expression in Escherichia coli in a Congo red binding assay. This screening result was further verified by in vitro enzymatic assay with purified full proteins or the GGDEF domains from representative functional and nonfunctional DGCs. In the same in vitro assay, a functional EAL domain catalyzing the degradation of c-di-GMP into pGpG was identified in a protein that has an inactive GGDEF domain but with an active phosphodiesterase (PDE) function. The identified functional DGCs generally exhibited low transcription levels in soybean nodules compared to free-living cultures, as revealed in transcriptomes. An engineered upregulation of a functional DGC in nodules led to a significant increase of c-di-GMP level and symbiotic defects, which were not observed when a functional EAL domain was upregulated at the same level. Further transcriptional analysis and gel shift assay demonstrated that these functional DGCs were all transcriptionally repressed in nodules by a global pleiotropic regulator, MucR1, that is essential in Sinorhizobium-soybean symbiosis. These findings shed novel insights onto the systematic regulation of c-di-GMP biosynthesis in mutualistic symbiosis.

Dynamics of biofilms on different polymeric membranes – A comparative study using five physiologically and genetically distinct bacteria

Biofilm formation potential of anaerobic microbes on polymeric membranes is widely acknowledged but barely investigated. In this study, the biofilm dynamics of five bacterial isolates from anaerobic sludge, against eight different polymeric membranes, was systematically characterized via scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Fibrous surfaces, i.e., polytetrafluoroethylene, polypropylene and polyvinyl chloride were more favorable for biofilms than the leveled porous substratum. Cellulose-based membranes, especially cellulose nitrate, and cellulose acetate-cellulose nitrate, with a relatively hydrophilic surface showed the highest resistance to biofilms among all membranes. The growth stage played a vital role in biofilm formation, as differences in biofilms of the same strain, in terms of biofilm volumes, thickness and surface coverage, across 8 different substrata, were only observed for mature biofilms. While for early-stage biofilms, the same biomass volume was developed regardless of substratum. This was largely attributed to the interactions between substrata and extracellular polymeric substances (EPS), which was not a prerequisite for initial bacterial adhesion but crucial for cell cohesion in mature biofilms, and thus deciding the morphology and thickness of mature biofilms. These findings highlight that quenching EPS production and diminishing EPS-substratum interaction are the optimum fouling control strategies in membrane-based water treatment systems.

Biofilm formation of Staphylococcus epidermidis imaged using atmospheric scanning electron microscopy.

Staphylococcus epidermidis are gram-positive bacteria that form a biofilm around implanted devices and develop an infection into a chronic state. Recently, it has been revealed that microvesicles have important roles in biofilm formation and intercellular communication among bacteria. However, biofilm formation of Staphylococcus epidermidis, and its relation to microvesicle secretion, is poorly understood because of the difficulty required to preserve the delicate water-rich morphology of biofilm for high-resolution observations. Here, we successfully imaged the microvesicles secreted from Staphylococcus epidermidis and the subsequent process of their integration into biofilm using liquid-phase imaging using atmospheric scanning electron microscopy (ASEM). In the biofilm, cells were connected by nanotube-like structures attached by microvesicles, and surrounded by extracellular polymeric substances. Cells cultured in the ASEM specimen holder were aldehyde-fixed and stained using positively charged nanogold labelling and/or using National Center for Microscopy and Imaging Research method. The samples immersed in aqueous radical scavenger glucose buffer were imaged by the inverted SEM of ASEM. Information regarding the morphologies of microvesicles, nanotube-like fibrils, and biofilm formed by Staphylococcus epidermidis is expected to be useful to elucidate the biological mechanism of biofilm formation and to develop a medicine against biofilms and their associated infections.

Escherichia coli Nissle 1917 secondary metabolism: aryl polyene biosynthesis and phosphopantetheinyl transferase crosstalk.

Escherichia coli Nissle 1917 (EcN) is a Gram-negative bacterium that is used to treat inflammatory bowel diseases. The probiotic character of EcN is not well-understood, but its ability to produce secondary metabolites plays an important role in its activity. The EcN genome encodes for an aryl polyene (APE) biosynthetic gene cluster (BGC), and APE products have a role in biofilm formation. We show here that this unusual polyketide assembly line synthase produces four APE molecules which are likely cis/trans isomers. Within the APE BGC, two acyl carrier proteins are involved in biosynthesis. Acyl carrier proteins require activation by post-translational modification with a phosphopantetheinyl transferase (PPTase). Through analysis of single, double, and triple mutants of three PPTases, the PPTase-BGC crosstalk relationship in EcN was characterized. Understanding PPTase-BGC crosstalk is important for the engineering of secondary metabolite production hosts and for targeting of PPTases with new antibiotics. KEY POINTS: • Escherichia coli Nissle 1917 biosynthesizes four aryl polyene isoforms. • Phosphopantetheinyl transferase crosstalk is important for biosynthesis.