Promote Biofilm Formation Research Articles

Impact of manganese on biofilm formation and cell morphology of Candida parapsilosis clinical isolates with different biofilm forming abilities.

ABSTRACTThe commensal species Candida parapsilosis is an emerging human pathogen that has the ability to form biofilms. In this study, we explored the impact of the divalent cations cobalt (Co2+), copper (Cu2+), iron (Fe3+), manganese (Mn2+), nickel (Ni2+) and zinc (Zn2+) on biofilm formation of clinical isolates of C. parapsilosis with no, low and high biofilm forming abilities at 30 and 37°C. All strains besides one isolate showed a concentration-dependent enhancement of biofilm formation at 30°C in the presence of Mn2+ with a maximum at 2 mM. The biofilm forming ability of no and low biofilm forming isolates was >2-fold enhanced in the presence of 2 mM Mn2+, while the effect in high biofilm forming isolate was significantly less pronounced. Of note, cells in the biofilms of no and low biofilm forming strains differentiated into yeast and pseudohyphal cells similar in morphology to high biofilm formers. The biofilm transcriptional activator BCR1 has a dual developmental role in the absence and presence of 2 mM Mn2+ as it promoted biofilm formation of no biofilm forming strains, and, surprisingly, suppressed cells of no biofilm forming strains to develop into pseudohyphae and/or hyphae. Thus, environmental conditions can significantly affect the amount of biofilm formation and cell morphology of C. parapsilosis with Mn2+ to overcome developmental blocks to trigger biofilm formation and to partially relieve BCR1 suppressed cell differentiation.

A ParDE-family toxin antitoxin system in major resistance plasmids of Enterobacteriaceae confers antibiotic and heat tolerance

Toxin-antitoxin (TA) systems were initially discovered as plasmid addiction systems on low-copy-number plasmids. Thousands of TA loci have since been identified on chromosomes, plasmids and mobile elements in bacteria and archaea with diverse roles in bacterial physiology and in maintenance of genetic elements. Here, we identified and characterised a plasmid mediated type II TA system in Enterobacteriaceae as a member of the ParDE super family. This system (hereafter, ParDEI) is distributed among IncI and IncF-type antibiotic resistance and virulence plasmids found in avian and human-source Escherichia coli and Salmonella. It is found that ParDEI is a plasmid stability and stress response module that increases tolerance of aminoglycoside, quinolone and β-lactam antibiotics in E. coli by ~100–1,000-fold, and thus to levels beyond those achievable in the course of antibiotic therapy for human infections. ParDEI also confers a clear survival advantage at 42 °C and expression of the ParEI toxin in trans induces the SOS response, inhibits cell division and promotes biofilm formation. This transmissible high-level antibiotic tolerance is likely to be an important factor in the success of the IncI and IncF plasmids which carry it and the important pathogens in which these are resident.

Transcriptome analysis of the fish pathogen Flavobacterium columnare in biofilm suggests calcium role in pathogenesis

BackgroundFlavobacterium columnare is the causative agent of columnaris disease that affects cultured freshwater fishes worldwide. F. columnare easily colonizes surfaces by forming biofilm, which helps the pathogen resist antibiotic and disinfectant treatments. Previously, we had shown that increasing concentrations of calcium (Ca2+) promoted biofilm formation by F. columnare. The objective of this study was to further characterize the role of Ca2+ on biofilm formation and to compare the transcriptome profiles of planktonic and biofilm cells.ResultsRNA-Seq analysis was conducted to identify genes that were differentially expressed between the following states: i) planktonic cells in control medium (P), ii) planktonic cells in calcium-enriched medium (P/Ca), and iii) biofilm cells in calcium-enriched medium (B/Ca). Overall, we identified 441 significant (FDR-adjusted p < 0.05, fold change > 2) differentially expressed genes (DEGs) between P and B/Ca samples; 112 significant DEGs between P/Ca and B/Ca samples, and 175 significant DEGs between P/Ca and P samples, corresponding to 15.87, 4.03 and 6.30% of the total protein-coding sequences, respectively. The significant DEGs fell into different functional categories including iron acquisition, oxidative stress response, extracellular protein secretion, and respiratory metabolism.ConclusionsOur results posit Ca2+ as a critical signal in regulating bacterial surface adhesion and biofilm formation in F. columnare. Living in biofilm elicited a shift in several metabolic pathways that allowed the cells to cope with oxidative stress and nutrient starvation. In addition, Ca2+ supplementation induced the expression of putative virulence factors in F. columnare, such as extracellular protein secretion and iron acquisition.

Biotechnologies used in wastewater treatment

One of the most efficient biological wastewater treatment process is based on the biofilm carriers’ utilization, on which the microorganisms attaches to form biofilm communities. The biological technology that uses artificial mobile supports is known as the Moving Bed Biofilm Reactor (MBBR) process. To have an efficient attachment of the biomass on the carriers, hydrophilic substrates are needed. To promote biofilm formation and development, the chemical nature of the biofilm carriers should be carefully selected. Most of the biofilm carriers are made from polyethylene. This material has hydrophobic properties. The authors propose a new material with more hydrophilic properties. Talcum is combined with polyethylene resulting a more hydrophilic material. The results regarding the biofilm adhesion on the new material are presented by the authors. Also, the paper presents a laboratory installation for wastewater treatment, on which can be compared different biological treatment processes.

Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix.

We developed a new approach that couples Southwestern blotting and mass spectrometry to discover proteins that bind extracellular DNA (eDNA) in bacterial biofilms. Using Staphylococcus aureus as a model pathogen, we identified proteins with known DNA-binding activity and uncovered a series of lipoproteins with previously unrecognized DNA-binding activity. We demonstrated that expression of these lipoproteins results in an eDNA-dependent biofilm enhancement. Additionally, we found that while deletion of lipoproteins had a minimal impact on biofilm accumulation, these lipoprotein mutations increased biofilm porosity, suggesting that lipoproteins and their associated interactions contribute to biofilm structure. For one of the lipoproteins, SaeP, we showed that the biofilm phenotype requires the lipoprotein to be anchored to the outside of the cellular membrane, and we further showed that increased SaeP expression correlates with more retention of high-molecular-weight DNA on the bacterial cell surface. SaeP is a known auxiliary protein of the SaeRS system, and we also demonstrated that the levels of SaeP correlate with nuclease production, which can further impact biofilm development. It has been reported that S. aureus biofilms are stabilized by positively charged cytoplasmic proteins that are released into the extracellular environment, where they make favorable electrostatic interactions with the negatively charged cell surface and eDNA. In this work we extend this electrostatic net model to include secreted eDNA-binding proteins and membrane-attached lipoproteins that can function as anchor points between eDNA in the biofilm matrix and the bacterial cell surface.IMPORTANCE Many bacteria are capable of forming biofilms encased in a matrix of self-produced extracellular polymeric substances (EPS) that protects them from chemotherapies and the host defenses. As a result of these inherent resistance mechanisms, bacterial biofilms are extremely difficult to eradicate and are associated with chronic wounds, orthopedic and surgical wound infections, and invasive infections, such as infective endocarditis and osteomyelitis. It is therefore important to understand the nature of the interactions between the bacterial cell surface and EPS that stabilize biofilms. Extracellular DNA (eDNA) has been recognized as an EPS constituent for many bacterial species and has been shown to be important in promoting biofilm formation. Using Staphylococcus aureus biofilms, we show that membrane-attached lipoproteins can interact with the eDNA in the biofilm matrix and promote biofilm formation, which suggests that lipoproteins are potential targets for novel therapies aimed at disrupting bacterial biofilms.

Host-specialized fibrinogen-binding by a bacterial surface protein promotes biofilm formation and innate immune evasion.

Fibrinogen is an essential part of the blood coagulation cascade and a major component of the extracellular matrix in mammals. The interface between fibrinogen and bacterial pathogens is an important determinant of the outcome of infection. Here, we demonstrate that a canine host-restricted skin pathogen, Staphylococcus pseudintermedius, produces a cell wall-associated protein (SpsL) that has evolved the capacity for high strength binding to canine fibrinogen, with reduced binding to fibrinogen of other mammalian species including humans. Binding occurs via the surface-expressed N2N3 subdomains, of the SpsL A-domain, to multiple sites in the fibrinogen α-chain C-domain by a mechanism analogous to the classical dock, lock, and latch binding model. Host-specific binding is dependent on a tandem repeat region of the fibrinogen α-chain, a region highly divergent between mammals. Of note, we discovered that the tandem repeat region is also polymorphic in different canine breeds suggesting a potential influence on canine host susceptibility to S. pseudintermedius infection. Importantly, the strong host-specific fibrinogen-binding interaction of SpsL to canine fibrinogen is essential for bacterial aggregation and biofilm formation, and promotes resistance to neutrophil phagocytosis, suggesting a key role for the interaction during pathogenesis. Taken together, we have dissected a bacterial surface protein-ligand interaction resulting from the co-evolution of host and pathogen that promotes host-specific innate immune evasion and may contribute to its host-restricted ecology.

Activity of Sodium Lauryl Sulfate, Rhamnolipids, and N-Acetylcysteine Against Biofilms of Five Common Pathogens.

Bacteria in biofilms are more resistant to antibacterial agents than bacteria in planktonic form. Hence, antibacterial agents should be able to eradicate biofilms to ensure the best outcomes. Little is known about how well many antibacterial agents can disrupt biofilms. In this study, we compared sodium lauryl sulfate (SDS), rhamnolipids (RHL), and N-acetylcysteine (NAC) for their ability to eradicate mature biofilms and inhibit new biofilm formation against Helicobacter pylori, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Streptococcus mutans. SDS and RHL effectively inhibited formation of five bacterial biofilms in a dose-dependent manner, even at concentrations below the minimal inhibitory concentrations (MICs), suggesting that their antibiofilm activities are unrelated to their antibacterial activities. In contrast, NAC at certain concentrations promoted biofilm formation by all bacteria except P. aeruginosa, whereas at supra-MIC concentrations, it inhibited biofilm formation against the four bacteria, suggesting that its antibiofilm activity depends on its antibacterial activity. NAC was ineffective at eradicating mature H. pylori biofilms, and it actually promoted their formation at concentrations >10 mg/mL. Our results suggest that RHL is superior at eradicating biofilms of H. pylori, E. coli, and S. mutans; SDS is more effective against S. aureus biofilms; and NAC is more effective against P. aeruginosa biofilms. Our results may help determine which antibiofilm agents are effective against certain bacterial strains and develop agents effective against specific bacterial threats.

Proline Increases Pigment Production to Improve Oxidative Stress Tolerance and Biocontrol Ability of Metschnikowia citriensis.

Utilizing antagonistic yeasts is a promising approach for managing postharvest decay of fruits. However, it is well established that various severe stresses encountered in the environment and production process cause the intracellular reactive oxygen species (ROS) accumulation in yeast cells, resulting in cell damage and loss of vitality. Here, proline has been shown to function as a cell protectant and inducer of biofilm formation able to increase the oxidative stress tolerance and the biocontrol ability of the antagonistic yeast Metschnikowia citriensis. Addition of proline to M. citriensis cells induced a significant rise in superoxide dismutase (SOD) and catalase (CAT) activity in the early and late stages of oxidative stress, respectively, and increased the maroon pigment production that directly reduced intracellular iron content and indirectly diminished intracellular ROS levels and thus inhibited ROS- and iron-induced apoptosis. Treating cells with iron chelator tropolone yielded similar results. Pigment production induced by proline also enhanced the capability of biofilm formation of M. citriensis. These results suggested an important role for pigment of M. citriensis in response to oxidative stress. The abilities of proline to scavenge intracellular ROS and inhibit apoptosis, increase pigment production, and promote biofilm formation contribute to the improvements in oxidative stress tolerance and biocontrol efficacy of M. citriensis.

3-Benzyl-Hexahydro-Pyrrolo[1,2-a]Pyrazine-1,4-Dione Extracted From Exiguobacterium indicum Showed Anti-biofilm Activity Against Pseudomonas aeruginosa by Attenuating Quorum Sensing.

Bacterial cell-to-cell communication promotes biofilm formation and can potentially lead to multidrug resistance development. Quorum sensing inhibition (QSI) is an effective and widely employed strategy against biofilm formation. The extract from Exiguobacterium indicum SJ16, a gram-positive bacterium, isolated from the rhizosphere of Cyperus laevigatus showed significant anti-quorum sensing activity (about 99%) against the reference Chromobacterium violaceum CV026 strain without exerting any antibacterial effect. The potentially active QSI compound identified in the SJ16 extract was 3-Benzyl-hexahydro-pyrrolo[1, 2-a]pyrazine-1,4-dione. The SJ16 extract containing this active compound showed significant anti-quorum sensing activity against a model quorum sensing bacterium strain Pseudomonas aeruginosa PAO1 and a clinical isolate P. aeruginosa PAH by preventing biofilm formation without attenuating the cell growth within the biofilm. More specifically, the SJ16 extract changed the topography and architecture of the biofilm, thus preventing bacterial adherence and further development of the biofilm. Furthermore, it decreased virulence factors (rhamnolipid and pyocyanin), the bacterial motility, as well as the elastase, and protease activities in P. aeruginosa. Microarray analysis revealed the differential expression of quorum sensing regulatory genes. Based on these results, we herein propose a hypothetical model, characterizing the role of this QSI agent in the transcriptional regulation of quorum sensing in P. aeruginosa PAO1, demonstrating that this compound has significant drug-development potential. Further research is required to delineate its possible applications in therapeutics in the context of biofilm forming bacterial infections.

Graphene‐dispersed silane compound used as a coating to sense immunity from biofilm formation

AbstractBiofilm is a substance that contains organic polymers. It is derived from bacteria, produces slime and forms on material's surfaces. Therefore, it is important to properly evaluate biofilm formation using several types of sensors. Since biofilm forms easily on graphene, it is possible for graphene to serve as a sensor for biofilm formation. In the past, graphene's sensitivity to biofilm formation was confirmed by using CVD (chemical vapour deposition) graphene and by using the tape‐exfoliation method. However, the CVD method was expensive and required special equipment. On the other hand, the exfoliation method was not so expensive, but the precision and reproducibility for graphene formation were problematic. In this experiment, silane‐based resin, with dispersed graphene, was coated on a substrate. This coated substrate can easily be used as a sensor and is an inexpensive way to promote biofilm formation. Glass was used as the substrate, for which various samples were prepared using different concentrations of dispersed graphene. Biofilms formed on the specimens’ surfaces were analysed by using Raman spectroscopy and the crystal violet staining method. The results showed that biofilm formation capability increased with the concentration of dispersed graphene. We confirmed that the sensitivity was evaluated by the colour value for stained surfaces by crystal violet.

Impact of the Food Additive Titanium Dioxide (E171) on Gut Microbiota-Host Interaction.

The interaction between gut microbiota and host plays a central role in health. Dysbiosis, detrimental changes in gut microbiota and inflammation have been reported in non-communicable diseases. While diet has a profound impact on gut microbiota composition and function, the role of food additives such as titanium dioxide (TiO2), prevalent in processed food, is less established. In this project, we investigated the impact of food grade TiO2 on gut microbiota of mice when orally administered via drinking water. While TiO2 had minimal impact on the composition of the microbiota in the small intestine and colon, we found that TiO2 treatment could alter the release of bacterial metabolites in vivo and affect the spatial distribution of commensal bacteria in vitro by promoting biofilm formation. We also found reduced expression of the colonic mucin 2 gene, a key component of the intestinal mucus layer, and increased expression of the beta defensin gene, indicating that TiO2 significantly impacts gut homeostasis. These changes were associated with colonic inflammation, as shown by decreased crypt length, infiltration of CD8+ T cells, increased macrophages as well as increased expression of inflammatory cytokines. These findings collectively show that TiO2 is not inert, but rather impairs gut homeostasis which may in turn prime the host for disease development.

(Invited) Materials Design for Microbial Bioelectrochemical Devices

Electrochemically interfacing bacteria with electrodes presents a dynamic new set of materials challenges. Bacteria naturally form biofilms on traditional electrode surfaces, but materials design can help promote extracellular electron transfer between the bacteria and the electrode within biofilms. This paper will discuss materials strategies for promoting biofilm formation, biofilm maintenance, and extracellular electron transfer within the film, as well as improving the lifetime of microbial bioelectrodes. We will discuss different strategies for biophotocurrent generation versus microbial fuel cells versus shock biosensors, including novel carbon materials, electrode treatments, redox polymers, and biopolymer engineering.

A Bifunctional UDP-Sugar 4-Epimerase Supports Biosynthesis of Multiple Cell Surface Polysaccharides in Sinorhizobium meliloti.

Sinorhizobium meliloti produces multiple extracellular glycans, including among others, lipopolysaccharides (LPS), and the exopolysaccharides (EPS) succinoglycan (SG) and galactoglucan (GG). These polysaccharides serve cell protective roles. Furthermore, SG and GG promote the interaction of S. meliloti with its host Medicago sativa in root nodule symbiosis. ExoB has been suggested to be the sole enzyme catalyzing synthesis of UDP-galactose in S. meliloti (A. M. Buendia, B. Enenkel, R. Köplin, K. Niehaus, et al. Mol Microbiol 5:1519-1530, 1991, https://doi.org/10.1111/j.1365-2958.1991.tb00799.x). Accordingly, exoB mutants were previously found to be affected in the synthesis of the galactose-containing glycans LPS, SG, and GG and consequently, in symbiosis. Here, we report that the S. meliloti Rm2011 uxs1-uxe-apsS-apsH1-apsE-apsH2 (SMb20458-63) gene cluster directs biosynthesis of an arabinose-containing polysaccharide (APS), which contributes to biofilm formation, and is solely or mainly composed of arabinose. Uxe has previously been identified as UDP-xylose 4-epimerase. Collectively, our data from mutational and overexpression analyses of the APS biosynthesis genes and in vitro enzymatic assays indicate that Uxe functions as UDP-xylose 4- and UDP-glucose 4-epimerase catalyzing UDP-xylose/UDP-arabinose and UDP-glucose/UDP-galactose interconversions, respectively. Overexpression of uxe suppressed the phenotypes of an exoB mutant, evidencing that Uxe can functionally replace ExoB. We suggest that under conditions stimulating expression of the APS biosynthesis operon, Uxe contributes to the synthesis of multiple glycans and thereby to cell protection, biofilm formation, and symbiosis. Furthermore, we show that the C2H2 zinc finger transcriptional regulator MucR counteracts the previously reported CuxR-c-di-GMP-mediated activation of the APS biosynthesis operon. This integrates the c-di-GMP-dependent control of APS production into the opposing regulation of EPS biosynthesis and swimming motility in S. melilotiIMPORTANCE Bacterial extracellular polysaccharides serve important cell protective, structural, and signaling roles. They have particularly attracted attention as adhesives and matrix components promoting biofilm formation, which significantly contributes to resistance against antibiotics. In the root nodule symbiosis between rhizobia and leguminous plants, extracellular polysaccharides have a signaling function. UDP-sugar 4-epimerases are important enzymes in the synthesis of the activated sugar substrates, which are frequently shared between multiple polysaccharide biosynthesis pathways. Thus, these enzymes are potential targets to interfere with these pathways. Our finding of a bifunctional UDP-sugar 4-epimerase in Sinorhizobium meliloti generally advances the knowledge of substrate promiscuity of such enzymes and specifically of the biosynthesis of extracellular polysaccharides involved in biofilm formation and symbiosis in this alphaproteobacterium.

Preclinical evaluation of a novel silver gelling fiber dressing on Pseudomonas aeruginosa in a porcine wound infection model.

The wound environment is a fertile ground for biofilm forming pathogens. Once biofilms form within the wound, they can be very challenging to eradicate. The purpose of this study was to examine the effect of a gelling fiber dressing with silver using a well-established porcine wound biofilm model. Deep partial thickness wounds were inoculated with Pseudomonas aeruginosa ATCC 27312 and covered with a polyurethane film dressing to promote biofilm formation. Wounds were then divided into treatment groups: gelling fiber dressing with silver, gelling fiber dressing without silver, hydrofiber dressing with silver, benzethonium chloride and ethylenediaminetetraacetic acid and compared to untreated control. Microbiological, biofilm, and histological wound assessments were performed on days 3, 5, and 7 postinfection. Treatment with gelling fiber dressing with silver resulted in significant reduction of P. aeruginosa biofilm when compared to all other treatment groups on every assessment time point. In addition, gelling fiber dressing with silver treatment resulted in detachment of biofilm from the wound, while wounds treated with gelling fiber dressing with and without silver showed more granulation tissue formation on day 3. Our data show that a new gelling fiber dressing with silver was effective in reducing biofilm associated P. aeruginosa in vivo. This study may have important clinical implications especially for wounds heavily colonized with gram-negative biofilm-forming bacteria.

Deletion of D-Lactate Dehydrogenase A in Neisseria meningitidis Promotes Biofilm Formation Through Increased Autolysis and Extracellular DNA Release.

Neisseria meningitidis is a Gram-negative bacterium that asymptomatically colonizes the human nasopharyngeal mucosa. Pilus-mediated initial adherence of N. meningitidis to the epithelial mucosa is followed by the formation of three-dimensional aggregates, called microcolonies. Dispersal from microcolonies contributes to the transmission of N. meningitidis across the epithelial mucosa. We have recently discovered that environmental concentrations of host cell-derived lactate influences N. meningitidis microcolony dispersal. Here, we examined the ability of N. meningitidis mutants deficient in lactate metabolism to form biofilms. A lactate dehydrogenease A (ldhA) mutant had an increased level of biofilm formation. Deletion of ldhA increased the N. meningitidis cell surface hydrophobicity and aggregation. In this study, we used FAM20, which belongs to clonal complex ST-11 that forms biofilms independently of extracellular DNA (eDNA). However, treatment with DNase I abolished the increased biofilm formation and aggregation of the ldhA-deficient mutant, suggesting a critical role for eDNA. Compared to wild-type, the ldhA-deficient mutant exhibited an increased autolytic rate, with significant increases in the eDNA concentrations in the culture supernatants and in biofilms. Within the ldhA mutant biofilm, the transcription levels of the capsule, pilus, and bacterial lysis genes were downregulated, while norB, which is associated with anaerobic respiration, was upregulated. These findings suggest that the absence of ldhA in N. meningitidis promotes biofilm formation and aggregation through autolysis-mediated DNA release.

Efficient hydrogen production using Ni-graphene oxide-dispersed laser-engraved 3D carbon micropillars as electrodes for microbial electrolytic cell

A nickel (Ni)-graphene oxide (GO) dispersed carbon film was prepared by the carbonization of the phenolic precursor-based polymer. Three dimensional (3D) micropillars were fabricated on the carbon film using laser ablation technique. The micropillars-engraved film was used as electrodes in a single chamber microbial electrolytic cell (MEC) for hydrogen (H2) production. Besides promoting biofilm formation at anode, the 3D micropillars provided relatively more exposure to the in situ dispersed electrocatalytic Ni nanoparticles (NPs) and electroconductive GO in the carbon film. The Tafel slope of ∼49 mV dec−1 indicated that Heyrovsky reaction or electrochemical desorption was the rate determining step for H2 evolution. A H2 production rate of 4.22 ± 0.21 m3m−3d−1 was measured at 0.8 V in the prepared electrode-based MEC. Whereas the overall H2 and cathodic recoveries were measured to be 92.3 ± 2.77% and 98.7 ± 0.99%, respectively, the Coulombic and overall energy efficiencies were determined to be 93.5 ± 2.81% and 72.2 ± 3.61%, respectively. The relatively higher efficiency of the MEC was ascribed to the synergistic contributions of the 3D micropillars, Ni NPs and GO, indicating the prepared electrode to be a viable alternative to the expensive noble metal-based electrodes used in MECs for H2 production.

Mig-14 may contribute to Salmonella enterica serovar Typhi resistance to polymyxin B by decreasing the permeability of the outer-membrane and promoting the formation of biofilm

Mig-14 is essential for Salmonella enterica serovar Typhimurium (S. Typhimurium) resistance to antimicrobial peptides, including polymyxin B (PB). However, the molecular mechanism is as yet unknown. In this study, we demonstrated that mig-14 also played a crucial role in Salmonella enterica serovar Typhi (S. Typhi) resistance to PB. A series of genes associated with drug-resistance controlled by Mig-14 were identified in the presence of PB. Among which, ompF and ompC were up-regulated 8 and 6 folds in mig-14 mutant (Δmig-14) strains, respectively. Further, the deletion of ompF or/and ompC in Δmig-14 strains decreased their sensitivity to PB. Besides, the biofilm formation ability was reduced in Δmig-14 strains. Our results indicate that Mig-14 may contribute to PB resistance in S. Typhi by decreasing the permeability of the outer membrane and promoting biofilm formation.

Hidden Gems in the Transcriptome Maps of Competent Streptococci.

Natural transformation is regarded as an important mechanism in bacteria that allows for adaptation to different environmental stressors by ensuring genome plasticity. Since the discovery of this phenomenon in Streptococcus pneumoniae, remarkable progress has been made in the understanding of the molecular mechanisms and pathways coordinating this process. Recently, the advent of high-throughput sequencing allows the posing of questions that address the system at a larger scale but also allow for the creation of high-resolution maps of transcription. Thus, while much is already known about genetic competence in streptococci, recent studies continue to reveal intricate novel regulation pathways and components. In this perspective article, we highlight the use of transcriptional profiling and mapping as a valuable resource in the identification and characterization of “hidden gems” pertinent to the natural transformation system. Such strategies have recently been employed in a variety of different species. In S. mutans, for example, genome editing combined with the power of promoter mapping and RNA-Seq allowed for the identification of a link between the ComCDE and the ComRS systems, a ComR positive feedback loop mediated by SigX, and the XrpA peptide, encoded within sigX, which inhibits competence. In S. pneumoniae, a novel member of the competence regulon termed BriC was found to be directly under control of ComE and to promote biofilm formation and nasopharyngeal colonization but not competence. Together these new technologies enable us to discover new links and to revisit old pathways in the compelling study of natural genetic transformation.

Trace organic contaminant removal in six full-scale integrated fixed-film activated sludge (IFAS) systems treating municipal wastewater

Trace organic contaminants (TrOCs) often pass through conventional activated sludge wastewater treatment plants (CAS-WWTPs) and are discharged into surface waters, where they can threaten aquatic ecosystems and human health, largely due to the hormone disrupting effects of certain TrOCs. The integrated fixed-film activated sludge (IFAS) process is a cost-effective means of upgrading CAS-WWTPs by adding free-floating carrier media, which promotes biofilm formation in the well-mixed suspended growth reactors, providing a potential niche for slow-growing microorganisms. Although IFAS upgrades are typically aimed at enhancing nutrient removal, limited bench- and pilot-scale data indicate that TrOC removal may also be improved. However, only limited reports which focus on a small number of compounds in individual full-scale IFAS-WWTPs have been published to date, and no data is available regarding the removal of estrogenic activity in full-scale IFAS-WWTPs. In this study, six full-scale IFAS-WWTPs were surveyed to quantify TrOC and estrogenic activity removal. Twenty-four hour composite samples of secondary influent and effluent (pre-disinfection) were analyzed for total suspended solids (TSS), chemical oxygen demand (COD), ammonia, total nitrogen (TN), total phosphorus (TP), estrogenic activity, and 98 TrOCs. The biomass distribution between the suspended growth phase (i.e. mixed liquor) and IFAS media was also assessed. All IFAS-WWTPs performed well in terms of TSS, COD, and ammonia removal. TN removal varied in accordance with nitrate removal. Total solids per liter of wetted reactor volume ranged from 2.5 to 7.6 g, with 40–60% attached to media. TrOCs with no detection (17) and those with high median removal (23, ≥90% average removal) were observed. Other TrOCs had lower and more variable removal efficiencies. Qualitative comparison with CAS literature shows potentially higher IFAS removal efficiencies for a number of compounds including several which have been previously indicated in bench- or pilot-scale studies (atenolol, diclofenac, gemfibrozil, DEET, 4-nonylphenol, and 4-tert-octylphenol), as well as the chlorinated flame retardants TCIPP and TDCIPP. Effluent estrogenic activity was found to be similar to that reported for full-scale CAS-WWTPs. These results provide the first survey of multiple full-scale IFAS-WWTPs employing mobile plastic carrier media in terms of basic chemical endpoints (removal of ammonia, TN, TP, and COD), the distribution of solids within the systems, and the removal of TrOCs and estrogenic activity.

Extraction and characterization of stratified extracellular polymeric substances in Geobacter biofilms

Extracellular polymeric substances (EPS) play crucial roles in promoting biofilm formation and contribute to electrochemical activities of biofilms in bioelectrochemical systems (BES). In this study, three stratified EPS fractions were extracted from Geobacter biofilms using EDTA-, ultrasound- and heating-based protocols and characterized with chemical, spectral and electrochemical analyses. Results suggested that, for Geobacter biofilms, ultrasound-based extraction protocol was more effective in EPS yield (62.1–66.5 mg C/g dry cell) than EDTA method, and had less cell lysis than heating method. The extraction methods greatly affected the proteins composition in the extracted EPS, indicated by the varied ratios of tryptophan/tyrosine protein-like substances. Electrochemical measurements demonstrated a good correlation between protein concentration and extracellular electron transfer function for both tightly-bound EPS and total EPS. This is the first study to extract and characterize stratified EPS fractions from Geobacter biofilms, and helpful for better understanding the function of EPS in BESs predominated by Geobacter.