Biofilm Response Research Articles

Discharge, legacy effects and nutrient availability as determinants of temporal patterns in biofilm metabolism and accrual in an arctic river

Summary Significant changes in the timing of thawing and freezing, annual discharge and nutrient supply are anticipated for arctic river ecosystems due to climate warming. Our understanding of the effects of such changes, however, is poor. Since biofilms are both critical to ecosystem processes and subject to control by combinations of freezing dynamics, discharge and nutrient supply, the effects of climate warming on arctic river ecosystems may be substantial. We studied biofilm accrual and metabolism in the Kuparuk River of Alaska with three objectives. First, we assessed biofilm activity during the open‐water season, including the poorly known ‘shoulder seasons’ (periods shortly after the spring thaw and then before autumn freezing). Second, we assessed how discharge and freezing dynamics may affect temporal patterns in biofilm activity. Third, we assessed biofilm response to increases in concentrations predicted as the climate warms. Our study consisted of an upstream reference reach and a downstream treatment reach where H3PO4 was experimentally added for two years (2011–2012; ˜0.3 μm increase). We used archived reference‐reach data from 1983 to 2014 to assess discharge–chlorophyll relationships. Mean biofilm gross primary production (GPP) in the reference reach during 2011 and 2012 was 15.4 ± 2.2 (mean ± S.E.) and 13.4 ± 2.5 mg C m−2 h−1, respectively, with spring shoulder season values being significantly higher than the summer values that are more routinely measured. An analysis of a 31‐year record from the reference reach revealed that biofilm chlorophyll biomass was significantly (R2 = 0.39, P < 0.001) related to the discharge regime of the previous summer. This ‘legacy effect’ is attributable to bedfast ice within the river channel during winter that protects biofilms from scour during the spring thaw. Before–after‐control–impact (BACI) analysis showed significant and positive effects on mean biofilm chl‐a biomass in the treatment reach. This treatment effect was associated with a parallel increase in GPP. High biofilm activity in the Kuparuk River during the shoulder seasons indicates its potential significance to the energetics of arctic river ecosystems, particularly in the context of climate change. The related ‘legacy effect’ probably underlies the scope for rapid recovery of biofilm activity during the spring. Finally, increases in at concentrations predicted as the climate warms are likely to result in increases in summer biofilm biomass and metabolism. Further studies of biofilm processes during the shoulder seasons, and how these are affected by shifts in discharge, freezing dynamics and nutrient supply, are required for a comprehensive understanding of the effects of climate warming on arctic river ecosystems.

Development of a multifunctional adhesive system for prevention of root caries and secondary caries

ObjectivesThe objectives of this study were to: (1) develop a novel adhesive for prevention of tooth root caries and secondary caries by possessing a combination of protein-repellent, antibacterial, and remineralization capabilities for the first time; and (2) investigate the effects of 2-methacryloyloxyethyl phosphorylcholine (MPC), dimethylaminohexadecyl methacrylate (DMAHDM), and nanoparticles of amorphous calcium phosphate (NACP) on dentin bond strength, protein-repellent properties, and dental plaque microcosm biofilm response. MethodsMPC, DMAHDM and NACP were added into Scotchbond Multi-Purpose primer and adhesive. Dentin shear bond strengths were measured. Adhesive coating thickness, surface texture and dentin–adhesive interfacial structure were examined. Protein adsorption onto adhesive resin surface was determined by the micro bicinchoninic acid method. A human saliva microcosm biofilm model was used to investigate biofilm metabolic activity, colony-forming unit (CFU) counts, and lactic acid production. ResultsThe resin with 7.5% MPC+5% DMAHDM+30% NACP did not adversely affect dentin shear bond strength (p>0.1). The resin with 7.5% MPC+5% DMAHDM+30% NACP produced a coating on root dentin with a thickness of approximately 70μm and completely sealed all the dentinal tubules. The resin with 7.5% MPC+5% DMAHDM+30% NACP had 95% reduction in protein adsorption, compared to SBMP control (p<0.05). The resin with 7.5% MPC+5% DMAHDM+30% NACP was strongly antibacterial, with biofilm CFU being four orders of magnitude lower than that of SBMP control.Significance The novel multifunctional adhesive with strong protein-repellent, antibacterial and remineralization properties is promising to coat tooth roots to prevent root caries and secondary caries. The combined use of MPC, DMAHDM and NACP may have wide applicability to bonding agents, cements, sealants and composites to inhibit caries.

Biofilm Formation As a Response to Ecological Competition.

Bacteria form dense surface-associated communities known as biofilms that are central to their persistence and how they affect us. Biofilm formation is commonly viewed as a cooperative enterprise, where strains and species work together for a common goal. Here we explore an alternative model: biofilm formation is a response to ecological competition. We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on biofilm formation. We show that strain mixing reliably increases biofilm formation compared to unmixed conditions. Importantly, strain mixing leads to strong competition: one strain dominates and largely excludes the other from the biofilm. Furthermore, we show that pyocins, narrow-spectrum antibiotics made by other P. aeruginosa strains, can stimulate biofilm formation by increasing the attachment of cells. Side-by-side comparisons using microfluidic assays suggest that the increase in biofilm occurs due to a general response to cellular damage: a comparable biofilm response occurs for pyocins that disrupt membranes as for commercial antibiotics that damage DNA, inhibit protein synthesis or transcription. Our data show that bacteria increase biofilm formation in response to ecological competition that is detected by antibiotic stress. This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded. Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.

Bioelectrochemical treatment of municipal waste liquor in microbial fuel cells for energy valorization

Microbial fuel cells (MFCs) are recognized as promising applications to produce bioelectricity by utilizing various waste materials. In this study, dual-chamber microbial fuel cells were employed for energy valorization of an untested substrate, the liquid fraction of pressed municipal solid waste (LPW). This by-product is potentially applicable as a substrate in MFCs because of its high organic matter content. In the course of the experiments, the anodic biofilm response and energy production efficiency have been investigated by experimental design approach, taking substrate and fresh inoculum – mesophilic anaerobic sludge (MAS) – addition into account as factors. It was observed that reinoculation could result in a negative effect on the energy production, especially at low substrate (LPW) dosing levels. However, when the LPW to fresh MAS ratio in the anode chamber exceeded a particular value, the biofilm-associated electrical utilization dominated against the degradation in the bulk phase. Furthermore, the results indicated that the highest energy yields (8–9 J g−1 ΔCOD d−1) could be attained at the lowest input COD concentrations. The maximal and average COD removal efficiencies were 94% and 87%, respectively, which indicate the excellent biodegradability of LPW. As for COD removal rate, 1.2–1.9 kg COD m−3 d−1 could be reached.

Phenol removal and biofilm response in coupling of visible-light-driven photocatalysis and biodegradation: Effect of hydrothermal treatment temperature

Intimate coupling of visible-light-responsive photocatalysis and biodegradation (VPCB) provides novel insights into removal of inhibitory pollutants. Under these reactions, microbes are prone to photocatalytic activity. In this study, microbial responses to photocatalytic activity in VPCB used for phenol degradation were investigated. VPCB was conducted with Er3+:YAlO3/TiO2 photocatalysts prepared at heat-treatment temperatures [HT] of 110 °C, 130 °C, and 150 °C to evaluate photocatalytic activities. The highest phenol removal rate (71.6%) was observed in photocatalysis [HT] 130, and particular microbial responses were elicited in VPCB [HT] 130. Dehydrogenase activity exhibited the highest decrease (90.7%) in VPCB [HT] 130. In this treatment, large amounts of biofilm were detached from the exterior of the carrier but the biofilm remained well protected in the interior. Extracellular polymeric substances with decreases of 65.6% and 31.4% in protein and polysaccharide levels, respectively, were not stimulated in VPCB [HT] 130 but were evidently stimulated in the other two protocols, which could be due to lower phenol inhibition. These results indicated that the most efficient removal rates of phenol and dissolved organic carbon reached 99.8% and 67.1%, respectively, in VPCB [HT] 130.

Herbicide toxicity on river biofilms assessed by pulse amplitude modulated (PAM) fluorometry

The use of Rapid light curves (RLCs) as a toxicity endpoint for river biofilms was examined in this study and compared to “classical fluorescence parameters” i.e. minimal fluorescence (F0), optimal and effective quantum yields of photosystem II (Fv/Fm and ФPSII). Measurements were performed after exposure to five concentrations of diuron (from 0.3 to 33.4μgL−1), its main degradation product (DCPMU) (from 1.0 to 1014μgL−1) and norflurazon (from 0.6 to 585μgL−1) with the lowest exposure concentrations corresponding to levels regularly encountered in chronically contaminated sites. Biofilm responses were evaluated after 1, 5, 7 and 14 days of exposure to the different toxicants.Overall, the responses of both “classical fluorescence parameters” and RLC endpoints were highly time dependent and related to the mode of action of the different compounds. Interestingly, parameters calculated from RLCs (α, ETRmax and Ik) were useful early markers of pesticide exposure since they revealed significant effects of all the tested toxicants from the first day of exposure. In comparison, classical fluorescence endpoints (F0 and Fv/Fm) measured at day 1 were only affected in the DCPMU treatment.Our results demonstrated the interest of RLCs as early markers of toxicant exposure particularly when working with toxicants with less specific mode of action than PSII inhibitors.

Urbanisation and earthquake disturbance influence microbial nutrient limitation in streams

Summary Nutrient loading as a consequence of urbanisation is a common phenomenon that can influence microbial nutrient limitation in streams. How nutrient loading and nutrient limitation relate to increasing urban intensity, and whether there is any coherence in patterns across cities, is not well known. Nutrient limitation may also be influenced by natural disturbances that alter urban infrastructure and processes in urban streams. To examine this, we measured microbial nutrient limitation across urbanisation gradients in two cities in New Zealand. We took advantage of recent large earthquakes in one of the cities to determine how natural disturbance influences processes in an urban system. Biofilm responses to nitrogen (N) and phosphorus (P) amendment were tested using nutrient diffusing substrata (NDS) in 24 streams, 12 in each of New Zealand's two largest cities (Auckland and Christchurch). Biofilm response was measured across a gradient of land‐use intensity in each city. Regression models were used to quantify the relationships among water chemistry, land use and degree of biofilm N and P limitation. Water column N:P (DIN:SRP) was a good predictor of response of community respiration on organic substrata, but not of response of chl a on inorganic substrata. Autotrophic response was often not nutrient‐limited and was not likely to have been limited by light, suggesting other factors governed the development of autotrophs. We found a switch in response of biofilm respiration on organic substrata from N to P limitation in Auckland, with N limitation increasing below a N:P ratio of 20.8 and P limitation increasing above a ratio of 14.5, roughly in line with the Redfield ratio. Similarly, in Christchurch microbes showed little response to nutrient enrichment at sites with elevated nutrient concentrations. In Auckland, increasing urban intensity was associated with increased nutrient concentrations, but N increased faster than P. This translated to a nonlinear response of biofilm nutrient limitation to increasing urbanisation indicated as population density. There was a breakpoint from N to P limitation at a relatively low population density of &gt;97 people per km2. Patterns of limitation with urbanisation were not as strong in Christchurch; however, responses were similar within the range of overlapping population densities in the two cities. Earthquake disturbance had noticeable effects on streams, including sevenfold lower DIN concentrations and inhibition by both N and P of community respiration on organic substrata. Urbanisation is a key driver of microbial nutrient limitation. Between cities, nutrient loading associated with urbanisation consistently affected the capacity of biofilms to process excess nutrients. Earthquake effects further influenced microbial limitation patterns with natural disturbance dramatically altering how urbanisation effects are manifested.

Lactotransferrin Gene Polymorphism Associated with Caries Experience

Dental caries is a common multifactorial disease, resulting from the interaction of biofilm, cariogenic diet and host response over time. Lactotransferrin (LTF) is a main salivary glycoprotein, which modulates the host immune-inflammatory and antibacterial response. Although a genetic component for caries outcome has been identified, little is known over the genetic aspects underlying its susceptibility. Thus, the aim of this study was to investigate the association between LTF polymorphisms and caries susceptibility. Six hundred seventy seven 12-year-old students were selected: 346 with (DMFT ≥ 1) and 331 without caries experience (DMFT = 0). Also, individuals concentrating higher levels of disease (polarization group, DMFT ≥ 2, n = 253) were tested against those with DMFT ≤ 1 (n = 424). Along with clinical parameters, three representative LTF tag SNPs (rs6441989, rs2073495, rs11716497) were genotyped and the results were evaluated using univariate and multivariate analyses. Allele A for tag SNP rs6441989 was found to be significantly less frequent in the polarization group, conferring a protective effect against caries experience [AA + AG × GG (OR: 0.710, 95% CI: 0.514-0.980, p = 0.045)], and remained significantly associated with caries protection in the presence of gingivitis (p = 0.020) and plaque (p = 0.035). These results might contribute to the understanding of the genetic control of caries susceptibility in humans.

Multicomponent model of deformation and detachment of a biofilm under fluid flow.

A novel biofilm model is described which systemically couples bacteria, extracellular polymeric substances (EPS) and solvent phases in biofilm. This enables the study of contributions of rheology of individual phases to deformation of biofilm in response to fluid flow as well as interactions between different phases. The model, which is based on first and second laws of thermodynamics, is derived using an energetic variational approach and phase-field method. Phase-field coupling is used to model structural changes of a biofilm. A newly developed unconditionally energy-stable numerical splitting schemeis implemented for computing the numerical solution of the model efficiently. Model simulations predict biofilm cohesive failure for the flow velocity between [Formula: see text] and [Formula: see text] m s(-1) which is consistent with experiments. Simulations predict biofilm deformation resulting in the formation of streamers for EPS exhibiting a viscous-dominated mechanical response and the viscosity of EPS being less than [Formula: see text]. Higher EPS viscosity provides biofilm with greater resistance to deformation and to removal by the flow. Moreover, simulations show that higher EPS elasticity yields the formation of streamers with complex geometries that are more prone to detachment. These model predictions are shown to be in qualitative agreement with experimental observations.

Contribution of stress responses to antibiotic tolerance in Pseudomonas aeruginosa biofilms.

Enhanced tolerance of biofilm-associated bacteria to antibiotic treatments is likely due to a combination of factors, including changes in cell physiology as bacteria adapt to biofilm growth and the inherent physiological heterogeneity of biofilm bacteria. In this study, a transcriptomics approach was used to identify genes differentially expressed during biofilm growth of Pseudomonas aeruginosa. These genes were tested for statistically significant overlap, with independently compiled gene lists corresponding to stress responses and other putative antibiotic-protective mechanisms. Among the gene groups tested were those associated with biofilm response to tobramycin or ciprofloxacin, drug efflux pumps, acyl homoserine lactone quorum sensing, osmotic shock, heat shock, hypoxia stress, and stationary-phase growth. Regulons associated with Anr-mediated hypoxia stress, RpoS-regulated stationary-phase growth, and osmotic stress were significantly enriched in the set of genes induced in the biofilm. Mutant strains deficient in rpoS, relA and spoT, or anr were cultured in biofilms and challenged with ciprofloxacin and tobramycin. When challenged with ciprofloxacin, the mutant strain biofilms had 2.4- to 2.9-log reductions in viable cells compared to a 0.9-log reduction of the wild-type strain. Interestingly, none of the mutants exhibited a statistically significant alteration in tobramycin susceptibility compared to that with the wild-type biofilm. These results are consistent with a model in which multiple genes controlled by overlapping starvation or stress responses contribute to the protection of a P. aeruginosa biofilm from ciprofloxacin. A distinct and as yet undiscovered mechanism protects the biofilm bacteria from tobramycin.

Response of intertidal benthic microalgal biofilms to a coupled light-temperature stress: evidence for latitudinal adaptation along the Atlantic coast of Southern Europe.

Although estuarine microphytobenthos (MPB) is frequently exposed to excessive light and temperature conditions, little is known on their interactive effects on MPB primary productivity. Laboratory and in situ experiments were combined to investigate the short-term joint effects of high light (HL) and high temperature (37 °C versus 27 °C) on the operating efficiency of photoprotective processes [vertical migration versus non-photochemical quenching (NPQ)] exhibited by natural benthic diatom communities from two intertidal flats in France (FR) and Portugal (PT). A clear latitudinal pattern was observed, with PT biofilms being more resistant to HL stress, regardless the effect of temperature, and displaying a lower relative contribution of vertical migration to photoprotection and a stronger NPQ in situ. However, higher temperature leads to comparable effects, with photoinhibition increasing to about three times (i.e. from 3% to 10% and from 8% to 22% in PT and FR sites respectively). By using a number of methodological novelties in MPB research (lipid peroxidation quantification, Lhcx proteins immunodetection), this study brings a physiological basis to the previously reported depression of MPB photosynthetic productivity in summer. They emphasize the joint role of temperature and light in limiting, at least transiently (i.e. during emersion), MPB photosynthetic activity in situ.

Inefficacy of vancomycin and teicoplanin in eradicating and killing Staphylococcus epidermidis biofilms in vitro

Biofilm-associated bacteria display a decreased susceptibility towards antibiotics. Routine assessment of antibiotic susceptibility of planktonic bacteria therefore offers an insufficient prediction of the biofilm response. In this study, in vitro biofilms of eight clinical Staphylococcus epidermidis strains were subjected to treatment with vancomycin, teicoplanin, oxacillin, rifampicin and gentamicin. In addition, the biofilms were subjected to combinations of an antibiotic with rifampicin. The effects on the biofilms were assessed by crystal violet staining to determine the total biofilm biomass, staining with XTT to determine bacterial cell viability, and microscopy. Combining these methods showed that treatment of S. epidermidis biofilms with glycopeptides increased the total biofilm biomass and that these antibiotics were not effective in killing bacteria embedded in biofilms. The decreased killing efficacy was more pronounced in biofilms produced by strains that were classified as ‘strong’ biofilm producers. Rifampicin, oxacillin and gentamicin effectively killed biofilm-associated bacteria of all tested strains. Combining antibiotics with rifampicin increased the killing efficacy without influencing the total biofilm biomass. When vancomycin or teicoplanin were combined with rifampicin, the increase in biofilm biomass was neutralised and also the killing efficacy was influenced in a positive way. We conclude that the combined methodology used in this study showed that glycopeptides were not effective in eradicating S. epidermidis biofilms but that combination with rifampicin improved the killing efficacy in vitro.

Pharmaceuticals and personal care products alter growth and function in lentic biofilms

Environmental context Pharmaceuticals and personal care products are routinely found in waters discharged from treatment plants and in surrounding aquatic ecosystems. Despite the widespread occurrence of these biologically active agents, there is limited understanding of their potential effects on key ecosystem processes such as primary production, ecosystem respiration and algal growth. This paper examines the effects of five common pharmaceuticals on the rates of these fundamental processes. Abstract Pharmaceutical diffusing substrates were used to study in situ responses of aquatic biofilms in an urbanised lentic ecosystem to five pharmaceutical and personal care products (PPCPs; caffeine, cimetidine, ciprofloxacin, diphenhydramine and metformin). The pharmaceutical diffusing substrates consisted of porous biofilm substrates placed atop a mass of agar amended with 2.5mM of the PPCP compound of interest. Over 21 days, biofilms growing on the substrata were exposed to slow diffusion of the PPCP through the agar and porous substrate. Algal biomass was suppressed by exposure to diphenhydramine (–81%) and ciprofloxacin (–50%). Gross primary production was completely suppressed by diphenhydramine exposure but stimulated by caffeine (+39%) and cimetidine (+46%). For heterotroph biofilms, community respiration was suppressed by exposure to diphenhydramine (–24%). To characterise PPCP exposure, rates of diffusion from the pharmaceutical diffusing substrates were measured at 10, 20 and 30°C. Diffusion was Fickian for all compounds and all temperatures. Diffusion coefficients, D, were in the range 1.5×10–10 to 1.1×10–9m2s–1. From diffusion data, average release rates over 21 days were typically 30–50ngmin–1cm–2 at 20°C. The results show that PPCPs can dramatically affect rates of key ecological processes, and the relationship between release rate and ambient concentration of PPCPs is discussed.

Co-expressional conservation in virulence and stress related genes of three Gammaproteobacterial species: Escherichia coli, Salmonella enterica and Pseudomonas aeruginosa.

Gene co-expression analysis is one of the main aspects of systems biology that uses high-throughput gene expression data. In the present study we applied cross-species co-expressional analysis on a module of biofilm and stress response associated genes. We addressed different kinds of stresses in three most intensively studied members of Gammaproteobacteria including Escherichia coli K12, Pseudomonas aeruginosa PAO1 and Salmonella enterica for which large sets of gene expression data are available. Our aim was to evaluate the presence of common stress response strategies adopted by these microorganisms that may be assigned to the other members of Gammaproteobacteria. Results of functional annotation analysis revealed distinct categories among co-expressed genes, most of which concerned biological processes associated with virulence and stress response. Transcriptional regulatory analysis of genes present in co-expressed modules showed that the global stress sigma factor, RpoS, besides several local transcription factors accounts for the observed co-expressional response, and that several cases of feed-forward loops exist between global regulators, local transcription factors and their targets. Our results lend partial support to our underlying assumption of the conservation of core biological processes and regulatory interactions among these related Gammaproteobacteria members. This has led to the implementation of transferring gene function annotations from well-studied Gammaproteobacterial species to less-characterized members. These findings can shed light on the discovery of new drug targets capable of controlling severe infections caused by these groups of bacteria.

Protein-repellent and antibacterial dental composite to inhibit biofilms and caries

ObjectivesBiofilm acids contribute to secondary caries, which is a main reason for dental restoration failures. The objectives of this study were to: (1) develop a protein-repellent and antibacterial composite, and (2) investigate the effects of combining 2-methacryloyloxyethyl phosphorylcholine (MPC) with quaternary ammonium dimethylaminohexadecyl methacrylate (DMAHDM) on composite mechanical properties and biofilm response for the first time. MethodsMPC, DMAHDM and glass particles were mixed into a dental resin composite. Mechanical properties were measured in three-point flexure. Protein adsorption onto the composites was measured by a micro bicinchoninic acid method. A human saliva microcosm model was used to grow biofilms on composites. Colony-forming unit (CFU) counts, live/dead assay, metabolic activity, and lactic acid production of biofilms were determined. ResultsIncorporation of 3% MPC and 1.5% DMAHDM into composite achieved protein-repellent and antibacterial capabilities without compromising the mechanical properties. Composite with 3% MPC+1.5% DMAHDM had protein adsorption that was 1/10 that of a commercial composite (p<0.05). The composite with 3% MPC+1.5% DMAHDM had much greater reduction in biofilm growth than using MPC or DMAHDM alone (p<0.05). Biofilm CFU counts on composite with 3% MPC+1.5% DMAHDM were more than three orders of magnitude lower than that of commercial control. ConclusionsDental composite with a combination of strong protein-repellent and antibacterial capabilities was developed for the first time. Composite containing MPC and DMAHDM greatly reduced biofilm growth and lactic acid production, without compromising mechanical properties of the composite.Clinical significance: Novel composite with MPC and DMAHDM greatly reduced biofilm activity and is promising to inhibit secondary caries. The dual agents of MPC plus DMAHDM may have wide applicability to other dental materials.

Community structure and nutrient level control the tolerance of autotrophic biofilm to silver contamination.

Autotrophic biofilms are complex and fundamental biological compartments of many aquatic ecosystems. Since microbial species differ in their sensitivity to stressors, biofilms have long been proposed for assessing the quality of aquatic ecosystems. Among the many stressors impacting aquatic ecosystems, eutrophication and metal pollution are certainly the most common. Despite that these stressors often occur together, their effects on biofilms have been far much studied separately than interactively. In this study, we evaluated the interactive effects of silver (Ag), a reemerging contaminant, and phosphorus (P), a nutrient often associated with freshwater eutrophication, on the structure and functioning of two types of autotrophic biofilms, one dominated by diatoms and another one dominated by cyanobacteria. We hypothesized that P would alleviate the toxic effects of Ag, either directly, through the contribution of P in metal detoxification processes, or indirectly, through P-mediated shifts in biofilm community compositions and associated divergences in metal tolerance. Results showed that Ag impacted biofilm community structure and functioning but only at unrealistic concentrations (50μg/L). P availability led to significant shifts in biofilm community composition, these changes being more pronounced in diatom- than those in cyanobacteria-dominated biofilm. In addition, P tended to reduce the impact of Ag but only for the cyanobacteria-dominated biofilm. More generally, our results highlight the preponderant role of the initial community structure and nutrient level on biofilm response to metallic pollutants.

Synthetic glycolipids and (p)ppGpp analogs: development of inhibitors for mycobacterial growth, biofilm and stringent response.

Bacterial pathogens are the major cause of mortality across the globe, as current use of antibiotics and vaccines is unable to prevent the spread of bacterial pathogens. Rapid evolution allowed bacteria to overcome the hostile environmental conditions. Survival strategies, such as, biofilm formation, stringent response, sporulation, cyst formation and horizontal transfer of resistance genes have made it even more difficult to treat the bacterial disease. Rate of bacterial evolution has subdued the pace of discovery of new antibiotics. Most of the antibiotics target the lag and log phases of bacterial growth. It is realized that for complete omission of bacterial pathogens, survival strategies have to be restricted. Under stress, bacteria give rise to stringent response which initiates various signalling cascades escalating the activation of different survival strategies, including biofilm formation. Synthetic glycolipids and (p)ppGpp analogs hold promise to potential therapeutics for impeding the survival strategies. We have noticed earlier that various analogs of stringent response modulator (p)ppGpp hold promise for therapeutic intervention during stress. In addition, several small molecules, which are overproduced during biofilm formation, may also be targeted. In this article, such attempts are discussed.

Surface display of roGFP for monitoring redox status of extracellular microenvironments in Shewanella oneidensis biofilms.

Biofilms are the most ubiquitous and resilient form of microbial life on earth. One most important feature of a biofilm is the presence of a self-produced matrix, which creates highly heterogeneous and dynamic microenvironments within biofilms. Redox status in biofilm microenvironments plays a critical role in biofilm development and function. However, there is a lack of non-intrusive tools to quantify extracellular redox status of microenvironments within a biofilm matrix. In this study, using Shewanella oneidensis as a model organism, we demonstrated a novel approach to monitor extracellular redox status in biofilm microenvironments. Specifically, we displayed a redox sensitive fluorescence protein roGFP onto the cell surface of S. oneidensis by fusing it to the C-terminus of BpfA, a large surface protein, and used the surface displayed roGFP as a sensor to quantify the extracellular redox status in the matrix of S. oneidensis biofilms. The fusion of roGFP into BpfA has no negative impacts on cell growth and biofilm formation. Upon exposure to oxidizing agents such as H2 O2 , Ag(+) , and SeO3 (2-) , S. oneidensis BpfA-roGFP cells exhibited a characteristic fluorescence of roGFP. Proteinase treatment assay and super-resolution structured illumination microscopy confirmed the surface localization of BpfA-roGFP. We further used the surface displayed roGFP monitored the extracellular redox status in the matrix at different depths of a biofilm exposed to H2 O2 . This study provides a novel approach to non-invasively monitor extracellular redox status in microenvironments within biofilms, which can be used to understand redox responses of biofilms to environmental perturbations.

Influence of the natural growth environment on the sensitivity of phototrophic biofilm to herbicide

Ecotoxicological experiments were performed in laboratory-scale microcosms to investigate community-level structural responses of river phototrophic biofilms from different environments to herbicide exposure. Biofilms were initially cultivated on artificial supports placed in situ for 4weeks at two sites, site M, located in an agricultural watershed basin and site S, located in a forested watershed basin. The biofilms were subsequently transferred to microcosms and, after an acclimatisation phase of 7days wereexposed to alachlor at 10 and 30μgL(-1) for 23days. Alachlor effects were assessed by a combination of structural parameters, including biomass (ash-free dry mass and chlorophyll a), molecular fingerprinting of the bacterial community (polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE)) and diatom species composition. Alachlor impacted the chlorophyll a and ash-free dry mass levels of phototrophic biofilms previously cultivated at site S. The structural responses of bacterial and diatom communities were difficult to distinguish from changes linked to the microcosm incubation period. Phototrophic biofilms from site S exposed at 30μgL(-1) alachlor were characterised by an increase of Achnanthidium minutissimum (K-z.) Czarnecki abundance, as well as a higher proportion of abnormal frustules. Thus, phototrophic biofilms with different histories, exhibited different responses to alachlor exposure demonstrating the importance of growth environment. These observations also confirm the problem of distinguishing changes induced by the stress of pesticide toxicity from temporal evolution of the community in the microcosm.

Foreign Body Infection Models to Study Host-Pathogen Response and Antimicrobial Tolerance of Bacterial Biofilm.

The number of implanted medical devices is steadily increasing and has become an effective intervention improving life quality, but still carries the risk of infection. These infections are mainly caused by biofilm-forming staphylococci that are difficult to treat due to the decreased susceptibility to both antibiotics and host defense mechanisms. To understand the particular pathogenesis and treatment tolerance of implant-associated infection (IAI) animal models that closely resemble human disease are needed. Applications of the tissue cage and catheter abscess foreign body infection models in the mouse will be discussed herein. Both models allow the investigation of biofilm and virulence of various bacterial species and a comprehensive insight into the host response at the same time. They have also been proven to serve as very suitable tools to study the anti-adhesive and anti-infective efficacy of different biomaterial coatings. The tissue cage model can additionally be used to determine pharmacokinetics, efficacy and cytotoxicity of antimicrobial compounds as the tissue cage fluid can be aspirated repeatedly without the need to sacrifice the animal. Moreover, with the advance in innovative imaging systems in rodents, these models may offer new diagnostic measures of infection. In summary, animal foreign body infection models are important tools in the development of new antimicrobials against IAI and can help to elucidate the complex interactions between bacteria, the host immune system, and prosthetic materials.