In situ detection and characterization of potable water biofilms on materials by microscopic, spectroscopic and electrochemistry methods

We studied biofilm formation on stainless steel occurring in a drinking water distribution system which operated in parallel at 20 and 37 °C, in order to focus on the effect of temperature rather than on other operational and water quality parameters. A surface conditioning step was followed as a function of time on this material until adhesion of bacterial colonies by using microscopic methods: scanning electron microscopy (SEM) and atomic force microscopy (AFM); a spectroscopic method: polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS), and an electrochemical method: rotating disk electrode (RDE). Correlations between surface analysis, the duration of immersion of the sample and the influence of temperature have been identified clearly before bacterial adhesion. In cold water, these results showed an initial conditioning step of surface occurring during the first 8 days, with detection of superficial acidic functions grafted on surface, until adsorption of proteins. After 12 days, formation of independent bacteria microcolonies, reaching 2-3 μm in length was observed. In tepid water, the first step was reduced to 2 days during which carbonates, acidic functions, and proteins were detected. After 90 days, the biofilm entered in a stable population state, which appeared as a bacteria rich film, including possibly Legionella. The spatial variation of the biofilm was limited as deduced from the thickness determination (about 4 μm for 3-month period), using a RDE. The combination of these different techniques confirms successive steps for biofilm formation on stainless steel in a tap water. Then, we scrutinized the external near environment of bacteria including extracellular polymeric substances (EPS) and then further characterize the morphology of dominant bacteria (shape, size, flagellum) on gold substrate by AFM in air.     

Three-Dimensional Stratification of Bacterial Biofilm Populations in a Moving Bed Biofilm Reactor for Nitritation-Anammox

Moving bed biofilm reactors (MBBRs) are increasingly used for nitrogen removal with nitritation-anaerobic ammonium oxidation (anammox) processes in wastewater treatment. Carriers provide protected surfaces where ammonia oxidizing bacteria (AOB) and anammox bacteria form complex biofilms. However, the knowledge about the organization of microbial communities in MBBR biofilms is sparse. We used new cryosectioning and imaging methods for fluorescence in situ hybridization (FISH) to study the structure of biofilms retrieved from carriers in a nitritation-anammox MBBR. The dimensions of the carrier compartments and the biofilm cryosections after FISH showed good correlation, indicating little disturbance of biofilm samples by the treatment. FISH showed that Nitrosomonas europaea/eutropha-related cells dominated the AOB and Candidatus Brocadia fulgida-related cells dominated the anammox guild. New carriers were initially colonized by AOB, followed by anammox bacteria proliferating in the deeper biofilm layers, probably in anaerobic microhabitats created by AOB activity. Mature biofilms showed a pronounced three-dimensional stratification where AOB dominated closer to the biofilm-water interface, whereas anammox were dominant deeper into the carrier space and towards the walls. Our results suggest that current mathematical models may be oversimplifying these three-dimensional systems and unless the multidimensionality of these systems is considered, models may result in suboptimal design of MBBR carriers.     

Acetate to enhance electrochemical activity of biofilms from garden compost

Dimensionally Stable Anodes embedded in garden compost and maintained under constant polarization at 0.50 V/SCE for several days progressively became covered by a microbial biofilm that gave them the capability to oxidize the organic matter contained in the compost. The effect of acetate supply on the electrochemical activity of biofilms was investigated either by adding acetate after biofilm formation or mixing it into the compost initially. Addition of acetate allowed the current density values to increase up to 545 mA/m². Six individually monitored electrodes set up in the same reactor showed very good reproducibility, indicating that discrepancies observed between the different experiments were mainly due the different batches of compost. A numerical treatment of the epifluorescent microscopy pictures allowed the biofilm coverage ratios to be assessed. Comparing the variations of current density during chronoamperometry with the biofilm surface coverage ratios and with the current obtained by cyclic voltammetry led us to propose a mechanism based on two different steps that corresponded to different time-scales: slow acetate oxidation through the cell metabolism and fast electron transfer between the cell and the electrode surface.     

Dental Chatter: Bacterial Cross-Talk in the Biofilm of the Oral Cavity

Dental biofilms are composed of hundreds of bacterial species. These biofilms are diverse biological structures due to the heterogeneity of the many different types of supports in the oral cavity. The bacteria immobilized in these biofilms are exposed to rapid environmental changes such as pH, temperature, nutrition and anti-plaque agents. One mode in which these bacteria adapt in the dental biofilm is by quorum sensing. This cell-cell communication regulates diverse sets of adhesion modes, physiological changes, virulence properties, allowing the bacteria to persist in the dental biofilm under rapid environmental changes. In this review, we will concentrate mostly on the cariogenic bacterium Streptococcus mutans as one of the pivotal microorganisms in the supra-gingival biofilm that plays a major role in dental caries.     

Anti-Biofilm Performance of Three Natural Products against Initial Bacterial Attachment

Marine bacteria contribute significantly towards the fouling consortium, both directly (modern foul release coatings fail to prevent “slime” attachment) and indirectly (biofilms often excrete chemical cues that attract macrofouling settlement). This study assessed the natural product anti-biofilm performance of an extract of the seaweed, Chondrus crispus, and two isolated compounds from terrestrial sources, (+)-usnic acid and juglone, against two marine biofilm forming bacteria, Cobetia marina and Marinobacter hydrocarbonoclasticus. Bioassays were developed using quantitative imaging and fluorescent labelling to test the natural products over a range of concentrations against initial bacterial attachment. All natural products affected bacterial attachment; however, juglone demonstrated the best anti-biofilm performance against both bacterial species at a concentration range between 5–20 ppm. In addition, for the first time, a dose-dependent inhibition (hormetic) response was observed for natural products against marine biofilm forming bacteria.     

Investigation and Modeling of Growth,Structure and Oxygen Penetration in Particle Supported Biofilms

Particle supported biofilms have been investigated with respect to biofilm formation, substrate transport and utilization. The investigated autotrophic and heterotrophic biofilms were cultivated in airlift suspension reactors. CLSM was used to describe the biofilm structure by recording volumes of bacteria and EPS glycoconjugates. Additionally, the microelectrode technique was used to measure transport and substrate utilization in the biofilm system. The experimental results on the microscopic scale were used to improve a mathematical model for biofilm growth. The oxygen profiles measured in the particle supported biofilms and the data from CLSM were used to optimize the model parameters.     

Cell-Wall-Targeting Antibiotics Cause Lag-Phase Bacteria to Form Surface-Mediated Filaments Promoting the Formation of Biofilms and Aggregates

Antibiotics are known to promote bacterial formation of enhanced biofilms, the mechanism of which is not well understood. Here, using biolayer interferometry, we have shown that bacterial cultures containing antibiotics that target cell walls cause biomass deposition on surfaces over time with a linear profile rather than the Langmuir-like profiles exhibited by bacterial adherence in the absence of antibiotics. We observed about three times the initial rate and 12 times the final biomass deposition on surfaces for cultures containing carbenicillin than without. Unexpectedly, in the presence of antibiotics, the rate of biomass deposition inversely correlated with bacterial densities from different stages of a culture. Detailed studies revealed that carbenicillin caused faster growth of filaments that were seeded on surfaces from young bacteria (from lag phase) than those from high-density fast-growing bacteria, with rates of filament elongation of about 0.58 and 0.13 μm min⁻¹, respectively. With surfaces that do not support bacterial adherence, few filaments were observed even in solution. These filaments aggregated in solution and formed increased amounts of biofilms on surfaces. These results reveal the lifestyle of antibiotic-induced filamentous bacteria, as well as one way in which the antibiotics promote biofilm formation.     

A whole cell electrochemical biosensor for water genotoxicity bio-detection

This work presents a novel micro-fluidic whole cell biosensor for water toxicity analysis. The biosensor presented here is based on bacterial cells that are genetically “tailored” to generate a sequence of biochemical reactions that eventually generate an electrical signal in the presence of genotoxicants. The bacterial assay was affected by toxicant contaminated water for an induction time that ranged between 30 min and 120 min. Enzymatic substrate (pAPP) was added to the assay generating the electrochemical active material (pAP) only when toxicants are sensed by the bacteria. The bacteria were integrated onto a micro-chip that was manufactured by MEMS technology and comprises various micro-chambers with volume ranging between 2.5 nl and 157 nl with electrode radius between 37.5 μm and 300 μm. We describe the biochip operation, its electrochemical response to calibration solutions as well as to the whole cell assays. The potential use of the whole cell biochip for toxicity detection of two different genotoxicants, nalidixic acid (NA) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), is demonstrated. We demonstrate minimal toxicant detection of 10 μg/ml for NA using 30 min for induction and 0.31 μM for IQ using 120 min for induction, both 3 min after the addition of the substrate material.     

SARS-CoV-2 Spike Protein and Mouse Coronavirus Inhibit Biofilm Formation by Streptococcus pneumoniae and Staphylococcus aureus

The presence of co-infections or superinfections with bacterial pathogens in COVID-19 patients is associated with poor outcomes, including increased morbidity and mortality. We hypothesized that SARS-CoV-2 and its components interact with the biofilms generated by commensal bacteria, which may contribute to co-infections. This study employed crystal violet staining and particle-tracking microrheology to characterize the formation of biofilms by Streptococcus pneumoniae and Staphylococcus aureus that commonly cause secondary bacterial pneumonia. Microrheology analyses suggested that these biofilms were inhomogeneous soft solids, consistent with their dynamic characteristics. Biofilm formation by both bacteria was significantly inhibited by co-incubation with recombinant SARS-CoV-2 spike S1 subunit and both S1 + S2 subunits, but not with S2 extracellular domain nor nucleocapsid protein. Addition of spike S1 and S2 antibodies to spike protein could partially restore bacterial biofilm production. Furthermore, biofilm formation in vitro was also compromised by live murine hepatitis virus, a related beta-coronavirus. Supporting data from LC-MS-based proteomics of spike–biofilm interactions revealed differential expression of proteins involved in quorum sensing and biofilm maturation, such as the AI-2E family transporter and LuxS, a key enzyme for AI-2 biosynthesis. Our findings suggest that these opportunistic pathogens may egress from biofilms to resume a more virulent planktonic lifestyle during coronavirus infections. The dispersion of pathogens from biofilms may culminate in potentially severe secondary infections with poor prognosis. Further detailed investigations are warranted to establish bacterial biofilms as risk factors for secondary pneumonia in COVID-19 patients.     

Nutrient Sensing and Biofilm Modulation: The Example of L-arginine in Pseudomonas

Bacterial biofilm represents a multicellular community embedded within an extracellular matrix attached to a surface. This lifestyle confers to bacterial cells protection against hostile environments, such as antibiotic treatment and host immune response in case of infections. The Pseudomonas genus is characterised by species producing strong biofilms difficult to be eradicated and by an extraordinary metabolic versatility which may support energy and carbon/nitrogen assimilation under multiple environmental conditions. Nutrient availability can be perceived by a Pseudomonas biofilm which, in turn, readapts its metabolism to finally tune its own formation and dispersion. A growing number of papers is now focusing on the mechanism of nutrient perception as a possible strategy to weaken the biofilm barrier by environmental cues. One of the most important nutrients is amino acid L-arginine, a crucial metabolite sustaining bacterial growth both as a carbon and a nitrogen source. Under low-oxygen conditions, L-arginine may also serve for ATP production, thus allowing bacteria to survive in anaerobic environments. L-arginine has been associated with biofilms, virulence, and antibiotic resistance. L-arginine is also a key precursor of regulatory molecules such as polyamines, whose involvement in biofilm homeostasis is reported. Given the biomedical and biotechnological relevance of biofilm control, the state of the art on the effects mediated by the L-arginine nutrient on biofilm modulation is presented, with a special focus on the Pseudomonas biofilm. Possible biotechnological and biomedical applications are also discussed.     

Electrochemical activity of Geobacter sulfurreducens biofilms on stainless steel anodes

Stainless steel was studied as anode for the biocatalysis of acetate oxidation by biofilms of Geobacter sulfurreducens. Electrodes were individually polarized at different potential in the range −0.20 V to +0.20 V vs. Ag/AgCl either in the same reactor or in different reactors containing acetate as electron donor and no electron acceptor except the working electrode. At +0.20 V vs. Ag/AgCl, the current increased after a 2-day lag period up to maximum current densities around 0.7 A m⁻² and 2.4 A m⁻² with 5 mM and 10 mM acetate, respectively. No current was obtained during chronoamperometry (CA) at potential values lower than 0.00 V vs. Ag/AgCl, while the cyclic voltammetries (CV) that were performed periodically always detected a fast electron transfer, with the oxidation starting around −0.25 V vs. Ag/AgCl. Epifluorescent microscopy showed that the current recorded by chronoamperometry was linked to the biofilm growth on the electrode surface, while CVs were more likely linked to the cells initially adsorbed from the inoculum. A model was proposed to explain the electrochemical behaviour of the biofilm, which appeared to be controlled by the pioneering adherent cells playing the role of “electrochemical gate” between the biofilm and the electrode surface.     

Novel Strategies for the Prevention and Treatment of Biofilm Related Infections

Biofilm formation by human bacterial pathogens on implanted medical devices causes major morbidity and mortality among patients, and leads to billions of dollars in healthcare cost. Biofilm is a complex bacterial community that is highly resistant to antibiotics and human immunity. As a result, novel therapeutic solutions other than the conventional antibiotic therapies are in urgent need. In this review, we will discuss the recent research in discovery of alternative approaches to prevent or treat biofilms. Current anti-biofilm technologies could be divided into two groups. The first group focuses on targeting the biofilm forming process of bacteria based on our understanding of the molecular mechanism of biofilm formation. Small molecules and enzymes have been developed to inhibit or disrupt biofilm formation. Another group of anti-biofilm technologies focuses on modifying the biomaterials used in medical devices to make them resistant to biofilm formation. While these novel anti-biofilm approaches are still in nascent phases of development, efforts devoted to these technologies could eventually lead to anti-biofilm therapies that are superior to the current antibiotic treatment.     

Decompostion of pharmaceuticals (sulfamethazine and sulfathiazole) using oxygen-based membrane biofilm reactor

The subject of this research was the decomposition of pharmaceuticals (sulfamethazine and sulfathiazole) using an oxygen-based membrane biofilm reactor. The influent concentrations in pharmaceuticals feed-medium were (in ppb): sulfamethazine (40) and sulfathiazole (85). The oxygen-based membrane biofilm reactor system consisted of two membrane modules connected to a recirculation loop. The main membrane module contained a bundle of 32 hydrophobic hollow-fiber membranes inside a polyvinyl-chloride pipe shell, and the other module contained a single fiber used to take biofilm samples. Pure O₂ was supplied to the inside of the hollow fibers through the manifold at the base, and the O₂ pressure for both reactors was 13 kPa. (1 kPa = 0.0099 atm = 0.145 psi). HRT was 3 h. The decomposition ratio of pharmaceuticals (sulfamethazine and sulfathiazole) using oxygen-based membrane biofilm reactor was (%): sulfamethazine (77 ± 2), and sulfathiazole (87 ± 2).     

Combined application of 13C NMR spectroscopy and confocal laser scanning microscopy—Investigation on biofilm structure and physico-chemical properties

Long-term biofilm processes are influenced by the interplay of biofilm accumulation and detachment, which in turn depend partially on the biofilm structure and composition. In this study a combination of confocal laser scanning microscopy (CLSM) and nuclear magnetic resonance (NMR) spectroscopy was applied to analyze biofilm structure, composition and molecular mobility. Whereas CLSM delivers information about the structure of biofilms the NMR measurement provides detailed but not locally resolved information about the chemical composition of biofilm constituents. Heterotrophic mixed-species biofilms were cultivated in rotating annular reactors exposed to different flow conditions and glucose concentrations in order to obtain biofilms with diverse architectural structures. The growth state of the biofilms appeared to influence the composition of biofilm and detached biomass. The difference in the ¹³C NMR spectra between the differently structured biofilms or between biofilm and detached biomass was small, except for the still exponential growing biofilm supplied with the highest glucose concentration. More information was gained from the mobility of specific molecular groups within the biofilm biomass. Molecules within the biofilm biomass of the non-filamentous biofilms were more strongly bound than the molecules within the respective detached biomass. Glucose starvation resulted in a reduction in the biofilm molecular mobility. The opposite was observed in the filamentous biofilm. In this case, the molecular mobility in the biofilm increased after starvation and the molecules in the detached biomass were bound more strongly than in the respective biofilm biomass. It could be shown that the combination of CLSM and ¹³C NMR spectroscopy is a promising approach to analyze the interactions between biofilm architecture, composition or growth state and biofilm detachment.     

Environmental Stimuli Shape Biofilm Formation and the Virulence of Periodontal Pathogens

Periodontitis is a common inflammatory disease affecting the tooth-supporting structures. It is initiated by bacteria growing as a biofilm at the gingival margin, and communication of the biofilms differs in health and disease. The bacterial composition of periodontitis-associated biofilms has been well documented and is under continual investigation. However, the roles of several host response and inflammation driven environmental stimuli on biofilm formation is not well understood. This review article addresses the effects of environmental factors such as pH, temperature, cytokines, hormones, and oxidative stress on periodontal biofilm formation and bacterial virulence.     

Electroactive biofilms of sulphate reducing bacteria

Biofilms formed from a pure strain of Desulfovibrio desulfuricans 27774 on stainless steel and graphite polarised surfaces were studied. The polarisation conditions applied were −0.4 V vs. SCE for different times. A cathodic current related with the biofilms growth was observed with a maximum intensity of −270 mA m⁻² that remained stable for several days using graphite electrodes. These sulphate reducing bacteria biofilms present electrocatalytic activity towards hydrogen and oxygen reduction reactions. Electrode polarisation has a selective effect on the catalytic activity. The biofilms were also observed by scanning electronic microscopy revealing the formation of homogeneous films on the surfaces.     

Accelerated low water corrosion of carbon steel in the presence of a biofilm harbouring sulphate-reducing and sulphur-oxidising bacteria recovered from a marine sediment

Investigations were undertaken to elucidate causes of accelerated low water corrosion (ALWC) of steel piling in a harbour in Southern England. Visual inspection revealed features characteristic of ALWC such as the presence of poorly adherent, thick corrosion products of varying morphology, often seen as large blisters randomly located on sections of the structure at the low water mark. Upon the removal of blisters, a bright surface covered with shallow pits was exposed. Representative samples of the corrosion products were collected from the structure and water and sediment specimens were retrieved from selected areas in the harbour for microbiological, chemical and microscopy testing. In the laboratory, field samples were enriched to detect and enumerate communities of sulphur-oxidising bacteria (SOB) and sulphate-reducing bacteria (SRB). Biofilms, comprising SRB and SOB populations isolated from a sediment sample were grown under static conditions on surfaces of electrodes manufactured from steel piling material. Linear polarisation resistance (LPR) measurements revealed that the corrosion rate of steel with biofilms (0.518 mm y⁻¹) was higher than that recorded in sterile seawater alone (0.054 mm y⁻¹) and in sterile seawater to which nutrient was added (0.218 mm y⁻¹). Scanning electron microscopy (SEM) imaging demonstrated enhanced pitting under biofilms. The results of our investigation revealed for the first time that the attack on steel piling in the presence of sediment SRB and SOB populations was characteristic of ALWC.     

Electroactive biofilms: new means for electrochemistry

This work demonstrates that electrochemical reactions can be catalysed by the natural biofilms that form on electrode surfaces dipping into drinking water or compost. In drinking water, oxygen reduction was monitored with stainless steel ultra-microelectrodes under constant potential electrolysis at −0.30 V/SCE for 13 days. 16 independent experiments were conducted in drinking water, either pure or with the addition of acetate or dextrose. In most cases, the current increased and reached 1.5–9.5 times the initial current. The current increase was attributed to biofilm forming on the electrode in a similar way to that has been observed in seawater. Epifluorescence microscopy showed that the bacteria size and the biofilm morphology depended on the nutrients added, but no quantitative correlation between biofilm morphology and current was established. In compost, the oxidation process was investigated using a titanium based electrode under constant polarisation in the range 0.10–0.70 V/SCE. It was demonstrated that the indigenous micro-organisms were responsible for the current increase observed after a few days, up to 60 mA m⁻². Adding 10 mm acetate to the compost amplified the current density to 145 mA m⁻² at 0.50 V/SCE. The study suggests that many natural environments, other than marine sediments, waste waters and seawaters that have been predominantly investigated until now, may be able to produce electrochemically active biofilms.     

Multiple thermosetting of semicrystalline polymers: Polyamide 66 and poly(ethylene terephthalate) fibers

Polyamide 66 fibers were thermoset in a torsion-bending deformation at various temperatures up to 240 °C. Some of the fibers were heat-set at constant length prior to the deformation at presetting temperatures of 150 °C and 200 °C to vary the structural state of the starting material. Fractional recovery was measured after various combinations of temperature and time. It was found that heat setting of PA66 is dominated by time-dependent stress relaxation exhibiting time-temperature equivalence. Increased crystallinity, and/or other molecular rearrangements occurring during presetting, impose additional constraints on molecular mobility, which delay onset of the flow regime and increase the time constant of relaxation at a given temperature. The thermosetting characteristics of PA66 fibers are very similar to those of poly(ethylene terephthalate) fibers. For both polymers, superposing the curves of fractional recovery vs. setting time at different temperatures produce satisfactory master curves, without the need for vertical shifting of the data. Arrhenius plots yield approximate activation energies for the thermosetting flow process of 35-65 kcal/mol in PA66 and 95-115 kcal/mol in PET.     

Clickable Zwitterionic Copolymer as a Universal Biofilm‐Resistant Coating

Hospital‐acquired infections are often caused by bacterial biofilms on medical devices. To prevent biofilm formation, herein, a universal coating of an antifouling polymer that inhibits the initial adhesion of bacteria is developed. This copolymer is made of methacryloyloxyethyl phosphorylcholine (MPC) and a methacrylate‐substituted dihydrolipoic acid (DHLA) (poly(MPC‐DHLA)). The MPC units provide the antifouling property, while the DHLA units offer cross‐linkable sites via thiol‐ene reactions to form the stable coated copolymer film. Without the requirement for covalent surface grafting, the poly(MPC‐DHLA) coating on various biomedically relevant substrates is investigated, where X‐ray photoelectron spectroscopy, water contact angle measurements, atomic force microscopy, and ellipsometry are used to confirm the success of the surface coating. Moreover, to mimic an actual clinical use, the copolymer coating is applied on a titanium dental substrate and the ability to inhibit biofilm formation by Staphylococcus aureus is quantified and visualized by crystal violet staining and scanning electron microscopy, respectively. As compared with the bare substrate, an effective reduction in bacterial adhesion and suppression of the subsequent biofilm formation is observed on the copolymer‐modified substrate. These features are maintained for up to 7 d indicating the durability as well as universal applicability of this coating approach.