Hydrodynamic deformation and removal of Staphylococcus epidermidis biofilms treated with urea, chlorhexidine, iron chloride, or DispersinB

The force-deflection and removal characteristics of bacterial biofilm were measured by two different techniques before and after chemical, or enzymatic, treatment. The first technique involved time lapse imaging of a biofilm grown in a capillary flow cell and subjected to a brief shear stress challenge imparted through increased fluid flow. Biofilm removal was determined by calculating the reduction in biofilm area from quantitative analysis of transmission images. The second technique was based on micro-indentation using an atomic force microscope. In both cases, biofilms formed by Staphylococcus epidermidis were exposed to buffer (untreated control), urea, chlorhexidine, iron chloride, or DispersinB. In control experiments, the biofilm exhibited force-deflection responses that were similar before and after the same treatment. The biofilm structure was stable during the post-treatment shear challenge (1% loss). Biofilms treated with chlorhexidine became less deformable after treatment and no increase in biomass removal was seen during the post-treatment shear challenge (2% loss). In contrast, biofilms treated with urea or DispersinB became more deformable and exhibited significant biofilm loss during the post-treatment flow challenge (71% and 40%, respectively). During the treatment soak phase, biofilms exposed to urea swelled. Biofilms exposed to iron chloride showed little difference from the control other than slight contraction during the treatment soak. These observations suggest the following interpretations: (1) chemical or enzymatic treatments, including those that are not frankly antimicrobial, can alter the cohesion of bacterial biofilm; (2) biocidal treatments (e.g., chlorhexidine) do not necessarily weaken the biofilm; and (3) biofilm removal following treatment with agents that make the biofilm more deformable (e.g., urea, DispersinB) depend on interaction between the moving fluid and the biofilm structure. Measurements such as those reported here open the door to development of new technologies for controlling detrimental biofilms by targeting biofilm cohesion rather than killing microorganisms.     

Inhibition and eradication of Salmonella Typhimurium biofilm using P22 bacteriophage,EDTA and nisin

Abstract

P22 phage >10⁵ PFU ml^?1 could be used to inhibit Salmonella Typhimurium biofilm formation by 55–80%. Concentrations of EDTA >1.25?mM and concentrations of nisin >1,200?µg ml^?1 were also highly effective in reducing S. Typhimurium biofilm formation (≥96% and ≥95% reductions were observed, respectively). A synergistic effect was observed when EDTA and nisin were combined whereas P22 phage in combination with nisin had no synergistic impact on biofilm formation. Triple combination of P22 phage, EDTA and nisin could be also used to inhibit biofilm formation (≥93.2%) at a low phage titer (10² PFU ml^?1), and low EDTA (1.25?mM) and nisin (9.375?µg ml^?1) concentrations. A reduction of 70% in the mature biofilm was possible when 10⁷ PFU ml^?1 of P22 phage, 20?mM of EDTA and 150?μg ml^?1 of nisin were used in combination. This study revealed that it could be possible to reduce biofilm formation by S. Typhimurium by the use of P22 phage, EDTA and nisin, either alone or in combination. Although, removal of the mature biofilm was more difficult, the triple combination could be successfully used for mature biofilm of S. Typhimurium.     

Continuum heterogeneous biofilm model--a simple and accurate method for effectiveness factor determination

We present a novel analytical approach to describe biofilm processes considering continuum variation of both biofilm density and substrate effective diffusivity. A simple perturbation and matching technique was used to quantify biofilm activity using the steady-state diffusion-reaction equation with continuum variable substrate effective diffusivity and biofilm density, along the coordinate normal to the biofilm surface. The procedure allows prediction of an effectiveness factor, η, defined as the ratio between the observed rate of substrate utilization (reaction rate with diffusion resistance) and the rate of substrate utilization without diffusion limitation. Main assumptions are that (i) the biofilm is a continuum, (ii) substrate is transferred by diffusion only and is consumed only by microorganisms at a rate according to Monod kinetics, (iii) biofilm density and substrate effective diffusivity change in the x direction, (iv) the substrate concentration above the biofilm surface is known, and (v) the substratum is impermeable. With this approach one can evaluate, in a fast and efficient way, the effect of different parameters that characterize a heterogeneous biofilm and the kinetics of the rate of substrate consumption on the behavior of the biological system. Based on a comparison of η profiles the activity of a homogeneous biofilm could be as much as 47.8% higher than that of a heterogeneous biofilm, under the given conditions. A comparison of η values estimated for first order kinetics and η values obtained by numerical techniques showed a maximum deviation of 1.75% in a narrow range of modified Thiele modulus values. When external mass transfer resistance, is also considered, a global effectiveness factor, η(0) , can be calculated. The main advantage of the approach lies in the analytical expression for the calculation of the intrinsic effectiveness factor η and its implementation in a computer program. For the test cases studied convergence was achieved quickly after four or five iterations. Therefore, the simulation and scale-up of heterogeneous biofilm reactors can be easily carried out.     

细菌生物膜的结构及形成机制研究进展

细菌生物膜是细菌在特定条件下形成的一种特殊细菌群体结构,菌体被包裹在其自身分泌的多聚物中。近年来,有关生物膜组成结构、形成机制、抗逆性机制及其应用防治等诸方面的研究工作进展迅速,本文主要针对细菌生物膜的结构及形成机制方面的研究进展进行了介绍。     

On the calculation of the elastic modulus of a biofilm streamer

Biofilm mechanical properties are essential in quantifying the rate of microbial detachment, a key process in determining the function and structure of biofilm systems. Although properties such as biofilm elastic moduli, yield stress and cohesive strength have been studied before, a wide range of values for the biofilm Young's modulus that differ by several orders of magnitude are reported in the literature. In this article, we use experimental data reported in Stoodley et al. [Stoodley et al., Biotechnol Bioeng (1999): 65(1):83-92] and present a methodology for the calculation of Young's modulus, which partially explains the large difference between the values reported in the literature.     

The roles of epithelial cell contact,respiratory bacterial interactions and phosphorylcholine in promoting biofilm formation by Streptococcus pneumoniae and nontypeable Haemophilus influenzae

Streptococcus pneumoniae and nontypeable Haemophilus influenzae (NTHi) often share a common niche within the nasopharynx, both associated with infections such as bronchitis and otitis media. This study investigated how the association between NTHi and S. pneumoniae and the host affects their propensity to form biofilms. We investigated a selection of bacterial strain and serotype combinations on biofilm formation, and the effect of contact with respiratory epithelial cells. Measurement of biofilm showed that co-infection with NTHi and S. pneumoniae increased biofilm formation following contact with epithelial cells compared to no contact demonstrating the role of epithelial cells in biofilm formation. Additionally, the influence of phosphorylcholine (ChoP) on biofilm production was investigated using the licD mutant strain of NTHi 2019 and found that ChoP had a role in mixed biofilm formation but was not the only requirement. The study highlights the complex interactions between microbes and the host epithelium during biofilm production, suggesting the importance of understanding why certain strains and serotypes differentially influence biofilm formation. A key contributor to increased biofilm formation was the upregulation of biofilm formation by epithelial cell factors.     

Ex vivo gingival-biofilm consortia

AIMS: To develop a protocol for harvesting ex vivo samples of gingival-biofilm consortia and to investigate their basic characteristics. METHODS AND RESULTS: Gingival epithelial cells with attached biofilm were collected from healthy subjects by taking a smear. The bacterial viability was estimated via the alteration of the membrane permeability and metabolic activity via the double/single-stranded nucleic acid ratio using a confocal laser-scanning microscope. Morphological analysis was performed by scanning and transmission electron microscopy. Additionally, microbiological estimations were made. The electron microscopy revealed fimbriae-mediated adhesion and the formation of a biofilm matrix. Most bacteria were viable and had a high metabolic activity. CONCLUSIONS: The presented study offers an easy to follow approach for harvesting samples of gingival-biofilm consortia. The latter differs considerably from the supragingival plaque in viability and zonal distribution. Related to free-living and in vitro-grown biofilms, the gingiva-associated biofilm revealed an atypically high metabolic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Biofilm fragments should possess the basic features of the entire gingiva-associated biofilm; which as yet cannot be simulated in vitro. Thus, samples of ex vivo gingival-biofilm consortia can be used to investigate the resistance of oral biofilms against antibiotics and biocides.     

Effective biofilm control in a membrane biofilm reactor using a quenching bacterium (Rhodococcus sp. BH4)

The biofilm thickness in membrane biofilm reactors (MBfRs) is an important factor affecting system performance because excessive biofilm formation on the membrane surface inhibits gas diffusion to the interior of the biofilm, resulting in a significant reduction in the performance of contaminant removal. This study provides innovative insights into the control of biofilm thickness in O₂-based MBfRs by using the quorum quenching (QQ) method. The study was carried out in MBfRs operated at different gas pressures and hydraulic retention times (HRTs) using QQ beads containing Rhodococcus sp. BH4 at different amounts. The highest performance was observed in reactors operated with 0.21 ml QQ bead/cm² membrane surface area, 12 HRTs and 1.40 atm. Over this period, the performance increase in chemical oxygen demand (COD) removal was 25%, while the biofilm thickness on the membrane surface was determined to be 250 μm. Moreover, acetate and equivalent oxygen flux results reached 6080 and 10 640 mg·m⁻²·d⁻¹ maximum values, respectively. The extracellular polymeric substances of the biofilm decreased significantly with the increase of gas pressure and QQ beads amount. Polymerase chain reaction denaturing gradient gel electrophoresis results showed that the microbial community in the MBfR system changed depending on operating conditions and bead amount. The results showed that the QQ method was an effective method to control the biofilm thickness in MBfR and provide insights for future research.     

Mathematical Model and Mechanisms for Biofilm Wastewater Treatment Systems

Summary Fundamental theoretical depiction is still lacking on biofilm wastewater treatment systems up to today. A mathematical model of biofilm wastewater treatment systems, taking account of suspended microorganisms and some factors influencing biofilm formation and stabilization, is developed in this paper. By theoretical and numerical analyses, the factors influencing biofilm formation and stabilization, such as the dilution rate, influent organic concentration, detachment and initial inoculum concentration etc, are discussed. Qualitative investigations were carried out and suggestions on industrial applications are then proposed. This paper not only plays an important role in understanding the physical mechanisms of biofilm dynamics, but also has far-reaching implications for industrial practices.     

Comparative susceptibility of planktonic and 3‐day‐old Salmonella Typhimurium biofilms to disinfectants

Aims: To compare the susceptibility of a 3‐day‐old biofilm and planktonic Salmonella to disinfectants at different exposure times. We hypothesize that Salmonella biofilms are more resilient to disinfectants compared to planktonic Salmonella. Methods and Results: The susceptibility of planktonic cells to disinfectants was tested by a modified version of the Council of Europe suspension test EN 1276. Salmonella biofilms were formed using the Calgary Biofilm Device. Results show that 3‐day‐old Salmonella biofilms are less susceptible to the disinfectants benzalkonium chloride, chlorhexidine gluconate, citric acid, quaternary ammonium compounds, sodium hypochlorite (SH) and ethanol, compared to planktonic Salmonella. Surprisingly, the results also demonstrate that low concentrations of SH were more effective against a 3‐day‐old biofilm compared to high concentrations of SH. Conclusions: While all the disinfectants evaluated were able to reduce biofilm‐associated cells at concentrations and contact times sufficient to eliminate planktonic cells, there were still sufficient viable cells remaining in the biofilm to cause further contamination and potential infection. Significance and Impact of the Study: Protocols for the use of chemical disinfectants need to include biofilm susceptibility testing. There is a requirement for an effective and standardized tool for determining the susceptibility of biofilms to disinfectants.     

Optical method for long-term and large-scale monitoring of spatial biofilm development

A method was developed that allows biofilm monitoring on the square centimeter scale over extended periods of time. The method is based on image acquisition using a desktop scanner and subsequent image analysis. It was shown that results from grey level analysis are highly correlated with physical properties of the biofilm like average biomass and biofilm thickness. The scanner method was applied to monitor overall biofilm growth, detachment, and surface roughness during two 3 and 4 week long experiments. Two significantly different growth dynamics during the biofilm development could be identified, depending on the biofilm history. Surface roughness on transects in flow direction was always higher than on transects perpendicular to the flow, reflecting the anisotropic characteristics of biofilms growing in a flow field.     

Biofilm characteristics and evaluation of the sanitation procedures of thermophilic Aeribacillus pallidus E334 biofilms

The ability of Aeribacillus pallidus E334 to produce pellicle and form a biofilm was studied. Optimal biofilm formation occurred at 60 °C, pH 7.5 and 1.5% NaCl. Extra polymeric substances (EPS) were composed of proteins and eDNA (21.4 kb). E334 formed biofilm on many surfaces, but mostly preferred polypropylene and glass. Using CLSM analysis, the network-like structure of the EPS was observed. The A. pallidus biofilm had a novel eDNA content. DNaseI susceptibility (86.8% removal) of eDNA revealed its importance in mature biofilms, but the purified eDNA was resistant to DNaseI, probably due to its extended folding outside the matrix. Among 15 cleaning agents, biofilms could be removed with alkaline protease and sodium dodecyl sulphate (SDS). The removal of cells from polypropylene and biomass on glass was achieved with combined SDS/alkaline protease treatment. Strong A. pallidus biofilms could cause risks for industrial processes and abiotic surfaces must be taken into consideration in terms of sanitation procedures.     

Gene expression in Bacillus subtilis surface biofilms with and without sporulation and the importance of yveR for biofilm maintenance

Five independent DNA microarray experiments were used to study the gene expression profile of a 5-day Bacillus subtilis air-liquid interface biofilm relative to planktonic cells. Both wild-type B. subtilis and its sporulation mutant (DeltaspoIIGB::erm) were investigated to discern the important biofilm genes (in the presence and absence of sporulation). The microarray results indicated that suspension cells were encountering anaerobic conditions, and the air-liquid interface biofilm was metabolically active. For the statistically significant differential expression (P < 0.05), there were 342 genes induced and 248 genes repressed in the wild-type biofilm, whereas 371 genes were induced and 128 genes were repressed in the sporulation mutant biofilm. The microarray results were confirmed with RNA dot blotting. A small portion of cells (1.5%) in the wild-type biofilm formed spores and sporulation genes were highly expressed. In the biofilm formed by the sporulation mutant, competence genes (comGA, srfAA, srfAB, srfAD, and comS) were induced which indicate a role for quorum sensing (bacterial gene expression controlled by sensing their population) in biofilms. There were 53 genes consistently induced in the biofilms of both the wild-type strain and its spoIIGB mutant-those genes have functions for transport, metabolism, antibiotic production-and 26 genes with unknown functions. Besides the large number of genes with known functions induced in the biofilm (121 genes in the wild-type biofilm and 185 genes in the sporulation mutant biofilm), some genes with unknown functions were also induced (221 genes in the wild-type biofilm and 186 genes in the sporulation mutant biofilm), such as the yve operon which appears to be involved in polysaccharide synthesis and the ybc operon which inhibits the growth of competitors for nutrients. A knockout mutant of yveR was constructed, and the mutant showed major defects in biofilm maintenance. Both the wild-type strain and its sporulation mutant formed normal biofilms, suggesting complete sporulation is not necessary for biofilm formation. The expression profiles of these two strains share more repressed genes than induced genes, suggesting that the biofilm cells repress similar pathways in response to starvation and high cell density.     

Structure and shear strength of microbial biofilms as determined with confocal laser scanning microscopy and fluid dynamic gauging using a novel rotating disc biofilm reactor

The cohesive strength of microbial biofilms cultivated on a rotating disc has been measured using fluid dynamic gauging (FDG). The thickness of heterotrophic mixed culture biofilms was found to depend on substrate concentration and shear force at the biofilm surface during the cultivation. For high substrate concentrations and low shear forces the biofilm thickness increased to several 100 microm within 7 days. Low substrate concentration and higher shear forces yielded thin biofilms of about 100 microm thickness. Independent from cultivation conditions and thickness of the biofilms their cohesive strength ranged between 6.0 and 7.7 N m(-2). The ratio between cohesive strength measured with FDG and shear forces applied during biofilm cultivation have ranged from 200 to 1,100. Higher concentrations of iron in the cultivation media has a positive effect on the stability of the biofilms cultivated. By using the CLSM technique a stable base biofilm with a high amount of stained EPS glycoconjugates could be visualized after gauging. The thickness of the base biofilm was about 100 microm for all biofilms cultivated and was not removable under the applied shear conditions used during FDG.     

Heterogeneity of biofilms in rotating annular reactors: Occurrence, structure, and consequences

A rotating annular reactor (Roto Torque) was used for qualitative and quantitative studied on biofilm heterogeneity. In contrast to the classic image of biofilms as smooth, homogeneous layers of biomass on a substratum, studies using various pure and mixed cultures consistently revealed more-dimensional structures that resembled dunes and ridges, among others. These heterogeneities were categorized and their underlying causes analyzed. Contrary to expectations, motility of the microorganisms not a decisive factor in determining biofilm homogeneity. Small Variations in substratum geometry homogeneity. Small variations in substratum geometry and flow patterns were clearly reflected in the biofilm pattern. Nonhomogeneous flow and shear patterns in the reactor, together with inadequate mixing resulted in significant, position-dependent differences in surface growth. It was therefore not possible to take representative samples of the attached biomass. Like many other types of reactors, the Roto Torque reactor is valuable for qualitative and morphological biofilm experiments but less suitable for quantitative physiological and kinetics studies using attached microorganisms. (c) 1994 John Wiley & Sons, Inc.     

Does biofilm origin matter? Biofilm responses to non-flow period in permanent and temporary streams

1. In some regions, climate change is increasing the variability of rainfall and the frequency of extreme events such as drought. Consequently, non-flow periods have grown in length and frequency, both in temporary and in formerly permanent streams. Water abstraction for human use may further prolong these dry periods.
2. We analysed the resistance and resilience of biofilms from permanent and temporary streams to non-flow conditions. This was achieved by exposing cobbles (collected from permanent and temporary streams) with intact biofilm to 31 days of non-flow, followed by 20 days of stream flow in artificial stream channels. Biofilm resistance and resilience were assessed at a structural (algal biomass, pigment composition, and algae and cyanobacteria composition) and functional level (photosynthetic efficiency and community metabolism).
3. Algal taxa in biofilms from permanent and temporary streams differed throughout the experiment. Biofilms from permanent streams were less resistant to non-flow than those from temporary streams at structural level. Permanent stream biofilms also presented lower resilience at a structural level, but responded similarly to temporary stream biofilms at a functional level.
4. Our investigation shows how the non-flow period disturbed permanent stream biofilms, and suggests that temporary stream biofilms will have greater adaptive capacity as hydroperiod becomes shorter due to climate change.

     

Kinetics of anaerobic methane oxidation coupled to denitrification in the membrane biofilm reactor

Anaerobic oxidation of methane coupled to denitrification (AOM-D) in a membrane biofilm reactor (MBfR), a platform used for efficiently coupling gas delivery and biofilm development, has attracted attention in recent years due to the low cost and high availability of methane. However, experimental studies have shown that the nitrate-removal flux in the CH₄-based MBfR (<1.0 g N/m²-day) is about one order of magnitude smaller than that in the H₂-based MBfR (1.1–6.7 g N/m²-day). A one-dimensional multispecies biofilm model predicts that the nitrate-removal flux in the CH₄-based MBfR is limited to <1.7 g N/m²-day, consistent with the experimental studies reported in the literature. The model also determines the two major limiting factors for the nitrate-removal flux: The methane half-maximum-rate concentration (K₂) and the specific maximum methane utilization rate of the AOM-D syntrophic consortium (k_max2), with k_max2 being more important. Model simulations show that increasing k_max2 to >3 g chemical oxygen demand (COD)/g cell-day (from its current 1.8 g COD/g cell-day) and developing a new membrane with doubled methane-delivery capacity (D_m) could bring the nitrate-removal flux to ≥4.0 g N/m²-day, which is close to the nitrate-removal flux for the H₂-based MBfR. Further increase of the maximum nitrate-removal flux can be achieved when D_m and k_max2 increase together.     

Biofilm formation by ica-positive and ica-negative strains of Staphylococcus epidermidis in vitro

Staphylococcus epidermidis is a clinically important opportunistic pathogen that forms biofilm infections on nearly all types of indwelling medical devices. The biofilm forming capability of S. epidermidis has been linked to the presence of the ica operon in the genome, and the amount of biofilm formation measured by the crystal violet (CV) adherence assay. Six S. epidermidis strains were characterized for their ica status using PCR, and their biofilm forming ability over 6 days, using the CV assay and a flow cell system. Ica-negative strains characterized as ‘negative for biofilm formation’ based on the CV assay were demonstrated to form strongly attached biofilms after 6 days. However, the biofilms were not as extensive as the ica-positive strains. It was concluded that ica is not required for biofilm formation, nor is the 24-h CV assay generalizable for predicting the 6-day biofilm-forming ability for all S. epidermidis strains.